Identification and functional analysis of downy mildew effectors in ...
Identification and functional analysis of downy mildew effectors in ...
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<strong>Identification</strong> <strong>and</strong> <strong>functional</strong> <strong>analysis</strong><br />
<strong>of</strong> <strong>downy</strong> <strong>mildew</strong> <strong>effectors</strong><br />
<strong>in</strong> lettuce <strong>and</strong> Arabidopsis<br />
H 2<br />
N<br />
N<br />
N<br />
H 2<br />
N<br />
N<br />
O<br />
N<br />
N<br />
N<br />
OH<br />
OH<br />
OH<br />
O<br />
O<br />
O<br />
O<br />
O<br />
O<br />
O<br />
P<br />
P<br />
P<br />
O<br />
-<br />
O<br />
O<br />
-<br />
O<br />
O<br />
-<br />
Joost H.M. Stassen
<strong>Identification</strong> <strong>and</strong> <strong>functional</strong> <strong>analysis</strong> <strong>of</strong><br />
<strong>downy</strong> <strong>mildew</strong> <strong>effectors</strong> <strong>in</strong> lettuce <strong>and</strong> Arabidopsis<br />
Joost H. M. Stassen
Cover <strong>and</strong> Layout: J.H.M. Stassen<br />
Pr<strong>in</strong>ted by:<br />
Proefschriftmaken.nl || Pr<strong>in</strong>tyourthesis.com<br />
ISBN: 978-90-393-5843-6
<strong>Identification</strong> <strong>and</strong> <strong>functional</strong> <strong>analysis</strong> <strong>of</strong><br />
<strong>downy</strong> <strong>mildew</strong> <strong>effectors</strong> <strong>in</strong> lettuce <strong>and</strong> Arabidopsis<br />
Identificatie en functionele analyse van<br />
valse meeldauw effectoren <strong>in</strong> sla en Arabidopsis<br />
(met een samenvatt<strong>in</strong>g <strong>in</strong> het Nederl<strong>and</strong>s)<br />
Proefschrift<br />
ter verkrijg<strong>in</strong>g van de graad van doctor aan de Universiteit Utrecht<br />
op gezag van de rector magnificus, pr<strong>of</strong>.dr. G.J. van der Zwaan,<br />
<strong>in</strong>gevolge het besluit van het college voor promoties<br />
<strong>in</strong> het openbaar te verdedigen<br />
op woensdag 24 oktober 2012 des ochtends te 10.30 uur<br />
door<br />
Johannes Hubertus Marie Stassen<br />
geboren op 1 november 1984 te Woerden
Promotor:<br />
Co-promotor:<br />
Pr<strong>of</strong>.dr.ir. C.M.J. Pieterse<br />
Dr. A.F.J.M. van den Ackerveken<br />
Dit proefschrift werd mede mogelijk gemaakt met f<strong>in</strong>anciële steun van het<br />
Technologisch Top<strong>in</strong>stituut Groene Genetica.
Contents<br />
Chapter 1: 7<br />
How do oomycete <strong>effectors</strong> <strong>in</strong>terfere with plant life<br />
Outl<strong>in</strong>e 23<br />
Chapter 2: 25<br />
<strong>Identification</strong> <strong>of</strong> Hyaloperonospora arabidopsidis transcript sequences<br />
expressed dur<strong>in</strong>g <strong>in</strong>fection reveals isolate-specific <strong>effectors</strong><br />
Chapter 3: 63<br />
Effector identification <strong>in</strong> the lettuce <strong>downy</strong> <strong>mildew</strong> Bremia lactucae by<br />
massively parallel transcriptome sequenc<strong>in</strong>g<br />
Chapter 4: 95<br />
Effectors <strong>of</strong> the lettuce <strong>downy</strong> <strong>mildew</strong> Bremia lactucae enhance host<br />
susceptibility<br />
Chapter 5: 131<br />
Two GKLR prote<strong>in</strong>s <strong>of</strong> the lettuce <strong>downy</strong> <strong>mildew</strong> Bremia lactucae <strong>in</strong>duce<br />
effector-triggered immunity<br />
Chapter 6: 165<br />
Discussion<br />
Summary 181<br />
Samenvatt<strong>in</strong>g 183<br />
Acknowledgements 185<br />
Curriculum vitae 187
9<br />
Chapter 1:<br />
How do oomycete <strong>effectors</strong> <strong>in</strong>terfere with<br />
plant life<br />
Joost HM Stassen 1 <strong>and</strong> Guido Van den Ackerveken 1,2<br />
1<br />
Plant-Microbe Interactions, Department <strong>of</strong> Biology, Utrecht University,<br />
Padualaan 8, 3584 CH Utrecht, The Netherl<strong>and</strong>s<br />
2<br />
Centre for BioSystems Genomics, 6700 AB, Wagen<strong>in</strong>gen, The Netherl<strong>and</strong>s<br />
Current Op<strong>in</strong>ion <strong>in</strong> Plant Biology (2011) 14: 407-14<br />
doi: 10.1016/j.pbi.2011.05.002
10 Chapter 1<br />
Introduction<br />
Oomycetes are fungal-like microorganisms that belong to the k<strong>in</strong>gdom Stramenopila<br />
<strong>and</strong> are evolutionary related to brown algae. Many well-known plant<br />
pathogens are oomycetes, rang<strong>in</strong>g from the necrotrophic broad host range Pythium<br />
species, through hemibiotrophic Phytophthora species, to obligate biotrophic<br />
species such as white rust (Albugo) <strong>and</strong> narrow host-range <strong>downy</strong> <strong>mildew</strong>s (e.g.<br />
Hyaloperonospora <strong>and</strong> Bremia species). These oomycete pathogens <strong>in</strong>terfere with<br />
plant life for successful <strong>in</strong>fection, utilis<strong>in</strong>g effector prote<strong>in</strong>s to manipulate their<br />
hosts. The sequenc<strong>in</strong>g <strong>of</strong> the genomes <strong>of</strong> Phytophthora ramorum <strong>and</strong> P. sojae [1] ,<br />
P. <strong>in</strong>festans [2] , Pythium ultimum [3] , <strong>and</strong> Hyaloperonospora arabidopsidis [4] has<br />
advanced the field enormously by enabl<strong>in</strong>g the identification <strong>of</strong> a large number<br />
<strong>of</strong> genes encod<strong>in</strong>g secreted effector prote<strong>in</strong>s <strong>in</strong> this diverse group <strong>of</strong> pathogens<br />
(Figure 1). In Phytophthora species many effector genes are found <strong>in</strong> gene-poor<br />
regions that have recently exp<strong>and</strong>ed, probably driven by repeats (transposons) [2,5] .<br />
These regions are l<strong>in</strong>ked to accelerated gene evolution after host jumps [5] <strong>and</strong> may<br />
expla<strong>in</strong> the evolutionary flexibility <strong>of</strong> these species. Also, more novel comb<strong>in</strong>ations<br />
<strong>of</strong> known prote<strong>in</strong> doma<strong>in</strong>s are present <strong>in</strong> oomycetes than <strong>in</strong> other species with<br />
a similar s<strong>in</strong>gle doma<strong>in</strong> repertoire. These comb<strong>in</strong>ations are enriched <strong>in</strong> secreted<br />
<strong>in</strong>fection-related prote<strong>in</strong>s, suggest<strong>in</strong>g that oomycetes have evolved a unique<br />
toolbox for host manipulation [6,7] .<br />
Figure 1: The lifestyle <strong>of</strong> oomycetes <strong>of</strong> which a full genome sequence is available <strong>and</strong> the number <strong>of</strong><br />
members per family <strong>of</strong> selected effector types reported [1–4] . The presumed evolutionary relationship<br />
between the species is <strong>in</strong>dicated <strong>in</strong> the phylogenetic tree.<br />
Here we discuss (summarised <strong>in</strong> Figure 2) recent advances <strong>in</strong> underst<strong>and</strong><strong>in</strong>g the<br />
roles <strong>of</strong> secreted oomycete <strong>effectors</strong> <strong>in</strong> successful <strong>in</strong>fection <strong>of</strong> host plants.<br />
Clear<strong>in</strong>g the way<br />
In early phases <strong>of</strong> the <strong>in</strong>teraction, the <strong>in</strong>vad<strong>in</strong>g oomycete needs to deal with<br />
biochemical barriers <strong>in</strong> the plant apoplast. Both pathogen <strong>and</strong> host secrete prote<strong>in</strong>s<br />
<strong>and</strong> metabolites to control the extracellular environment. Three types <strong>of</strong> apoplastic<br />
<strong>effectors</strong> from oomycetes have been shown to <strong>in</strong>terfere with plant processes; <strong>in</strong>hib-
How do oomycete <strong>effectors</strong> <strong>in</strong>terfere with plant life<br />
11<br />
Figure 2: Schematic representation <strong>of</strong> how oomycetes <strong>in</strong>terfere with plant life. Details are described<br />
<strong>in</strong> the ma<strong>in</strong> text under the head<strong>in</strong>gs; (a) Clear<strong>in</strong>g the way, (b) Break<strong>in</strong>g <strong>in</strong>, (c) Quell<strong>in</strong>g resistance, <strong>and</strong><br />
(d) Sweet rewards. Prote<strong>in</strong>s produced <strong>in</strong> the oomycete cytoplasm (OC) are secreted over the oomycete<br />
membrane (OM) <strong>and</strong> cell wall (OW). The haustorium (H) has breached the plant cell wall (PW) <strong>and</strong><br />
<strong>in</strong>vag<strong>in</strong>ated the host plasma membrane (PM). RXLR <strong>and</strong> Cr<strong>in</strong>kler <strong>effectors</strong> (RXLRs <strong>and</strong> CRNs) are<br />
secreted from the haustorium <strong>and</strong> arrive, for example <strong>in</strong> the plant cytoplasm (PC) <strong>and</strong> nucleus (N)<br />
where they exert their activity on host cell processes.<br />
itors <strong>of</strong> host enzymes, RGD (Arg<strong>in</strong><strong>in</strong>e–Glyc<strong>in</strong>e–Aspartic acid)-conta<strong>in</strong><strong>in</strong>g prote<strong>in</strong>s,<br />
<strong>and</strong> tox<strong>in</strong>s that lead to host cell death.<br />
Enzyme <strong>in</strong>hibitors counter hydrolytic enzymes (e.g. chit<strong>in</strong>ases, glucanases,<br />
<strong>and</strong> proteases) that are secreted by the host. Plant proteases contribute to pathogen<br />
defence, for example susceptibility <strong>of</strong> Nicotiana benthamiana to P. <strong>in</strong>festans is<br />
<strong>in</strong>creased when the apoplastic protease C14 genes are silenced [8] . Furthermore,<br />
tomato cyste<strong>in</strong>e protease rcr3 mutants are more susceptible to P. <strong>in</strong>festans <strong>in</strong>fection<br />
than wild-type tomato [9] . Inhibit<strong>in</strong>g apoplastic proteases is therefore an effective<br />
virulence strategy <strong>of</strong> pathogens. Rcr3 activity is reduced by the extracellular protease<br />
<strong>in</strong>hibitors EPIC1 <strong>and</strong> EPIC2B, which also <strong>in</strong>hibit other proteases, for example<br />
C14 [8,9] <strong>and</strong> PIP1 [10] . The EPICs show signs <strong>of</strong> recent evolution <strong>in</strong> P. <strong>in</strong>festans [10] ,<br />
<strong>and</strong> also its host target, potato C14, is under diversify<strong>in</strong>g selection [8] , <strong>in</strong>dicat<strong>in</strong>g<br />
these apoplastic <strong>effectors</strong> are locked <strong>in</strong> an evolutionary arms race. Whether<br />
oomycetes secrete <strong>in</strong>hibitors to arm themselves aga<strong>in</strong>st host hydrolytic enzymes
12 Chapter 1<br />
or to protect their secreted prote<strong>in</strong>acious <strong>effectors</strong> from degradation rema<strong>in</strong>s to be<br />
determ<strong>in</strong>ed.<br />
A second type <strong>of</strong> apoplastic <strong>effectors</strong> <strong>in</strong>terferes with adhesion, <strong>and</strong> possibly<br />
signall<strong>in</strong>g, between host cell wall <strong>and</strong> plasma membrane, for example IPI-O <strong>of</strong><br />
P. <strong>in</strong>festans that seems to act at two cellular locations; <strong>in</strong>side the host cell, to<br />
suppress defence, <strong>and</strong> extracellularly. The IPI-O RGD-motif disrupts the adhesion<br />
between cell wall <strong>and</strong> plasma membrane [11] , possibly by b<strong>in</strong>d<strong>in</strong>g to the Arabidopsis<br />
legume-like lect<strong>in</strong> receptor k<strong>in</strong>ase LecRK-I.9 [12] . Arabidopsis lecRK-I.9 mutants<br />
<strong>and</strong> IPI-O overexpression l<strong>in</strong>es are more susceptible to Phytophthora brassicae,<br />
are altered <strong>in</strong> cell wall-<strong>in</strong>tegrity, <strong>and</strong> impaired <strong>in</strong> callose deposition [13] . IPI-O<br />
mediated disruption <strong>of</strong> plasma membrane-cell wall contacts could <strong>in</strong>terfere with<br />
cell-wall-associated defences, thereby promot<strong>in</strong>g <strong>in</strong>fection.<br />
A third type <strong>of</strong> apoplastic <strong>effectors</strong> represents tox<strong>in</strong>s that are produced by<br />
oomycetes that are necrotrophic (e.g. certa<strong>in</strong> Pythium species) or hemibiotrophic<br />
(most Phytophthora species). These tox<strong>in</strong>s act <strong>in</strong> an <strong>of</strong>fensive way by trigger<strong>in</strong>g<br />
host cell death that could favour the necrotrophic phase <strong>of</strong> development. Two<br />
families <strong>of</strong> toxic prote<strong>in</strong>s are encoded <strong>in</strong> the genomes <strong>of</strong> most oomycetes; the<br />
PcF/SCR prote<strong>in</strong>s that are small, secreted, hydroxyprol<strong>in</strong>e-conta<strong>in</strong><strong>in</strong>g prote<strong>in</strong>s,<br />
<strong>and</strong> the NEP1-like prote<strong>in</strong>s (NLPs), that are related to the necrosis <strong>and</strong> ethylene<br />
<strong>in</strong>duc<strong>in</strong>g peptide (NEP1) from the fungus Fusarium oxysporum. NLPs can <strong>in</strong>duce<br />
cell death <strong>in</strong> dicots by act<strong>in</strong>g on the outside <strong>of</strong> the host cell membrane, as shown<br />
for NLP Pp<br />
<strong>of</strong> Phytophthora parasitica [14] . The crystal structure <strong>of</strong> NLP Pya<br />
from the<br />
necrotroph Pythium aphanidermatum revealed structural homology to cytolytic<br />
act<strong>in</strong>opor<strong>in</strong>s [15] , suggest<strong>in</strong>g that NLPs <strong>in</strong>sert <strong>in</strong>to the host membrane to form pores.<br />
The cytolytic activity could be responsible for the necrotic response, however, cell<br />
death <strong>in</strong>duction by NLPs requires host defence signall<strong>in</strong>g <strong>and</strong> active host metabolism<br />
[14,16] . It was surpris<strong>in</strong>g to f<strong>in</strong>d a family <strong>of</strong> NLP genes <strong>in</strong> the obligate biotroph<br />
H. arabidopsidis, as this pathogen is dependent on liv<strong>in</strong>g plant cells <strong>and</strong> does not<br />
<strong>in</strong>duce host cell death. One <strong>of</strong> n<strong>in</strong>e H. arabidopsidis NLPs (HaNLP3) belongs to<br />
the clade <strong>of</strong> necrosis-<strong>in</strong>duc<strong>in</strong>g NLPs, but is not capable <strong>of</strong> <strong>in</strong>duc<strong>in</strong>g cell death [4] .<br />
Also Phytophthora species have many non-cytolytic NLPs that, like HaNLP3,<br />
could have an alternative function. One proposed alternative function is attachment<br />
to the host, as NLP Pya<br />
also shows structural homology to fungal lect<strong>in</strong>s that could<br />
b<strong>in</strong>d surface-exposed compounds such as glucans [15] .<br />
Break<strong>in</strong>g <strong>in</strong><br />
The (hemi)biotrophic oomycetes engage <strong>in</strong> an <strong>in</strong>timate relation with plant cells by<br />
form<strong>in</strong>g haustoria that serve a dual role; nutrient uptake from the host <strong>and</strong> delivery<br />
<strong>of</strong> <strong>effectors</strong> to the host. Haustoria penetrate the host cell wall, <strong>in</strong>vag<strong>in</strong>ate the host
How do oomycete <strong>effectors</strong> <strong>in</strong>terfere with plant life<br />
13<br />
membrane, conta<strong>in</strong> specific membrane prote<strong>in</strong>s required for pathogenicity [17] , <strong>and</strong><br />
have been implicated as a site <strong>of</strong> effector production <strong>and</strong> secretion [18] . Effectors<br />
are secreted from the pathogen, <strong>and</strong> those that carry host-translocation signals are<br />
transported <strong>in</strong>to the plant cell.<br />
Two classes <strong>of</strong> host-translocated <strong>effectors</strong> are currently known <strong>in</strong> oomycetes.<br />
The first, the RXLR-<strong>effectors</strong>, are named after a four am<strong>in</strong>o acid (Arg<strong>in</strong><strong>in</strong>e, any<br />
am<strong>in</strong>o acid, Leuc<strong>in</strong>e, Arg<strong>in</strong><strong>in</strong>e; <strong>in</strong> short: RXLR) motif that was found <strong>in</strong> 2005 to<br />
be common among all then known oomycete avirulence (AVR) prote<strong>in</strong>s, which are<br />
recognised <strong>in</strong>side the host cell [19] . The RXLR-<strong>effectors</strong> have an N-term<strong>in</strong>al doma<strong>in</strong><br />
consist<strong>in</strong>g <strong>of</strong> a signal peptide, an RXLR-like motif, an optional am<strong>in</strong>o acid motif<br />
(consist<strong>in</strong>g <strong>of</strong> two glutamic acid residues <strong>and</strong> an arg<strong>in</strong><strong>in</strong>e residue, <strong>of</strong>ten preceded<br />
by an aspartic acid residue) known as the dEER-motif, <strong>and</strong> a C-term<strong>in</strong>al effector<br />
doma<strong>in</strong>. A stunn<strong>in</strong>g 563 RXLR-<strong>effectors</strong> are predicted for P. <strong>in</strong>festans [2] , whereas<br />
they are absent <strong>in</strong> Pythium ultimum [3] <strong>and</strong> Aphanomyces euteiches [20] , suggest<strong>in</strong>g<br />
these <strong>effectors</strong> have recently evolved with<strong>in</strong> the Peronosporales [3] .<br />
RXLR-<strong>effectors</strong> <strong>of</strong> different species show extensive sequence divergence,<br />
though <strong>in</strong> P. sojae <strong>and</strong> P. ramorum most have been suggested to belong to a s<strong>in</strong>gle<br />
superfamily [21] . There is little overlap <strong>in</strong> the RXLR gene repertoire between the<br />
different sequenced Phytophthora species, but also compared to H. arabidopsidis,<br />
suggest<strong>in</strong>g recent species-specific evolution <strong>and</strong> expansion [21] . Accelerated evolution<br />
<strong>of</strong> effector genes was confirmed by <strong>analysis</strong> <strong>of</strong> genomes <strong>of</strong> four P. <strong>in</strong>festans<br />
sister species [5] .<br />
The RXLR-motif has been shown to be <strong>in</strong>volved <strong>in</strong> translocation <strong>in</strong>to the<br />
host [18,22] . A possible mechanism by which this occurs is by b<strong>in</strong>d<strong>in</strong>g <strong>of</strong> the motif<br />
to phosphatidyl<strong>in</strong>ositol-3-phosphate (PI3P) <strong>and</strong> subsequent lipid-raft dependent<br />
uptake [23] . However, this mechanism <strong>and</strong> its support<strong>in</strong>g experiments are strongly<br />
debated. Other mechanisms <strong>of</strong> b<strong>in</strong>d<strong>in</strong>g/entry <strong>of</strong> <strong>effectors</strong>, for example via other<br />
negatively charged molecules, are be<strong>in</strong>g proposed but are, however, not yet<br />
published. Entry after b<strong>in</strong>d<strong>in</strong>g phospholipids has been shown for <strong>effectors</strong> from<br />
different pathogens, <strong>in</strong>clud<strong>in</strong>g fungi [23] . However, for one <strong>of</strong> the tested fungal<br />
<strong>effectors</strong>, AvrL567, lipid b<strong>in</strong>d<strong>in</strong>g could not be reproduced by another laboratory,<br />
though background b<strong>in</strong>d<strong>in</strong>g <strong>of</strong> phospholipids was observed [24] . Such lipid b<strong>in</strong>d<strong>in</strong>g<br />
assays appear technically dem<strong>and</strong><strong>in</strong>g <strong>and</strong> condition-dependent, warrant<strong>in</strong>g caution<br />
when <strong>in</strong>terpret<strong>in</strong>g their results. Furthermore, lipid b<strong>in</strong>d<strong>in</strong>g properties <strong>of</strong> AvrM<br />
can be separated from the sequence required for uptake [24] . These observations on<br />
AvrL567 <strong>and</strong> AvrM do not support the idea that lipid b<strong>in</strong>d<strong>in</strong>g by the RXLR-like<br />
am<strong>in</strong>o acid motifs is required for translocation <strong>of</strong> fungal <strong>effectors</strong>. Indications<br />
that the RXLR-motif may not be essential for uptake <strong>of</strong> oomycete <strong>effectors</strong> are<br />
found <strong>in</strong> <strong>effectors</strong> <strong>of</strong> Pseudoperonospora cubensis, <strong>in</strong> which the EER-motif has a<br />
more important role than the RXLR-motif or RXLR-like-motif QXLR, which is
14 Chapter 1<br />
prevalent <strong>in</strong> this species [25] . Furthermore ATR5 <strong>of</strong> H. arabidopsidis has no clear<br />
RXLR-motif, but does have an EER-motif <strong>and</strong> is translocated as it is recognised<br />
<strong>in</strong>tracellularly [26] . More examples <strong>of</strong> variants <strong>of</strong> the RXLR-motif or EER-motif<br />
found <strong>in</strong> prote<strong>in</strong>s that can translocate <strong>in</strong>to the host cell will undoubtedly emerge<br />
<strong>in</strong> the near future, enlarg<strong>in</strong>g the known effector arsenal <strong>of</strong> oomycete species even<br />
further.<br />
The second class <strong>of</strong> host <strong>in</strong>tracellular <strong>effectors</strong> encompasses the Cr<strong>in</strong>klers.<br />
These <strong>effectors</strong> have conserved N-term<strong>in</strong>i, which conta<strong>in</strong> a signal peptide, an<br />
LXLFLAK-motif followed by a conserved DWL-doma<strong>in</strong> <strong>and</strong> an HVLVXXP-motif<br />
[2] . The LXLFLAK-motif has been shown to be required for host <strong>in</strong>tracellular<br />
localisation <strong>of</strong> the Cr<strong>in</strong>klers, as this N-term<strong>in</strong>al motif, but not mutated forms,<br />
enable transport <strong>of</strong> the C-term<strong>in</strong>al half <strong>of</strong> AVR3a <strong>in</strong>to the plant cell, where AVR3a<br />
triggers cytoplasmic recognition <strong>in</strong> the presence <strong>of</strong> the resistance prote<strong>in</strong> R3a [27] .<br />
Like the RXLR-<strong>effectors</strong>, the modular C-term<strong>in</strong>al effector doma<strong>in</strong>s <strong>of</strong> the Cr<strong>in</strong>klers<br />
are highly diverse. Several C-term<strong>in</strong>al doma<strong>in</strong>s <strong>of</strong> P. <strong>in</strong>festans Cr<strong>in</strong>klers have been<br />
shown to target the host nucleus, <strong>and</strong>/or <strong>in</strong>duce cell death when expressed with<strong>in</strong><br />
plant cells [2,27] . The rapid evolution <strong>and</strong> expansion <strong>of</strong> genes encod<strong>in</strong>g host-translocated<br />
<strong>effectors</strong> stresses the presumed importance <strong>of</strong> these prote<strong>in</strong>s <strong>in</strong> establish<strong>in</strong>g a<br />
successful <strong>in</strong>fection. The positive selection, which is observed for many <strong>effectors</strong>,<br />
highlights the evolutionary adaptation to overcome the immune system <strong>of</strong> the plant.<br />
Box 1: Plant defence <strong>in</strong> brief<br />
A first l<strong>in</strong>e <strong>of</strong> activated plant defence aga<strong>in</strong>st <strong>in</strong>vad<strong>in</strong>g microorganisms is triggered<br />
by pathogen-associated molecular patterns (PAMPs) that are recognised by pattern<br />
recognition receptors (PRRs), <strong>in</strong>duc<strong>in</strong>g PAMP-triggered immunity (PTI) [45] .<br />
Oomycetes secrete several prote<strong>in</strong>s that can act as PAMPs, for example elicit<strong>in</strong>s,<br />
transglutam<strong>in</strong>ase, <strong>and</strong> cellulose-b<strong>in</strong>d<strong>in</strong>g prote<strong>in</strong>s [46] . Oomycete cell wall derived<br />
fragments, for example β-glucan fragments, can also act as PAMPs [47] . The BAK1<br />
PRR co-receptor is required for immunity to P. <strong>in</strong>festans <strong>in</strong> Nicotiana benthamiana<br />
[48] , however, no plant genes encod<strong>in</strong>g PRRs that detect oomycete PAMPs<br />
have been cloned so far. Pathogens have evolved effector prote<strong>in</strong>s to suppress PTI<br />
<strong>and</strong> have thereby established effector-triggered susceptibility (ETS). Plants have<br />
evolved resistance prote<strong>in</strong>s to recognise these <strong>effectors</strong> or their activity. This second<br />
l<strong>in</strong>e <strong>of</strong> defence, named effector-triggered immunity (ETI), confers race-specific<br />
resistance with<strong>in</strong> plant species <strong>and</strong> can be very effectively phenotyped. This has<br />
been <strong>in</strong>strumental <strong>in</strong> the clon<strong>in</strong>g <strong>of</strong> many oomycete effector genes (see Table 1),<br />
which are also named avirulence (AVR) genes as they confer <strong>in</strong>compatibility on<br />
host plants that carry the correspond<strong>in</strong>g resistance (R) gene.
How do oomycete <strong>effectors</strong> <strong>in</strong>terfere with plant life<br />
15<br />
Quell<strong>in</strong>g resistance<br />
The suppression <strong>of</strong> plant defence, that is PAMP-triggered immunity (PTI) that is<br />
activated upon <strong>in</strong>vasion <strong>of</strong> pathogens (see Box 1), is key to successful <strong>in</strong>fection <strong>of</strong><br />
the host. This is well documented for bacterial pathogens that use host-translocated<br />
(type III-secreted) <strong>effectors</strong> to <strong>in</strong>terfere with host defence responses. It is evident<br />
that oomycete pathogens actively suppress <strong>in</strong>nate immunity too. A strik<strong>in</strong>g example<br />
is the suppression <strong>of</strong> host cell death <strong>in</strong> the Arabidopsis lesion mimic mutant lsd1 by<br />
the white rust pathogen Albugo c<strong>and</strong>ida [28] . Moreover, for many <strong>effectors</strong> that have<br />
been predicted from oomycete genomes a putative defence-suppressive or susceptibility-<strong>in</strong>duc<strong>in</strong>g<br />
activity has been found. However, the molecular mechanisms by<br />
which host-translocated <strong>effectors</strong> <strong>in</strong>terfere with plant life are still largely unknown.<br />
Table 1 gives an overview <strong>of</strong> oomycete host-translocated <strong>effectors</strong> that are active <strong>in</strong><br />
plant cells, either as suppressors or as <strong>in</strong>ducers <strong>of</strong> defence.<br />
A challeng<strong>in</strong>g goal is to determ<strong>in</strong>e the function <strong>of</strong> oomycete <strong>effectors</strong>, as the<br />
availability <strong>of</strong> powerful bioassays is limited. Most currently used assays monitor<br />
the suppression <strong>of</strong> PTI. An elegant method is the use <strong>of</strong> Pseudomonas bacteria for<br />
type III delivery <strong>of</strong> oomycete <strong>effectors</strong> <strong>in</strong> the host. Bacteria that translocate the H.<br />
arabidopsidis ATR1 <strong>and</strong> ATR13 prote<strong>in</strong>s were able to grow to higher densities <strong>in</strong>dicat<strong>in</strong>g<br />
that susceptibility <strong>of</strong> the plant was enhanced [29] . ATR13 suppresses PTI as<br />
transgenic plants showed reduced PAMP-triggered callose deposition [29] . A similar<br />
activity was observed for the RXLR29 prote<strong>in</strong>, that is an isolate-specific effector<br />
<strong>of</strong> H. arabidopsidis [30] . Another method to suppress PTI responses is by Agrobacterium-mediated<br />
transient expression <strong>of</strong> effector genes. Co-expression <strong>of</strong> the P.<br />
<strong>in</strong>festans AVR3a prote<strong>in</strong> was shown to suppress PTI-associated cell death <strong>in</strong>duced<br />
by the elicit<strong>in</strong> INF1 [31] . Expression <strong>of</strong> the C-term<strong>in</strong>al doma<strong>in</strong> only, without signal<br />
peptide <strong>and</strong> RXLR-DEER doma<strong>in</strong>, was sufficient for AVR3a effector function. An<br />
alternative way to test <strong>effectors</strong> <strong>in</strong> planta is by particle bombardment, for example<br />
by monitor<strong>in</strong>g the suppression <strong>of</strong> cell death <strong>in</strong>duced by the mouse pro-apoptotic<br />
prote<strong>in</strong> BAX. Reduction <strong>of</strong> cell death was observed by co-bombardment with the<br />
P. sojae AVR1b gene which encodes a prote<strong>in</strong> with conserved C-term<strong>in</strong>al W <strong>and</strong><br />
Y motifs that are present <strong>in</strong> many Phytophthora <strong>effectors</strong> [32] . These motifs are<br />
required for suppression <strong>of</strong> cell death, suggest<strong>in</strong>g that this activity is a major function<br />
<strong>of</strong> oomycete <strong>effectors</strong>. Cell death-suppression assays have been used by many<br />
groups to test c<strong>and</strong>idate <strong>effectors</strong>. A screen <strong>of</strong> 32 P. <strong>in</strong>festans RXLR <strong>effectors</strong> for<br />
suppression <strong>of</strong> elicit<strong>in</strong> INF1-<strong>in</strong>duced cell death was used to reveal effector activity<br />
<strong>of</strong> the PexRD8 <strong>and</strong> PexRD36 45‐1<br />
RXLR prote<strong>in</strong>s [33] . One would expect that the<br />
suppression <strong>of</strong> defence is vital to the virulence <strong>of</strong> oomycetes, however, <strong>in</strong> vivo data<br />
that support this idea are miss<strong>in</strong>g for most <strong>effectors</strong> as knock<strong>in</strong>g out genes is not an<br />
established method <strong>in</strong> oomycete research. Nevertheless, gene silenc<strong>in</strong>g <strong>of</strong> Avr3a <strong>in</strong>
16 Chapter 1<br />
Table 1: Host-translocated <strong>effectors</strong> <strong>of</strong> oomycete pathogens with described <strong>in</strong> planta effects. Class:<br />
CRN; Cr<strong>in</strong>kler effector, RXLR; RXLR-motif effector, RXLR; RXLR-like motif effector. ETI:<br />
correspond<strong>in</strong>g <strong>in</strong> planta R–gene. Species (Sp.): Ae; Aphanomyces euteiches, Ha; H. arabidopsidis, Pi;<br />
P. <strong>in</strong>festans, Ps; P. sojae<br />
Effector Sp. Class ETI Effects Target Ref.<br />
AeCRN5 Ae CRN Cell death (via nucleus)<br />
[27]<br />
ATR1 Ha RXLR AtRPP1 Contributes to virulence<br />
[19]<br />
ATR13 Ha RXLR AtRPP13<br />
Suppresses callose<br />
deposition <strong>and</strong> ROS<br />
secretion<br />
[49]<br />
AVR1 Pi RXLR R1<br />
[50]<br />
AVR2 Pi RXLR R2<br />
[50]<br />
AVR3a Pi RXLR R3a Stabilises host E3-ligase CMPG1<br />
[34,51]<br />
AVR3b/10/11 Pi RXLR R3b/R10/R11<br />
[52]<br />
AVR4 Pi RXLR R4<br />
[53]<br />
AVRBlb2 Pi RXLR Rpi-Blb2<br />
Interferes with protease<br />
secretion<br />
[50]<br />
IPI-O1 Pi RXLR<br />
Rpi-Blb1/Rpi- Disruption PM-CW<br />
Sto1/Rpi-Pta1 <strong>in</strong>tegrity<br />
IPI-O2 Pi RXLR<br />
Rpi-Blb1/Rpi-<br />
Sto1/Rpi-Pta1<br />
[36]<br />
IPI-O4 Pi RXLR<br />
Suppresses IPI-O1/IPI-O2<br />
ETI<br />
[37]<br />
PexRD36 45‐1<br />
Pi RXLR Suppresses INF1 PTI<br />
[33]<br />
PexRD8 Pi RXLR Suppresses INF1 PTI<br />
[33]<br />
SNE1 Pi RXLR<br />
Suppresses Avr3a ETI <strong>and</strong><br />
NLP-<strong>in</strong>duced cell death<br />
[38]<br />
Various CRNs<br />
(e.g. CRN Pi CRN Cell death (via nucleus)<br />
[2,27]<br />
1,2,8,16)<br />
AVR1a Ps RXLR Rps1a<br />
[54]<br />
AVR1b-1 Ps RXLR Rps1b<br />
Suppresses BAX-<strong>in</strong>duced<br />
cell death<br />
[32][55]<br />
AVR1k Ps RXLR Rps1k<br />
[50]<br />
AVR3a Ps RXLR Rps3a<br />
[54]<br />
AVR3c Ps RXLR Rps3c<br />
[56]<br />
AVR4/6 Ps RXLR Rps4/Rps6<br />
[57]<br />
PsCRN115 Ps CRN<br />
Suppresses PsCRN63/<br />
NLP-<strong>in</strong>duced cell death<br />
[35]<br />
PsCRN63 Ps CRN Cell death (via nucleus)<br />
[35]<br />
LecRK-I.9 [12,13,36]
How do oomycete <strong>effectors</strong> <strong>in</strong>terfere with plant life<br />
17<br />
P. <strong>in</strong>festans [34] , <strong>and</strong> <strong>of</strong> the Cr<strong>in</strong>klers PsCRN115 <strong>and</strong> PsCRN63 <strong>in</strong> P. sojae resulted <strong>in</strong><br />
reduced virulence [35] .<br />
Other RXLR <strong>effectors</strong> are able to suppress cell death that is associated with<br />
effector triggered immunity (ETI, see Box 1). IPI-O4, a sequence divergent<br />
member <strong>of</strong> the P. <strong>in</strong>festans IPI-O family, can suppress cell death <strong>in</strong>duced by variants<br />
<strong>of</strong> IPI-O1 <strong>and</strong> IPI-O2 (now renamed to AVR-blb1) <strong>in</strong> potato plants carry<strong>in</strong>g<br />
the resistance gene Rpi-blb1 [36,37] . A broad ETI suppressive activity was detected<br />
for suppressor <strong>of</strong> necrosis 1 (SNE1), a P. <strong>in</strong>festans prote<strong>in</strong> with a RXLR-like<br />
motif (RQLG) [38] . SNE1 was able to suppress ETI triggered by the recognition<br />
<strong>of</strong> avirulence prote<strong>in</strong>s from oomycetes (AVR3a), bacteria (AvrPto), fungi (Avr9)<br />
<strong>and</strong> viruses (PVX coat prote<strong>in</strong>). Furthermore, it also suppressed NLP-<strong>in</strong>duced<br />
cell death. As SNE1 expression is high dur<strong>in</strong>g the biotrophic stage, <strong>and</strong> drops<br />
when NLP expression rises, SNE1 could be important <strong>in</strong> tim<strong>in</strong>g the onset <strong>of</strong> the<br />
necrotrophic phase.<br />
For most <strong>of</strong> the <strong>effectors</strong> discussed a virulence target <strong>in</strong> the host cell has<br />
not yet been identified. An exception is the well studied P. <strong>in</strong>festans AVR3a<br />
prote<strong>in</strong> that targets a host ubiquit<strong>in</strong> E3-ligase, CMPG1 [34] . CMPG1 is one <strong>of</strong><br />
three host ubiquit<strong>in</strong> E3-ligases known to be <strong>in</strong>volved <strong>in</strong> plant defence [39] . AVR3a<br />
manipulates the host ubiquit<strong>in</strong> proteasome system by stabilis<strong>in</strong>g CMPG1 <strong>and</strong><br />
suppresses plant immunity. More oomycete <strong>effectors</strong> are expected to act on the<br />
ubiquit<strong>in</strong>ation systems <strong>of</strong> the host, however, recognisable doma<strong>in</strong>s are absent <strong>in</strong><br />
predicted host-translocated <strong>effectors</strong>. Effectors could have structural similarities to<br />
ubiquit<strong>in</strong>ation-related prote<strong>in</strong>s, for example the bacterial AvrPtoB effector that was<br />
recognised as an E3 ligase based on its structure, rather than homology [40] .<br />
Sweet rewards<br />
Interfer<strong>in</strong>g with plant metabolism is another anticipated activity <strong>of</strong> oomycete<br />
pathogens, especially <strong>in</strong> biotrophic <strong>in</strong>teractions. In contrast to hemibiotrophs, that<br />
switch to necrotrophy <strong>in</strong> the course <strong>of</strong> <strong>in</strong>fection possibly by us<strong>in</strong>g NLPs <strong>and</strong> other<br />
<strong>effectors</strong> that trigger cell death, obligate biotrophs do not kill their host cells, but<br />
rather keep plants cells alive while they retrieve nutrients from the host. Analysis<br />
<strong>of</strong> metabolic pathways, based on predicted oomycete proteomes has confirmed that<br />
the Phytophthora <strong>and</strong> Pythium species are autotrophic (besides their requirement<br />
<strong>of</strong> sterols) <strong>and</strong> can hence make use <strong>of</strong> a broad range <strong>of</strong> nutrients that are available<br />
<strong>in</strong> the host [1‐3] . By contrast, the obligate biotroph H. arabidopsidis lacks several<br />
important enzymes <strong>in</strong> nitrate <strong>and</strong> sulfate metabolism suggest<strong>in</strong>g that it is dependent<br />
on the host to provide other sources <strong>of</strong> reduced nitrogen <strong>and</strong> sulfur [4] .<br />
One can envision that <strong>effectors</strong> not only act on plant defence pathways, but<br />
also <strong>in</strong>terfere with host metabolic pathways or transporters, redirect<strong>in</strong>g nutrients
18 Chapter 1<br />
<strong>and</strong> chang<strong>in</strong>g host metabolism. Bacterial <strong>and</strong> fungal pathogens manipulate host<br />
sugar transport by activat<strong>in</strong>g SWEET transporter genes dur<strong>in</strong>g <strong>in</strong>fection. In rice, a<br />
bacterial effector <strong>of</strong> the TALE class b<strong>in</strong>ds to the OsSWEET11 promoter to activate<br />
the sugar transporter gene [41] . Changes <strong>in</strong> host physiology <strong>and</strong> metabolism that are<br />
possibly manipulated by pathogen <strong>effectors</strong> have been observed <strong>in</strong> oomycete-<strong>in</strong>fected<br />
plants, for example the repression <strong>of</strong> photosynthesis <strong>and</strong> <strong>in</strong>duction <strong>of</strong> s<strong>in</strong>k<br />
status <strong>in</strong>creas<strong>in</strong>g the availability <strong>of</strong> assimilates for pathogens [42] . Genome-wide<br />
expression studies <strong>of</strong> host responses dur<strong>in</strong>g <strong>in</strong>fection have, unfortunately, not<br />
provided a clear picture <strong>of</strong> transcriptional changes related to host metabolism.<br />
Comparison <strong>of</strong> a compatible <strong>and</strong> <strong>in</strong>compatible (plant is resistant) <strong>in</strong>teraction <strong>of</strong><br />
Arabidopsis <strong>in</strong>oculated with H. arabidopsidis showed a large overlap <strong>in</strong> expression<br />
changes <strong>of</strong> defence-associated genes between the treatments. By contrast, only<br />
a limited number <strong>of</strong> 17 compatible-specific genes were identified <strong>of</strong> which none<br />
could be directly l<strong>in</strong>ked to metabolism [43] , suggest<strong>in</strong>g that pathogen-<strong>in</strong>duced<br />
metabolic reprogramm<strong>in</strong>g <strong>of</strong> the host is ma<strong>in</strong>ly post-transcriptional.<br />
Conclusion<br />
Recent f<strong>in</strong>d<strong>in</strong>gs highlight the unique complement <strong>of</strong> <strong>effectors</strong> that make the oomycetes<br />
such remarkable pathogens. More <strong>and</strong> more <strong>effectors</strong> that act <strong>in</strong>side the host<br />
cell are be<strong>in</strong>g identified while the debate on the mechanism <strong>of</strong> their translocation<br />
cont<strong>in</strong>ues. The next step is to unravel their effect on host processes <strong>and</strong> the molecular<br />
mechanisms by which they carry out their virulence function. The discovery <strong>of</strong><br />
CMPG1 as a host target is an important first step towards underst<strong>and</strong><strong>in</strong>g the activity<br />
<strong>of</strong> the oomycete effector complement. Major challenges rema<strong>in</strong> the development <strong>of</strong><br />
robust effector bioassays <strong>and</strong> gene knockout protocols for oomycetes, <strong>in</strong> particular<br />
for the obligate biotrophs. Emerg<strong>in</strong>g studies on host prote<strong>in</strong>-effector <strong>in</strong>teractors<br />
<strong>and</strong> structural data on <strong>effectors</strong> will be <strong>in</strong>strumental <strong>in</strong> elucidat<strong>in</strong>g the functions<br />
<strong>of</strong> <strong>effectors</strong>. Plant-oomycete <strong>in</strong>teractions are reach<strong>in</strong>g a complexity that requires<br />
a systems biology approach [44] that, as a bonus, will also provide better <strong>in</strong>sights <strong>in</strong><br />
the physiology <strong>of</strong> the plant <strong>and</strong> the host immune network.<br />
Acknowledgements<br />
JHMS is supported by a grant from TTI-Green Genetics. We thank the reviewers<br />
<strong>and</strong> editors for constructive comments on the manuscript.
How do oomycete <strong>effectors</strong> <strong>in</strong>terfere with plant life<br />
19<br />
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14 Qutob D, Kemmerl<strong>in</strong>g B, Brunner F, Küfner I, Engelhardt S, Gust AA, Luberacki B, Seitz HU,<br />
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15 Ottmann C, Luberacki B, Küfner I, Koch W, Brunner F, Wey<strong>and</strong> M, Matt<strong>in</strong>en L, Pirhonen M,<br />
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16 Kanneganti T-D, Huitema E, Cakir C & Kamoun S (2006) Synergistic <strong>in</strong>teractions <strong>of</strong> the plant<br />
cell death pathways <strong>in</strong>duced by Phytophthora <strong>in</strong>festans Nepl-like prote<strong>in</strong> PiNPP1.1 <strong>and</strong> INF1<br />
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17 Avrova AO, Boev<strong>in</strong>k PC, Young V, Grenville-Briggs LJ, Van West P, Birch PRJ & Whisson SC<br />
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18 Whisson SC, Boev<strong>in</strong>k PC, Moleleki L, Avrova AO, Morales JG, Gilroy EM, Armstrong MR,<br />
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How do oomycete <strong>effectors</strong> <strong>in</strong>terfere with plant life<br />
21<br />
19 Rehmany AP, Gordon A, Rose LE, Allen RL, Armstrong MR, Whisson SC, Kamoun S, Tyler BM,<br />
Birch PRJ & Beynon JL (2005) Differential recognition <strong>of</strong> highly divergent <strong>downy</strong> <strong>mildew</strong><br />
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20 Gaul<strong>in</strong> E, Madoui M-A, Bott<strong>in</strong> A, Jacquet C, Mathé C, Couloux A, W<strong>in</strong>cker P & Dumas B (2008)<br />
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21 Jiang RHY, Tripathy S, Govers F & Tyler BM (2008) RXLR effector reservoir <strong>in</strong> two<br />
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22 Dou D, Kale SD, Wang X, Jiang RHY, Bruce NA, Arredondo FD, Zhang X & Tyler BM (2008)<br />
RXLR-mediated entry <strong>of</strong> Phytophthora sojae effector Avr1b <strong>in</strong>to soybean cells does not require<br />
pathogen-encoded mach<strong>in</strong>ery. The Plant Cell 20, 1930-47.<br />
23 Kale SD, Gu B, Capelluto DGS, Dou D, Feldman E, Rumore A, Arredondo FD, Hanlon R,<br />
Fudal I, Rouxel T, Lawrence CB, Shan W & Tyler BM (2010) External lipid PI3P mediates entry<br />
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24 Gan PHP, Rafiqi M, Ellis JG, Jones DA, Hardham AR & Dodds PN (2010) Lipid b<strong>in</strong>d<strong>in</strong>g<br />
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25 Tian M, W<strong>in</strong> J, Savory E, Burkhardt A, Held M, Br<strong>and</strong>izzi F & Day B (2011) 454 Genome<br />
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26 Bailey K, Cevik V, Holton NJ, Byrne-Richardson J, Sohn KH, Coates M, Woods-Tör A,<br />
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27 Schornack S, Van Damme M, Bozkurt TO, Cano LM, Smoker M, Th<strong>in</strong>es M, Gaul<strong>in</strong> E, Kamoun S<br />
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28 Cooper AJ, Latunde-Dada AO, Woods-Tör A, Lynn J, Lucas JA, Crute IR & Holub EB (2008)<br />
Basic compatibility <strong>of</strong> Albugo c<strong>and</strong>ida <strong>in</strong> Arabidopsis thaliana <strong>and</strong> Brassica juncea causes broadspectrum<br />
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29 Sohn KH, Lei R, Nemri A & Jones JDG (2007) The <strong>downy</strong> <strong>mildew</strong> effector prote<strong>in</strong>s ATR1 <strong>and</strong><br />
ATR13 promote disease susceptibility <strong>in</strong> Arabidopsis thaliana. The Plant Cell 19, 4077-90.<br />
30 Cabral A, Stassen JHM, Seidl MF, Bautor J, Parker JE & Van den Ackerveken G (2011)<br />
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reveals isolate-specific <strong>effectors</strong>. PLoS ONE 6, e19328.<br />
31 Bos JIB, Kanneganti T-D, Young C, Cakir C, Huitema E, W<strong>in</strong> J, Armstrong MR, Birch PRJ<br />
& Kamoun S (2006) The C-term<strong>in</strong>al half <strong>of</strong> Phytophthora <strong>in</strong>festans RXLR effector AVR3a is<br />
sufficient to trigger R3a-mediated hypersensitivity <strong>and</strong> suppress INF1-<strong>in</strong>duced cell death <strong>in</strong><br />
Nicotiana benthamiana. The Plant Journal 48, 165-76.
22 Chapter 1<br />
32 Dou D, Kale SD, Wang XX, Chen Y, Wang Q, Jiang RHY, Arredondo FD, Anderson RG,<br />
Thakur PB, McDowell JM, Wang Y & Tyler BM (2008) Conserved C-term<strong>in</strong>al motifs required for<br />
avirulence <strong>and</strong> suppression <strong>of</strong> cell death by Phytophthora sojae effector Avr1b. The Plant Cell 20,<br />
1118-33.<br />
33 Oh S-K, Young C, Lee M, Oliva R, Bozkurt TO, Cano LM, W<strong>in</strong> J, Bos JIB, Liu H-Y, Van<br />
Damme M, Morgan W, Choi D, Van der Vossen EAG, Vleeshouwers VGAA & Kamoun S (2009)<br />
In planta expression screens <strong>of</strong> Phytophthora <strong>in</strong>festans RXLR <strong>effectors</strong> reveal diverse phenotypes,<br />
<strong>in</strong>clud<strong>in</strong>g activation <strong>of</strong> the Solanum bulbocastanum disease resistance prote<strong>in</strong> Rpi-blb2. The Plant<br />
Cell 21, 2928-47.<br />
34 Bos JIB, Armstrong MR, Gilroy EM, Boev<strong>in</strong>k PC, He<strong>in</strong> I, Taylor RM, Zhendong T, Engelhardt S,<br />
Vetukuri RR, Harrower B, Dixelius C, Bryan G, Sadan<strong>and</strong>om A, Whisson SC, Kamoun S<br />
& Birch PRJ (2010) Phytophthora <strong>in</strong>festans effector AVR3a is essential for virulence <strong>and</strong><br />
manipulates plant immunity by stabiliz<strong>in</strong>g host E3 ligase CMPG1. Proceed<strong>in</strong>gs <strong>of</strong> the National<br />
Academy <strong>of</strong> Sciences <strong>of</strong> the United States <strong>of</strong> America 107, 9909-14.<br />
35 Liu T, Ye W, Ru Y, Yang X, Gu B, Tao K, Lu S, Dong S, Zheng X, Shan W, Wang Y & Dou D<br />
(2011) Two host cytoplasmic <strong>effectors</strong> are required for pathogenesis <strong>of</strong> Phytophthora sojae by<br />
suppression <strong>of</strong> host defenses. Plant Physiology 155, 490-501.<br />
36 Vleeshouwers VGAA, Rietman H, Krenek P, Champouret N, Young C, Oh S-K, Wang M,<br />
Bouwmeester K, Vosman B, Visser RGF, Jacobsen E, Govers F, Kamoun S & Van der<br />
Vossen EAG (2008) Effector genomics accelerates discovery <strong>and</strong> <strong>functional</strong> pr<strong>of</strong>il<strong>in</strong>g <strong>of</strong> potato<br />
disease resistance <strong>and</strong> phytophthora <strong>in</strong>festans avirulence genes. PLoS ONE 3, e2875.<br />
37 Halterman DA, Chen Y, Sopee J, Berduo-S<strong>and</strong>oval J & Sánchez-Pérez A (2010) Competition<br />
between Phytophthora <strong>in</strong>festans <strong>effectors</strong> leads to <strong>in</strong>creased aggressiveness on plants conta<strong>in</strong><strong>in</strong>g<br />
broad-spectrum late blight resistance. PLoS ONE 5, e10536.<br />
38 Kelley BS, Lee S-J, Damasceno CMB, Chakravarthy S, Kim B-D, Mart<strong>in</strong> GB & Rose JKC (2010)<br />
A secreted effector prote<strong>in</strong> (SNE1) from Phytophthora <strong>in</strong>festans is a broadly act<strong>in</strong>g suppressor <strong>of</strong><br />
programmed cell death. The Plant Journal 62, 357-66.<br />
39 Van den Burg HA, Tsitsigiannis DI, Rowl<strong>and</strong> O, Lo J, Rallapalli G, Maclean D, Takken FLW &<br />
Jones JDG (2008) The F-box prote<strong>in</strong> ACRE189/ACIF1 regulates cell death <strong>and</strong> defense responses<br />
activated dur<strong>in</strong>g pathogen recognition <strong>in</strong> tobacco <strong>and</strong> tomato. The Plant Cell 20, 697-719.<br />
40 Abramovitch RB, Janjusevic R, Stebb<strong>in</strong>s CE & Mart<strong>in</strong> GB (2006) Type III effector AvrPtoB<br />
requires <strong>in</strong>tr<strong>in</strong>sic E3 ubiquit<strong>in</strong> ligase activity to suppress plant cell death <strong>and</strong> immunity.<br />
Proceed<strong>in</strong>gs <strong>of</strong> the National Academy <strong>of</strong> Sciences <strong>of</strong> the United States <strong>of</strong> America 103, 2851-6.<br />
41 Chen L-Q, Hou B-H, Lalonde S, Takanaga H, Hartung ML, Qu X-Q, Guo W-J, Kim J-G,<br />
Underwood W, Chaudhuri B, Chermak D, Antony G, White FF, Somerville SC, Mudgett MB &<br />
Frommer WB (2010) Sugar transporters for <strong>in</strong>tercellular exchange <strong>and</strong> nutrition <strong>of</strong> pathogens.<br />
Nature 468, 527-32.<br />
42 Berger S, S<strong>in</strong>ha AK & Roitsch T (2007) Plant Physiology meets phytopathology: plant primary<br />
metabolism <strong>and</strong> plant-pathogen <strong>in</strong>teractions. Journal <strong>of</strong> Experimental Botany 58, 4019-26.<br />
43 Huibers RP, De Jong M, Dekter RW & Van den Ackerveken G (2009) Disease-specific expression<br />
<strong>of</strong> host genes dur<strong>in</strong>g <strong>downy</strong> <strong>mildew</strong> <strong>in</strong>fection <strong>of</strong> Arabidopsis. Molecular Plant-Microbe<br />
Interactions 22, 1104-15.
How do oomycete <strong>effectors</strong> <strong>in</strong>terfere with plant life<br />
23<br />
44 Pritchard L & Birch P (2011) A systems biology perspective on plant-microbe <strong>in</strong>teractions:<br />
biochemical <strong>and</strong> structural targets <strong>of</strong> pathogen <strong>effectors</strong>. Plant Science 180, 584-603.<br />
45 Jones JDG & Dangl JL (2006) The plant immune system. Nature 444, 323-9.<br />
46 Kamoun S (2006) A catalogue <strong>of</strong> the effector secretome <strong>of</strong> plant pathogenic oomycetes. Annual<br />
Review Phytopathology 44, 41-60.<br />
47 Daxberger A, Nemak A, Mithöfer A, Fliegmann J, Ligter<strong>in</strong>k W, Hirt H & Ebel J (2007) Activation<br />
<strong>of</strong> members <strong>of</strong> a MAPK module <strong>in</strong> beta-glucan elicitor-mediated non-host resistance <strong>of</strong> soybean.<br />
Planta 225, 1559-71.<br />
48 Chaparro-Garcia A, Wilk<strong>in</strong>son RC, Gimenez-Ibanez S, F<strong>in</strong>dlay K, C<strong>of</strong>fey MD, Zipfel C,<br />
Rathjen JP, Kamoun S & Schornack S (2011) The receptor-like k<strong>in</strong>ase SERK3/BAK1 is<br />
required for basal resistance aga<strong>in</strong>st the late blight pathogen phytophthora <strong>in</strong>festans <strong>in</strong> Nicotiana<br />
benthamiana. PLoS ONE 6, e16608.<br />
49 Allen RL, Bittner-Eddy PD, Grenville-Briggs LJ, Meitz JC, Rehmany AP, Rose LE & Beynon JL<br />
(2004) Host-parasite coevolutionary conflict between Arabidopsis <strong>and</strong> <strong>downy</strong> <strong>mildew</strong>. Science<br />
306, 1957-60.<br />
50 Tyler BM (2009) Effectors. In Oomycete Genetics <strong>and</strong> Genomics: Diversity, Plant <strong>and</strong> Animal<br />
Interactions, <strong>and</strong> Toolbox pp. 361–86.<br />
51 Armstrong MR, Whisson SC, Pritchard L, Bos JIB, Venter E, Avrova AO, Rehmany AP,<br />
Böhme U, Brooks K, Cherevach I, Haml<strong>in</strong> N, White B, Fraser A, Lord A, Quail MA, Churcher C,<br />
Hall N, Berriman M, Huang S, Kamoun S, Beynon JL & Birch PRJ (2005) An ancestral oomycete<br />
locus conta<strong>in</strong>s late blight avirulence gene Avr3a, encod<strong>in</strong>g a prote<strong>in</strong> that is recognized <strong>in</strong> the host<br />
cytoplasm. Proceed<strong>in</strong>gs <strong>of</strong> the National Academy <strong>of</strong> Sciences <strong>of</strong> the United States <strong>of</strong> America 102,<br />
7766-71.<br />
52 Jiang RHY, Weide R, Van de Vondervoort PJI & Govers F (2006) Amplification generates<br />
modular diversity at an avirulence locus <strong>in</strong> the pathogen Phytophthora. Genome Research 16,<br />
827-40.<br />
53 Van Poppel PMJA, Guo J, Van de Vondervoort PJI, Jung MWM, Birch PRJ, Whisson SC &<br />
Govers F (2008) The Phytophthora <strong>in</strong>festans avirulence gene Avr4 encodes an RXLR-dEER<br />
effector. Molecular Plant-Microbe Interactions 21, 1460-70.<br />
54 Qutob D, Tedman-Jones J, Dong S, Kuflu K, Pham H, Wang Y, Dou D, Kale SD, Arredondo FD,<br />
Tyler BM & Gijzen M (2009) Copy number variation <strong>and</strong> transcriptional polymorphisms <strong>of</strong><br />
Phytophthora sojae RXLR effector genes Avr1a <strong>and</strong> Avr3a. PLoS ONE 4, e5066.<br />
55 Shan W, Cao M, Leung D & Tyler BM (2004) The Avr1b locus <strong>of</strong> Phytophthora sojae encodes an<br />
elicitor <strong>and</strong> a regulator required for avirulence on soybean plants carry<strong>in</strong>g resistance gene Rps1b.<br />
Molecular Plant-Microbe Interactions 17, 394-403.<br />
56 Dong S, Qutob D, Tedman-Jones J, Kuflu K, Wang Y, Tyler BM & Gijzen M (2009) The<br />
Phytophthora sojae avirulence locus Avr3c encodes a multi-copy RXLR effector with sequence<br />
polymorphisms among pathogen stra<strong>in</strong>s. PLoS ONE 4, e5556.<br />
57 Dou D, Kale SD, Liu T, Tang Q, Wang X, Arredondo FD, Basnayake S, Whisson S, Drenth A,<br />
Maclean D & Tyler BM (2010) Different doma<strong>in</strong>s <strong>of</strong> Phytophthora sojae effector Avr4/6 are<br />
recognized by soybean resistance genes Rps4 <strong>and</strong> Rps6. Molecular Plant-Microbe Interactions<br />
23, 425-35.
25<br />
Outl<strong>in</strong>e<br />
The aim <strong>of</strong> the research described <strong>in</strong> this thesis was to identify <strong>and</strong> <strong>functional</strong>ly<br />
analyse <strong>effectors</strong> <strong>of</strong> <strong>downy</strong> <strong>mildew</strong>s, <strong>and</strong> was performed as part <strong>of</strong> a larger research<br />
project entitled “Novel approaches for resistance breed<strong>in</strong>g us<strong>in</strong>g pathogen <strong>effectors</strong><br />
<strong>and</strong> their host plant targets: towards durable resistance to Bremia <strong>in</strong> lettuce”.<br />
Effectors were first identified <strong>in</strong> transcript sequences <strong>of</strong> the <strong>downy</strong> <strong>mildew</strong><br />
pathogen <strong>of</strong> Arabidopsis, H. arabidopsidis (Chapter 2). Transcriptome sequenc<strong>in</strong>g<br />
proved to be a powerful tool for effector identification <strong>and</strong> revealed that the effector<br />
arsenal differs between different isolates <strong>of</strong> the same pathogen species.<br />
The Bremia lactucae – Lactuca sativa <strong>in</strong>teraction was then <strong>in</strong>vestigated us<strong>in</strong>g<br />
the 454 sequenc<strong>in</strong>g technology to analyse the transcriptome <strong>of</strong> Bremia-<strong>in</strong>fected<br />
lettuce <strong>and</strong> <strong>downy</strong> <strong>mildew</strong> spores, allow<strong>in</strong>g for deep sampl<strong>in</strong>g <strong>of</strong> the Bremia<br />
transcriptome (Chapter 3). Additionally, genomic DNA from Bremia spores was<br />
sequenced to enable accurate selection <strong>of</strong> Bremia transcripts from the assembled<br />
sequences. From these data >16,000 Bremia transcripts were assembled, <strong>of</strong> which<br />
1023 were predicted to encode prote<strong>in</strong>s that are secreted by Bremia <strong>and</strong> thus potentially<br />
act on the host. Predicted <strong>effectors</strong> <strong>in</strong>clude 77 potential host-translocated<br />
RXLR <strong>effectors</strong> <strong>and</strong> various types <strong>of</strong> potential apoplastic <strong>effectors</strong>.<br />
The role <strong>of</strong> 34 c<strong>and</strong>idate RXLR <strong>effectors</strong> <strong>in</strong> stimulat<strong>in</strong>g susceptibility was<br />
<strong>in</strong>vestigated <strong>and</strong> described <strong>in</strong> Chapter 4. Transient expression <strong>of</strong> effector c<strong>and</strong>idates<br />
BLR16 <strong>and</strong> BLR27 significantly enhanced susceptibility <strong>of</strong> lettuce to Bremia<br />
<strong>in</strong>fection. A negative effect on host susceptibility was observed <strong>in</strong> leaf discs<br />
transiently express<strong>in</strong>g BLR03. Analysis <strong>of</strong> the temporal expression <strong>of</strong> <strong>effectors</strong><br />
revealed the presence <strong>of</strong> BLR16 <strong>and</strong> BLR27 transcripts throughout <strong>in</strong>fection, <strong>and</strong><br />
<strong>in</strong>dicated that BLR03 expression strongly decreases from early <strong>in</strong>fection onwards.<br />
Effectors did not enhance susceptibility by repression <strong>of</strong> the biotic stress-response<br />
genes <strong>of</strong> lettuce that were selected from transcriptome data. The identification <strong>of</strong><br />
susceptibility-enhanc<strong>in</strong>g <strong>effectors</strong> <strong>of</strong> Bremia opens up the way to <strong>in</strong>vestigate their<br />
activity <strong>and</strong> host targets.<br />
The recognition <strong>of</strong> Bremia <strong>effectors</strong> <strong>in</strong> Lactuca breed<strong>in</strong>g l<strong>in</strong>es was <strong>in</strong>vestigated<br />
<strong>and</strong> described <strong>in</strong> chapter five. The <strong>effectors</strong> were transiently expressed <strong>in</strong> almost<br />
130 different l<strong>in</strong>es <strong>of</strong> Lactuca species that potentially conta<strong>in</strong> novel sources <strong>of</strong><br />
resistance to Bremia. Two <strong>effectors</strong> conta<strong>in</strong><strong>in</strong>g a GXLR variant <strong>of</strong> the RXLR motif<br />
were found to be recognised, <strong>and</strong> trigger ETI; BLG01 be<strong>in</strong>g recognised <strong>in</strong> many
26 Outl<strong>in</strong>e<br />
L. saligna l<strong>in</strong>es, whilst BLG03 was recognised <strong>in</strong> two Dm2-conta<strong>in</strong><strong>in</strong>g L. sativa<br />
l<strong>in</strong>es. BLG01 ETI was mapped to the short arm <strong>of</strong> chromosome 3 <strong>and</strong> BLG03 ETI<br />
was shown to be l<strong>in</strong>ked to the Dm2 locus. Surpris<strong>in</strong>gly, though these loci conferred<br />
effector recognition, they did not confer resistance to the B. lactucae isolate from<br />
which they were isolated.<br />
The f<strong>in</strong>al chapter concerns a discussion <strong>of</strong> the work described <strong>in</strong> the thesis<br />
<strong>and</strong> the use <strong>of</strong> <strong>effectors</strong> <strong>in</strong> the development <strong>of</strong> novel breed<strong>in</strong>g strategies for more<br />
durable resistance to <strong>downy</strong> <strong>mildew</strong>.
27<br />
Chapter 2:<br />
<strong>Identification</strong> <strong>of</strong> Hyaloperonospora arabidopsidis<br />
transcript sequences expressed dur<strong>in</strong>g <strong>in</strong>fection<br />
reveals isolate-specific <strong>effectors</strong><br />
Adriana Cabral 1 , Joost H. M. Stassen 1 , Michael F. Seidl 2,3 ,<br />
Jaquel<strong>in</strong>e Bautor 4 , Jane E. Parker 4 , Guido Van den Ackerveken 1,3<br />
1<br />
Department <strong>of</strong> Plant-Microbe Interactions, Department <strong>of</strong> Biology,<br />
Utrecht University, Utrecht, The Netherl<strong>and</strong>s<br />
2<br />
Theoretical Biology <strong>and</strong> Bio<strong>in</strong>formatics, Department <strong>of</strong> Biology,<br />
Utrecht University, Utrecht, The Netherl<strong>and</strong>s<br />
3<br />
Centre for BioSystems Genomics (CBSG), Wagen<strong>in</strong>gen, The Netherl<strong>and</strong>s<br />
4<br />
Department <strong>of</strong> Plant-Microbe Interactions,<br />
Max Planck Institute for Plant Breed<strong>in</strong>g Research, Cologne, Germany<br />
PLoS ONE (2011) 6: e19328.<br />
doi: 10.1371/journal.pone.0019328
28 Chapter 2<br />
Abstract<br />
Biotrophic plant pathogens secrete effector prote<strong>in</strong>s that are important for <strong>in</strong>fection<br />
<strong>of</strong> the host. The aim <strong>of</strong> this study was to identify <strong>effectors</strong> <strong>of</strong> the <strong>downy</strong> <strong>mildew</strong><br />
pathogen Hyaloperonospora arabidopsidis (Hpa) that are expressed dur<strong>in</strong>g<br />
<strong>in</strong>fection <strong>of</strong> its natural host Arabidopsis thaliana. Infection-related transcripts<br />
were identified from Expressed Sequence Tags (ESTs) derived from leaves <strong>of</strong> the<br />
susceptible Arabidopsis Ws eds1-1 mutant <strong>in</strong>oculated with the highly virulent<br />
Hpa isolate Waco9. Assembly <strong>of</strong> 6364 ESTs yielded 3729 unigenes, <strong>of</strong> which<br />
2164 were Hpa-derived. From the translated Hpa unigenes, 198 predicted secreted<br />
prote<strong>in</strong>s were identified. Of these, 75 were found to be Hpa-specific <strong>and</strong> six<br />
isolate Waco9-specific. Among 42 putative <strong>effectors</strong> identified there were three<br />
Elicit<strong>in</strong>-like prote<strong>in</strong>s, 16 Cyste<strong>in</strong>e-rich prote<strong>in</strong>s <strong>and</strong> 18 host-translocated RXLR<br />
<strong>effectors</strong>. Sequenc<strong>in</strong>g <strong>of</strong> alleles <strong>in</strong> different Hpa isolates revealed that five RXLR<br />
genes show signatures <strong>of</strong> diversify<strong>in</strong>g selection. Thus, EST <strong>analysis</strong> <strong>of</strong> Hpa-<strong>in</strong>fected<br />
Arabidopsis is prov<strong>in</strong>g to be a powerful method for identify<strong>in</strong>g pathogen<br />
effector c<strong>and</strong>idates expressed dur<strong>in</strong>g <strong>in</strong>fection. Delivery <strong>of</strong> the Waco9-specific<br />
prote<strong>in</strong> RXLR29 <strong>in</strong> planta revealed that this effector can suppress PAMP-triggered<br />
immunity <strong>and</strong> enhance disease susceptibility. We propose that differences <strong>in</strong> host<br />
colonization can be conditioned by isolate-specific <strong>effectors</strong>.
Isolate-specific <strong>effectors</strong> from Hyaloperonospora ESTs<br />
29<br />
Introduction<br />
Plant pathogens secrete an arsenal <strong>of</strong> effector molecules that modulate host<br />
responses to enable successful <strong>in</strong>fection. Effector prote<strong>in</strong>s constitute part <strong>of</strong> the<br />
secretome <strong>of</strong> the <strong>in</strong>vad<strong>in</strong>g organism <strong>and</strong> are regarded as be<strong>in</strong>g crucial for pathogenicity<br />
[1] . Pathogen-derived <strong>effectors</strong> target different sites <strong>in</strong> host plant tissues.<br />
While apoplastic <strong>effectors</strong> are secreted <strong>in</strong>to the plant extracellular space, host-translocated<br />
<strong>effectors</strong> are delivered <strong>in</strong>to host cells after secretion from the pathogen [2–4] .<br />
For successful colonization, several layers <strong>of</strong> defence have to be overcome by<br />
the pathogen. An <strong>in</strong>itial barrier is conferred by host membrane-resident receptors<br />
recogniz<strong>in</strong>g pathogen-associated molecular patterns (PAMPs), molecules that are<br />
structurally conserved among related pathogenic microorganisms <strong>and</strong> not present <strong>in</strong><br />
the host [5–7] . PAMP-triggered immunity (PTI) is a resistance response that generally<br />
protects plants aga<strong>in</strong>st a broad range <strong>of</strong> non-adapted microorganisms. Several<br />
bacterial effector prote<strong>in</strong>s delivered to host cells by the type III secretion system<br />
(TTSS) have been shown to suppress PTI [8,9] . A second layer <strong>of</strong> defence (effector-triggered<br />
immunity, ETI) can be activated through recognition <strong>of</strong> particular<br />
pathogen <strong>effectors</strong> or their actions on host targets by Resistance (R) prote<strong>in</strong>s. ETI<br />
is a more acute plant reaction <strong>of</strong>ten <strong>in</strong>volv<strong>in</strong>g programmed cell death at <strong>in</strong>fection<br />
sites. Effectors can modulate the ETI response or mutate to circumvent recognition<br />
result<strong>in</strong>g <strong>in</strong> a co-evolutionary battle <strong>in</strong> which the pathogen attempts to evade host<br />
resistance <strong>and</strong> new plant R genes evolve to restrict further pathogen growth [10,11] .<br />
Therefore, pathogen <strong>effectors</strong> with high rates <strong>of</strong> gene loss, duplication or diversification<br />
are likely to be elicitors <strong>and</strong>/or modulators <strong>of</strong> plant immunity depend<strong>in</strong>g on<br />
the host genetic background they encounter [12,13] .<br />
Some <strong>of</strong> the most highly co-evolved <strong>in</strong>teractions are between plants <strong>and</strong><br />
biotrophic pathogens. At one end <strong>of</strong> the biotroph spectrum are hemibiotrophic<br />
species that <strong>in</strong>itially colonize liv<strong>in</strong>g cells but then switch to necrotrophy. At the<br />
other end are obligate biotrophs that ma<strong>in</strong>ta<strong>in</strong> host cell <strong>in</strong>tegrity <strong>and</strong> depend<br />
entirely on their host for growth <strong>and</strong> completion <strong>of</strong> their life cycle. Obligate<br />
biotrophs have evolved sophisticated mechanisms for host cell manipulation <strong>and</strong><br />
defence suppression [14] . Characteriz<strong>in</strong>g the activities <strong>and</strong> targets <strong>of</strong> biotroph-secreted<br />
<strong>effectors</strong> should therefore provide <strong>in</strong>sights to how host-adapted pathogens<br />
promote disease <strong>and</strong> avoid recognition.<br />
The obligate biotroph Hyaloperonospora arabidopsidis (Hpa) naturally <strong>in</strong>fects<br />
the model plant Arabidopsis thaliana, caus<strong>in</strong>g <strong>downy</strong> <strong>mildew</strong> disease [15] . Hpa is<br />
a highly specialized oomycete pathogen with a narrow host range. Analysis <strong>of</strong><br />
the Arabidopsis-Hpa <strong>in</strong>teraction has been particularly <strong>in</strong>formative because <strong>of</strong> the<br />
extensive genetic variation <strong>in</strong> responses <strong>of</strong> different Arabidopsis accessions to a<br />
correspond<strong>in</strong>gly diverse set <strong>of</strong> natural pathogen isolates [16,17] . Strong differences
30 Chapter 2<br />
<strong>in</strong> resistance <strong>of</strong> Arabidopsis accessions to particular Hpa isolates were found<br />
to be conferred by R genes <strong>of</strong>ten resid<strong>in</strong>g at polymorphic loci [18–21] . Although<br />
plant-<strong>in</strong>fect<strong>in</strong>g oomycetes exhibit a fungal-like morphology <strong>and</strong> feed<strong>in</strong>g structures<br />
(haustoria), they form a phylogenetically dist<strong>in</strong>ct group <strong>of</strong> eukaryotic pathogens<br />
that, together with brown algae <strong>and</strong> diatoms, belong to the Stramenopile l<strong>in</strong>eage<br />
(heterokonts) [22] . The genomes <strong>of</strong> several agronomically important hemibiotrophic<br />
oomycete pathogen species have been sequenced, such as Phytophthora sojae<br />
(caus<strong>in</strong>g soybean root <strong>and</strong> stem rot), P. ramorum (sudden oak death) <strong>and</strong> P.<br />
<strong>in</strong>festans (potato late blight) [23,24] . Recently, the genome sequence <strong>of</strong> the Hpa<br />
isolate Emoy2 has also become available [25] . Extensive oomycete genome sequence<br />
<strong>in</strong>formation comb<strong>in</strong>ed with data from expression pr<strong>of</strong>il<strong>in</strong>g <strong>of</strong> pathogen <strong>and</strong> host<br />
genes at different <strong>in</strong>fection stages is now serv<strong>in</strong>g as a basis to identify potentially<br />
important pathogen effector genes [26–28] . To date, a number <strong>of</strong> oomycete effector<br />
prote<strong>in</strong>s trigger<strong>in</strong>g ETI have been identified [11,29–33] . Notably, these molecules<br />
are characterized by a secretion signal followed by a conserved “RXLR” motif<br />
(where X can be any am<strong>in</strong>o acid residue) <strong>and</strong> <strong>of</strong>ten a stretch <strong>of</strong> acidic am<strong>in</strong>o acids<br />
end<strong>in</strong>g with the motif “EER”. Whereas the RXLR motif is required for effector<br />
translocation <strong>in</strong>to the host cell, the C-term<strong>in</strong>al region comprises a more variable<br />
“effector doma<strong>in</strong>” [34–37] . ATR1 <strong>and</strong> ATR13 are two RXLR type <strong>effectors</strong> <strong>of</strong> Hpa<br />
that are recognized by Arabidopsis R genes [11,30] . High levels <strong>of</strong> ATR1 <strong>and</strong> ATR13<br />
polymorphism among Hpa isolates suggest that diversify<strong>in</strong>g selection has driven<br />
the evolution <strong>of</strong> both genes [11,13,30] .<br />
Previous studies aimed at identify<strong>in</strong>g Hpa pathogenicity genes by isolat<strong>in</strong>g<br />
pathogen transcripts expressed dur<strong>in</strong>g <strong>in</strong>fection have so far resulted <strong>in</strong> the isolation<br />
<strong>of</strong> only a few c<strong>and</strong>idate <strong>effectors</strong>. A differential cDNA-amplified fragment<br />
length polymorphism (AFLP) <strong>analysis</strong> was used by van der Biezen et al. [38] on<br />
Hpa-<strong>in</strong>fected Arabidopsis leaves lead<strong>in</strong>g to identification <strong>of</strong> 10 Hpa-derived cDNA<br />
fragments. In another study, a suppression subtractive hybridization (SSH) strategy<br />
was used to identify 25 Hpa-expressed genes from <strong>in</strong>fected Arabidopsis [39] .<br />
Here we report a transcript sequenc<strong>in</strong>g approach based on Expressed Sequence<br />
Tag (EST) <strong>analysis</strong> <strong>of</strong> Hpa-<strong>in</strong>fected Arabidopsis leaves. To <strong>in</strong>crease the proportion<br />
<strong>of</strong> Hpa transcripts <strong>in</strong> a mixed sample <strong>of</strong> plant <strong>and</strong> pathogen RNA we used a highly<br />
virulent Hpa isolate (Waco9) to <strong>in</strong>fect a hyper-susceptible Arabidopsis mutant<br />
(eds1) [40] <strong>and</strong> collected tissues at a time po<strong>in</strong>t <strong>of</strong> maximum hyphal <strong>and</strong> haustorial<br />
growth. From a set <strong>of</strong> 2164 Hpa unigenes we have identified <strong>and</strong> classified<br />
Hpa-secreted prote<strong>in</strong>s <strong>in</strong>to several effector categories based on the presence <strong>of</strong><br />
characteristic doma<strong>in</strong>s/motifs. Eighteen <strong>of</strong> the Hpa prote<strong>in</strong>s belong to the class <strong>of</strong><br />
host-targeted RXLR <strong>effectors</strong>. While several effector c<strong>and</strong>idates are shared with
Isolate-specific <strong>effectors</strong> from Hyaloperonospora ESTs<br />
31<br />
other Hpa isolates, <strong>and</strong> <strong>in</strong> some cases oomycete species, six predicted effector prote<strong>in</strong>s<br />
from Waco9 are not detected <strong>in</strong> the genome <strong>of</strong> the sequenced isolate Emoy2,<br />
highlight<strong>in</strong>g the dynamic nature <strong>of</strong> Hpa pathogenicity.<br />
Results<br />
EST sequenc<strong>in</strong>g <strong>of</strong> Hpa-<strong>in</strong>fected Arabidopsis tissue<br />
Hpa isolates Emoy2, Emwa1, Noco2 <strong>and</strong> Waco9 were assessed for growth on the<br />
Arabidopsis enhanced disease susceptibility 1 mutant Ws eds1-1 [40] . Microscopic<br />
<strong>analysis</strong> <strong>of</strong> <strong>in</strong>fected leaves after trypan blue sta<strong>in</strong><strong>in</strong>g revealed the extent <strong>of</strong><br />
pathogen growth. A higher level <strong>of</strong> colonization <strong>of</strong> Arabidopsis leaves, with<br />
abundant hyphal growth <strong>and</strong> haustoria projections formed <strong>in</strong> adjacent plant cells,<br />
was observed for Hpa isolate Waco9 compared to Emoy2 (Fig. S1) <strong>and</strong> other<br />
isolates tested (results not shown). Waco9-<strong>in</strong>fected leaves harvested at 5 dpi (before<br />
sporulation) (Fig. 1A) were therefore used for the construction <strong>of</strong> a cDNA library.<br />
Total RNA isolated from the Waco9-<strong>in</strong>fected material revealed ribosomal<br />
RNA (rRNA) peaks <strong>of</strong> Hpa <strong>and</strong> Arabidopsis (Fig. 1B). The observed peaks were<br />
validated as rRNA orig<strong>in</strong>at<strong>in</strong>g from Hpa or Arabidopsis by compar<strong>in</strong>g pr<strong>of</strong>iles<br />
<strong>of</strong> <strong>in</strong>fected leaves with those <strong>of</strong> RNA from Hpa conidiospores mixed <strong>in</strong> different<br />
proportions with Arabidopsis leaf RNA (Fig. S2). This <strong>analysis</strong> showed that<br />
~50% <strong>of</strong> RNA extracted from the Waco9-<strong>in</strong>fected leaves was derived from Hpa.<br />
Poly (A+) RNA was isolated from the <strong>in</strong>fected leaf total RNA preparations <strong>and</strong><br />
size-fractionated cDNAs rang<strong>in</strong>g from 500 to 5000 bp were used for cDNA library<br />
construction.<br />
7680 sequenc<strong>in</strong>g reactions correspond<strong>in</strong>g to the 5′ end <strong>of</strong> the cDNAs were<br />
performed. After vector trimm<strong>in</strong>g <strong>and</strong> removal <strong>of</strong> low quality sequences <strong>and</strong><br />
chimeras, 6364 ESTs rema<strong>in</strong>ed rang<strong>in</strong>g <strong>in</strong> length from 50 to 1000 nucleotides (nt).<br />
A<br />
0<br />
20 30 40 50<br />
Migration time (sec)<br />
Figure 1: Hpa isolateWaco9-<strong>in</strong>fected Arabidopsis seedl<strong>in</strong>gs used for cDNA library construction. (A)<br />
Trypan blue sta<strong>in</strong><strong>in</strong>g <strong>of</strong> <strong>in</strong>fected Arabidopsis leaves (Ws eds1-1) show<strong>in</strong>g extensive hyphal growth <strong>in</strong><br />
the absence <strong>of</strong> asexual sporulation at 5 dpi. (B) Bioanalyzer pr<strong>of</strong>ile <strong>of</strong> total RNA obta<strong>in</strong>ed from Hpa<br />
Waco9-<strong>in</strong>fected leaf material. The 28S rRNA peaks <strong>of</strong> Arabidopsis (At) <strong>and</strong> Waco9 (Hpa) are <strong>in</strong>dicated.<br />
B<br />
Fluorescence<br />
80<br />
60<br />
40<br />
20<br />
At<br />
Hpa
32 Chapter 2<br />
The majority (84%; 5321 sequences) <strong>of</strong> ESTs had a read length <strong>of</strong> more than 500 nt<br />
(Fig. 2A). Assembly <strong>of</strong> ESTs yielded a total <strong>of</strong> 3729 unique sequences (unigenes)<br />
consist<strong>in</strong>g <strong>of</strong> 2810 (~75%) s<strong>in</strong>gletons <strong>and</strong> 919 (~25%) contigs with two or more<br />
ESTs (Fig. 2B).<br />
To def<strong>in</strong>e the orig<strong>in</strong> <strong>of</strong> the unigene sequences, BLASTN searches were<br />
performed aga<strong>in</strong>st the genome sequences <strong>of</strong> Arabidopsis, Hpa, three oomycete<br />
pathogens (P. <strong>in</strong>festans, P. sojae <strong>and</strong> P. ramorum) <strong>and</strong> the NCBI nr nucleotide<br />
database. Almost all unigenes (3722; ~99.8%) showed significant similarity<br />
(E
Isolate-specific <strong>effectors</strong> from Hyaloperonospora ESTs<br />
33<br />
rema<strong>in</strong><strong>in</strong>g unigenes two had high identity scores with sequences <strong>of</strong> other non-plant<br />
<strong>and</strong> non-oomycete organisms <strong>and</strong> seven did not show significant homology to<br />
any sequence <strong>in</strong> the databases (unknown sequences <strong>in</strong> Fig. 2C), nor conta<strong>in</strong>ed a<br />
signal peptide <strong>in</strong> the predicted open read<strong>in</strong>g frame <strong>and</strong> were therefore not further<br />
analysed. Altogether, we def<strong>in</strong>ed 2164 unigenes as Hpa-derived to use them for<br />
subsequently <strong>functional</strong> classification.<br />
<strong>Identification</strong> <strong>of</strong> putative secreted Hpa prote<strong>in</strong>s<br />
Secreted prote<strong>in</strong>s <strong>of</strong> fungal <strong>and</strong> oomycete plant pathogens have been shown to<br />
function as pathogenicity factors [4,41,42] . We therefore performed a comprehensive<br />
search for Hpa transcripts encod<strong>in</strong>g secreted prote<strong>in</strong>s. From the 2164 Hpa unigenes<br />
a set <strong>of</strong> 198 unigenes (822 ESTs, 9.15% <strong>of</strong> unigenes) encoded prote<strong>in</strong>s with a predicted<br />
signal peptide as analyzed with SignalP s<strong>of</strong>tware [43,44] , <strong>and</strong> without putative<br />
transmembrane doma<strong>in</strong>s. The EST-derived unigenes are significantly enriched<br />
(p = 4e−6) for transcripts encod<strong>in</strong>g predicted secreted prote<strong>in</strong>s when compared to<br />
the genome wide percentage <strong>of</strong> 6.8% (Table S2).<br />
To def<strong>in</strong>e which <strong>of</strong> the Hpa secreted prote<strong>in</strong>s are shared with other organisms,<br />
the 198 unigenes were compared to the genome sequences <strong>of</strong> three oomycete<br />
pathogens (Phytophthora <strong>in</strong>festans, P. sojae, <strong>and</strong> P. ramorum) <strong>and</strong> sequences <strong>in</strong><br />
GenBank, exclud<strong>in</strong>g Hpa. Significant blast hits (E75%) were obta<strong>in</strong>ed for 123 unigenes (Fig. 3). The rema<strong>in</strong><strong>in</strong>g 75 sequences<br />
(38%) did not have a significant hit with other oomycetes or non-Hpa sequences<br />
<strong>in</strong> GenBank, suggest<strong>in</strong>g that these genes are Hpa-specific. As the Hpa genome<br />
sequence is derived from isolate Emoy2 [25] <strong>and</strong> the ESTs isolated here are from<br />
isolate Waco9, we searched for possible differences between these two isolates.<br />
The 75 Hpa-specific unigenes were queried aga<strong>in</strong>st the assembled Emoy2 genome<br />
sequence <strong>and</strong> manually compared to Hpa trace files (http://www.ncbi.nlm.nih.<br />
gov/BLAST) to search for sequences that might be miss<strong>in</strong>g from the Hpa genome<br />
assembly. No significant hits were obta<strong>in</strong>ed for six sequences, suggest<strong>in</strong>g that these<br />
unigenes are present <strong>in</strong> Hpa isolate Waco9 but not <strong>in</strong> Emoy2.<br />
A <strong>functional</strong> annotation <strong>of</strong> the 198 Hpa unigenes predicted to encode secreted<br />
prote<strong>in</strong>s was performed by comparison to the Pfam database <strong>of</strong> prote<strong>in</strong> doma<strong>in</strong>s [45]<br />
<strong>and</strong> by manual annotation to search for specific classes <strong>of</strong> <strong>effectors</strong>. No function<br />
could be def<strong>in</strong>ed for 108 sequences that were therefore classified as unknowns.<br />
Table S3 shows the putative functions or doma<strong>in</strong>s assigned to 90 unigene-encoded<br />
prote<strong>in</strong>s. A selection <strong>of</strong> categories potentially associated with pathogenicity<br />
(adapted from Tyler et al. [23] ), <strong>and</strong> the number <strong>of</strong> ESTs <strong>and</strong> unigenes belong<strong>in</strong>g<br />
to each group, is shown <strong>in</strong> Table 1. Unigenes encod<strong>in</strong>g putative pathogenicity<br />
prote<strong>in</strong>s (58 sequences) were classified <strong>in</strong>to three categories: hydrolase enzymes,
34 Chapter 2<br />
198 unigenes<br />
NCBI nr database<br />
(exclud<strong>in</strong>g Hpa - Emoy2)<br />
Oomycetes<br />
(proteome/assembly)<br />
(exclud<strong>in</strong>g Hpa - Emoy2)<br />
evalue cut<strong>of</strong>f 1e-5; >75% coverage<br />
123 unigenes<br />
75 unigenes<br />
Hpa - specific<br />
Hpa - Emoy2<br />
(proteome/assembly/<br />
trace files)<br />
evalue cut<strong>of</strong>f 1e-10, identity ratio >80%<br />
69 unigenes<br />
6 unigenes<br />
Hpa - Waco9 - specific<br />
Figure 3: Scheme show<strong>in</strong>g categorization <strong>of</strong> Hpa Waco9 unigenes encod<strong>in</strong>g predicted secreted<br />
prote<strong>in</strong>s. Sequences with homologues <strong>in</strong> oomycetes (exclud<strong>in</strong>g Hpa) <strong>and</strong> other organisms are grouped<br />
(123 sequences). The rema<strong>in</strong><strong>in</strong>g 75 unigenes were used to search for homologues <strong>in</strong> the assembled<br />
Emoy2 genome <strong>and</strong> trace files. A set <strong>of</strong> six unigenes with no significant similarity to any other sequence<br />
is def<strong>in</strong>ed as Waco9-specific.<br />
protection aga<strong>in</strong>st oxidative stress, <strong>and</strong> <strong>effectors</strong>. These 58 unigenes, together with<br />
the 108 sequences with unknown function, were then divided accord<strong>in</strong>g to the<br />
group <strong>of</strong> organisms <strong>in</strong> which homologues were identified. From the 108 unknown<br />
sequences, 51 are categorized as Hpa-specific (Table 1). Homologues <strong>in</strong> other<br />
oomycetes were identified for all unigenes predicted to function as hydrolases or <strong>in</strong><br />
protection aga<strong>in</strong>st oxidative stress (Table 1), <strong>in</strong>dicat<strong>in</strong>g that prote<strong>in</strong>s <strong>in</strong> these classes<br />
are conserved among oomycetes. We looked for various families <strong>of</strong> <strong>effectors</strong> that<br />
are associated with the trigger<strong>in</strong>g or manipulation <strong>of</strong> host cell defences [46–48] . Putative<br />
effector prote<strong>in</strong>s formed the largest <strong>functional</strong> group identified, compris<strong>in</strong>g 42<br />
unigenes. In this category we assigned 16 prote<strong>in</strong>s as Cyste<strong>in</strong>e-rich (CR) prote<strong>in</strong>s<br />
<strong>and</strong> 18 as belong<strong>in</strong>g to the RXLR family. The majority <strong>of</strong> both effector types (23<br />
out <strong>of</strong> 34 unigenes) appear to be Hpa-specific s<strong>in</strong>ce no related sequences were<br />
detected <strong>in</strong> other oomycetes. Moreover, two CR prote<strong>in</strong>s <strong>and</strong> four RXLRs were
Isolate-specific <strong>effectors</strong> from Hyaloperonospora ESTs<br />
35<br />
Table 1: Classification <strong>of</strong> predicted Hpa secreted prote<strong>in</strong>s. The distribution <strong>in</strong> different pathogenicity<br />
categories <strong>and</strong> organisms <strong>in</strong> which homologues were identified are shown. Functional classification is<br />
based on Pfam searches <strong>and</strong> manual annotation.<br />
Total<br />
ESTs<br />
Unigenes<br />
Oomycetes specific<br />
specific<br />
Other Hpa-<br />
Waco9-<br />
Emoy2<br />
Hydrolases 41 9 9 0 0 0<br />
Cell Wall Degrad<strong>in</strong>g Enzymes 32 4 4 0 0 0<br />
Ser<strong>in</strong>e proteases 5 1 1 0 0 0<br />
Ser<strong>in</strong>e carboxypeptidases 2 2 2 0 0 0<br />
Aspartyl prote<strong>in</strong>ase/proteases 1 1 1 0 0 0<br />
Cyste<strong>in</strong>e protease/prote<strong>in</strong>ases 1 1 1 0 0 0<br />
Protection aga<strong>in</strong>st oxidative stress 35 7 7 0 0 0<br />
Glutathione s-transferases 1 1 1 0 0 0<br />
Reductases 16 2 2 0 0 0<br />
Glutaredox<strong>in</strong>s 3 1 1 0 0 0<br />
Thioredox<strong>in</strong>s 15 3 3 0 0 0<br />
Effectors 342 42 17 24 18 6<br />
RXLRs 40 18 4 14 10 4<br />
Cyste<strong>in</strong>e-rich prote<strong>in</strong>s 279 16 * 6 9 7 2<br />
Elicit<strong>in</strong>s 9 4 3 1 ** 1 0<br />
Necrosis-<strong>in</strong>duc<strong>in</strong>g like prote<strong>in</strong>s 3 2 2 0 0 0<br />
Cr<strong>in</strong>klers 1 1 1 0 0 0<br />
Cyclophil<strong>in</strong>s 2 1 1 0 0 0<br />
unknowns 361 108 55 51 51 0<br />
*<br />
One predicted Cyste<strong>in</strong>e-rich c<strong>and</strong>idate has homology to Tetrahymena thermophila <strong>and</strong> to Hpa<br />
sequences. No homologue was found <strong>in</strong> other oomycetes.<br />
**<br />
Elicit<strong>in</strong> HaELL2 is classified as Hpa-specific due to its extended specific C-term<strong>in</strong>al doma<strong>in</strong>.<br />
not identified <strong>in</strong> the Emoy2 genome suggest<strong>in</strong>g they are isolate Waco9-specific.<br />
Other classes <strong>of</strong> predicted effector prote<strong>in</strong>s such as Necrosis-<strong>in</strong>duc<strong>in</strong>g like prote<strong>in</strong>s,<br />
elicit<strong>in</strong>s, Cr<strong>in</strong>klers <strong>and</strong> cyclophil<strong>in</strong> had homologues with<strong>in</strong> other oomycetes, suggest<strong>in</strong>g<br />
that these sequences belong to a common core <strong>of</strong> putative <strong>effectors</strong> present<br />
<strong>in</strong> oomycete organisms. The Cr<strong>in</strong>kler identified carries a variation <strong>of</strong> the LxLFLAK<br />
translocation motif (Fig. S3), which has also been described to be present <strong>in</strong> several<br />
Cr<strong>in</strong>klers <strong>of</strong> P. <strong>in</strong>festans [24] .<br />
Elicit<strong>in</strong> signatures <strong>in</strong> Hpa<br />
Elicit<strong>in</strong>s (ELIs) are extracellular <strong>effectors</strong> characterized by a 98 am<strong>in</strong>o acid<br />
conserved doma<strong>in</strong> with a core <strong>of</strong> six cyste<strong>in</strong>es <strong>in</strong> a specific spac<strong>in</strong>g pattern<br />
allow<strong>in</strong>g their classification <strong>in</strong>to different groups. Elicit<strong>in</strong>-like prote<strong>in</strong>s (ELLs)
36 Chapter 2<br />
have more variation <strong>in</strong> the size <strong>and</strong> sequence <strong>of</strong> their elicit<strong>in</strong> doma<strong>in</strong>s [49] . Although<br />
Pfam searches identified four ELL unigenes <strong>in</strong> Waco9 (Table 1), only three <strong>of</strong><br />
these sequences were further analysed (Fig. 4) s<strong>in</strong>ce the fourth conta<strong>in</strong>ed only<br />
five cyste<strong>in</strong>es <strong>and</strong> therefore did not classify as an ELI/ELL. The HaELL1-, 2- <strong>and</strong><br />
3-encoded prote<strong>in</strong>s have fewer than 5% cyste<strong>in</strong>e residues <strong>and</strong> are classified as<br />
alpha-elicit<strong>in</strong>s due to their acidic pI [50] . The different size <strong>and</strong> cyste<strong>in</strong>e spac<strong>in</strong>g<br />
<strong>in</strong> the elicit<strong>in</strong> doma<strong>in</strong> <strong>of</strong> these three HaELLs is shown <strong>in</strong> Fig. 4. While HaELL2<br />
resembles Phytophthora ELL-1 prote<strong>in</strong>s [49] , the Cys pattern <strong>in</strong> HaELL1 <strong>and</strong> 3<br />
appears to be specific for Hpa. All three prote<strong>in</strong>s have an extended C-term<strong>in</strong>al<br />
region follow<strong>in</strong>g the elicit<strong>in</strong> doma<strong>in</strong>, a feature <strong>of</strong> other oomycete ELLs [49] . This<br />
region is smaller <strong>in</strong> HaELL1 <strong>and</strong> 3 (57 <strong>and</strong> 53 am<strong>in</strong>o acids, respectively) <strong>in</strong> comparison<br />
to HaELL2 (137 am<strong>in</strong>o acids). The C-term<strong>in</strong>al doma<strong>in</strong> appears to have a<br />
biased am<strong>in</strong>o acid composition as it is rich <strong>in</strong> threon<strong>in</strong>e, alan<strong>in</strong>e <strong>and</strong> ser<strong>in</strong>e (Fig. 4).<br />
The high abundance <strong>of</strong> threon<strong>in</strong>e <strong>and</strong> ser<strong>in</strong>e residues <strong>in</strong> the C-term<strong>in</strong>al part <strong>of</strong><br />
HaELL1, 2 <strong>and</strong> 3 po<strong>in</strong>ts to numerous potential sites for O-glycosylation (predicted<br />
by NetOGlyc 3.1) that could l<strong>in</strong>k the prote<strong>in</strong>s to the cell wall. A hydrophobic<br />
region conta<strong>in</strong><strong>in</strong>g a GPI anchor was predicted <strong>in</strong> the C-term<strong>in</strong>al part <strong>of</strong> HaELL2,<br />
suggest<strong>in</strong>g that this prote<strong>in</strong> might be anchored to the plasma membrane. O-glycosylation<br />
sites <strong>and</strong> GPI anchor regions have been described for other Phytophthora<br />
ELIs/ELLs [49] .<br />
HaELL1<br />
(175 aa)<br />
C-23-C-23-C-4-C-14-C-22-C<br />
97 aa<br />
C<br />
HaELL2<br />
(175 aa)<br />
N<br />
C-16-C-22-C-4-C-14-C-18-C<br />
85 aa<br />
C<br />
HaELL3<br />
(175 aa)<br />
N C-24-C-23-C-4-C-14-C-23-C<br />
C1 C2<br />
99 aa<br />
Figure 4: Schematic representation <strong>of</strong> prote<strong>in</strong> doma<strong>in</strong>s <strong>of</strong> three identified Hpa HaELLs. The predicted<br />
signal peptide is shown <strong>in</strong> black. A region between the signal peptide <strong>and</strong> the elicit<strong>in</strong> doma<strong>in</strong> found<br />
<strong>in</strong> HaELL2 <strong>and</strong> 3, with respectively 23 <strong>and</strong> 8 am<strong>in</strong>o acids, is shown (N). The pattern <strong>of</strong> cyste<strong>in</strong>e<br />
spac<strong>in</strong>g <strong>and</strong> the size <strong>of</strong> the elicit<strong>in</strong> doma<strong>in</strong> are depicted. The percentage <strong>of</strong> highly abundant am<strong>in</strong>o acid<br />
residues <strong>in</strong> the C-term<strong>in</strong>us is: HaELL1: T: 14%, A: 12%, S: 7%; HaELL2: T: 28%, A: 13%, S: 10%.<br />
The C-term<strong>in</strong>us <strong>of</strong> HaELL3 is divided <strong>in</strong>to two regions (C1 <strong>and</strong> C2). C1: S: 51%, T: 19%; C2: D;56%,<br />
E:31%.<br />
Hpa derived Cyste<strong>in</strong>e-rich (CR) prote<strong>in</strong>s<br />
A number <strong>of</strong> CR prote<strong>in</strong>s secreted by fungal pathogens have been shown to be<br />
recognized by R prote<strong>in</strong>s <strong>in</strong> resistant host plants [51–53] . The cyste<strong>in</strong>es <strong>in</strong> these prote<strong>in</strong>s<br />
form disulphide bridges that contribute to their structural stability <strong>in</strong> the plant<br />
apoplast, an environment rich <strong>in</strong> proteases. To identify HaCR c<strong>and</strong>idates among
Isolate-specific <strong>effectors</strong> from Hyaloperonospora ESTs<br />
37<br />
the putative Hpa secreted prote<strong>in</strong>s, we determ<strong>in</strong>ed the relative number <strong>of</strong> cyste<strong>in</strong>e<br />
residues <strong>in</strong> the full-length translated sequences. A total <strong>of</strong> 16 HaCR prote<strong>in</strong>s were<br />
identified which conta<strong>in</strong> more than 5% cyste<strong>in</strong>es <strong>and</strong> range <strong>in</strong> size from 72 to 405<br />
am<strong>in</strong>o acids (Table 2). N<strong>in</strong>e <strong>of</strong> the HaCR c<strong>and</strong>idates could be placed <strong>in</strong>to two<br />
groups based on the pattern <strong>and</strong> spac<strong>in</strong>g <strong>of</strong> the cyste<strong>in</strong>es (Fig. 5). The cyste<strong>in</strong>e<br />
motif <strong>in</strong> group I <strong>and</strong> II is repeated one or more times <strong>in</strong> most group members. As<br />
shown <strong>in</strong> Tables 1 <strong>and</strong> 2, six HaCR c<strong>and</strong>idates have homologues <strong>in</strong> other oomycete<br />
pathogens. C<strong>and</strong>idates from Groups I <strong>and</strong> II do not have an obvious oomycete<br />
counterpart. However for HaCR2 significant homology (blast E value
38 Chapter 2<br />
Figure 5: Two groups <strong>of</strong> HaCR prote<strong>in</strong>s identified with dist<strong>in</strong>ct cyste<strong>in</strong>e spac<strong>in</strong>g patterns. The boxes<br />
show the pattern repeated <strong>in</strong> each prote<strong>in</strong>.<br />
HaCR1, represented by the highest number <strong>of</strong> ESTs (84) among the secreted<br />
prote<strong>in</strong>s, is a homologue <strong>of</strong> Ppat14 from Hpa isolate Maks9 [39] .<br />
Classification <strong>of</strong> host-translocated Hpa RXLR prote<strong>in</strong>s<br />
RXLR prote<strong>in</strong>s comprise a class <strong>of</strong> oomycete <strong>effectors</strong> that are translocated <strong>in</strong>to the<br />
plant cell [36] . All known oomycete effector prote<strong>in</strong>s that are recognized by specific<br />
plant R prote<strong>in</strong>s belong to this class [11,29–33] . The Hpa unigenes encod<strong>in</strong>g putative<br />
secreted prote<strong>in</strong>s were therefore m<strong>in</strong>ed for c<strong>and</strong>idate RXLR <strong>effectors</strong>. Prote<strong>in</strong>s conta<strong>in</strong><strong>in</strong>g<br />
either an RXLR or RXLQ/RXLG motif <strong>in</strong> the mature prote<strong>in</strong> were selected.<br />
Variations <strong>in</strong> the last arg<strong>in</strong><strong>in</strong>e <strong>of</strong> the RXLR still allows prote<strong>in</strong> translocation <strong>in</strong>to<br />
the host cell [37] . We identified 18 RXLR c<strong>and</strong>idates (<strong>in</strong>clud<strong>in</strong>g one RXLQ conta<strong>in</strong><strong>in</strong>g<br />
prote<strong>in</strong>) encod<strong>in</strong>g relatively small prote<strong>in</strong>s rang<strong>in</strong>g from 115 to 340 am<strong>in</strong>o<br />
acids (Table 3). The distance between the RXLR/Q motif <strong>and</strong> the signal peptide<br />
cleavage site varied from 15 to 51 am<strong>in</strong>o acids <strong>in</strong>dicat<strong>in</strong>g that the RXLR/Q motif
Isolate-specific <strong>effectors</strong> from Hyaloperonospora ESTs<br />
39<br />
Table 3: Sequence features <strong>of</strong> c<strong>and</strong>idate Waco9 RXLR effector prote<strong>in</strong>s.<br />
Name<br />
Hpa RXLR<br />
Size RXLR<br />
ESTs<br />
gene IDs 1 (am<strong>in</strong>o acids) distance 2 EER 3 Homologues 4<br />
RXLR3 - 1 129 29 - Hpa-specific<br />
RXLR4 - 3 134 29 - Waco9<br />
RXLR5 - 1 340 30 - Oomycetes<br />
RXLR6 HaRXL80 1 129 27 + (44) Hpa-specific<br />
RXLR7 HaRXL17 1 285 20 + (32) Hpa-specific<br />
RXLR9 HaRXL78 2 150 28 - Hpa-specific<br />
RXLR12 - 3 125 28 - Waco9<br />
RXLR13 HaRXL76 5 286 32 + (49) Hpa-specific<br />
RXLR15 HaRXL77 3 129 15 + (37) Oomycetes<br />
RXLR16<br />
HaRXL30;<br />
HaRXL79<br />
3 198 28 + (41) Waco9<br />
RXLR17 (RXLQ) HaRXL42 2 135 28 + (41) Hpa-specific<br />
RXLR18 - 2 209 51 - Oomycetes<br />
RXLR19 - 1 299 31 - Hpa-specific<br />
RXLR20 HaRXL10 1 241 23 - Hpa-specific<br />
RXLR21<br />
HaRXL37;<br />
HaRXL75<br />
1 115 29 + (44) Oomycetes<br />
RXLR22 - 1 137 29 - Waco9<br />
RXLR23 HaRXL4 5 304 32 + (49) Hpa-specific<br />
RXLR29 - 4 132 28 - Hpa-specific<br />
1<br />
Gene IDs refer to RXLR sequences identified <strong>in</strong> Emoy2 [25]<br />
2<br />
Distance to signal peptide cleavage site.<br />
3<br />
In brackets the distance to signal peptide cleavage site.<br />
4<br />
Homologues found based on blast searches as described for Figure 6.<br />
is near the N-term<strong>in</strong>us. The presence <strong>of</strong> an acidic region (EER) downstream <strong>of</strong> the<br />
RXLR motif was identified <strong>in</strong> eight RXLR c<strong>and</strong>idates (Table 3) <strong>and</strong> two (RXLR3<br />
<strong>and</strong> RXLR6) have a putative nuclear localization signal as predicted by Psort.<br />
From the 18 RXLR sequences identified here, only four prote<strong>in</strong>s have homology<br />
with other oomycetes, <strong>in</strong>dicat<strong>in</strong>g that the majority <strong>of</strong> the RXLR c<strong>and</strong>idates are<br />
Hpa-specific (Tables 1 <strong>and</strong> 3). Among these four RXLR prote<strong>in</strong>s, RXLR5 <strong>and</strong> 18<br />
showed a high degree <strong>of</strong> similarity with homologues <strong>in</strong> P. <strong>in</strong>festans (75% <strong>and</strong> 72%<br />
identity, respectively, with 100% sequence coverage). From the 14 Hpa-specific<br />
prote<strong>in</strong>s, blast searches revealed that 10 RXLRs have correspond<strong>in</strong>g genes <strong>in</strong><br />
Emoy2 (Tables 1 <strong>and</strong> 3). Further <strong>analysis</strong> <strong>of</strong> these sequences revealed that RXLR29<br />
is represented by a null allele <strong>in</strong> Hpa Emoy2 caused by a frame-shift mutation<br />
result<strong>in</strong>g <strong>in</strong> a premature stop codon (Fig. S4). For RXLR4, 12, 16 <strong>and</strong> 22 no significant<br />
blast hits with the Emoy2 genome sequence were obta<strong>in</strong>ed (Tables 1 <strong>and</strong> 3). It<br />
is possible that these RXLR genes are either highly polymorphic or absent from the<br />
Emoy2 genome.
40 Chapter 2<br />
Allelic diversity <strong>of</strong> RXLR effector c<strong>and</strong>idate genes<br />
Cod<strong>in</strong>g sequences <strong>of</strong> the 18 RXLR c<strong>and</strong>idate <strong>effectors</strong> were deduced from seven<br />
Hpa isolates. Table 4 shows the number <strong>of</strong> prote<strong>in</strong> variants found for each RXLR<br />
<strong>and</strong> their distribution among the different Hpa isolates. For example, RXLR3 is<br />
represented by two allelic forms: Waco9, Noks1, Emco5 <strong>and</strong> Cala2 share variant<br />
A, Emoy2 <strong>and</strong> Maks9 carry prote<strong>in</strong> variant B <strong>and</strong> H<strong>in</strong>d2 has no RXLR3 gene.<br />
Heterozygosity was also observed for some RXLRs (e.g. RXLR6 <strong>in</strong> Cala2 conta<strong>in</strong>s<br />
both prote<strong>in</strong> variants A <strong>and</strong> B). RXLR5 was the least variable gene with identical<br />
prote<strong>in</strong>s <strong>in</strong> all seven Hpa isolates.<br />
Table 4: Allelic variants identified for 18 Hpa RXLR c<strong>and</strong>idate prote<strong>in</strong>s <strong>and</strong> their distribution among 7<br />
Hpa isolates. The letters <strong>in</strong>dicate prote<strong>in</strong> variants present at each isolate (prote<strong>in</strong> level). When 2 letters<br />
are present it means that the Hpa isolate is heterozygous for that locus <strong>and</strong> has one copy <strong>of</strong> each allele.<br />
Allele at the <strong>in</strong>dicated Hpa isolate<br />
Gene No. Alleles Cala2 Emco5 Emoy2 H<strong>in</strong>d2 Maks9 Noks1 Waco9<br />
RXLR3 2 A A B - B A A<br />
RXLR4 1 - - - A - - A<br />
RXLR5 1 A A A A A A A<br />
RXLR6 4 A,B C B D C C A<br />
RXLR7 3 B C B B B B A<br />
RXLR9 5 B;C E D B E D A<br />
RXLR12 1 - - - - - - A<br />
RXLR13 6 B A D E F C A<br />
RXLR15 2 B B A;B B A B A<br />
RXLR16 6 B C B;C D E F A<br />
RXLR17 2 B B B B B B A<br />
RXLR18 2 B A A A A B A<br />
RXLR19 5 B C D E B D A<br />
RXLR20 4 - A;B A C C D A<br />
RXLR21 3 A A A;B C B B A<br />
RXLR22 1 - - - - - - A<br />
RXLR23 6 C D D E A;B F A<br />
RXLR29 3 B B B B B;C B A<br />
No allele sequences were amplified for RXLR12 <strong>and</strong> 22 <strong>in</strong> Emoy2 or <strong>in</strong> any<br />
<strong>of</strong> the five other Hpa isolates, <strong>in</strong>dicat<strong>in</strong>g that these are Waco9-specific. RXLR4<br />
was only amplified from isolates H<strong>in</strong>d2 <strong>and</strong> Waco9. Although blast searches did<br />
not reveal a significant hit for RXLR16 <strong>in</strong> the Emoy2 genome (Table 3), allele<br />
sequences were obta<strong>in</strong>ed for Emoy2 as well as the other isolates. Due to a high<br />
level <strong>of</strong> polymorphism between the RXLR16 prote<strong>in</strong> sequences <strong>in</strong> Waco9 <strong>and</strong><br />
Emoy2, the blast hit obta<strong>in</strong>ed was excluded based on cut-<strong>of</strong>f values.
Isolate-specific <strong>effectors</strong> from Hyaloperonospora ESTs<br />
41<br />
The RXLR29 gene was <strong>of</strong> particular <strong>in</strong>terest because Waco9 is the only isolate<br />
<strong>of</strong> these analysed that conta<strong>in</strong>s an <strong>in</strong>tact full-length ORF. In the rema<strong>in</strong><strong>in</strong>g six Hpa<br />
isolates, RXLR29 displayed <strong>in</strong>sertions/deletions giv<strong>in</strong>g rise to frame shifts result<strong>in</strong>g<br />
<strong>in</strong> null alleles (Fig. S4). In Maks9 we also found two different truncated versions<br />
<strong>of</strong> RXLR29. These results show that a <strong>functional</strong> RXLR29 prote<strong>in</strong> is only present<br />
<strong>in</strong> Waco9 suggest<strong>in</strong>g that it has been counter-selected <strong>in</strong> the other Hpa isolates<br />
exam<strong>in</strong>ed.<br />
Of the 13 RXLR c<strong>and</strong>idate <strong>effectors</strong> with at least two different prote<strong>in</strong> variants,<br />
we def<strong>in</strong>ed the percentage <strong>of</strong> variable sites with<strong>in</strong> the prote<strong>in</strong> sequences (Fig. 6).<br />
RXLR9, 13, 16, 19 <strong>and</strong> 23 are the most polymorphic effector prote<strong>in</strong>s with more<br />
than 10% variable sites among the isolates sequenced. The ratio <strong>of</strong> >1 non-synonymous<br />
(dN) to synonymous (dS) nucleotide substitutions [54] <strong>in</strong> RXLR 9, 13, 16,<br />
19 <strong>and</strong> 23 suggests that these genes are under positive selection to ma<strong>in</strong>ta<strong>in</strong> am<strong>in</strong>o<br />
acid diversity (Table 5).<br />
Figure 6: Polymorphic Hpa RXLR prote<strong>in</strong>s. Numbers <strong>of</strong> variable sites were determ<strong>in</strong>ed us<strong>in</strong>g Mega4<br />
for RXLR prote<strong>in</strong>s possess<strong>in</strong>g two or more prote<strong>in</strong> variants.<br />
Hpa RXLR29 suppresses pathogen-<strong>in</strong>duced callose deposition<br />
S<strong>in</strong>ce only isolate Waco9 appears to have reta<strong>in</strong>ed a <strong>functional</strong> RXLR29 prote<strong>in</strong>,<br />
we selected this gene to determ<strong>in</strong>e a potential effector activity. We used a bacterial<br />
effector delivery vector (EDV) system which was previously shown to successfully<br />
deliver the Hpa effector ATR13 to Arabidopsis leaf cells by fusion to the N-term<strong>in</strong>al<br />
portion <strong>of</strong> a TTSS bacterial effector [42] . RXLR29 was cloned <strong>in</strong> the EDV system<br />
<strong>and</strong> expressed <strong>in</strong> the Pseudomonas syr<strong>in</strong>gae pv tomato (Pst) DC3000ΔCEL mutant<br />
stra<strong>in</strong> which lacks the conserved effector locus (CEL) <strong>and</strong> is therefore unable to<br />
efficiently suppress PTI [55,56] . Pathogen-<strong>in</strong>duced deposition <strong>of</strong> callose at the cell<br />
wall is used as a marker <strong>of</strong> PTI [57] . We therefore measured whether RXLR29<br />
delivery affects the capacity <strong>of</strong> Pst DC3000ΔCEL to <strong>in</strong>duce callose deposition<br />
after <strong>in</strong>filtrat<strong>in</strong>g bacteria <strong>in</strong>to leaves. In these experiments, YFP <strong>and</strong> ATR13 were
42 Chapter 2<br />
Table 5: Ratio <strong>of</strong> non-synonymous (dN) <strong>and</strong> synonymous (dS) substitutions <strong>of</strong> RXLR genes. Nucleotide<br />
sequences obta<strong>in</strong>ed for each RXLR c<strong>and</strong>idate <strong>of</strong> Hpa isolates were used to calculate the ratio dN/dS.<br />
Genes under positive selection exhibit a dN/dS ratio higher than 1 <strong>and</strong> a P-value
Isolate-specific <strong>effectors</strong> from Hyaloperonospora ESTs<br />
43<br />
A<br />
ATR13 RXLR29 YFP<br />
B<br />
Amount callose spots<br />
250<br />
200<br />
150<br />
100<br />
50<br />
0<br />
YFP<br />
RXLR29 ATR13<br />
Bacterial counts [Log (CFU/mg)]<br />
7<br />
6<br />
5<br />
4<br />
3<br />
2<br />
1<br />
0<br />
YFP<br />
ATR13<br />
RXLR29<br />
day 0 day 3<br />
Figure 7: RXLR29 suppresses callose deposition<br />
<strong>in</strong> bacteria-<strong>in</strong>oculated Arabidopsis leaves. Fiveweek-old<br />
Col-0 leaves were h<strong>and</strong>-<strong>in</strong>filtrated with<br />
1 x 108 cfu/ml Pst DC3000ΔCEL carry<strong>in</strong>g YFP,<br />
RXLR29 or ATR13, as <strong>in</strong>dicated. Leaf samples<br />
were taken at 12 h after <strong>in</strong>fection <strong>and</strong> sta<strong>in</strong>ed with<br />
anil<strong>in</strong>e blue to visualize callose (A). The number<br />
<strong>of</strong> callose spots was quantified <strong>in</strong> each sample<br />
(B). The experiment was repeated three times<br />
with similar results.<br />
Figure 8: RXLR29 enhances bacterial growth<br />
<strong>in</strong> Arabidopsis leaves. Five-week-old Col-0<br />
leaves were h<strong>and</strong>-<strong>in</strong>filtrated with 5×105 cfu/<br />
ml Pst DC3000-LUX deliver<strong>in</strong>g YFP, ATR13<br />
or RXLR29, as <strong>in</strong>dicated. Bacterial growth <strong>in</strong><br />
leaves at 0 <strong>and</strong> 3 dpi was measured by number <strong>of</strong><br />
colony form<strong>in</strong>g units (cfu) per mg <strong>of</strong> leaf tissue.<br />
Error bars <strong>in</strong>dicate the st<strong>and</strong>ard error <strong>of</strong> bacterial<br />
counts. Enhanced growth was observed at day 3<br />
for Pst deliver<strong>in</strong>g ATR13 or RXLR29 compared<br />
to YFP (T-test p value
44 Chapter 2<br />
frequency <strong>of</strong> ESTs encod<strong>in</strong>g secreted prote<strong>in</strong>s <strong>in</strong> <strong>in</strong>teraction libraries [27] .<br />
From the 198 Hpa secreted prote<strong>in</strong>s, a putative function could be assigned to<br />
90 sequences based on the presence <strong>of</strong> known doma<strong>in</strong>s/motifs, specific features<br />
(e.g. relative number <strong>of</strong> cyste<strong>in</strong>e residues) or similarity to known prote<strong>in</strong>s (Table<br />
S3). A set <strong>of</strong> 58 sequences compris<strong>in</strong>g potential pathogenicity factors was further<br />
classified <strong>in</strong>to different categories <strong>of</strong> which the largest group (42 unigenes)<br />
corresponded to putative effector prote<strong>in</strong>s (Table 1). EST <strong>analysis</strong> has been shown<br />
to be an effective method to identify pathogen genes encod<strong>in</strong>g effector prote<strong>in</strong>s.<br />
For example, Cr<strong>in</strong>klers were first described by analys<strong>in</strong>g ESTs from Phytophthora<br />
<strong>in</strong>festans [58] . Also, 25 Cr<strong>in</strong>kler-like sequences <strong>and</strong> 17 ELIs/ELLs were identified <strong>in</strong><br />
a P. sojae EST data set [27] . Moreover, <strong>analysis</strong> <strong>of</strong> a haustorium specific EST library<br />
<strong>of</strong> Melampsora l<strong>in</strong>i, a fungal rust pathogen <strong>of</strong> flax, has enabled identification <strong>of</strong><br />
three secreted effector prote<strong>in</strong>s that are recognized by specific R prote<strong>in</strong>s [59] .<br />
It is notable that no putative function could be assigned for 108 unigenes<br />
correspond<strong>in</strong>g to ~55% <strong>of</strong> the predicted secreted prote<strong>in</strong>s identified <strong>in</strong> our study,<br />
<strong>and</strong> therefore these sequences were classified as unknowns (Table 1). Further<br />
characterization <strong>of</strong> the secreted prote<strong>in</strong>s <strong>of</strong> unknown function should reveal new<br />
aspects <strong>of</strong> the biology <strong>of</strong> oomycete pathogens as well as processes specific to Hpa<br />
biotrophy, s<strong>in</strong>ce ~47% <strong>of</strong> the unknowns (51 unigenes) appear to be Hpa-specific<br />
sequences.<br />
From the set <strong>of</strong> secreted Hpa prote<strong>in</strong>s with a predicted function we identified<br />
three Elicit<strong>in</strong>-like prote<strong>in</strong>s. Although ELIs <strong>and</strong> ELLs have been described <strong>in</strong><br />
Phytophthora <strong>and</strong> Pythium [49,60] , it is likely that the number <strong>of</strong> oomycete species<br />
<strong>in</strong> which these prote<strong>in</strong>s are expressed will exp<strong>and</strong> as more sequences become<br />
available. Indeed, our blast searches identified a homologue <strong>of</strong> HaELL3 <strong>in</strong><br />
Saprolegnia parasitica. The cyste<strong>in</strong>e spac<strong>in</strong>g pattern allowed classification <strong>of</strong><br />
Phytophthora ELIs <strong>and</strong> ELLs <strong>in</strong> different groups [49] . Interest<strong>in</strong>gly, HaELL2 has<br />
the same pattern as some Phytophthora ELLs <strong>in</strong>clud<strong>in</strong>g INL1 (P. <strong>in</strong>festans) <strong>and</strong><br />
SOL1A (P. sojae) that belong to ELL-1 group. The spac<strong>in</strong>g pattern <strong>of</strong> HaELL1 <strong>and</strong><br />
3 was not observed <strong>in</strong> Phytophthora. Further <strong>analysis</strong> <strong>of</strong> elicit<strong>in</strong>s from different<br />
oomycete species should establish if there are species-specific patterns <strong>of</strong> cyste<strong>in</strong>e<br />
spac<strong>in</strong>g. Two beta-elicit<strong>in</strong>s (cryptoge<strong>in</strong> <strong>and</strong> c<strong>in</strong>namom<strong>in</strong>) have been shown to<br />
b<strong>in</strong>d lipids [61,62] . The majority <strong>of</strong> the residues <strong>of</strong> cryptoge<strong>in</strong> that <strong>in</strong>teract with<br />
ergosterol [63] are divergent <strong>in</strong> the three alpha HaELLs identified here as well as <strong>in</strong><br />
Phytophthora ELLs [49] . Therefore, ELLs may not b<strong>in</strong>d sterols <strong>and</strong> their function <strong>in</strong><br />
the <strong>in</strong>fection process rema<strong>in</strong>s unclear.<br />
The 16 Cyste<strong>in</strong>e-rich prote<strong>in</strong>s identified <strong>in</strong> our survey belong to a specific class<br />
<strong>of</strong> <strong>effectors</strong> with a high relative number <strong>of</strong> cyste<strong>in</strong>e residues (>5%) <strong>and</strong> no homology<br />
to other <strong>effectors</strong> [49,64] . The HaCRs are a heterogeneous group s<strong>in</strong>ce some prote<strong>in</strong>s<br />
have homologues <strong>in</strong> other oomycetes, although the majority are Hpa-specific
Isolate-specific <strong>effectors</strong> from Hyaloperonospora ESTs<br />
45<br />
(Fig. 5; Table 2). So far no function could be assigned to the HaCR prote<strong>in</strong>s based<br />
on known doma<strong>in</strong>s. Blast searches aga<strong>in</strong>st the Emoy2 genome sequence revealed<br />
a high level <strong>of</strong> conservation <strong>of</strong> several HaCR prote<strong>in</strong>s between these two isolates.<br />
Analysis <strong>of</strong> more Hpa isolates will reveal whether this degree <strong>of</strong> conservation is<br />
ma<strong>in</strong>ta<strong>in</strong>ed <strong>in</strong> other isolates. Only HaCR7 was more divergent between the isolates<br />
Waco9 <strong>and</strong> Emoy2 (Table 2). It is worth not<strong>in</strong>g that HaCR6 <strong>and</strong> 16 are not found<br />
<strong>in</strong> the Emoy2 genome, suggest<strong>in</strong>g that a different comb<strong>in</strong>ation <strong>of</strong> HaCR prote<strong>in</strong>s<br />
might be present <strong>in</strong> each Hpa isolate.<br />
The majority <strong>of</strong> the 18 RXLR c<strong>and</strong>idate <strong>effectors</strong> identified <strong>in</strong> our study were<br />
Hpa-specific (Table 1). Five RXLR c<strong>and</strong>idates comprise divergent prote<strong>in</strong> variants<br />
<strong>in</strong> seven Hpa isolates (Fig. 6) <strong>and</strong> significant numbers <strong>of</strong> non-synonymous relative<br />
to synonymous nucleotide substitutions. These effector c<strong>and</strong>idates add to a number<br />
<strong>of</strong> <strong>in</strong>terest<strong>in</strong>g RXLR genes that appear to be under diversify<strong>in</strong>g selection [12] <strong>in</strong>clud<strong>in</strong>g<br />
the two Hpa <strong>effectors</strong> ATR1 <strong>and</strong> ATR13, known to that trigger ETI [11,30] .<br />
We identified variations <strong>in</strong> the set <strong>of</strong> <strong>effectors</strong> produced by different Hpa<br />
isolates. Six Waco9 <strong>effectors</strong> (two HaCRs <strong>and</strong> four HaRXLRs) are not present <strong>in</strong><br />
Emoy2, as determ<strong>in</strong>ed by comparison with the Emoy2 genome sequence. Also,<br />
allele sequenc<strong>in</strong>g <strong>in</strong> seven Hpa isolates revealed that three RXLRs are so far only<br />
found <strong>in</strong> Waco9. The presence <strong>of</strong> an effector <strong>in</strong> some but not all isolates suggests<br />
that the gene has become lost dur<strong>in</strong>g host-pathogen co-evolution to avoid trigger<strong>in</strong>g<br />
ETI. This has been described for <strong>effectors</strong> <strong>of</strong> different pathogens, <strong>in</strong>clud<strong>in</strong>g<br />
Avr4 from Phytophthora <strong>in</strong>festans [65] <strong>and</strong> Avr2 from Cladosporium fulvum [66] , <strong>of</strong><br />
which truncated forms that are not recognized by R prote<strong>in</strong>s have been identified<br />
<strong>in</strong> several isolates [65] . However, delivery <strong>of</strong> RXLRs 12, 22 <strong>and</strong> 29 to leaves <strong>of</strong> 12<br />
different Arabidopsis accessions us<strong>in</strong>g the EDV system did not reveal an obvious<br />
host cell death response which would typify ETI (unpublished data). This may be<br />
because the bacterial delivery system dampens recognition or doesn’t permit full<br />
expression <strong>of</strong> ETI responses to specific Hpa <strong>effectors</strong>. Alternatively, isolate-specific<br />
<strong>effectors</strong> might affect colonization by different Hpa isolates by <strong>in</strong>terfer<strong>in</strong>g mostly<br />
with host PAMP-triggered defences. Our f<strong>in</strong>d<strong>in</strong>g that RXLR29, an effector that is<br />
present only <strong>in</strong> the highly virulent Hpa isolate Waco9, is able to suppress PTI <strong>and</strong><br />
enhance susceptibility to bacterial <strong>in</strong>fection, supports this idea. The categorization<br />
<strong>of</strong> different effector prote<strong>in</strong> types expressed by Hpa Waco9 dur<strong>in</strong>g Arabidopsis<br />
leaf tissue colonization <strong>and</strong> before the developmental transition to asexual <strong>and</strong><br />
sexual sporulation has allowed us to select effector c<strong>and</strong>idates for further <strong>functional</strong><br />
studies on <strong>in</strong>terference with the host immune system. The activities <strong>and</strong> <strong>in</strong>teractions<br />
<strong>of</strong> Waco9-derived <strong>effectors</strong> as well as the extensive allelic diversity <strong>of</strong> <strong>in</strong>dividual<br />
RXLR genes among Hpa isolates <strong>and</strong> oomycete pathogen species provides a<br />
rich source <strong>of</strong> material to trace the co-evolutionary history <strong>of</strong> this plant-biotroph<br />
system.
46 Chapter 2<br />
Materials <strong>and</strong> Methods<br />
Hpa isolates <strong>and</strong> plant material<br />
Hpa isolates used <strong>in</strong> this study were orig<strong>in</strong>ally collected from Arabidopsis populations<br />
with<strong>in</strong> the UK (isolates Cala2, Emwa1, Emco5, Emoy2, H<strong>in</strong>d2, Maks9,<br />
Noco2, Noks1) <strong>and</strong> the Netherl<strong>and</strong>s (Waco9) [16,18,67,68] . Arabidopsis plants were<br />
grown at 22°C with ~75% relative humidity (RH) <strong>and</strong> a 10 h light period. For Hpa<br />
<strong>in</strong>fections, conidiospore suspensions (5×104 conidiospores.ml -1 ) were spray <strong>in</strong>oculated<br />
on 2-week-old Arabidopsis seedl<strong>in</strong>gs. Plants were allowed to dry for 1 h <strong>and</strong><br />
kept at 100% RH for 24 h <strong>in</strong> a growth chamber with 10 h light at 16°C. Plants were<br />
then moved to ~75% RH for an additional 4 to 6 days to delay asexual sporulation.<br />
Hpa growth on Arabidopsis leaves was visualized on whole-leaf mounts sta<strong>in</strong>ed<br />
with trypan blue as described previously [67] <strong>and</strong> exam<strong>in</strong>ed by differential-<strong>in</strong>terference<br />
contrast microscopy.<br />
RNA isolation <strong>and</strong> cDNA library construction<br />
For RNA isolations, <strong>in</strong>fected leaf material was ground to a f<strong>in</strong>e powder <strong>in</strong> liquid<br />
nitrogen. Total RNA <strong>of</strong> Waco9-<strong>in</strong>fected Ws eds1-1 plants was isolated us<strong>in</strong>g the<br />
RNeasy Plant M<strong>in</strong>i Kit (Qiagen), follow<strong>in</strong>g manufacturer’s <strong>in</strong>structions. Poly (A+)<br />
RNA was purified from 1.3 mg <strong>of</strong> total RNA us<strong>in</strong>g dynabeads oligo (dT)25 (Dynal<br />
Biotech). To elim<strong>in</strong>ate rRNA contam<strong>in</strong>ation, the mRNA was purified twice. RNA<br />
concentrations were determ<strong>in</strong>ed on a UV m<strong>in</strong>i 1240 spectrophotometer (Shimadzu)<br />
<strong>and</strong> quality was assessed with the bioanalyzer 2100 us<strong>in</strong>g the RNA 6000 Nano<br />
Assay kit (Agilent Technologies). Poly (A+) RNA (5 µg) was used to construct a<br />
directional cDNA library <strong>in</strong> the λ-Zap vector (ZAP-cDNA synthesis, GigapackIII<br />
clon<strong>in</strong>g kit, Agilent Technologies) follow<strong>in</strong>g manufacturer’s <strong>in</strong>structions. cDNA<br />
was synthesized conta<strong>in</strong><strong>in</strong>g EcoRI <strong>and</strong> XhoI sites at the 5′ <strong>and</strong> 3′ ends, respectively,<br />
allow<strong>in</strong>g unidirectional clon<strong>in</strong>g <strong>of</strong> cDNA. Size fractionation <strong>of</strong> the synthesized<br />
cDNA’s was performed <strong>and</strong> 12 fractions were collected <strong>and</strong> precipitated with 100%<br />
ethanol. The pellet was resuspended <strong>in</strong> RNase-free water <strong>and</strong> verified on the Bioanalyzer<br />
2100 us<strong>in</strong>g the DNA 7500 LabChip Kit (Agilent Technologies). Phagemid<br />
DNA was excised without library amplification. DNA isolations <strong>and</strong> sequenc<strong>in</strong>g<br />
were done by Macrogen (Korea). Sequenc<strong>in</strong>g reactions were performed from the 5′<br />
end us<strong>in</strong>g a T3 promoter primer.
Isolate-specific <strong>effectors</strong> from Hyaloperonospora ESTs<br />
47<br />
Sequence process<strong>in</strong>g <strong>and</strong> <strong>analysis</strong><br />
Base call<strong>in</strong>g, quality clipp<strong>in</strong>g <strong>and</strong> vector screen<strong>in</strong>g were performed with pregap4,<br />
which is part <strong>of</strong> the Staden sequence <strong>analysis</strong> package [69] . ESTs were assembled<br />
<strong>in</strong>to contigs us<strong>in</strong>g CAP3 [70] . To def<strong>in</strong>e sequence orig<strong>in</strong>s, the assembled sequences<br />
were queried (BLASTN, e-value cut<strong>of</strong>f 1 e-5, no low complexity filter<strong>in</strong>g) [71]<br />
aga<strong>in</strong>st versions 6, 8.3 <strong>and</strong> trace files <strong>of</strong> the Hpa genome [25] , version 8 <strong>of</strong> the TAIR<br />
Arabidopsis genome [72] , version 1 <strong>of</strong> the Phytophthora <strong>in</strong>festans [24] , P. ramorum<br />
<strong>and</strong> P. sojae [23] genomes <strong>and</strong> NCBI non-redundant (nr) nucleotide database [73] . EST<br />
sequences were then split <strong>in</strong>to subsets based on the best blast match. ESTs from Ha<br />
were submitted to dbEST (NCBI).<br />
The most likely ORF <strong>of</strong> the Hpa set <strong>of</strong> sequences was identified by translat<strong>in</strong>g<br />
the assembled EST sequences <strong>and</strong> s<strong>in</strong>gletons <strong>in</strong> 3 positive frames <strong>and</strong> select<strong>in</strong>g the<br />
longest ORF. Prote<strong>in</strong> predictions shorter than 10 am<strong>in</strong>o acids were discarded. For<br />
selection <strong>of</strong> the secreted prote<strong>in</strong>s, all prote<strong>in</strong> models were trimmed to start with a<br />
methion<strong>in</strong>e. Signal peptide predictions were performed by SignalP version 3.0 [43,44] ,<br />
us<strong>in</strong>g both the neural network method <strong>and</strong> the hidden markov model methods at<br />
default cut<strong>of</strong>fs. Sequences with predicted transmembrane helices downstream<br />
<strong>of</strong> the signal peptide determ<strong>in</strong>ed by TMHMM version 2 (http://www.cbs.dtu.<br />
dk/services/TMHMM/) [74] were discarded. Putative functions were assigned<br />
by doma<strong>in</strong> composition after scann<strong>in</strong>g the sequences aga<strong>in</strong>st Pfam [45] us<strong>in</strong>g the<br />
gather<strong>in</strong>g threshold as cut-<strong>of</strong>f. Additionally, functions were manually assigned<br />
based on homology derived by similarity matches to NCBI nr (us<strong>in</strong>g blast), am<strong>in</strong>o<br />
acid composition or presence <strong>of</strong> an RLXR, RXLQ or RXLG motif after the signal<br />
peptide cleavage site (only consider<strong>in</strong>g prote<strong>in</strong>s with 40 or more am<strong>in</strong>o acids after<br />
the RXL sequence). The number <strong>of</strong> <strong>effectors</strong> encoded <strong>in</strong> the Hpa genome was<br />
either taken from the Hpa genome paper [25] , determ<strong>in</strong>ed by Pfam searches, or by<br />
manual annotation (e.g. for the number <strong>of</strong> cyste<strong>in</strong>e residues). For the elicit<strong>in</strong>s, the<br />
pI was determ<strong>in</strong>ed at http://www3.embl.de/cgi/pi-wrapper.pl. GPI anchor sites<br />
were predicted by the program big-PI Plant Predictor [75] <strong>and</strong> potential sites for<br />
O-glycosylation were predicted by NetOGlyc 3.1 [76] . Psort prediction (http://psort.<br />
hgc.jp/) [77] was used to identify putative nuclear localization signals <strong>in</strong> RXLR<br />
prote<strong>in</strong>s. Sequences <strong>of</strong> the HaELL, HaCR <strong>and</strong> HaRXLR genes were submitted to<br />
GenBank (accession numbers JF800099-JF800135).<br />
<strong>Identification</strong> <strong>of</strong> Hpa- <strong>and</strong> isolate Waco9-specific sequences<br />
For identification <strong>of</strong> secreted prote<strong>in</strong>s specific to Hpa or to isolate Waco9,<br />
similarity searches were conducted with BLASTP <strong>and</strong> TBLASTN with disabled<br />
low complexity filter<strong>in</strong>g <strong>and</strong> a fixed database size <strong>of</strong> 1.000.000 [70] . Sequences were
48 Chapter 2<br />
queried aga<strong>in</strong>st a local NCBI nr prote<strong>in</strong> <strong>and</strong> nucleotide databases (downloaded<br />
23rd June 2010) <strong>and</strong> a set <strong>of</strong> three oomycete genome sequences (P. <strong>in</strong>festans<br />
version 1 [24] , P. sojae version 2, <strong>and</strong> P. ramorum version 2 [23] .Sequences with no<br />
significant similarity aga<strong>in</strong>st other organisms (Hpa sequences were excluded;<br />
e-value cut<strong>of</strong>f 1e−5; covered for >75% to exclude hits with only local similarity)<br />
were selected <strong>and</strong> def<strong>in</strong>ed as Hpa-specific sequences. The predicted proteome <strong>and</strong><br />
the genome assembly <strong>of</strong> Hpa isolate Emoy2 (versions 6.0 <strong>and</strong> 8.3) was searched<br />
to identify sequences potentially specific for isolate Waco9. All sequences with<br />
a significant hit (e-value cut<strong>of</strong>f 1e−10) <strong>and</strong> an identity ratio <strong>of</strong> >80% ((identity%<br />
<strong>of</strong> alignment*length alignment)/length query) [26] were filtered. If two Waco9<br />
sequences had the same Emoy2 sequence as a best hit, we excluded the better hit<br />
<strong>and</strong> reta<strong>in</strong>ed the other one. The rema<strong>in</strong><strong>in</strong>g sequences were compared manually to<br />
the Hpa Emoy2 trace files that also conta<strong>in</strong> reads not present <strong>in</strong> genome assembly.<br />
Sequences without significant blast hits were def<strong>in</strong>ed as Waco9-specific.<br />
Hpa allele sequences <strong>and</strong> <strong>analysis</strong><br />
To sequence the RXLR alleles <strong>of</strong> Hpa isolates Cala2, Emco5, Emoy2, H<strong>in</strong>d2,<br />
Maks9 <strong>and</strong> Noks1, genomic DNA was isolated from Hpa-<strong>in</strong>fected Ws eds1-1<br />
leaves us<strong>in</strong>g DNeasy Plant M<strong>in</strong>i Kit (Qiagen). Primer pairs (Table S1) flank<strong>in</strong>g<br />
or with<strong>in</strong> the cod<strong>in</strong>g sequence were designed from Waco9 assembled sequences<br />
<strong>and</strong> used to amplify alleles <strong>of</strong> the Hpa isolates from genomic DNA. If a s<strong>in</strong>gle<br />
amplification product was obta<strong>in</strong>ed, the PCR product was sequenced directly<br />
us<strong>in</strong>g the amplification primers. If multiple PCR fragments were amplified from a<br />
s<strong>in</strong>gle isolate these were <strong>in</strong>dividually purified from gel us<strong>in</strong>g NucleoSp<strong>in</strong> Extract<br />
II (Machery-Nagel) <strong>and</strong> sequenced. Obta<strong>in</strong>ed sequences that were not readable due<br />
to amplification <strong>of</strong> different products <strong>of</strong> the same size, Phusion (F<strong>in</strong>nzymes)-amplified<br />
PCR products were cloned <strong>in</strong> pENTR/D-TOPO (Invitrogen) <strong>and</strong> sequenced.<br />
In cases where the obta<strong>in</strong>ed sequences were not full-length (3′or 5′ sequences<br />
miss<strong>in</strong>g), new primers were designed based on the Emoy2 genome sequence<br />
(Versions 6 <strong>and</strong> 8.3). When no PCR product was amplified, new reactions were<br />
performed us<strong>in</strong>g up to three new primer sets.<br />
The RXLR DNA <strong>and</strong> predicted prote<strong>in</strong> sequences were analysed us<strong>in</strong>g Mega4<br />
s<strong>of</strong>tware [78] . Alignments <strong>of</strong> the obta<strong>in</strong>ed allele sequences (prote<strong>in</strong> level) were performed<br />
us<strong>in</strong>g the ClustalW option [79] <strong>and</strong> manually edited. The number <strong>of</strong> variable<br />
sites was analysed by the Sequence Data Explorer tool. Alignments <strong>of</strong> the allele<br />
sequences (nucleotide level) were used to calculate the rate <strong>of</strong> non-synonymous<br />
(dN) <strong>and</strong> synonymous (dS) substitutions. The rates <strong>of</strong> substitutions were calculated<br />
us<strong>in</strong>g the Nei <strong>and</strong> Gojobori’s method [80] , as implemented <strong>in</strong> the MEGA4 program.<br />
St<strong>and</strong>ard error was determ<strong>in</strong>ed by 500 bootstrap replications. The null hypothesis
Isolate-specific <strong>effectors</strong> from Hyaloperonospora ESTs<br />
49<br />
<strong>of</strong> no selection (H0: dN = dS) versus the positive selection hypothesis (H1: dN ><br />
dS) were tested us<strong>in</strong>g the Z-test: Z = (dN-dS)/ √(Var(dS)+Var(dN)).<br />
RXLR29 delivery by Pseudomonas syr<strong>in</strong>gae<br />
The cod<strong>in</strong>g sequences <strong>of</strong> Hpa Waco9 RXLR29 (without signal peptide) <strong>and</strong> YFP<br />
were amplified by PCR <strong>and</strong> cloned <strong>in</strong> pENTR/D-TOPO (Invitrogen) us<strong>in</strong>g primers:<br />
CACCATGGAGGTGGTCCTGATC (forward) <strong>and</strong> TTACTTGCCAGGACGCGC<br />
(reverse) for RXLR29 <strong>and</strong> CACCATGGTGAGCAAGGGCGAGGAGCTGTTC<br />
(forward) <strong>and</strong> AGTCTAGAGCTCTTACTTGTACAGCTCGTCCATGC (reverse)<br />
for YFP. Follow<strong>in</strong>g Gateway clon<strong>in</strong>g procedures these genes were cloned <strong>in</strong>to<br />
pEDV6, a variant <strong>of</strong> the previously described pEDV3 [42] that has a gateway cassette<br />
<strong>in</strong>stead <strong>of</strong> a multiple clon<strong>in</strong>g site (k<strong>in</strong>dly provided by K. Sohn, G. Fabro <strong>and</strong> J.<br />
Jones, Sa<strong>in</strong>sbury Laboratory, Norwich, UK). Plasmids were mobilized from E. coli<br />
DH5α to Pst DC3000ΔCEL or Pst DC3000-LUX, which has stable chromosomal<br />
<strong>in</strong>tegration <strong>of</strong> the luxCDABE operon from Photorhabdus lum<strong>in</strong>escens [81] , by<br />
st<strong>and</strong>ard triparental mat<strong>in</strong>g us<strong>in</strong>g E. coli HB101 (pRK2013) as a helper stra<strong>in</strong>. Pst<br />
DC3000ΔCEL-ATR13 (Emco5) <strong>and</strong> Pst DC3000-LUX-ATR13 (Emco5) were<br />
k<strong>in</strong>dly provided by G. Fabro <strong>and</strong> J. Jones.<br />
Callose sta<strong>in</strong><strong>in</strong>g <strong>and</strong> microscopic <strong>analysis</strong><br />
Leaves <strong>of</strong> 5-week-old Arabidopsis accession Col-0 plants were h<strong>and</strong>-<strong>in</strong>filtrated<br />
with 1×10 8 cfu/ml Pst DC3000ΔCEL suspensions. A total <strong>of</strong> ~48 leaf samples were<br />
taken for callose sta<strong>in</strong><strong>in</strong>g 12–14 h after <strong>in</strong>filtration. Leaves were cleared with 100%<br />
ethanol, <strong>and</strong> re-hydrated <strong>and</strong> sta<strong>in</strong>ed with anil<strong>in</strong>e blue (0.05% <strong>in</strong> phosphate buffer<br />
pH 8.0) for 24 h. Images were taken us<strong>in</strong>g an Olympus AX70 Microscope with UV<br />
filter. Callose spots were counted us<strong>in</strong>g ImageJ (http://rsb.<strong>in</strong>fo.nih.gov/ij/) [82] .<br />
Bacterial growth assay<br />
Leaves <strong>of</strong> 5-week-old Arabidopsis accession Col-0 were h<strong>and</strong>-<strong>in</strong>filtrated with<br />
a bacterial <strong>in</strong>oculum <strong>of</strong> 5×10 5 cfu/ml. Initially, PstDC3000-LUX was used for<br />
assessment <strong>of</strong> bacterial growth by measur<strong>in</strong>g <strong>in</strong>creased luciferase activity as previously<br />
published [81] . For colony count<strong>in</strong>g, <strong>in</strong>fected leaves were collected at 0 <strong>and</strong><br />
3 days after bacterial <strong>in</strong>filtration. A total <strong>of</strong> 12 leaves divided <strong>in</strong>to three biological<br />
replicates were harvested per sample <strong>and</strong> time po<strong>in</strong>t. Bacterial growth was measured<br />
by gr<strong>in</strong>d<strong>in</strong>g <strong>in</strong>fected leaves <strong>and</strong> plat<strong>in</strong>g serial dilutions on solid KB medium<br />
with appropriate antibiotics. Similar results were obta<strong>in</strong>ed <strong>in</strong> two <strong>in</strong>dependent<br />
experiments.
50 Chapter 2<br />
Acknowledgments<br />
We thank Sh<strong>in</strong>pei Katou, Joyce Elberse <strong>and</strong> Tale Sliedrecht for technical assistance,<br />
Aless<strong>and</strong>ro Guida <strong>and</strong> Henk van den Toorn for bio<strong>in</strong>formatics assistance, <strong>and</strong> Kee<br />
Sohn, Georg<strong>in</strong>a Fabro, <strong>and</strong> Jonathan Jones for k<strong>in</strong>dly provid<strong>in</strong>g the EDV6 vector<br />
<strong>and</strong> Pst ΔCEL mutant.
Isolate-specific <strong>effectors</strong> from Hyaloperonospora ESTs<br />
51<br />
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67 Van Damme M, Andel A, Huibers RP, Panstruga R, Weisbeek PJ & Van den Ackerveken G<br />
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Hyaloperonospora parasitica. Molecular Plant-Microbe Interactions 18, 583-92.<br />
68 Bittner-Eddy P, Can C, Gunn N, P<strong>in</strong>el M, Tör M, Crute I, Holub EB & Beynon J (1999) Genetic<br />
<strong>and</strong> physical mapp<strong>in</strong>g <strong>of</strong> the RPP13 locus, <strong>in</strong> Arabidopsis, responsible for specific recognition <strong>of</strong><br />
several Peronospora parasitica (<strong>downy</strong> <strong>mildew</strong>) isolates. Molecular Plant-Microbe Interactions<br />
12, 792-802.<br />
69 Staden R, Beal KF & Bonfield JK (2000) The Staden package, 1998. Methods <strong>in</strong> Molecular<br />
Biology 132, 115-30.<br />
70 Huang X & Madan A (1999) CAP3: A DNA sequence assembly program. Genome Research 9,<br />
868-77.<br />
71 Altschul SF, Gish W, Miller W, Myers EW & Lipman DJ (1990) Basic local alignment search<br />
tool. Journal <strong>of</strong> Molecular Biology 215, 403-10.<br />
72 Rhee SY (2003) The Arabidopsis Information Resource (TAIR): a model organism database<br />
provid<strong>in</strong>g a centralized, curated gateway to Arabidopsis biology, research materials <strong>and</strong><br />
community. Nucleic Acids Research 31, 224-8.<br />
73 Pruitt KD, Tatusova T & Maglott DR (2005) NCBI Reference Sequence (RefSeq): a curated nonredundant<br />
sequence database <strong>of</strong> genomes, transcripts <strong>and</strong> prote<strong>in</strong>s. Nucleic Acids Research 33,<br />
D501-4.<br />
74 Krogh A, Larsson B, von Heijne G & Sonnhammer EL (2001) Predict<strong>in</strong>g transmembrane prote<strong>in</strong><br />
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Biology 305, 567-80.<br />
75 Eisenhaber B, Wildpaner M, Schultz CJ, Borner GHH, Dupree P & Eisenhaber F (2003)<br />
Glycosylphosphatidyl<strong>in</strong>ositol lipid anchor<strong>in</strong>g <strong>of</strong> plant prote<strong>in</strong>s. Sensitive prediction from<br />
sequence- <strong>and</strong> genome-wide studies for Arabidopsis <strong>and</strong> rice. Plant Physiology 133, 1691-701.<br />
76 Julenius K, Mølgaard A, Gupta R & Brunak S (2005) Prediction, conservation <strong>analysis</strong>, <strong>and</strong><br />
structural characterization <strong>of</strong> mammalian muc<strong>in</strong>-type O-glycosylation sites. Glycobiology 15,<br />
153-64.<br />
77 Nakai K & Horton P (1999) PSORT: a program for detect<strong>in</strong>g sort<strong>in</strong>g signals <strong>in</strong> prote<strong>in</strong>s <strong>and</strong><br />
predict<strong>in</strong>g their subcellular localization. Trends <strong>in</strong> Biochemical Sciences 24, 34-6.
Isolate-specific <strong>effectors</strong> from Hyaloperonospora ESTs<br />
57<br />
78 Tamura K, Dudley J, Nei M & Kumar S (2007) MEGA4: Molecular Evolutionary Genetics<br />
Analysis (MEGA) s<strong>of</strong>tware version 4.0. Molecular Biology <strong>and</strong> Evolution 24, 1596-9.<br />
79 Thompson JD, Higg<strong>in</strong>s DG & Gibson TJ (1994) CLUSTAL W: improv<strong>in</strong>g the sensitivity <strong>of</strong><br />
progressive multiple sequence alignment through sequence weight<strong>in</strong>g, position-specific gap<br />
penalties <strong>and</strong> weight matrix choice. Nucleic Acids Research 22, 4673-80.<br />
80 Nei M & Gojobori T (1986) Simple methods for estimat<strong>in</strong>g the numbers <strong>of</strong> synonymous <strong>and</strong><br />
nonsynonymous nucleotide substitutions. Molecular Biology <strong>and</strong> Evolution 3, 418-26.<br />
81 Fan J, Crooks C & Lamb C (2008) High-throughput quantitative lum<strong>in</strong>escence assay <strong>of</strong> the<br />
growth <strong>in</strong> planta <strong>of</strong> Pseudomonas syr<strong>in</strong>gae chromosomally tagged with Photorhabdus lum<strong>in</strong>escens<br />
luxCDABE. The Plant Journal 53, 393-9.<br />
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International 11, 36-42.
58 Chapter 2<br />
Supplemental Information<br />
A<br />
B<br />
C<br />
D<br />
Figure S1: Growth <strong>of</strong> Hpa Emoy2<br />
<strong>and</strong> Waco9 isolates <strong>in</strong> Arabidopsis.<br />
The level <strong>of</strong> colonization <strong>of</strong><br />
cotyledons <strong>of</strong> 2-week-old Arabidopsis<br />
Ws eds1-1 seedl<strong>in</strong>gs <strong>in</strong>fected with<br />
Hpa isolates Emoy2 (A,B) <strong>and</strong> Waco9<br />
(C,D) at 7 dpi is visualized by trypan<br />
blue-sta<strong>in</strong><strong>in</strong>g <strong>and</strong> light microscopy.<br />
Fluorescence<br />
Fluorescence<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
0<br />
120<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
At<br />
120<br />
25% Hpa 20% Hpa<br />
100<br />
20 25 30 35 40 45 50 55<br />
Migration time (sec)<br />
Hpa<br />
80<br />
50% Hpa 33% Hpa<br />
Fluorescence<br />
20 25 30 35 40 45 50 55 20 25 30 35 40 45 50 55<br />
Migration time (sec)<br />
Migration time (sec)<br />
Fluorescence<br />
60<br />
40<br />
20<br />
0<br />
80<br />
60<br />
40<br />
20<br />
0<br />
20 25 30 35 40 45 50 55<br />
Migration time (sec)<br />
Figure S2: Determ<strong>in</strong>ation <strong>of</strong> Hpa<br />
rRNA peaks on the Bioanalyzer 2100.<br />
Total RNA isolated from Hpa<br />
conidiospores <strong>and</strong> Arabidopsis<br />
leaves was mixed <strong>in</strong> different<br />
proportions to allow identification<br />
<strong>of</strong> the correspond<strong>in</strong>g rRNA peaks <strong>of</strong><br />
Hpa <strong>and</strong> Arabidopsis <strong>in</strong> bioanalyzer<br />
pr<strong>of</strong>iles. Peak sizes correlated with the<br />
relative amount <strong>of</strong> plant <strong>and</strong> pathogen<br />
total RNA.<br />
HaCr<strong>in</strong>kler<br />
(393 aa)<br />
SP LRLYVAKR C<br />
Figure S3: Schematic representation<br />
<strong>of</strong> the Hpa Cr<strong>in</strong>kler identified <strong>in</strong> the<br />
Waco9 cDNA library.<br />
The signal peptide (SP), the variable<br />
CRN motif (LYVAK) <strong>and</strong> the<br />
C-term<strong>in</strong>al doma<strong>in</strong> (C) are shown.
Isolate-specific <strong>effectors</strong> from Hyaloperonospora ESTs<br />
59<br />
RXLR29_Emoy ATGCGTCTGTCTGC-ATCCTGTTCTTGATTGTGGCTCCCCTGCATCTTTGTGTCGGCGAG 59<br />
RXLR29_Noks ATGCGTCTGTCTGC-ATCCTGTTCTTGATTGTGGCTCCCCTGCATCTTTGTGTCGGCGAG 59<br />
RXLR29_Cala ATGCGTCTGTCTGC-ATCCTGTTCTTGATTGTGGCTCCCCTGCATCTTTGTGTCGGCGAG 59<br />
RXLR29_Emco ATGCGTCTGTCTGC-ATCCTGTTCTTGATTGTGGCTCCCCTGCATCTTTGTGTCGGCGAG 59<br />
RXLR29_H<strong>in</strong>d ATGCGTCTGTCTGC-ATCCTGTTCTTGATTGTGGCTCCCCTGCATCTTTGTGTCGGCGAG 59<br />
RXLR29_Maks1 ATGCGTCTGTCTGC-ATCCTGTTCTTGATTGTGGCTCCCCTGCATCTTTGTGTCGGCGAG 59<br />
RXLR29_Maks2 ATGCGTCTGTCTGCTATCCTGCCCTTGATTGTGGCTCCC-TGCATCTTTGTGTCGGCGAG 59<br />
RXLR29_Waco ATGCGTCTGTCTGCTATCCTGCCCTTGATTGTGGCTCCCCTGCATCTTTGTGTCGGCGAG 60<br />
************** ****** **************** ********************<br />
RXLR29_Emoy GTGGTTCTGATCCCTGCGACGATGGAGAACCCACTTCTTCGTTCGGCTTCTTCTGCTGGT 119<br />
RXLR29_Noks GTGGTTCTGATCCCTGCGACGATGGAGAACCCACTTCTTCGTTCGGCTTCTTCTGCTGGT 119<br />
RXLR29_Cala GTGGTTCTGATCCCTGCGACGATGGAGAACCCACTTCTTCGTTCGGCTTCTTCTGCTGGT 119<br />
RXLR29_Emco GTGGTTCTGATCCCTGCGACGATGGAGAACCCACTTCTTCGTTCGGCTTCTTCTGCTGGT 119<br />
RXLR29_H<strong>in</strong>d GTGGTTCTGATCCCTGCGACGATGGAGAACCCACTTCTTCGTTCGGCTTCTTCTGCTGGT 119<br />
RXLR29_Maks1 GTGGTTCTGATCCCTGCGACGATGGAGAACCCACTTCTTCGTTCGGCTTCTTCTGCTGGT 119<br />
RXLR29_Maks2 GTGGTCCTGATCCCTGCGACGATGGAGAACCCACTTCTTCGTTCGGCTTCTTCTCCTGGT 119<br />
RXLR29_Waco GTGGTCCTGATCCCTGCGACGATGGAGAACCCACTTCTTCGTTCGGCTTCTTCTGCTGGT 120<br />
***** ************************************************ *****<br />
RXLR29_Emoy GCTGG-GCACGCAACGACGGCCGATCCCTGCGAGAACTAAATTCAGCGGCTGGTATTGGC 178<br />
RXLR29_Noks GCTGG-GCACGCAACGACGGCCGATCCCTGCGAGAACTAAATTCAGCGGCTGGTATTGGC 178<br />
RXLR29_Cala GCTGG-GCACGCAACGACGGCCGATCCCTGCGAGAACTAAATTCAGCGGCTGGTATTGGC 178<br />
RXLR29_Emco GCTGG-GCACGCAACGACGGCCGATCCCTGCGAGAACTAAATTCAGCGGCTGGTATTGGC 178<br />
RXLR29_H<strong>in</strong>d GCTGG-GCACGCAACGACGGCCGATCCCTGCGAGAACTAAATTCAGCGGCTGGTATTGGC 178<br />
RXLR29_Maks1 GCTGG-GCACGCAACGACGGCCGATCCCTGCGAGAACTAAATTCAGCGGCTGGTATTGGC 178<br />
RXLR29_Maks2 GCTGGTGCACGCAACGACGGCCGATCCCTGCGAGAACTAAATTCAGCGGCTGGTATTGGC 179<br />
RXLR29_Waco GCTGGTGCACGCAACGACGGCCGATCCCTGCGAGAACTAAATTCAGCGGCTGGTATTGGC 180<br />
***** ******************************************************<br />
RXLR29_Emoy GCGGAAATGTCGAAAGTAGTCTCCGAGTTCGGAGGACATTTCAAAGGGACAACAGGCACT 238<br />
RXLR29_Noks GCGGAAATGTCGAAAGTAGTCTCCGAGTTCGGAGGACATTTCAAAGGGACAACAGGCACT 238<br />
RXLR29_Cala GCGGAAATGTCGAAAGTAGTCTCCGAGTTCGGAGGACATTTCAAAGGGACAACAGGCACT 238<br />
RXLR29_Emco GCGGAAATGTCGAAAGTAGTCTCCGAGTTCGGAGGACATTTCAAAGGGACAACAGGCACT 238<br />
RXLR29_H<strong>in</strong>d GCGGAAATGTCGAAAGTAGTCTCCGAGTTCGGAGGACATTTCAAAGGGACAACAGGCACT 238<br />
RXLR29_Maks1 GCGGAAATGTCGAAAGTAGTCTCCGAGTTCGGAGGACATTTCAAAGGGACAACAGGCACT 238<br />
RXLR29_Maks2 GCGGAAATGTCGAAAGTAGCCTCCAAGTTTGGAGGACATTTCAAAGGGACAACAGGCACT 239<br />
RXLR29_Waco GGGGAAATGTCGAAAGTAGTTTCCGAGTTCGGAGGACATTTCAAAGGGACAACAGGCACT 240<br />
* ***************** *** **** ******************************<br />
RXLR29_Emoy TCGGAGAGTGTTGCATCAAAGAAAGCCGAAGATGCAGCAATGGGGGCAGCAGATCGTGAT 298<br />
RXLR29_Noks TCGGAGAGTGTTGCATCAAAGAAAGCCGAAGATGCAGCAATGGGGGCAGCAGATCGTGAT 298<br />
RXLR29_Cala TCGGAGAGTGTTGCATCAAAGAAAGCCGAAGATGCAGCAATGGGAGCAGCAGATCGTGAT 298<br />
RXLR29_Emco TCGGAGAGTGTTGCATCAAAGAAAGCCGAAGATGCAGCAATGGGAGCAGCAGATCGTGAT 298<br />
RXLR29_H<strong>in</strong>d TCGGAGAGTGTTGCATCAAAGAAAGCCGAAGATGCAGCAATGGGAGCAGCAGATCGTGAT 298<br />
RXLR29_Maks1 TCGGAGAGTGTTGCATCAAAGAAAGCCGAAGATGCAGCAATGGGGGCAGCAGATCGTGAT 298<br />
RXLR29_Maks2 TCGGAGAGTGTCGCATCAAAGAAAGCCGAAGATGCAGCAATGAGGGCAGCAGATGATGAT 299<br />
RXLR29_Waco TCGGAGAGTGTCGCATCAAAGAAAGCCAAAGATGCAGCAATGAGGGCAGCAGATGATGAT 300<br />
*********** *************** ************** * ********* ****<br />
RXLR29_Emoy GAAGGACCCATTTGGGCGACGAGAAAAGACCTCGAAGATGTTTTGAATGTTGGAAAAAAA 358<br />
RXLR29_Noks GAAGGACCCATTTGGGCGACGAGAAAAGACCTCGAAGATGTTTTGAATGTTGGAAAAAAA 358<br />
RXLR29_Cala GAAGGACCCATTTGGGCGACGAGAAAAGACCTCGAAGATGTTTTGAATGTTGGAAAAAAA 358<br />
RXLR29_Emco GAAGGACCCATTTGGGCGACGAGAAAAGACCTCGAAGATGTTTTGAATGTTGGAAAAAAA 358<br />
RXLR29_H<strong>in</strong>d GAAGGACCCATTTGGGCGACGAGAAAAGACCTCGAAGATGTTTTGAATGTTGGAAAAAAA 358<br />
RXLR29_Maks1 GAAGGACCCATTTGGGCGACGAGAAAAGACCTCGAAGATGTTTTGAATGTTGGAAAAAAA 358<br />
RXLR29_Maks2 GAAGGACCCATTTTTGCGACGAGAGAAGACCTCGATGATGTTATGAAAGCTGGAAAAGAA 359<br />
RXLR29_Waco GAAGGACCCATTTTTGCGACGAAAGAAGACCTCGCTGATGTTTTGGCTGTTGGAAAAGAT 360<br />
************* ******* * ********* ****** ** * ******* *<br />
RXLR29_Emoy AAACTGAC-AAGAAAAAAA-GGCGCGTCCTGGCAAGTAA 395<br />
RXLR29_Noks AAACTGAC-AAGAAAAAAA-GGCGCGTCCTGGCAAGTAA 395<br />
RXLR29_Cala AAACTGAC-AAGAAAAAAAAGGCGCGTCCTGGCAAGTAA 396<br />
RXLR29_Emco AAACTGAC-AAGAAAAAAAAGGCGCGTCCTGGCAAGTAA 396<br />
RXLR29_H<strong>in</strong>d AAACTGAC-AAGAAAAAAAAGGCGCGTCCTGGCAAGTAA 396<br />
RXLR29_Maks1 AA-CTGAC-AAGAAAAAAA-GGCGCGTCCTGGCAAGTAA 394<br />
RXLR29_Maks2 AAACCGGCCAGGAAAAAAAAGGTGCGTCCTGGCAAGTAA 398<br />
RXLR29_Waco AAACCGGCCAGGAAAAAAAAGGCGCGTCCTGGCAAGTAA 399<br />
** * * * * ******** ** ****************<br />
Figure S4: Multiple alignment <strong>of</strong> RXLR29 sequences from 7 Hpa isolates. Stop codons are shaded grey.
60 Chapter 2<br />
Table S1: Primers used for RXLR allele sequenc<strong>in</strong>g.<br />
Gene Forward Reverse<br />
RXLR3 AGATGCGTCTTCACATCCTG TTGTTGTTATTCTGCTGCTGCT<br />
RXLR4 GGAAAGATGCGTCTTCACAT ATTGTTGTTATTCTGCTGCTGCT<br />
RXLR5 TTTTTCGTTGACAACCATGC CAATAACCTCGCTGGCTTACA<br />
RXLR6 CACGTCGTAAGTTGACCATG ACATCGATTGCTCGCATCAC<br />
RXLR7 CGTCCCGTCCGACATACTTA GTAGCTACCGCCACCCAGT<br />
RXLR9 TCTCTTAAGCTTGTGCGATCTTC AAGTCCTCACCTCCAGTCCA<br />
RXLR13 TCGACCCCTTCACCTGTTAC GACATCAGGTCTGCGTCTCA<br />
RXLR15 CACAACGTCAAACCGACATC GGGTTTATTGCCGTTTCGTA<br />
RXLR16 ATGCTGCCAGCTCGCGCAG TCAAATCGCCGCATTGATGTC<br />
RXLR17 CACCCACCGAGAACGAGTAT TGAATGTCTATCCGCCGTCT<br />
RXLR18 CAATTTAATAATAGAGGGAGAGTCACG AGGCTGCCAAAGCTTCAAGT<br />
RXLR19 CGATTCATCGCCCTCACC CGGTTTCATCACCCACTACG<br />
RXLR20 CTTTCACCTTGGGTCCTTCC AAGCACACTTAAGTTCTACTATTACGC<br />
RXLR20 CGATATGCGACGGAGAGCTG CAGCTCTCCGTCGCATATCG<br />
RXLR21 CGACGATGTAGGATCAGATACG TTTGTGTTTTAGCTGCTACGG<br />
RXLR23 TCGACCCCTTCACCTGTTAC CCACGCACTACCTTAGCACA<br />
RXLR29 AAGTCCTCACCTCCAGTCCA CATTGCCGTAGCTCCTTTGT
Isolate-specific <strong>effectors</strong> from Hyaloperonospora ESTs<br />
61<br />
Table S2: Enrichment <strong>of</strong> prote<strong>in</strong> class members <strong>in</strong> the EST project compared to the genome-wide<br />
occurunce.<br />
A hypergeometric propability value
62 Chapter 2<br />
Table S3: Functional classification <strong>of</strong> 90 unigenes based on Pfam doma<strong>in</strong> predictions, BLASTX <strong>and</strong><br />
manual annotation (cont<strong>in</strong>ued on next page). For manual annotations the IDs are given <strong>in</strong> lists.<br />
Pfam doma<strong>in</strong> predictions<br />
ID Pfam ID Pfam name E-value<br />
Pro3526IW PF00012.13 HSP70 2.8e-98<br />
Pro1991IW PF00026.16 Asp 5e-58<br />
Pro2490IW PF00043.18 GST_C 2.9e-06<br />
Pro3738IW PF00056.16 Ldh_1_N 1.2e-35<br />
Pro2968IW PF00085.13 Thioredox<strong>in</strong> 6.1e-27<br />
Pro2968IW PF00085.13 Thioredox<strong>in</strong> 2e-27<br />
Pro2875IW PF00085.13 Thioredox<strong>in</strong> 2e-28<br />
Pro2875IW PF00085.13 Thioredox<strong>in</strong> 1.7e-26<br />
Pro3759IW PF00085.13 Thioredox<strong>in</strong> 6e-31<br />
Pro3759IW PF00085.13 Thioredox<strong>in</strong> 2.2e-31<br />
TC1311MERGE PF00106.18 adh_short 1.3e-10<br />
Pro2915IW PF00149.21 Metallophos 0.00014<br />
Pro3807IW PF00150.11 Cellulase 6.1e-12<br />
Pro3351IW PF00160.14 Pro_isomerase 4.9e-46<br />
Pro3776IW PF00245.13 Alk_phosphatase 7.2e-76<br />
Pro3221IW PF00254.21 FKBP_C 4.3e-32<br />
Pro3019IW PF00300.15 PGAM 3.5e-10<br />
Pro2270IW PF00400.25 WD40 1.3<br />
Pro2270IW PF00400.25 WD40 0.00033<br />
Pro2270IW PF00400.25 WD40 2.4e-10<br />
Pro3224IW PF00428.12 Ribosomal_60s 2.2e-20<br />
Pro3566IW PF00445.11 Ribonuclease_T2 5.6e-32<br />
Pro1764IW PF00450.15 Peptidase_S10 2e-81<br />
Pro2843IW PF00450.15 Peptidase_S10 1.5e-80<br />
Pro534IW PF00462.17 Glutaredox<strong>in</strong> 9.9e-20<br />
Pro93IW PF00487.17 FA_desaturase 1.7e-23<br />
Pro2024IW PF00614.15 PLDc 2.9e-09<br />
Pro3222IW PF00639.14 Rotamase 1.1e-15<br />
Pro3222IW PF00639.14 Rotamase 1.8e-16<br />
Pro203IW PF00742.12 Homoser<strong>in</strong>e_dh 3.8e-39<br />
Pro2119IW PF00899.14 ThiF 1.6e-36<br />
Pro3133IW PF00964.10 Elicit<strong>in</strong> 4.7e-07<br />
HaELL1_Waco PF00964.10 Elicit<strong>in</strong> 4.6e-15<br />
HaELL2_Waco PF00964.10 Elicit<strong>in</strong> 6.2e-15<br />
HaELL3_Waco PF00964.10 Elicit<strong>in</strong> 3.8e-13<br />
Pro3815IW PF01011.14 PQQ 1.5e-05<br />
Pro3584IW PF01095.12 Pect<strong>in</strong>esterase 1.2e-48<br />
Pro3481IW PF01263.13 Aldose_epim 2.5e-19<br />
Pro3763IW PF01263.13 Aldose_epim 6.1e-17<br />
Pro5506MERGE PF01341.10 Glyco_hydro_6 2.1e-53<br />
Pro245IW PF01435.11 Peptidase_M48 1.7e-55<br />
Pro2157IW PF01451.14 LMWPc 1e-25
Isolate-specific <strong>effectors</strong> from Hyaloperonospora ESTs<br />
63<br />
Pfam doma<strong>in</strong> predictions<br />
ID Pfam ID Pfam name E-value<br />
Pro776IW PF02219.10 MTHFR 2.7e-83<br />
Pro2250IW PF02466.12 Tim17 6.2e-12<br />
Pro2490IW PF02798.13 GST_N 3.7e-16<br />
Pro9IW PF02815.12 MIR 3.2e-12<br />
Pro3738IW PF02866.11 Ldh_1_C 7.6e-34<br />
Pro1884IW PF03330.11 DPBB_1 1.4e-09<br />
Pro2846IW PF03372.16 Exo_endo_phos 4.7e-10<br />
Pro35IW PF03388.6 Lect<strong>in</strong>_leg-like 2.4e-30<br />
Pro985IW PF03446.8 NAD_b<strong>in</strong>d<strong>in</strong>g_2 1.4e-29<br />
Pro203IW PF03447.9 NAD_b<strong>in</strong>d<strong>in</strong>g_3 1e-17<br />
Pro2681IW PF03813.7 Nrap 1.7e-40<br />
Pro1930IW PF04597.7 Ribophor<strong>in</strong>_I 3e-23<br />
Pro1396IW PF04616.7 Glyco_hydro_43 5.4e-27<br />
Pro3361IW PF04981.6 NMD3 8.9e-70<br />
Pro3632IW PF05162.6 Ribosomal_L41 2.2e-08<br />
HaNLP1_Waco9 PF05630.4 NPP1 2.4e-48<br />
HaNLP2_Waco9 PF05630.4 NPP1 2.6e-45<br />
Pro964IW PF07542.4 ATP12 2.2e-13<br />
Pro3759IW PF07749.5 ERp29 3.1e-13<br />
Pro3232IW PF09360.3 zf-CDGSH 1.6e-09<br />
Pro2891IW PF10988.1 DUF2807 8e-07<br />
Pro388IW PF11380.1 DUF3184 5.2e-17<br />
Pro547IW PF11779.1 DUF3317 4.8e-07<br />
Manual annotation (ID lists)<br />
Cyste<strong>in</strong>erich<br />
searches<br />
BlastX<br />
RXLR<br />
HaCR1 RXLR3 Cr<strong>in</strong>kler<br />
HaCR2 RXLR4<br />
HaCR3 RXLR5<br />
HaCR4 RXLR6<br />
HaCR5 RXLR7<br />
HaCR6 RXLR9<br />
HaCR7 RXLR12<br />
HaCR8 RXLR13<br />
HaCR9 RXLR15<br />
HaCR10 RXLR16<br />
HaCR11 RXLR17<br />
HaCR12 RXLR18<br />
HaCR13 RXLR19<br />
HaCR14 RXLR20<br />
HaCR15 RXLR21<br />
HaCR16 RXLR22<br />
RXLR23<br />
RXLR29
65<br />
Chapter 3:<br />
Effector identification <strong>in</strong> the lettuce <strong>downy</strong><br />
<strong>mildew</strong> Bremia lactucae by massively parallel<br />
transcriptome sequenc<strong>in</strong>g<br />
Joost H. M. Stassen 1 , Michael F. Seidl 2,3 , Pim W. J. Vergeer 1 ,<br />
Isaäc J. Nijman 4 , Berend Snel 2,3 , Edw<strong>in</strong> Cuppen 4 ,<br />
Guido Van den Ackerveken 1,3<br />
1<br />
Plant-Microbe Interactions, Department <strong>of</strong> Biology, Utrecht University,<br />
Padualaan 8, 3508 CH Utrecht, the Netherl<strong>and</strong>s<br />
2<br />
Theoretical Biology <strong>and</strong> Bio<strong>in</strong>formatics, Department <strong>of</strong> Biology,<br />
Utrecht University, Padualaan 8, 3508 CH Utrecht, the Netherl<strong>and</strong>s<br />
3<br />
Centre for BioSystems Genomics (CBSG), Wagen<strong>in</strong>gen University,<br />
B<strong>in</strong>nenhaven 5, 6709 PD Wagen<strong>in</strong>gen, the Netherl<strong>and</strong>s<br />
4<br />
Hubrecht Institute, Developmental Biology <strong>and</strong> Stem Cell Research,<br />
KNAW <strong>and</strong> University Medical Center Utrecht, Uppsalalaan 8, Utrecht,<br />
the Netherl<strong>and</strong>s<br />
Molecular Plant Pathology.<br />
doi: 10.1111/j.1364-3703.2011.00780.x
66 Chapter 3<br />
Abstract<br />
Lettuce <strong>downy</strong> <strong>mildew</strong> (Bremia lactucae) is a rapidly adapt<strong>in</strong>g oomycete pathogen<br />
affect<strong>in</strong>g commercial lettuce cultivation. Oomycetes are known to use a diverse<br />
arsenal <strong>of</strong> secreted prote<strong>in</strong>s (<strong>effectors</strong>) to manipulate their hosts. Two classes <strong>of</strong><br />
effector are known to be translocated by the host: the RXLRs <strong>and</strong> Cr<strong>in</strong>klers. To<br />
ga<strong>in</strong> <strong>in</strong>sight <strong>in</strong>to the repertoire <strong>of</strong> <strong>effectors</strong> used by B. lactucae to manipulate its<br />
host, we performed massively parallel sequenc<strong>in</strong>g <strong>of</strong> cDNA derived from B. lactucae<br />
spores <strong>and</strong> <strong>in</strong>fected lettuce (Lactuca sativa) seedl<strong>in</strong>gs. From over 2.3 million<br />
454 GS FLX reads, 59 618 contigs were assembled represent<strong>in</strong>g both plant <strong>and</strong><br />
pathogen transcripts. Of these, 19 663 contigs were determ<strong>in</strong>ed to be <strong>of</strong> B. lactucae<br />
orig<strong>in</strong> as they matched pathogen genome sequences (SOLiD) that were obta<strong>in</strong>ed<br />
from >270 million reads <strong>of</strong> spore-derived genomic DNA. After correction <strong>of</strong> cDNA<br />
sequenc<strong>in</strong>g errors with SOLiD data, translation <strong>in</strong>to prote<strong>in</strong> models <strong>and</strong> filter<strong>in</strong>g,<br />
16 372 prote<strong>in</strong> models rema<strong>in</strong>ed, 1023 <strong>of</strong> which were predicted to be secreted.<br />
This secretome <strong>in</strong>cluded elicit<strong>in</strong>s, necrosis <strong>and</strong> ethylene-<strong>in</strong>duc<strong>in</strong>g peptide 1-like<br />
prote<strong>in</strong>s, glucanase <strong>in</strong>hibitors <strong>and</strong> lect<strong>in</strong>s, <strong>and</strong> was enriched <strong>in</strong> cyste<strong>in</strong>e-rich<br />
prote<strong>in</strong>s. C<strong>and</strong>idate host-translocated <strong>effectors</strong> <strong>in</strong>cluded 77 prote<strong>in</strong> models with<br />
RXLR effector features. In addition, we found <strong>in</strong>dications for an unknown number<br />
<strong>of</strong> Cr<strong>in</strong>kler-like sequences. Similarity cluster<strong>in</strong>g <strong>of</strong> secreted prote<strong>in</strong>s revealed<br />
additional effector c<strong>and</strong>idates. We provide a first look at the transcriptome <strong>of</strong><br />
B. lactucae <strong>and</strong> its encoded effector arsenal.
Effector identification <strong>in</strong> B. lactucae<br />
67<br />
Introduction<br />
Oomycete plant pathogens cause devastat<strong>in</strong>g diseases on a wide variety <strong>of</strong> crops.<br />
These organisms resemble fungi, but are more closely related to brown algae. Wellknown<br />
oomycete pathogens are the obligate biotrophic <strong>downy</strong> <strong>mildew</strong>s <strong>and</strong> the<br />
hemibiotrophic Phytophthora species, <strong>in</strong>clud<strong>in</strong>g P. <strong>in</strong>festans, the causative agent<br />
<strong>of</strong> the Irish potato fam<strong>in</strong>e, <strong>and</strong> P. ramorum, which causes sudden oak death. The<br />
<strong>downy</strong> <strong>mildew</strong>s have a narrow host range, for <strong>in</strong>stance Hyaloperonospora arabidopsidis<br />
grows only on liv<strong>in</strong>g Arabidopsis thaliana plants, Plasmopara viticola is<br />
an important grape pathogen <strong>and</strong> Bremia lactucae is the most important pathogen<br />
<strong>of</strong> lettuce (Lactuca sativa). The control <strong>of</strong> B. lactucae is an <strong>in</strong>creas<strong>in</strong>gly difficult<br />
task as fungicides have been phased out because <strong>of</strong> environmental concerns, <strong>and</strong><br />
fungicide resistance is becom<strong>in</strong>g more widespread [1] . Genetically controlled resistance<br />
to B. lactucae is present <strong>in</strong> most commercial lettuce varieties, but is quickly<br />
overcome by new rapidly evolv<strong>in</strong>g B. lactucae races.<br />
The B. lactucae life cycle starts from a spore l<strong>and</strong><strong>in</strong>g on the plant’s epidermis,<br />
followed by penetration, hyphal colonization <strong>of</strong> host tissue <strong>and</strong> sporulation, lead<strong>in</strong>g<br />
to the release <strong>of</strong> large numbers <strong>of</strong> spores. At all stages, B. lactucae is <strong>in</strong> contact<br />
with its host, so that it can <strong>in</strong>terfere with defence <strong>and</strong> manipulate host processes<br />
to obta<strong>in</strong> nutrients. In contrast with many oomycetes, B. lactucae <strong>in</strong>fection<br />
usually starts with the direct germ<strong>in</strong>ation <strong>of</strong> asexual spores, without a zoospore<br />
<strong>in</strong>termediate stage. B. lactucae also does not rely on entry via stomata, but, <strong>in</strong>stead,<br />
usually penetrates directly through the cuticula <strong>in</strong>to an epidermal cell. A primary<br />
vesicle <strong>and</strong>, later, a secondary vesicle are then formed <strong>in</strong> the epidermal cell, after<br />
which hyphae grow <strong>in</strong> the <strong>in</strong>tercellular space <strong>of</strong> the mesophyll tissue. Haustoria<br />
are formed <strong>in</strong> most mesophyll <strong>and</strong> epidermal cells encountered by the hyphae<br />
(reviewed by Lebeda et al. [2] ). The B. lactucae–lettuce <strong>in</strong>teraction is a classic<br />
example <strong>of</strong> a gene-for-gene <strong>in</strong>teraction, <strong>in</strong> which s<strong>in</strong>gle dom<strong>in</strong>ant avirulence genes<br />
<strong>in</strong> B. lactucae are genetically recognized by dom<strong>in</strong>ant resistance genes <strong>in</strong> lettuce<br />
(reviewed by Michelmore <strong>and</strong> Wong [3] ). Knowledge on the molecular biology <strong>and</strong><br />
basis <strong>of</strong> B. lactucae pathogenicity, however, is mostly lack<strong>in</strong>g.<br />
Studies <strong>of</strong> oomycete–plant <strong>in</strong>teractions at the molecular level are now reveal<strong>in</strong>g<br />
more <strong>and</strong> more details on the molecular toolbox used by these remarkable<br />
pathogens. The genomes <strong>of</strong> the sequenced oomycete species Albugo c<strong>and</strong>ida [4] ,<br />
A. laibachii [5] , H. arabidopsidis [6] , P. <strong>in</strong>festans [7] , P. sojae [8] , P. ramorum [8] ,<br />
Pseudoperonospora cubensis [9] <strong>and</strong> Pythium ultimum [10] represent a treasure<br />
trove <strong>of</strong> <strong>in</strong>formation on the effector repertoires secreted by these pathogens to<br />
manipulate their hosts. Various types <strong>of</strong> <strong>effectors</strong> are predicted to act <strong>in</strong> the apoplast<br />
<strong>and</strong>, <strong>in</strong> addition, two classes <strong>of</strong> oomycete effector translocate <strong>in</strong>to the host cell<br />
(reviewed by Stassen <strong>and</strong> Van den Ackerveken [11] ). Effectors from one <strong>of</strong> the
68 Chapter 3<br />
host-translocated classes are referred to as RXLR <strong>effectors</strong>, after the RXLR am<strong>in</strong>o<br />
acid motif conta<strong>in</strong>ed by the first characterized <strong>effectors</strong> <strong>of</strong> this class [12] , although<br />
variations <strong>in</strong> this motif also permit host translocation [13] . Examples <strong>of</strong> variants <strong>of</strong><br />
RXLR motifs <strong>in</strong>clude the QXLR motif found <strong>in</strong> host-translocat<strong>in</strong>g <strong>effectors</strong> <strong>of</strong><br />
the oomycete pathogen <strong>of</strong> cucumber, Ps. cubensis [9] , <strong>and</strong> the GXLR motif found<br />
<strong>in</strong> the P. <strong>in</strong>festans effector SNE1 [14] . Furthermore, ATR5 <strong>of</strong> H. arabidopsidis is<br />
recognized <strong>in</strong>side host cells <strong>and</strong> has homology to the RXLR motif-conta<strong>in</strong><strong>in</strong>g<br />
<strong>effectors</strong>, but does not conta<strong>in</strong> an RXLR motif [15] . A second class <strong>of</strong> host-translocated<br />
<strong>effectors</strong> are the Cr<strong>in</strong>klers, which conta<strong>in</strong> two conserved am<strong>in</strong>o acid motifs<br />
preced<strong>in</strong>g a modular C-term<strong>in</strong>al section. In a number <strong>of</strong> Cr<strong>in</strong>klers, the doma<strong>in</strong>s<br />
that form the modules <strong>in</strong> this C-term<strong>in</strong>al section <strong>in</strong>duce cell death when expressed<br />
<strong>in</strong> Nicotiana benthamiana [7] . Two Cr<strong>in</strong>klers from P. sojae are even thought to<br />
be <strong>in</strong>dispensable for successful <strong>in</strong>fection <strong>of</strong> soybean [16] . Although the effector<br />
repertoire is generally highly divergent between species, features such as motifs<br />
associated with host-translocated <strong>effectors</strong> allow for the identification <strong>of</strong> potential<br />
<strong>effectors</strong> encoded by gene models or mRNA sequences.<br />
Access to the catalogue <strong>of</strong> the <strong>effectors</strong> <strong>of</strong> a species allows for the clon<strong>in</strong>g<br />
<strong>and</strong> <strong>in</strong>vestigation <strong>of</strong> <strong>in</strong>dividual effector genes. This has led to the identification <strong>of</strong><br />
various activities <strong>of</strong> <strong>effectors</strong> on host processes. Examples <strong>in</strong>clude the disruption<br />
<strong>of</strong> host cell wall–membrane adhesion [17] , suppression <strong>of</strong> defensive protease activity<br />
[18‐20]<br />
<strong>and</strong> suppression <strong>of</strong> effector-triggered immunity (e.g. [14,16] ). In addition, the<br />
first cytoplasmic target <strong>and</strong> mode <strong>of</strong> action <strong>of</strong> a host-translocated effector has been<br />
identified; host E3 ligase CMPG1 is stabilized by P. <strong>in</strong>festans effector AVR3a<br />
[21]<br />
, reveal<strong>in</strong>g more details about the mechanisms by which oomycetes establish a<br />
successful <strong>in</strong>fection <strong>of</strong> their hosts.<br />
In this article, we <strong>in</strong>vestigate the potential effector arsenal <strong>of</strong> B. lactucae by<br />
cDNA sequenc<strong>in</strong>g <strong>and</strong> de novo assembly <strong>of</strong> transcript sequences. Transcripts<br />
obta<strong>in</strong>ed from B. lactucae-<strong>in</strong>fected lettuce were compared with short reads <strong>of</strong><br />
B. lactucae genomic DNA (gDNA) to select for B. lactucae transcripts. From the<br />
predicted B. lactucae secretome, we identified homologues <strong>of</strong> known effector<br />
families as well as new groups <strong>of</strong> potential effector prote<strong>in</strong>s.<br />
Results<br />
Bremia lactucae transcriptome sequenc<strong>in</strong>g<br />
Two sources <strong>of</strong> RNA were used for B. lactucae transcriptome sequenc<strong>in</strong>g: RNA<br />
isolated from asexual B. lactucae conidiospores <strong>and</strong> RNA isolated from B. lactucae-<strong>in</strong>fected<br />
lettuce leaves. Many effector genes are known to be differentially<br />
expressed dur<strong>in</strong>g different stages <strong>of</strong> <strong>in</strong>fection, with transcripts <strong>of</strong> early-stage effec-
Effector identification <strong>in</strong> B. lactucae<br />
69<br />
tors be<strong>in</strong>g present <strong>in</strong> spores, <strong>and</strong> transcripts <strong>of</strong> later stage (hyphal growth) <strong>effectors</strong><br />
be<strong>in</strong>g expressed <strong>in</strong> B. lactucae grow<strong>in</strong>g <strong>in</strong> planta. To <strong>in</strong>crease the number <strong>of</strong><br />
different <strong>in</strong>fection stages <strong>and</strong>, as a result, transcript diversity, lettuce seedl<strong>in</strong>gs were<br />
<strong>in</strong>oculated twice with a 3-day <strong>in</strong>terval (see time l<strong>in</strong>e <strong>in</strong> Fig. 1A). Lettuce seedl<strong>in</strong>gs<br />
were <strong>in</strong>oculated with conidiospores (Fig. 1B) that germ<strong>in</strong>ate on the leaf surface,<br />
penetrate through epidermal cells <strong>and</strong> then cont<strong>in</strong>ue to grow <strong>in</strong>tercellular hyphae<br />
<strong>in</strong> the mesophyll, form<strong>in</strong>g haustoria <strong>in</strong> plant cells (Fig. 1C). Plants were kept under<br />
low relative humidity <strong>and</strong>, as a result, when material was harvested for RNA isolation<br />
(day 7), no B. lactucae sporulation was observed, although abundant growth <strong>of</strong><br />
<strong>in</strong>tercellular hyphae <strong>and</strong> the formation <strong>of</strong> haustoria were visible (Fig. 1D).<br />
Figure 1: Preparation <strong>of</strong> Bremia lactucae-<strong>in</strong>fected lettuce leaves for transcriptome <strong>analysis</strong>. (A) Time<br />
l<strong>in</strong>e <strong>of</strong> sampl<strong>in</strong>g. Light grey bars represent periods <strong>in</strong> which <strong>in</strong>fected lettuce was kept at high humidity.<br />
(B) Spores were spray <strong>in</strong>oculated at time po<strong>in</strong>t 0 <strong>and</strong> at a second time po<strong>in</strong>t at 3 days post-<strong>in</strong>oculation<br />
(dpi). (C) Bremia lactucae establishes an <strong>in</strong>fection <strong>and</strong> forms hyphae between plant cells, creat<strong>in</strong>g<br />
haustoria <strong>in</strong> adjacent cells. (D) Leaf material heavily colonized with B. lactucae hyphae <strong>and</strong> haustoria<br />
is harvested for RNA isolation at 7 dpi.<br />
Transcript sequenc<strong>in</strong>g was performed us<strong>in</strong>g 454 sequenc<strong>in</strong>g technology on<br />
cDNA <strong>of</strong> spores <strong>and</strong> B. lactucae-<strong>in</strong>fected cotyledons generated by either oligo<br />
dT prim<strong>in</strong>g <strong>and</strong> 5′ enrichment, or by r<strong>and</strong>om prim<strong>in</strong>g <strong>and</strong> normalization. An<br />
overview <strong>of</strong> the number <strong>of</strong> obta<strong>in</strong>ed sequences per approach is given <strong>in</strong> Table 1.<br />
Normalization selectively removes sequences present <strong>in</strong> relatively high abundance,<br />
improv<strong>in</strong>g the sampl<strong>in</strong>g <strong>of</strong> rare transcripts. It is important to sample transcripts<br />
that are present <strong>in</strong> low abundance, as <strong>effectors</strong> may be produced very locally <strong>and</strong><br />
can therefore be present at low abundance <strong>in</strong> the overall sample. We generated<br />
r<strong>and</strong>om-primed cDNA to more evenly spread coverage over the entire length <strong>of</strong> the<br />
transcripts. Pools 3 <strong>and</strong> 4 (Table 1) are r<strong>and</strong>om primed <strong>and</strong> normalized, <strong>and</strong> were<br />
used to generate the majority <strong>of</strong> reads (>90%). We also sequenced the 450–650-bp
70 Chapter 3<br />
fragments from both the 3′ <strong>and</strong> 5′ ends, as the expected ~200-bp read size <strong>of</strong> the<br />
454 sequenc<strong>in</strong>g technology (GS FLX) used would otherwise probably miss the 3′<br />
ends <strong>of</strong> transcripts. In addition, we sequenced the 5′-enriched non-normalized pools<br />
to provide stronger coverage <strong>of</strong> the 5′ ends <strong>of</strong> transcripts that are most important<br />
when look<strong>in</strong>g for N-term<strong>in</strong>al signal peptides <strong>of</strong> secreted prote<strong>in</strong>s.<br />
Table 1: Materials <strong>and</strong> method <strong>of</strong> preparation <strong>of</strong> cDNA for transcriptome sequenc<strong>in</strong>g. The contribution<br />
<strong>of</strong> each pool to the number <strong>of</strong> reads <strong>and</strong> number <strong>of</strong> bases is <strong>in</strong>dicated.<br />
Pool Material Strategy 5’ Reads 1 Total 5’ nt 3’ Reads 2 Total 3’ nt<br />
1 Spores 5’ sequenc<strong>in</strong>g 86 352 22 212 388 nd nd<br />
2 Interaction 5’ sequenc<strong>in</strong>g 115 413 30 335 206 nd nd<br />
3 Interaction<br />
R<strong>and</strong>om-primed +<br />
normalized<br />
701 499 182 312 369<br />
4 Mixed<br />
R<strong>and</strong>om-primed +<br />
normalized<br />
637 709 165 624 293<br />
1 013 411 262 824 074<br />
1<br />
The DNA code <strong>in</strong> the adapter that l<strong>in</strong>ks a read to a pool could not be resolved for 19,422 reads that are<br />
not <strong>in</strong>cluded <strong>in</strong> this table.<br />
2<br />
The DNA code was not sequenced <strong>in</strong> 3’ Reads.<br />
A total <strong>of</strong> 523 477 146 bases from 2 349 338 cDNA reads (91.28% <strong>of</strong> all available<br />
reads) was assembled <strong>in</strong>to 59 618 contigs. The total length <strong>of</strong> contig consensus<br />
sequences was 36 217 753 bases <strong>and</strong> the average coverage <strong>of</strong> these sequences was<br />
14.6-fold. The average length <strong>of</strong> contig consensus sequences was 607.5 bases.<br />
Further statistics relat<strong>in</strong>g to the assembled sequences can be found <strong>in</strong> Table 2.<br />
Table 2: Assembly process statistics regard<strong>in</strong>g the number <strong>of</strong> <strong>in</strong>put reads, reads after filter<strong>in</strong>g <strong>and</strong><br />
rema<strong>in</strong><strong>in</strong>g s<strong>in</strong>gletons. The number <strong>and</strong> total length <strong>of</strong> contigs are given for all <strong>and</strong> large (>500<br />
nucleotide) contigs.<br />
Transcriptome assembly<br />
Reads<br />
Total number <strong>of</strong> reads 2 573 806<br />
Number <strong>of</strong> reads after filter<strong>in</strong>g 2 349 338 (91.28%)<br />
Number <strong>of</strong> bases <strong>in</strong> reads after filter<strong>in</strong>g 523 477 146 (90.65%)<br />
S<strong>in</strong>gletons rema<strong>in</strong><strong>in</strong>g after assembly 82 194 (3.19%)<br />
Contigs<br />
Contigs 59 618<br />
Bases <strong>in</strong> consensus sequences 36 217 753<br />
Large contigs (>500bp) 26 358 (44.21%)<br />
Bases <strong>in</strong> large contigs 26 543 633 (73.29%)<br />
Average size <strong>of</strong> large contigs 1 007<br />
N50 size <strong>of</strong> large contigs 1 087
Effector identification <strong>in</strong> B. lactucae<br />
71<br />
The assembled contigs are derived from both B. lactucae <strong>and</strong> lettuce, as most<br />
transcript sequences were derived from RNA <strong>of</strong> <strong>in</strong>fected plants. To determ<strong>in</strong>e<br />
which transcripts are derived from B. lactucae <strong>and</strong> to correct 454 sequenc<strong>in</strong>g<br />
errors, we generated additional sequence <strong>in</strong>formation by SOLiD sequenc<strong>in</strong>g <strong>of</strong><br />
B. lactucae gDNA. Conidiospores were used for gDNA isolation as they are the<br />
only life stage <strong>of</strong> B. lactucae that can be well separated from its lettuce host. We<br />
sequenced gDNA rather than mRNA from spores, as the transcript pool <strong>in</strong> spores<br />
most probably lacks many transcripts that are <strong>in</strong>volved <strong>in</strong> the <strong>in</strong>teraction with the<br />
host plant. The B. lactucae gDNA SOLiD sequenc<strong>in</strong>g data obta<strong>in</strong>ed consist <strong>of</strong><br />
173 428 926 reads <strong>of</strong> ~50 bp.<br />
By mapp<strong>in</strong>g B. lactucae gDNA reads to the assembled 454 reads, contigs<br />
represent<strong>in</strong>g B. lactucae transcripts can be differentiated from lettuce-derived or<br />
poorly assembled sequences. Transcripts with high average gDNA read coverage<br />
probably represent B. lactucae transcriptome sequences, whereas low or no coverage<br />
<strong>in</strong>dicates a lettuce or contam<strong>in</strong>ant orig<strong>in</strong>. A total <strong>of</strong> 20 925 <strong>of</strong> 59 618 contigs, or<br />
35.2%, had at least one SOLiD gDNA read mapped to the sequence. The average<br />
gDNA read coverage per contig peaked at 37–40-fold coverage, as shown <strong>in</strong> Fig. 2.<br />
The shoulder seen at approximately half this coverage (18–20-fold coverage) <strong>in</strong><br />
Fig. 2 may represent alleles that have been assembled as separate transcripts. We<br />
def<strong>in</strong>ed the B. lactucae transcriptome set as the 19 663 contigs with a 10-fold<br />
or higher average SOLiD-sequenced gDNA read coverage. The vast majority<br />
<strong>of</strong> excluded sequences had no gDNA read coverage <strong>and</strong>, <strong>of</strong> those that did, most<br />
had an average gDNA read coverage <strong>of</strong> less than one-fold (Fig. 2). The average<br />
length <strong>of</strong> the sequences <strong>in</strong> our B. lactucae transcriptome set <strong>of</strong> 19 663 contigs was<br />
736.7 bp, up from 607.5 bp <strong>in</strong> the overall set, probably as a result <strong>of</strong> the exclusion<br />
<strong>of</strong> short contam<strong>in</strong>ant sequences. Of all the assembled spore-derived reads (pool 1),<br />
over 93% were found <strong>in</strong> consensus sequences that were classified as B. lactucae.<br />
Figure 2: Number <strong>of</strong> consensus sequences with <strong>in</strong>dicated average genomic DNA (gDNA) read<br />
coverage. Read coverages are assigned <strong>in</strong>to groups by tak<strong>in</strong>g the <strong>in</strong>teger + 1 <strong>of</strong> the actual average read<br />
coverage.
72 Chapter 3<br />
For the non-normalized, <strong>in</strong>teraction-derived reads (pool 2), we found 20% <strong>of</strong><br />
reads <strong>in</strong> B. lactucae contigs, whereas, from normalized pools (pools 3 <strong>and</strong> 4), 47%<br />
matched to B. lactucae. This suggests that the normalization procedure <strong>in</strong>creased<br />
the proportion <strong>of</strong> B. lactucae transcript sequences <strong>in</strong> the <strong>in</strong>teraction cDNA. To<br />
assess whether lettuce transcripts were successfully excluded, we compared our<br />
assembled sequences with a collection <strong>of</strong> 226 050 expressed sequence tags (ESTs)<br />
from Lactuca species (The Compositae Genome Project; http://cgpdb.ucdavis.edu).<br />
For the sequences excluded from the B. lactucae transcriptome, we found match<strong>in</strong>g<br />
ESTs for ~70% <strong>of</strong> our sequences. More importantly, we found only 151 sequences<br />
(
Effector identification <strong>in</strong> B. lactucae<br />
73<br />
tively. The B. lactucae transcripts represent the genes expressed dur<strong>in</strong>g <strong>in</strong>fection,<br />
<strong>and</strong> may therefore not represent all genes that are present <strong>in</strong> the genome. With a<br />
match <strong>of</strong> 79%, the B. lactucae transcriptome is well represented.<br />
The prote<strong>in</strong> sequences correspond<strong>in</strong>g to a set <strong>of</strong> 119 core orthologous genes<br />
were used to determ<strong>in</strong>e the place <strong>of</strong> B. lactucae <strong>in</strong> oomycete phylogeny by<br />
comparison with six pathogenic oomycete species <strong>and</strong> four nonpathogenic<br />
stramenochromes for which predicted proteomes are available. As depicted <strong>in</strong><br />
Fig. 3, the stramenochromes branch <strong>of</strong>f first, after which Saprolegnia parasitica<br />
<strong>and</strong> then Py. ultimum branch <strong>of</strong>f <strong>in</strong> the evolutionary tree. We then f<strong>in</strong>d B. lactucae,<br />
followed by H. arabidopsidis <strong>and</strong>, f<strong>in</strong>ally, three Phytophthora species. We can<br />
therefore compare B. lactucae with the earlier branch<strong>in</strong>g necrotrophic oomycete<br />
Py. ultimum <strong>and</strong> the later branch<strong>in</strong>g biotrophic <strong>downy</strong> <strong>mildew</strong> H. arabidopsidis <strong>and</strong><br />
hemibiotrophic Phytophthora species.<br />
Saprolegnia parasitica<br />
100<br />
Pythium ultimum<br />
100<br />
Bremia lactucae<br />
100<br />
Hyaloperonospora<br />
arabidopsidis<br />
96<br />
93<br />
Phytophthora<br />
spp.<br />
0.2<br />
Figure 3: Phylogeny <strong>of</strong> Bremia lactucae <strong>and</strong> related stramenochromes determ<strong>in</strong>ed from a multiple<br />
sequence alignment <strong>of</strong> 119 concatenated orthologous sequences. The scale bar represents 0.2<br />
substitutions per am<strong>in</strong>o acid. Numbers represent the bootstrap support for the given branch.<br />
To def<strong>in</strong>e the B. lactucae secretome, we determ<strong>in</strong>ed which prote<strong>in</strong> models<br />
conta<strong>in</strong>ed a signal peptide but no transmembrane helix predictions. Of the 16 372<br />
models considered, 1023 were predicted to be secreted. The size <strong>of</strong> the secretome<br />
is close to that predicted for the <strong>downy</strong> <strong>mildew</strong> H. arabidopsidis (1016 predicted<br />
secreted prote<strong>in</strong>s) <strong>and</strong> Py. ultimum (1123 secreted prote<strong>in</strong>s), as shown <strong>in</strong> Fig. 4.<br />
The 1023 c<strong>and</strong>idate secreted B. lactucae prote<strong>in</strong>s were further analysed for prote<strong>in</strong><br />
doma<strong>in</strong>s <strong>and</strong> other features related to <strong>effectors</strong>.
74 Chapter 3<br />
B. lactucae<br />
H. arabidopsidis<br />
P. <strong>in</strong>festans<br />
P. ramorum<br />
P. sojae<br />
P. ultimum<br />
S. parasitica<br />
Figure 4: The size <strong>of</strong> the secretome<br />
encoded <strong>in</strong> the genomes <strong>of</strong> different<br />
oomycetes, as determ<strong>in</strong>ed from the<br />
presence <strong>of</strong> signal peptides <strong>and</strong> the<br />
absence <strong>of</strong> transmembrane helices <strong>in</strong> the<br />
prote<strong>in</strong> models <strong>of</strong> each species.<br />
Doma<strong>in</strong> composition <strong>of</strong> the B. lactucae secretome<br />
An <strong>in</strong>itial approach to catalogue the secretome was to determ<strong>in</strong>e which Pfam<br />
doma<strong>in</strong>s, families <strong>and</strong> repeats were present. We obta<strong>in</strong>ed a total <strong>of</strong> 348 Pfam<br />
annotations for 295 <strong>of</strong> the 1023 secretome prote<strong>in</strong> models. Prevalent doma<strong>in</strong>s <strong>in</strong><br />
the B. lactucae secretome (Table 3) <strong>in</strong>cluded various doma<strong>in</strong>s that may be l<strong>in</strong>ked<br />
to pathogenicity. The most prevalent doma<strong>in</strong> <strong>in</strong> the B. lactucae secretome was the<br />
elicit<strong>in</strong> doma<strong>in</strong>. Elicit<strong>in</strong>s are small highly conserved secreted prote<strong>in</strong>s characterized<br />
by a doma<strong>in</strong> that conta<strong>in</strong>s six cyste<strong>in</strong>e residues important for structure. Of the 12<br />
B. lactucae prote<strong>in</strong> models with elicit<strong>in</strong> doma<strong>in</strong> matches, n<strong>in</strong>e conta<strong>in</strong>ed at least<br />
six cyste<strong>in</strong>es <strong>in</strong> the predicted mature peptide. Phytophthora elicit<strong>in</strong>s trigger a<br />
hypersensitive response <strong>in</strong> host plants, an effect that is l<strong>in</strong>ked to sterol b<strong>in</strong>d<strong>in</strong>g <strong>and</strong><br />
transport<strong>in</strong>g activity for which a surface-exposed polar residue is important [23‐28] .<br />
Elicit<strong>in</strong>s may play a role <strong>in</strong> the uptake <strong>of</strong> sterols by oomycetes that do not synthesize<br />
their own, as the presence <strong>of</strong> elicit<strong>in</strong>s appears to be correlated with the loss <strong>of</strong><br />
the sterol biosynthetic pathway [28‐30] . Elicit<strong>in</strong>s are found <strong>in</strong> higher numbers <strong>in</strong> the<br />
hemibiotroph P. <strong>in</strong>festans <strong>and</strong> the necrotroph Py. ultimum than <strong>in</strong> H. arabidopsidis<br />
<strong>and</strong> B. lactucae (Table 3), suggest<strong>in</strong>g that they may be selected aga<strong>in</strong>st <strong>in</strong> biotrophs<br />
that adapt a more stealthy <strong>in</strong>fection strategy. All <strong>of</strong> the B. lactucae elicit<strong>in</strong>s are<br />
α-elicit<strong>in</strong>s [31] , which generally provoke a less marked host response than do<br />
β-elicit<strong>in</strong>s.<br />
Tryps<strong>in</strong> doma<strong>in</strong>s, found <strong>in</strong> peptidases, are present <strong>in</strong> B. lactucae <strong>in</strong> numbers<br />
similar to those <strong>in</strong> P. <strong>in</strong>festans <strong>and</strong> Py. ultimum, whereas fewer are found <strong>in</strong><br />
H. arabidopsidis. The role <strong>of</strong> oomycete proteases <strong>in</strong> pathogenesis has not been<br />
determ<strong>in</strong>ed, with only a s<strong>in</strong>gle report <strong>of</strong> a P. <strong>in</strong>festans protease with an <strong>in</strong>tact
Effector identification <strong>in</strong> B. lactucae<br />
75<br />
Table 3: Number <strong>of</strong> prote<strong>in</strong> models <strong>in</strong> Bremia lactucae, Phytophthora <strong>in</strong>festans, Hyaloperonospora<br />
arabidopsidis, <strong>and</strong> Pythium ultimum that are predicted to conta<strong>in</strong> the doma<strong>in</strong>s that are most prevalent<br />
<strong>in</strong> the B. lactucae secretome.<br />
Pfam Doma<strong>in</strong> B. lactucae H. arabidopsidis P. <strong>in</strong>festans P. ultimum<br />
Elicit<strong>in</strong> 12 9 28 37<br />
Tryps<strong>in</strong> 11 4 13 14<br />
Jacal<strong>in</strong> 7 4 4 0<br />
DnaJ 6 3 5 5<br />
Cyste<strong>in</strong>e-rich secretory prote<strong>in</strong> family 6 7 14 9<br />
Ric<strong>in</strong>-B-lect<strong>in</strong> 4 2 1 0<br />
catalytic triad that is expressed dur<strong>in</strong>g <strong>in</strong>fection [32] . More common are catalytically<br />
<strong>in</strong>active tryps<strong>in</strong>-like prote<strong>in</strong>s that act as glucanase <strong>in</strong>hibitors [33] . The B. lactucae<br />
prote<strong>in</strong>s with tryps<strong>in</strong> matches do not have <strong>in</strong>tact catalytic residues <strong>in</strong> the active site<br />
<strong>and</strong> could therefore function as glucanase <strong>in</strong>hibitors. Host glucanases can damage<br />
the oomycete cell wall, imped<strong>in</strong>g oomycete growth <strong>and</strong> releas<strong>in</strong>g glucan-oligosaccharide<br />
elictors [34] , <strong>and</strong> are therefore a potentially useful target for <strong>in</strong>hibition by an<br />
<strong>in</strong>vad<strong>in</strong>g oomycete.<br />
Jacal<strong>in</strong> doma<strong>in</strong>s <strong>and</strong> Ric<strong>in</strong>-B-lect<strong>in</strong> doma<strong>in</strong>s, both found <strong>in</strong> lect<strong>in</strong>s, are present<br />
<strong>in</strong> larger numbers than <strong>in</strong> the other oomycetes. A Jacal<strong>in</strong> doma<strong>in</strong>-match<strong>in</strong>g<br />
oomycete prote<strong>in</strong> has been proposed to act <strong>in</strong> cell wall degradation [32] , <strong>and</strong> roles<br />
<strong>in</strong> host attachment have been suggested on the basis <strong>of</strong> homology to lect<strong>in</strong>s [35] <strong>and</strong><br />
lect<strong>in</strong>-like activities [36] . Host lect<strong>in</strong>s, however, act <strong>in</strong> defence <strong>and</strong> may recognize<br />
oomycete-derived carbohydrates or glycosylation features. Pathogen lect<strong>in</strong>s could<br />
therefore also be <strong>in</strong>strumental <strong>in</strong> mask<strong>in</strong>g these signals, as has been demonstrated<br />
for the ECP6 effector <strong>of</strong> the plant-pathogenic fungus Cladosporium fulvum, which<br />
masks chit<strong>in</strong> oligosaccharides that normally trigger plant immune responses [37] .<br />
Cyste<strong>in</strong>e-rich secreted prote<strong>in</strong>s <strong>and</strong> prote<strong>in</strong>s mediat<strong>in</strong>g disulphide bond formation<br />
may play an important role <strong>in</strong> deal<strong>in</strong>g with the protease-rich conditions <strong>in</strong><br />
the extracellular environment. Cyste<strong>in</strong>e residues can form disulphide bridges that<br />
may contribute to prote<strong>in</strong> stability <strong>and</strong> protect aga<strong>in</strong>st proteases. In addition, DnaJ<br />
prote<strong>in</strong>s may act as molecular chaperones to further stabilize apoplastic prote<strong>in</strong>s.<br />
Many prote<strong>in</strong> families <strong>in</strong>volved <strong>in</strong> pathogenicity, found <strong>in</strong> other oomycetes, are<br />
small cyste<strong>in</strong>e-rich prote<strong>in</strong>s. Examples <strong>in</strong>clude the aforementioned elicit<strong>in</strong>s,<br />
Phytophthora PcFs [38‐40] <strong>and</strong> H. arabidopsidis PPATs [41] <strong>and</strong> HaCRs [42] . Although<br />
there are six prote<strong>in</strong>s match<strong>in</strong>g the Pfam cyste<strong>in</strong>e-rich secretory doma<strong>in</strong> model,
76 Chapter 3<br />
these prote<strong>in</strong> models conta<strong>in</strong> few cyste<strong>in</strong>e residues. Nonetheless, 135 cyste<strong>in</strong>e-rich<br />
prote<strong>in</strong>s (>5% cyste<strong>in</strong>e) are found <strong>in</strong> the B. lactucae secretome. The majority<br />
<strong>of</strong> these are short (
Effector identification <strong>in</strong> B. lactucae<br />
77<br />
Host-translocated <strong>effectors</strong><br />
To f<strong>in</strong>d potential host-translocated RXLR <strong>effectors</strong>, a search for the am<strong>in</strong>o acid<br />
motif RXLR was performed on the predicted B. lactucae secretome. This search<br />
identified 44 potential RXLR <strong>effectors</strong>, count<strong>in</strong>g only hits with the RXLR located<br />
between positions 30 <strong>and</strong> 60 <strong>in</strong> prote<strong>in</strong>s <strong>of</strong> at least 65 am<strong>in</strong>o acids. As variants<br />
<strong>of</strong> the RXLR motif also allow host entry, we exp<strong>and</strong>ed our search to <strong>in</strong>clude<br />
prote<strong>in</strong> models that are similar to known RXLRs. We therefore performed a blast<br />
search with all H. arabidopsidis [6] <strong>and</strong> Phytophthora spp. [7] RXLRs aga<strong>in</strong>st the<br />
B. lactucae secretome. To rule out matches based on homology to conserved<br />
signal peptide sequences, they were removed from the B. lactucae prote<strong>in</strong>s. In this<br />
search, 38 matches were found <strong>in</strong> the B. lactucae secretome, irrespective <strong>of</strong> the<br />
presence or absence <strong>of</strong> an RXLR or RXLR-like motif. In a third approach to f<strong>in</strong>d<br />
RXLR <strong>effectors</strong>, we selected the RXLR motif <strong>and</strong> 20 residues on either side <strong>in</strong> the<br />
sequences that were identified by the motif search, <strong>and</strong> used these as <strong>in</strong>put for a<br />
jackhmmer search. jackhmmer is an iterative search that uses matches to an <strong>in</strong>itial<br />
<strong>in</strong>put sequence to construct an alignment pr<strong>of</strong>ile which is employed to search for<br />
additional matches <strong>in</strong> the target database <strong>and</strong> to further ref<strong>in</strong>e the pr<strong>of</strong>ile. Seven<br />
new c<strong>and</strong>idates were identified <strong>in</strong> a set <strong>of</strong> 52 that <strong>in</strong>cluded 42 <strong>in</strong>put sequences<br />
<strong>and</strong> three sequences that were found by blast. Ten prote<strong>in</strong>s were predicted by all<br />
three methods (Fig. 5). The comb<strong>in</strong>ed set consists <strong>of</strong> 77 unique potential RXLR<br />
<strong>effectors</strong>, the details <strong>of</strong> which are provided <strong>in</strong> Table 4. We also found prote<strong>in</strong><br />
models by blast comparison that did not have an RXLR motif or variant there<strong>of</strong>,<br />
but did have an EER motif, albeit slightly more N-term<strong>in</strong>al <strong>of</strong> the location at which<br />
the RXLR motif would be expected. For 11 B. lactucae contigs encod<strong>in</strong>g RXLR<br />
<strong>and</strong> RXLR-like prote<strong>in</strong>s, we observed clear<br />
differential expression based on transcript<br />
reads between spores <strong>and</strong> <strong>in</strong> planta stages<br />
(Table S1). Most <strong>of</strong> the B. lactucae RXLR<br />
<strong>and</strong> RXLR-like <strong>effectors</strong> were not found <strong>in</strong><br />
the genomes <strong>of</strong> other <strong>downy</strong> <strong>mildew</strong>s. Bidirectional<br />
blast matches (E-value < 1e-3 both<br />
ways) were found for only seven B. lactucae<br />
versus eight H. arabidopsidis RXLRs, <strong>and</strong><br />
none for Ps. cubensis. In addition, between<br />
Ps. cubensis <strong>and</strong> H. arabidopsidis only three<br />
RXLR matches were found.<br />
23<br />
2<br />
10<br />
32<br />
Motif<br />
3<br />
7<br />
Blast<br />
HMM<br />
Figure 5: Venn diagram depict<strong>in</strong>g the<br />
overlap <strong>of</strong> the three methods used for the<br />
prediction <strong>of</strong> RXLR <strong>effectors</strong>. HMM,<br />
hidden Markov model.
78 Chapter 3<br />
Table 4 (cont<strong>in</strong>ued next page): RXLR <strong>and</strong> RXLR-like motifs found <strong>in</strong> Bremia lactucae secretome<br />
prote<strong>in</strong> models identified as potential RXLR <strong>effectors</strong> based on a motif search, BLAST comparison<br />
with RXLRs <strong>of</strong> other oomycetes or a jackhmmer search with B. lactucae RXLR motifs <strong>and</strong> surround<strong>in</strong>g<br />
am<strong>in</strong>o acids as <strong>in</strong>put. All sequence coord<strong>in</strong>ates are given <strong>in</strong> am<strong>in</strong>o acids. Evidence codes are built up <strong>of</strong><br />
M (motif search), B (BLAST comparison) <strong>and</strong> H (jackhmmer search)<br />
contig ID<br />
RXLR<br />
EER(like)<br />
RXLR EER start<br />
start<br />
motif 1 Stop codon Evidence 2<br />
16131 44 RRLR 55 EER 363 MBH<br />
16134 41 RRLR 50 EER 454 MBH<br />
23155 49 RLLR 61 EQEER - MBH<br />
27267 55 RALR 68 EER - MBH<br />
35642 42 RRLR 60 EDR - MBH<br />
40355 50 RRLR 64 DER 270 MBH<br />
42290 45 RGLR 200 NEK 333 MBH<br />
43449 35 RMLR - - MBH<br />
45396 53 RLLR - 130 MBH<br />
49490 44 RRLR 57 DER - MBH<br />
19377 49 RRLR - 269 MB<br />
22293 45 RFLR 65 DEK - MB<br />
07991 43 RQLR 78 EER 161 MH<br />
08247 52 RSLR - 103 MH<br />
08248 52 RSLR - 146 MH<br />
08983 49 RRLR - - MH<br />
16394 46 RRLR 59 QNDER - MH<br />
18684 50 RRLR - 282 MH<br />
18840 49 RPLR 61 NQEER - MH<br />
20700 56 RELR - 252 MH<br />
21364 47 RALR 58 EDK - MH<br />
23333 35 RFLR - - MH<br />
24965 57 RSLR 62 DENR 107 MH<br />
29191 46 RKLR - 75 MH<br />
31910 45 RLLR 54 DNNEER 160 MH<br />
32917-1 47 RSLR 60 NDER - MH<br />
33962 39 RILR - 74 MH<br />
36117 38 RDLR - 424 MH<br />
36219 45 RHLR 60 ENR 201 MH<br />
38529 40 RQLR 91 EDK - MH<br />
39974 41 RQLR 50 DER - MH<br />
40514 44 RLLR 56 EER 126 MH<br />
41799 42 RALR - 90 MH<br />
41935 44 RLLR 53 DDNDER - MH<br />
43687 55 RSLR - 91 MH<br />
43968 33 RGLR - 185 MH
Effector identification <strong>in</strong> B. lactucae<br />
79<br />
contig ID<br />
RXLR<br />
EER(like)<br />
RXLR EER start<br />
start<br />
motif 1 Stop codon Evidence 2<br />
45726 42 RLLR - - MH<br />
46714 45 RSLR 82 NQR - MH<br />
48006 46 RALR 55 NEDR 92 MH<br />
48013 55 RALR - 82 MH<br />
50216 47 RSLR 60 DEER 102 MH<br />
53213 43 RGLR 61 EER - MH<br />
53460 47 RSLR 60 DEER - MH<br />
59265 49 RLLR 62 EE - MH<br />
22259 - 52 EER 375 BH<br />
29756 36 RSLLQ 51 DER - BH<br />
56090 42 RSLQ 55 EER - BH<br />
07727 45 RRLK 63 EER - B<br />
07952 - 65 NEER 304 B<br />
08155 55 RRLQ 109 EEK 321 B<br />
09272 - 58 DEER 346 B<br />
09828 - 48 EEK 652 B<br />
11813 - 49 EER 629 B<br />
12298 63 RLKREQ - 612 B<br />
13165 114 RSLR 360 DEK - B<br />
14316 - 50 EER 365 B<br />
16501 481 RKLR - 485 B<br />
17177 - - - B<br />
18477 42 QNLR - - B<br />
19297 57 KVLR - 253 B<br />
24419 - 54 DEER 434 B<br />
24503 - - 520 B<br />
28334 43 EKLR 53 ENR 479 B<br />
29771 - - 263 B<br />
35494 - - - B<br />
35745 169 RSLR 72 EER - B<br />
48427 62 RLLD - - B<br />
51693 - 49 EER - B<br />
51725 39 PSLR - - B<br />
56746 - 49 EER - B<br />
23857 42 GKLR 55 DER 243 H<br />
25695 44 GKLR 57 DER 336 H<br />
26201 58 RSLR 63 DENR 111 H<br />
31920 44 GRLR 57 DER 233 H<br />
38296 63 RRLR - - H<br />
46806 43 QLRLR 57 DER - H<br />
50948 41 GRLR 54 DER - H<br />
1<br />
Determ<strong>in</strong>ed as [DENQ]{2,}[RK]<br />
2<br />
Evidence codes are as follows: M = Motif, B = Blast, H = jackhmmer.
80 Chapter 3<br />
Us<strong>in</strong>g the collection <strong>of</strong> Cr<strong>in</strong>kler <strong>effectors</strong> <strong>of</strong> Phytophthora species [7] , we performed<br />
similarity searches to identify these <strong>effectors</strong> <strong>in</strong> the B. lactucae secretome.<br />
We found six c<strong>and</strong>idates, three <strong>of</strong> which conta<strong>in</strong>ed an LXFLA am<strong>in</strong>o acid motif<br />
that was similar to the motifs reported <strong>in</strong> Phytophthora species. Interest<strong>in</strong>gly,<br />
gDNA read coverage was higher than expected for four c<strong>and</strong>idates: two conta<strong>in</strong><strong>in</strong>g<br />
the LXFLA motif (six- <strong>and</strong> three-fold) <strong>and</strong> two without this sequence (22- <strong>and</strong><br />
40-fold). This suggests that more gene copies or pseudogenes may be present <strong>in</strong><br />
the genome than are detected <strong>in</strong> our transcriptome set, or that multiple genes have<br />
been assembled <strong>in</strong>to a s<strong>in</strong>gle sequence. The sequences conta<strong>in</strong><strong>in</strong>g an LXFLA motif<br />
are not complete, suggest<strong>in</strong>g problems with assembly. We therefore used the HMM<br />
models <strong>of</strong> Cr<strong>in</strong>kler doma<strong>in</strong>s [7] to screen all predicted peptides for the potential<br />
presence <strong>of</strong> Cr<strong>in</strong>kler doma<strong>in</strong>s. All peptides were <strong>in</strong>cluded as many <strong>of</strong> the potential<br />
Phytophthora Cr<strong>in</strong>klers do not have a signal peptide <strong>and</strong> signal peptides may have<br />
been assembled separately. We found a total <strong>of</strong> 75 sequences that had homology<br />
to one or more <strong>of</strong> the reported Cr<strong>in</strong>kler doma<strong>in</strong>s. In total, we found matches to 14<br />
different C-term<strong>in</strong>al Cr<strong>in</strong>kler doma<strong>in</strong>s <strong>in</strong> B. lactucae (Table 5), aga<strong>in</strong> with higher<br />
than expected gDNA coverage. Interest<strong>in</strong>gly, we also found traces <strong>of</strong> D2, DC <strong>and</strong><br />
DBF doma<strong>in</strong>s, which are also found <strong>in</strong> cell death-<strong>in</strong>duc<strong>in</strong>g P. <strong>in</strong>festans Cr<strong>in</strong>klers [7] .<br />
Table 5: Matches to hidden Markov models <strong>of</strong> Cr<strong>in</strong>kler<br />
doma<strong>in</strong>s (as def<strong>in</strong>ed by Haas et al [7] ) <strong>in</strong> the Bremia<br />
lactucae transcriptome, <strong>and</strong> the predicted copy numbers<br />
there<strong>of</strong>.<br />
Doma<strong>in</strong><br />
Peptide<br />
predictions<br />
Predicted<br />
copy number 1<br />
N-term<strong>in</strong>al<br />
LFLAK 15 64<br />
DWL 13 101<br />
C-term<strong>in</strong>al<br />
D2 11 11<br />
DAB 2 4<br />
DBE 4 22<br />
DBF 3 3<br />
DC 2 1<br />
DDB 3 2<br />
DDC 2 1<br />
DFA 2 2<br />
DFB 8 111<br />
DFC 9 68<br />
DN17 1 1<br />
DN7 1 1<br />
DX9 1 1<br />
DXX 15 135<br />
1<br />
The predicted copy number is determ<strong>in</strong>ed<br />
by divid<strong>in</strong>g the observed average genomic<br />
DNA coverage <strong>of</strong> the assembled transcripts<br />
<strong>in</strong> which the <strong>in</strong>dicated doma<strong>in</strong>s are encoded<br />
by the average genomic coverage <strong>of</strong> all<br />
assembled B. lactucae transcripts.
Effector identification <strong>in</strong> B. lactucae<br />
81<br />
Exp<strong>and</strong>ed sequence families <strong>in</strong> B. lactucae<br />
The cluster<strong>in</strong>g <strong>of</strong> B. lactucae secretome sequences with those <strong>of</strong> six other oomycete<br />
species <strong>and</strong> four stramenopile species was performed on the basis <strong>of</strong> a comparison<br />
<strong>of</strong> all sequences with each other. A cluster <strong>of</strong> closely related genes may <strong>in</strong>dicate<br />
a recently exp<strong>and</strong>ed family <strong>of</strong> genes that plays an important role <strong>in</strong> pathogenesis<br />
<strong>and</strong> adaptation to the host. Cluster<strong>in</strong>g revealed 85 groups <strong>of</strong> sequences conta<strong>in</strong><strong>in</strong>g<br />
multiple B. lactucae members. We focused on eight clusters with three or more<br />
B. lactucae members that conta<strong>in</strong>ed significantly more B. lactucae members than<br />
would be expected on the basis <strong>of</strong> the presence <strong>of</strong> members <strong>of</strong> six other oomycetes<br />
<strong>and</strong> four other stramenopiles.<br />
Two clusters could be categorized on the basis <strong>of</strong> Pfam doma<strong>in</strong> hits. The first is<br />
a B. lactucae-unique cluster conta<strong>in</strong><strong>in</strong>g a three-member subset <strong>of</strong> the Pfam elicit<strong>in</strong><br />
doma<strong>in</strong>-match<strong>in</strong>g sequences. The second cluster, with 11 B. lactucae members <strong>and</strong><br />
two H. arabidopsidis, five P. <strong>in</strong>festans, 10 P. sojae <strong>and</strong> 18 P. ramorum members,<br />
conta<strong>in</strong>s all seven Jacal<strong>in</strong> doma<strong>in</strong>-conta<strong>in</strong><strong>in</strong>g prote<strong>in</strong>s. The members <strong>of</strong> this cluster<br />
have blast hits <strong>in</strong> the NCBI NR prote<strong>in</strong> database to P. <strong>in</strong>festans hypothetical<br />
conserved prote<strong>in</strong>s, but also to NPP1, an NLP, <strong>and</strong> putative adhes<strong>in</strong>s. The<br />
sequences are dist<strong>in</strong>ct from the sequences with a Pfam NLP prediction, as those<br />
cluster together <strong>in</strong> another cluster, with eight B. lactucae, n<strong>in</strong>e H. arabidopsidis, 25<br />
P. <strong>in</strong>festans, 40 P. sojae <strong>and</strong> 56 P. ramorum members.<br />
Of the rema<strong>in</strong><strong>in</strong>g six clusters, five consist <strong>of</strong> three B. lactucae sequences each,<br />
<strong>and</strong> one cluster consists <strong>of</strong> four B. lactucae sequences. All but one cluster could<br />
be l<strong>in</strong>ked to identified RXLR effector c<strong>and</strong>idates. The four-member cluster most<br />
closely represents the ‘classic’ RXLR effector, with an RXLR motif <strong>in</strong> all members<br />
<strong>and</strong> a DER motif <strong>in</strong> one <strong>of</strong> them. The four clusters <strong>of</strong> three sequences share<br />
similar sequence features that are more apparent when these clusters are aligned<br />
(Fig. 6). No significant similarity to sequences <strong>in</strong> the NCBI NR prote<strong>in</strong> database<br />
was found for sequences <strong>in</strong> these four clusters. All members <strong>of</strong> the C1 cluster had<br />
already been identified as potential RXLR <strong>effectors</strong>, as well as a s<strong>in</strong>gle member<br />
<strong>of</strong> each <strong>of</strong> two other clusters (C4 contig18477 <strong>and</strong> C3 contig28334). One member<br />
<strong>of</strong> C1, contig49490, has an <strong>in</strong>tact RXLR motif that aligns with similar features <strong>in</strong><br />
sequences <strong>in</strong> all sequence clusters. The presence <strong>of</strong> these sequences <strong>in</strong> groups <strong>of</strong><br />
related sequences, with features similar to those found <strong>in</strong> known RXLR <strong>effectors</strong>,<br />
suggests that these prote<strong>in</strong>s may be host-translocated <strong>effectors</strong>. The cluster that<br />
could not be l<strong>in</strong>ked to RXLR <strong>effectors</strong> has no RXLR-like motif (not shown) <strong>and</strong><br />
conta<strong>in</strong>s no members that were identified as potential RXLR <strong>effectors</strong>, although all<br />
members conta<strong>in</strong> an EER motif.
82 Chapter 3<br />
Figure 6: Alignment <strong>of</strong> the sequences <strong>of</strong> four Bremia lactucae-specific clusters <strong>of</strong> predicted secreted<br />
prote<strong>in</strong>s. The columns <strong>in</strong> which potential RXLR <strong>and</strong> dEER-like motifs can be found are <strong>in</strong>dicated<br />
above the alignment.<br />
The cluster<strong>in</strong>g <strong>of</strong> secreted sequences highlights three elicit<strong>in</strong>s specific for B. lactucae.<br />
It also shows that the family <strong>of</strong> jacal<strong>in</strong> doma<strong>in</strong> prote<strong>in</strong>s occurs <strong>in</strong> a cluster<br />
with vary<strong>in</strong>g numbers <strong>of</strong> family members <strong>in</strong> other oomycete species, suggest<strong>in</strong>g<br />
that they might fulfil a species-specific role. F<strong>in</strong>ally, it identifies other potential<br />
host-translocated <strong>effectors</strong> that were not predicted by other methods.<br />
Discussion<br />
The B. lactucae transcriptome sequences that have been generated by next-generation<br />
sequenc<strong>in</strong>g methods provide a first look at the toolbox used by B. lactucae to<br />
manipulate its host. Potential apoplastic <strong>and</strong> host-translocated <strong>effectors</strong> could be<br />
predicted from the assembled transcript sequences. We showed that 79% <strong>of</strong> a set <strong>of</strong><br />
conserved eukaryotic genes used to assess genome completion are represented <strong>in</strong><br />
the B. lactucae transcriptome, underl<strong>in</strong><strong>in</strong>g a broad sampl<strong>in</strong>g <strong>of</strong> the transcriptome.<br />
The transcriptome is far more plastic than the genome, <strong>and</strong> the rema<strong>in</strong><strong>in</strong>g 21% <strong>of</strong><br />
conserved eukaryotic genes may not have been sampled because not all developmental<br />
stages <strong>of</strong> B. lactucae were represented <strong>in</strong> our material (e.g. sporangiophore<br />
formation, but also sexual reproduction). Another possibility is that the transcripts<br />
have been sampled, but are not assembled as full-length transcripts <strong>and</strong> therefore<br />
do not satisfy the cut-<strong>of</strong>f values for the models <strong>of</strong> the conserved eukaryotic genes.<br />
Prote<strong>in</strong> predictions were based on the use <strong>of</strong> homology to known prote<strong>in</strong>s to<br />
select the most probable open read<strong>in</strong>g frame (ORF) from the contigs. In 432 cases,<br />
this suggested a model that spanned multiple ORFs <strong>in</strong> a s<strong>in</strong>gle contig, <strong>in</strong> which<br />
case these ORFs were taken together as a s<strong>in</strong>gle prote<strong>in</strong> model. Manual <strong>in</strong>spection<br />
revealed that this may be a result <strong>of</strong> the <strong>in</strong>clusion <strong>of</strong> unprocessed <strong>in</strong>trons <strong>in</strong> the<br />
cDNA sample. Although <strong>in</strong>trons are thought to be rare <strong>in</strong> oomycetes, one study [45]
Effector identification <strong>in</strong> B. lactucae<br />
83<br />
found that, <strong>of</strong> a total <strong>of</strong> 128 alternative splic<strong>in</strong>g events detected <strong>in</strong> P. sojae, <strong>in</strong>tron<br />
skipp<strong>in</strong>g was the most common event, expla<strong>in</strong><strong>in</strong>g 97 cases.<br />
Sequenc<strong>in</strong>g <strong>of</strong> plant-pathogenic oomycetes has revealed a variable number <strong>of</strong><br />
RXLR <strong>effectors</strong> <strong>in</strong> different organisms. We identified 77 potential RXLR <strong>effectors</strong><br />
<strong>in</strong> the B. lactucae secretome, 43 <strong>of</strong> which conta<strong>in</strong>ed a conserved RXLR motif. This<br />
is a relatively small number compared with the 134 found <strong>in</strong> H. arabidopsidis [6]<br />
<strong>and</strong> the 563 found <strong>in</strong> P. <strong>in</strong>festans [7] , but close to the 32 RXLR <strong>effectors</strong> found <strong>in</strong><br />
Ps. cubensis [9] . In addition, 29 potential <strong>effectors</strong> with a QXLR motif were found <strong>in</strong><br />
Ps. cubensis. There is evidence that QXLR motif prote<strong>in</strong>s can also translocate <strong>in</strong>to<br />
the host [9] , illustrat<strong>in</strong>g that strict conformation to the RXLR motif is not required.<br />
In B. lactucae, we identified four clusters <strong>of</strong> three related B. lactucae sequences<br />
that all conta<strong>in</strong> sequence features rem<strong>in</strong>iscent <strong>of</strong> RXLR motifs.<br />
We reported traces <strong>of</strong> Cr<strong>in</strong>kler prote<strong>in</strong>s <strong>in</strong> the B. lactucae transcriptome. These<br />
sequences were not completely assembled <strong>and</strong> had a higher than expected coverage<br />
<strong>of</strong> genomic DNA reads. This may <strong>in</strong>dicate that our sampl<strong>in</strong>g strategy is suboptimal<br />
for captur<strong>in</strong>g Cr<strong>in</strong>klers; EST sequenc<strong>in</strong>g <strong>of</strong> H. arabidopsidis material collected <strong>in</strong> a<br />
similar manner revealed a s<strong>in</strong>gle partially assembled Cr<strong>in</strong>kler [42] . It may be that, <strong>in</strong><br />
these obligate biotrophs, Cr<strong>in</strong>klers are expressed <strong>in</strong> smaller numbers or <strong>in</strong> a more<br />
limited timeframe. Alternatively, key Cr<strong>in</strong>kler-derived reads may be lost dur<strong>in</strong>g<br />
normalization <strong>and</strong> assembly because <strong>of</strong> high sequence similarity.<br />
Sequenc<strong>in</strong>g <strong>of</strong> the transcriptome <strong>of</strong> B. lactucae has provided a wealth <strong>of</strong><br />
<strong>in</strong>formation about the prote<strong>in</strong>s expressed dur<strong>in</strong>g <strong>in</strong>fection <strong>of</strong> the host. The prote<strong>in</strong><br />
models described are an extensive source <strong>of</strong> potential <strong>effectors</strong>. C<strong>and</strong>idate <strong>effectors</strong><br />
can now be cloned <strong>and</strong> tested to study their role <strong>in</strong> the <strong>in</strong>fection process, e.g. as<br />
suppressors <strong>of</strong> host immunity. An important next challenge is to determ<strong>in</strong>e how<br />
these prote<strong>in</strong>s manipulate host processes. Ultimately, host susceptibility <strong>and</strong><br />
resistance genes can then be identified from Lactuca resources, allow<strong>in</strong>g for novel<br />
strategies <strong>in</strong> breed<strong>in</strong>g for resistance aga<strong>in</strong>st B. lactucae.<br />
Experimental procedures<br />
454 sequenc<strong>in</strong>g<br />
Bremia lactucae isolate BL24 was grown on Lactuca sativa cultivar Ol<strong>of</strong> <strong>and</strong><br />
the enhanced B. lactucae-susceptible Lactuca sativa cv. Ol<strong>of</strong> × Lactuca saligna<br />
recomb<strong>in</strong>ant <strong>in</strong>bred l<strong>in</strong>e BIL4.4 [61,62] . Plants were kept at 17 °C under 9 h light per<br />
day (100 µE/m 2 /s). Bremia lactucae spores were spray <strong>in</strong>oculated at 150 spores/µL<br />
suspension on 7-day-old soil-grown lettuce seedl<strong>in</strong>gs until run-<strong>of</strong>f was imm<strong>in</strong>ent.<br />
Inoculation was repeated at 3 days after the <strong>in</strong>itial <strong>in</strong>oculation. After each <strong>in</strong>oculation,<br />
plants were kept under high humidity for 24 h <strong>and</strong> thereafter at low humidity.
84 Chapter 3<br />
Trypan blue sta<strong>in</strong><strong>in</strong>g was performed by boil<strong>in</strong>g <strong>in</strong> sta<strong>in</strong><strong>in</strong>g solution for 5 m<strong>in</strong>, <strong>and</strong><br />
otherwise as described for H. arabidopsidis [46] . Material was harvested 7 days after<br />
the <strong>in</strong>itial <strong>in</strong>oculation. For spore isolations, <strong>in</strong>oculated plants were kept at high<br />
humidity for 7 days, after which spores were r<strong>in</strong>sed <strong>of</strong>f leaves <strong>in</strong> water. Spores<br />
were filtered through a double layer <strong>of</strong> miracloth <strong>and</strong> then spun down (3650 g) <strong>and</strong><br />
washed <strong>in</strong> water three times. Spores <strong>and</strong> <strong>in</strong>fected leaf material were snap frozen<br />
<strong>and</strong> ground us<strong>in</strong>g a mortar <strong>and</strong> pestle. RNA was isolated from ground material<br />
us<strong>in</strong>g the Qiagen (Venlo, The Netherl<strong>and</strong>s) plant RNA m<strong>in</strong>i kit employ<strong>in</strong>g RLC<br />
buffer accord<strong>in</strong>g to the manufacturer’s <strong>in</strong>structions.<br />
Isolated RNA was sent for preparation for sequenc<strong>in</strong>g to Vertis Biotechnologie<br />
AG (Freis<strong>in</strong>g, Germany). Poly(A) RNA was selected from all samples. Two<br />
cDNA synthesis strategies were then performed. In the first, the poly(A) RNA was<br />
<strong>in</strong>cubated with calf <strong>in</strong>test<strong>in</strong>e phosphatase (CIP) <strong>and</strong> tobacco acid pyrophosphatase<br />
(TAP), followed by ligation <strong>of</strong> an RNA adapter to the 5′-phosphate <strong>of</strong> decapped<br />
mRNAs. First-str<strong>and</strong> cDNA synthesis was then performed with an adapter-r<strong>and</strong>om<br />
hexamer primer <strong>and</strong> M-MLV-RNase H reverse transcriptase. The result<strong>in</strong>g cDNAs<br />
were amplified with 21 cycles <strong>of</strong> long <strong>and</strong> accurate polymerase cha<strong>in</strong> reaction<br />
(LA-PCR). In the second approach, first-str<strong>and</strong> cDNA synthesis was primed with<br />
a r<strong>and</strong>om hexamer primer, after which 454 adapters A <strong>and</strong> B were ligated to the 5′<br />
<strong>and</strong> 3′ ends <strong>of</strong> the cDNA. The cDNA was f<strong>in</strong>ally amplified with PCR us<strong>in</strong>g a pro<strong>of</strong><br />
read<strong>in</strong>g enzyme (17 cycles). To normalize samples, one cycle <strong>of</strong> denaturation <strong>and</strong><br />
reassociation <strong>of</strong> the cDNA <strong>and</strong> subsequent separation <strong>of</strong> s<strong>in</strong>gle-str<strong>and</strong>ed cDNA<br />
(normalized cDNA) <strong>of</strong> double-str<strong>and</strong>ed RNA by hydroxylapatite chromatography<br />
were performed. Normalized cDNA was amplified with eight PCR cycles. For both<br />
approaches, cDNA <strong>in</strong> the size range 450–650 bp was eluted from agarose gel us<strong>in</strong>g<br />
the Macherey & Nagel (Düren, Germany) NucleoSp<strong>in</strong> Extract II kit. Sequenc<strong>in</strong>g<br />
was performed on the 454 GS FLX system, us<strong>in</strong>g st<strong>and</strong>ard reagents.<br />
SOLiD sequenc<strong>in</strong>g<br />
Lettuce leaves were surface sterilized <strong>in</strong> 4% bleach for 3 m<strong>in</strong>, r<strong>in</strong>sed with water<br />
<strong>and</strong> placed abaxial side up on wet filter paper. The leaves were then spray <strong>in</strong>oculated<br />
(200 spores/µL) with B. lactucae isolate BL24 <strong>and</strong> kept at high humidity<br />
for 8 days. Spores were snap frozen <strong>and</strong> lysed by bead beat<strong>in</strong>g <strong>and</strong> <strong>in</strong>cubation<br />
<strong>in</strong> cetyltrimethylammonium bromide (CTAB). DNA was extracted with phenol–<br />
chlor<strong>of</strong>orm; 7 µg <strong>of</strong> DNA was sheared <strong>in</strong>to 100–110-bp mean size fragments,<br />
end-repaired with the End-It DNA End-Repair Kit (Epicentre Biotechnology,<br />
Madison, WI, USA) accord<strong>in</strong>g to the manufacturer’s <strong>in</strong>structions, <strong>and</strong> cleaned by<br />
phenol–chlor<strong>of</strong>orm extraction. Adapters were ligated as recommended by Applied<br />
Biosystems (Carlsbad, CA, USA) <strong>and</strong> cleaned by phenol–chlor<strong>of</strong>orm extraction.
Effector identification <strong>in</strong> B. lactucae<br />
85<br />
Fragments <strong>of</strong> 150–200 bp were excised from a 2% agarose electrophoresis gel <strong>and</strong><br />
purified us<strong>in</strong>g the QIAquick Gel Extraction Kit (Qiagen). Further steps <strong>in</strong> sample<br />
preparation <strong>and</strong> sequenc<strong>in</strong>g were taken as recommended by Applied Biosystems.<br />
Sequence <strong>analysis</strong><br />
The 454 transcriptome sequences were assembled us<strong>in</strong>g Roche (Basel, Switzerl<strong>and</strong>)<br />
gsAssembler (version 2.0.00) s<strong>of</strong>tware with an expected coverage <strong>of</strong> 15-fold<br />
(based on <strong>in</strong>itial assembly coverage levels) <strong>and</strong> otherwise default sett<strong>in</strong>gs. SOLiD<br />
reads were mapped to the assembled 454 reads by Burrows–Wheeler Aligner<br />
(BWA) [47] (version 0.5.8 r1442; maximum edit distance, 10; first 25 bases as seed;<br />
maximally two mismatches <strong>in</strong> the seed; otherwise default sett<strong>in</strong>gs for colourspace<br />
alignment). Assembled sequences were corrected us<strong>in</strong>g <strong>in</strong>sertion/deletion<br />
<strong>in</strong>formation from the mapp<strong>in</strong>g process. Correction effectiveness was determ<strong>in</strong>ed<br />
by comparison with the NCBI NR database by blastx [48] (E-value < 1e-3, best hit).<br />
Sequences with >10-fold average SOLiD read coverage were kept as B. lactucae<br />
sequences. Prote<strong>in</strong>s were predicted from these sequences by select<strong>in</strong>g an ORF<br />
based on the blastx comparison with the NCBI NR database or, fail<strong>in</strong>g matches,<br />
select<strong>in</strong>g the ORF(s) <strong>of</strong> greatest length. In cases <strong>in</strong> which blast comparison led to<br />
the selection <strong>of</strong> adjacent ORFs, these ORFs were concatenated <strong>in</strong>to a s<strong>in</strong>gle model.<br />
ORF lengths were determ<strong>in</strong>ed from methion<strong>in</strong>e to stop unless the ORF was at the<br />
5′ or 3′ end, <strong>in</strong> which case the length was determ<strong>in</strong>ed from the 5′ or up to the 3′,<br />
respectively, as sequences may be <strong>in</strong>complete. Sequences were trimmed to start<br />
with a methion<strong>in</strong>e <strong>and</strong> sequences shorter than 20 am<strong>in</strong>o acids were removed before<br />
signal peptide prediction by SignalP (3.0a) [49,50] (first 70 am<strong>in</strong>o acids, eukaryote,<br />
default cut-<strong>of</strong>fs). Transmembrane helices were predicted us<strong>in</strong>g TMHMM (2.0)<br />
(http://www.cbs.dtu.dk/services/TMHMM/). Secretome criteria were the prediction<br />
<strong>of</strong> a signal peptide by both neural network <strong>and</strong> HMM prediction methods <strong>and</strong> no<br />
predicted transmembrane helices (unless overlapp<strong>in</strong>g at least 10 am<strong>in</strong>o acids <strong>of</strong><br />
the signal peptide). All blast comparisons were performed with an E-value cut-<strong>of</strong>f<br />
<strong>of</strong> 1e-3. HMM <strong>and</strong> jackhmmer (version 2.3.2; http://hmmer.org/) searches were<br />
performed with an E-value cut-<strong>of</strong>f <strong>of</strong> 1e-3 <strong>and</strong> otherwise default sett<strong>in</strong>gs, except<br />
for the genome completeness models, for which bit-score cut-<strong>of</strong>fs were provided.<br />
For jackhmmer searches us<strong>in</strong>g B. lactucae RXLRs, the RXLR motif <strong>and</strong> the 20<br />
am<strong>in</strong>o acids N- <strong>and</strong> C-term<strong>in</strong>al there<strong>of</strong> were used as <strong>in</strong>put. Pfam [51] searches were<br />
performed at the gather<strong>in</strong>g threshold.<br />
To obta<strong>in</strong> a phylogeny <strong>of</strong> NLP prote<strong>in</strong>s <strong>in</strong> B. lactucae <strong>and</strong> other oomycetes, we<br />
predicted the presence <strong>of</strong> the NLP doma<strong>in</strong> (PF05630, Pfam v24; http://pfam.sanger.<br />
ac.uk/ [51] ) <strong>in</strong> their proteomes us<strong>in</strong>g hmmer3 (http://hmmer.org/) (gather<strong>in</strong>g cut-<strong>of</strong>f).<br />
To predict full-length NLP doma<strong>in</strong>s, we retrieved the seed alignments from the
86 Chapter 3<br />
Pfam database <strong>and</strong> created a calibrated hmmer2 model. hmmer2 (gather<strong>in</strong>g cut-<strong>of</strong>f)<br />
was used with this model on the hmmer3-identified c<strong>and</strong>idates. Short doma<strong>in</strong><br />
hits (50% <strong>of</strong> both target <strong>and</strong> query sequences, <strong>and</strong> had >20% effective coverage<br />
(fraction <strong>of</strong> covered am<strong>in</strong>o acids), were taken <strong>in</strong>to account, with the exception<br />
that B. lactucae sequences did not need to span 50% <strong>of</strong> non-B. lactucae sequences.<br />
Bremia lactucae sequences may be truncated <strong>and</strong> therefore not span 50% <strong>of</strong> the<br />
non-B. lactucae sequence. The similarity network was then clustered <strong>in</strong>to families<br />
us<strong>in</strong>g the Markov-Cluster-Algorithm (MCL; http://micans.org/mcl/, v09-308,<br />
1.008, <strong>in</strong>flation = 3) [57,58] . Clustered sequences were aligned <strong>in</strong> Jalview [59] us<strong>in</strong>g the<br />
MAFFT web service [60] . We predicted a reliable species phylogeny utiliz<strong>in</strong>g a multi-prote<strong>in</strong><br />
marker conta<strong>in</strong><strong>in</strong>g 119 concatenated 1 : 1 : 1 families retrieved from the<br />
prote<strong>in</strong> clusters determ<strong>in</strong>ed beforeh<strong>and</strong>. These families were aligned us<strong>in</strong>g mafft<br />
(v6.713b; maxiterate 1000; localpair) [60] <strong>and</strong> subsequently concatenated. Positions<br />
conta<strong>in</strong><strong>in</strong>g >20% gaps, as well as less conserved adjacent positions, were removed<br />
until a conserved column with a median <strong>of</strong> the pairwise BLOSUM62 <strong>of</strong> ≥0 was<br />
reached. The phylogenetic tree was predicted us<strong>in</strong>g RAxML [52] (v7.0.4; gamma<br />
model <strong>of</strong> <strong>in</strong>variant sites, WAG substitution model, fast bootstrap approximation)<br />
<strong>and</strong> the robustness <strong>of</strong> the topology was assessed by 1000 bootstrap replicates.
Effector identification <strong>in</strong> B. lactucae<br />
87<br />
Accession numbers<br />
Bremia lactucae sequences longer than 200 nucleotides were deposited <strong>in</strong> the<br />
NCBI Transcriptome Shotgun Assembly sequence database under accession<br />
numbers JP948721-JP965883. All nucleotide data <strong>and</strong> prote<strong>in</strong> translations are<br />
available at http://web.science.uu.nl/pmi/data/bremia/.<br />
Acknowledgements<br />
This project was supported by the foundation TTI Green Genetics (TTI GG) <strong>in</strong><br />
collaboration with Wagen<strong>in</strong>gen University (Wagen<strong>in</strong><strong>in</strong>gen, The Netherl<strong>and</strong>s),<br />
Enza zaden (Enkhuizen, The Netherl<strong>and</strong>s), Nunhems (Nunhem, The Netherl<strong>and</strong>s),<br />
Syngenta (Enkhuizen, The Netherl<strong>and</strong>s), Rijk-Zwaan (De Lier, The Netherl<strong>and</strong>s)<br />
<strong>and</strong> Vilmor<strong>in</strong> (La Ménitré, France). We thank Ewart de Bruijn <strong>and</strong> Patrick van Zon<br />
for excellent technical assistance.
88 Chapter 3<br />
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61 Zhang NW, L<strong>in</strong>dhout P, Niks RE & Jeuken MJW (2009) Genetic dissection <strong>of</strong> Lactuca saligna<br />
nonhost resistance to <strong>downy</strong> <strong>mildew</strong> at various lettuce developmental stages. Plant Pathology 58,<br />
923-32.<br />
62 Zhang NW, Pelgrom K, Niks RE, Visser RGF & Jeuken MJW (2009) Three comb<strong>in</strong>ed<br />
quantitative trait loci from nonhost Lactuca saligna are sufficient to provide complete resistance<br />
<strong>of</strong> lettuce aga<strong>in</strong>st Bremia lactucae. Molecular plant-microbe <strong>in</strong>teractions 22, 1160-8.
94 Chapter 3
Effector identification <strong>in</strong> B. lactucae<br />
95<br />
Supplemental Information<br />
Figure S1 (previous page): Phylogenetic relationship between NLPs [necrosis <strong>and</strong> ethylene-<strong>in</strong>duc<strong>in</strong>g<br />
peptide 1 (NEP1)-like prote<strong>in</strong>s] from various oomycete plant pathogens.<br />
Four <strong>of</strong> the NLPs <strong>in</strong> the Bremia lactucae secretome provided full-length NLP doma<strong>in</strong> matches <strong>and</strong><br />
are <strong>in</strong>cluded <strong>in</strong> the ma<strong>in</strong> tree, <strong>in</strong>dicated <strong>in</strong> purple. The positions <strong>of</strong> fragments <strong>of</strong> the doma<strong>in</strong>s found<br />
<strong>in</strong> the complete B. lactucae transcriptome were determ<strong>in</strong>ed separately (<strong>in</strong>dicated <strong>in</strong> the ma<strong>in</strong> tree by<br />
stars), as they are difficult to locate based on the alignment <strong>of</strong> the full-length doma<strong>in</strong>. Bremia lactucae<br />
NLPs are spread more widely across the oomycete NLP gene tree than are the Hyaloperonospora<br />
arabidopsidis NLPs (<strong>in</strong>dicated <strong>in</strong> bold as clade or <strong>in</strong>dividual common names), <strong>and</strong> do not fall with<strong>in</strong><br />
H. arabidopsidis or Pythium ultimum subclades. Bremia lactucae NLPs cluster close to the two H.<br />
arabidopsidis NLPs outside <strong>of</strong> the H. arabidopsidis-specific subclade. NLP names <strong>in</strong>dicated <strong>in</strong> blue<br />
belong to NLPs that have been shown to <strong>in</strong>duce necrosis when expressed <strong>in</strong> tobacco leaves. Scale bar<br />
<strong>in</strong>dicates 0.1 substitutions per am<strong>in</strong>o acid. Stars <strong>in</strong>dicate the location <strong>of</strong> subtrees that conta<strong>in</strong> short<br />
fragments (<strong>in</strong>complete assembly) <strong>of</strong> Bremia NLPs that were assessed separately.<br />
Table S1 (cont<strong>in</strong>ued next page): Number <strong>of</strong> reads from spore <strong>and</strong> <strong>in</strong>fection sequenc<strong>in</strong>g pools per contig<br />
for those that show a five-fold or greater difference <strong>in</strong> the number <strong>of</strong> reads between spore <strong>and</strong> <strong>in</strong>fection<br />
stages as determ<strong>in</strong>ed from 5′ end sequences <strong>of</strong> the non-normalized sequenc<strong>in</strong>g pools.<br />
Differential expression was observed for contigs correspond<strong>in</strong>g to prote<strong>in</strong> models <strong>of</strong> all the <strong>in</strong>dicated<br />
categories, except for those encod<strong>in</strong>g for DnaJ doma<strong>in</strong> prote<strong>in</strong>s <strong>and</strong> NLPs [necrosis <strong>and</strong> ethylene<strong>in</strong>duc<strong>in</strong>g<br />
peptide 1 (NEP1)-like prote<strong>in</strong>s] for which no difference <strong>in</strong> abundance was found.<br />
Overal Spores Interaction 1<br />
Reads considered 67615 20169<br />
Contigs conta<strong>in</strong><strong>in</strong>g reads 8667 6567<br />
Average reads /<br />
contig conta<strong>in</strong><strong>in</strong>g reads<br />
7.8 3.1<br />
Controls Spores Interaction<br />
Act<strong>in</strong><br />
contig12283 3 9<br />
Tubul<strong>in</strong><br />
contig12868 4 5<br />
18s Ribosomal<br />
contig01002 25 32<br />
Phospholipase A2<br />
contig36115 2 3<br />
1<br />
(count<strong>in</strong>g only Bremia)
96 Chapter 3<br />
Spores Interaction Spores Interaction<br />
Elicit<strong>in</strong>s<br />
Tryps<strong>in</strong><br />
contig32983 26 1 contig34965 26 0<br />
contig43325 0 11 contig56967 49 1<br />
contig46242 20 1 >5% cyste<strong>in</strong>e<br />
contig48912 16 1 contig08246 54 2<br />
Jacal<strong>in</strong> contig10442 20 1<br />
contig06799 11 1 contig16641 6 1<br />
contig12503 15 0 contig17422 17 2<br />
contig14955 48 2 contig19602 0 8<br />
CAP contig28850 6 0<br />
contig36558 9 0 contig30126 1 9<br />
Ric<strong>in</strong>-B-Lect<strong>in</strong> contig31077 10 0<br />
contig22692 16 1 contig38644 46 5<br />
contig28297 33 2 contig39684 14 1<br />
RXLR <strong>and</strong> RXLR-likes contig40030 8 1<br />
contig07991 1 8 contig40488 16 0<br />
contig08248 35 3 contig44287 8 0<br />
contig09272 2 14 contig44488 0 6<br />
contig16131 6 0 contig44717 0 11<br />
contig18840 17 0 contig44719 0 23<br />
contig23155 135 0 contig50965 6 1<br />
contig24965 0 29 contig52084 0 14<br />
contig40514 0 19 contig56102 5 0<br />
contig41935 11 0 contig57102 6 0<br />
contig43968 0 12 contig57464 5 0<br />
contig51725 6 0 contig57969 12 1<br />
Table S2: Versions <strong>and</strong> sources <strong>of</strong> genomes used for <strong>analysis</strong>.<br />
Species name Version Source TaxonID<br />
Number<br />
predicted References<br />
prote<strong>in</strong>s<br />
Aureococcus anophagefferens 1 JGI 44056 11501<br />
[56]<br />
Phaeodactylum tricornutum 2 JGI 2850 10402<br />
[54]<br />
Thalassiosira pseudonana 3 JGI 35128 11776<br />
[53]<br />
Ectocarpus siliculosus 1 Gent 2880 16582<br />
[55]<br />
Phytophthora <strong>in</strong>festans 1 BROAD 4787 18138<br />
[7]<br />
Phytophthora ramorum 1 BROAD 164328 14394<br />
[8]<br />
Phytophthora sojae 1 BROAD 67593 16989<br />
[8]<br />
Pythium ultimum 4 BROAD 65071 15323<br />
[10]<br />
Hyaloperonospora arabidopsidis 8.3 VBI 272952 14910<br />
[6]<br />
Saprolegnia parasitica 1 BROAD 101203 18729<br />
A<br />
A<br />
www.broad<strong>in</strong>stitute.org/annotation/genome/Saprolegnia_parasitica/MultiHome.html
97<br />
Chapter 4:<br />
Effectors <strong>of</strong> the lettuce <strong>downy</strong> <strong>mildew</strong> Bremia<br />
lactucae enhance host susceptibility<br />
Joost H. M. Stassen, Pim W. J. Vergeer, Annemiek Andel,<br />
Guido Van den Ackerveken<br />
Plant-Microbe Interactions, Department <strong>of</strong> Biology, Faculty <strong>of</strong> Science,<br />
Utrecht University, Utrecht, The Netherl<strong>and</strong>s
98 Chapter 4<br />
Abstract<br />
Bremia lactucae is an obligate biotrophic oomycete that causes <strong>downy</strong> <strong>mildew</strong>, an<br />
important disease <strong>of</strong> lettuce. Bremia uses an arsenal <strong>of</strong> effector prote<strong>in</strong>s that are<br />
thought to manipulate the host <strong>and</strong> establish a successful <strong>in</strong>fection. Among these<br />
are RXLR <strong>effectors</strong> that are proposed to be host-translocated <strong>and</strong> to act <strong>in</strong>side<br />
the host cell. A set <strong>of</strong> 34 potential RXLR <strong>effectors</strong> <strong>of</strong> Bremia was cloned <strong>and</strong><br />
sequence verified by Sanger sequenc<strong>in</strong>g. The contribution <strong>of</strong> each effector to host<br />
susceptibility was <strong>in</strong>vestigated by Agrobacterium-mediated transient expression<br />
<strong>of</strong> effector genes <strong>in</strong> lettuce leaf discs <strong>and</strong> subsequent <strong>in</strong>oculation with Bremia.<br />
The number <strong>of</strong> spores formed by Bremia on these leaf discs provides a measure <strong>of</strong><br />
host susceptibility towards Bremia. A trend towards enhanc<strong>in</strong>g susceptibility was<br />
observed for most potential <strong>effectors</strong>. Transient expression <strong>of</strong> two effector genes,<br />
BLR16 <strong>and</strong> BLR27 significantly <strong>in</strong>creased host susceptibility, whilst transient<br />
expression <strong>of</strong> BLR03 significantly reduced host susceptibility. The temporal expression<br />
<strong>of</strong> selected <strong>effectors</strong> dur<strong>in</strong>g <strong>in</strong>fection <strong>of</strong> lettuce by Bremia was determ<strong>in</strong>ed,<br />
reveal<strong>in</strong>g that BLR03 expression was limited to early <strong>in</strong>fection stages, whereas<br />
BLR16 <strong>and</strong> BLR27 were expressed at all <strong>in</strong>fection stages. To analyse the enhanced<br />
susceptibility phenotype, a set <strong>of</strong> biotic stress-associated markers was developed<br />
from lettuce transcripts that are highly abundant dur<strong>in</strong>g <strong>in</strong>fection. C<strong>and</strong>idate<br />
markers were selected based on homology to A. thaliana genes that are annotated<br />
as biotic stress-responsive or were found to be responsive to biotic stress <strong>in</strong> data<br />
from publicly available microarray experiments. Strong <strong>in</strong>duction <strong>of</strong> five lettuce<br />
biotic stress-associated markers was found <strong>in</strong> response to <strong>in</strong>fection by both Bremia<br />
<strong>and</strong> Agrobacterium. Us<strong>in</strong>g these markers, however, no evidence was found for<br />
strong suppression <strong>of</strong> biotic stress-responses <strong>in</strong> leaf material transiently express<strong>in</strong>g<br />
susceptibility-enhanc<strong>in</strong>g Bremia effector c<strong>and</strong>idates. This work clearly shows that<br />
several c<strong>and</strong>idate <strong>effectors</strong> <strong>of</strong> Bremia enhance disease susceptibility. The identification<br />
<strong>of</strong> these <strong>effectors</strong> <strong>of</strong> Bremia now opens the way to <strong>in</strong>vestigate their activity <strong>and</strong><br />
targets <strong>in</strong> the lettuce host.
Bremia <strong>effectors</strong> enhance host susceptibility<br />
99<br />
Introduction<br />
Bremia lactucae is an obligate biotrophic oomycete that causes <strong>downy</strong> <strong>mildew</strong><br />
<strong>of</strong> lettuce. Losses <strong>in</strong> susceptible crops are large, <strong>and</strong> considerable effort is put<br />
<strong>in</strong>to breed<strong>in</strong>g resistant lettuce cultivars. Many agricultural crops are attacked by<br />
oomycetes, a diverse group <strong>of</strong> organisms with fungal-like features belong<strong>in</strong>g to the<br />
Stramenopiles. Oomycete pathogens differ <strong>in</strong> lifestyle, rang<strong>in</strong>g from necrotrophic<br />
species such as Pythium ultimum through hemibiotrophic Phytophthora species<br />
(e.g. P. <strong>in</strong>festans <strong>and</strong> P. sojae) to the obligate biotrophic <strong>downy</strong> <strong>mildew</strong>s. Examples<br />
<strong>of</strong> crops affected by <strong>downy</strong> <strong>mildew</strong> are grapev<strong>in</strong>e (caused by Plasmopara<br />
viticola), sunflower (caused by Plasmopara halstedii) <strong>and</strong> cucurbits (caused by<br />
Pseudoperonospora cubensis). Much <strong>of</strong> our current underst<strong>and</strong><strong>in</strong>g <strong>of</strong> the molecular<br />
biology <strong>of</strong> <strong>downy</strong> <strong>mildew</strong>-host <strong>in</strong>teractions has been learnt from the <strong>in</strong>teraction<br />
between Hyaloperonospora arabidopsidis <strong>and</strong> Arabidopsis thaliana, which is a<br />
prime host-pathogen model system (reviewed by Coates <strong>and</strong> Beynon [1] ), whereas<br />
the lettuce-Bremia <strong>in</strong>teraction has been ma<strong>in</strong>ly studied as model for gene-for-gene<br />
<strong>in</strong>teractions (reviewed by Michelmore <strong>and</strong> Wong [2] ). The lifestyle <strong>of</strong> H. arabidopsidis<br />
is similar to that <strong>of</strong> Bremia, though there are clear differences. Bremia <strong>and</strong><br />
H. arabidopsidis <strong>in</strong>fections start from a sporangio- or conidiospore germ<strong>in</strong>at<strong>in</strong>g<br />
on plant leaves. Bremia then forms an appressorium that serves to penetrate the<br />
epidermis, allow<strong>in</strong>g the establishment <strong>of</strong> a primary vesicle, <strong>and</strong> later a secondary<br />
one, <strong>in</strong> an epidermal cell [3] . H. arabidopsidis does not penetrate through epidermal<br />
cells, rather it forces a hyphae through the anticl<strong>in</strong>al wall between two epidermal<br />
cells. Both pathogens then grow hyphae between mesophyll cells, penetrat<strong>in</strong>g the<br />
cell walls <strong>and</strong> <strong>in</strong>vag<strong>in</strong>at<strong>in</strong>g the host membrane to form protrusions called haustoria.<br />
In order to successfully <strong>in</strong>teract with their hosts, oomycetes use an arsenal <strong>of</strong><br />
different <strong>effectors</strong>. A number <strong>of</strong> these <strong>effectors</strong> act on the outside <strong>of</strong> the host cell,<br />
whilst other types are translocated across the host cell wall <strong>and</strong> cell membrane <strong>in</strong>to<br />
<strong>in</strong>tracellular compartments <strong>of</strong> the host (reviewed by Göhre <strong>and</strong> Robatzek [4] ). The<br />
different lifestyles <strong>of</strong> oomycetes are reflected <strong>in</strong> the <strong>effectors</strong> <strong>of</strong> different groups<br />
<strong>of</strong> pathogens. Whereas necrotrophic <strong>and</strong> hemibiotrophic oomycete species encode<br />
<strong>effectors</strong> that are thought to act similar to tox<strong>in</strong>s, biotrophs do not kill their host<br />
<strong>and</strong> are thought to adapt a more stealthy lifestyle [5] . This requires extensive <strong>in</strong>terference<br />
with host cell processes to promote disease susceptibility <strong>in</strong> the broadest<br />
sense. Effectors can serve to suppress immunity, but also to promote or alter host<br />
metabolism <strong>and</strong> to redirect nutrient transport <strong>in</strong> favour <strong>of</strong> the pathogen.<br />
Suppression <strong>of</strong> plant immunity by <strong>effectors</strong> <strong>of</strong> bacterial pathogens is a<br />
well-studied phenomenon (Reviewed by Göhre <strong>and</strong> Robatzek [6] <strong>and</strong> Rodríguez-<br />
Herva et al. [7] ). Gram-negative bacteria, e.g. those belong<strong>in</strong>g to genera <strong>of</strong><br />
Pseudomonas or Xanthomonas, deliver <strong>effectors</strong> <strong>in</strong>to host cells via a Type Three
100 Chapter 4<br />
Secretion System (TTSS) to <strong>in</strong>terfere with plant immunity by act<strong>in</strong>g on host<br />
targets. A recent example <strong>of</strong> surpris<strong>in</strong>g targets <strong>in</strong>clude the tomato PsbQ prote<strong>in</strong>,<br />
which is a member <strong>of</strong> the oxygen evolv<strong>in</strong>g complex <strong>of</strong> photosystem II [8] . Target<strong>in</strong>g<br />
<strong>of</strong> this prote<strong>in</strong> by the P. syr<strong>in</strong>gae pv tomato DC3000 effector HopN1 compromised<br />
the production <strong>of</strong> defence-related reactive oxygen species <strong>and</strong> programmed cell<br />
death. Effectors can also act on processes not related to plant immunity to enhance<br />
disease susceptibility. For example, the PthXO1 effector <strong>of</strong> Xanthomonas oryzae<br />
pv oryzae PXO99 <strong>in</strong>duces the ectopic expression <strong>of</strong> the rice OsSWEET11 sugar<br />
transporter by directly b<strong>in</strong>d<strong>in</strong>g to the promoter <strong>of</strong> its encod<strong>in</strong>g gene. Apparently<br />
the activation <strong>of</strong> the sugar transporter is important for nutrient acquisition by the<br />
<strong>in</strong>vad<strong>in</strong>g pathogen [9] .<br />
Oomycetes do not deploy a TTSS to <strong>in</strong>troduce <strong>effectors</strong> <strong>in</strong>to the host cell, but<br />
rather use a two-step procedure. Effectors <strong>of</strong> oomycetes are thought to be secreted<br />
from the haustorium, after which they are taken up or translocate across the hostcell<br />
membrane [10] . Two motifs that are associated with host-cell entry have been<br />
described <strong>in</strong> multiple oomycete species. Based on the presence <strong>of</strong> these motifs<br />
the RXLR <strong>effectors</strong> <strong>and</strong> the Cr<strong>in</strong>kler <strong>effectors</strong> are dist<strong>in</strong>guished as major classes<br />
<strong>of</strong> <strong>effectors</strong>. In <strong>downy</strong> <strong>mildew</strong>s fewer Cr<strong>in</strong>kler <strong>effectors</strong> appear to be expressed<br />
compared to RXLR <strong>effectors</strong> [11–13] . The RXLR <strong>effectors</strong> conta<strong>in</strong> a signal peptide for<br />
secretion from the pathogen, <strong>and</strong> a translocation doma<strong>in</strong> that conta<strong>in</strong>s the RXLR<br />
motif, or a variant there<strong>of</strong>, <strong>and</strong> an optional EER motif. The region C-term<strong>in</strong>al <strong>of</strong> the<br />
translocation doma<strong>in</strong> is referred to as the effector doma<strong>in</strong>. Uptake <strong>of</strong> these <strong>effectors</strong><br />
by host cells appears to take place <strong>in</strong> absence <strong>of</strong> a pathogen-encoded mach<strong>in</strong>ery<br />
<strong>and</strong> is associated with the RXLR motif [10,14,15] , though there are also reports <strong>of</strong><br />
translocation <strong>of</strong> <strong>effectors</strong> with a less conserved RXLR motifs or only an EER motif<br />
[16–18]<br />
. The exact mechanism <strong>of</strong> host-cell entry by RXLR <strong>effectors</strong> is under debate,<br />
with reports <strong>of</strong> b<strong>in</strong>d<strong>in</strong>g to cell surface-exposed phosphatidyl<strong>in</strong>ositol-3-phosphate <strong>in</strong><br />
plant <strong>and</strong> human cells [19] , <strong>and</strong> tyros<strong>in</strong>e-O-sulphate-modified cell surface molecules<br />
<strong>in</strong> fish [15] as <strong>in</strong>itial steps <strong>in</strong> host entry.<br />
RXLR <strong>effectors</strong> have been predicted from the genomes <strong>of</strong> oomycetes based on<br />
the presence <strong>of</strong> predicted signal peptides <strong>and</strong> RXLR motifs. Many <strong>of</strong> these <strong>effectors</strong><br />
have been <strong>functional</strong>ly analysed for suppression <strong>of</strong> plant immunity. Two <strong>of</strong><br />
32 tested P. <strong>in</strong>festans <strong>effectors</strong> were found to suppress Infest<strong>in</strong>-1 (INF-1) <strong>in</strong>duced<br />
hypersensitive cell death when expressed by Agrobacterium transient transformation<br />
24 hours before challenge <strong>in</strong>oculation with an Agrobacterium stra<strong>in</strong> carry<strong>in</strong>g<br />
an INF-1 construct [20] . In a similar setup, the majority <strong>of</strong> a large collection <strong>of</strong> P.<br />
sojae <strong>effectors</strong> was shown to suppress cell death <strong>in</strong>duced by the mouse pro-apoptotic<br />
prote<strong>in</strong> BAX or by the INF-1 <strong>in</strong> Nicotiana benthamiana [21] . Constructs <strong>of</strong><br />
RXLR <strong>effectors</strong> <strong>of</strong> the <strong>downy</strong> <strong>mildew</strong> H. arabidopsidis fused with TTSS target<strong>in</strong>g<br />
signal have been expressed <strong>in</strong> Pseudomonas syr<strong>in</strong>gae <strong>and</strong> targeted to the host
Bremia <strong>effectors</strong> enhance host susceptibility<br />
101<br />
cytoplasm via the TTSS. This revealed a surpris<strong>in</strong>gly high number <strong>of</strong> <strong>effectors</strong> that<br />
enhanced susceptibility to P. syr<strong>in</strong>gae <strong>in</strong>fection <strong>and</strong> decreased defence-associated<br />
callose deposition by the host [11,22,23] .<br />
Details on the molecular mechanisms underly<strong>in</strong>g the actions <strong>of</strong> host-translocated<br />
oomycetes are largely unknown, but are beg<strong>in</strong>n<strong>in</strong>g to be elucidated. Most<br />
potential <strong>effectors</strong> do not conta<strong>in</strong> any known doma<strong>in</strong>s or motifs other than the<br />
motifs associated with translocation. An exception is Avr3b, a P. sojae-specific<br />
effector, which is a <strong>functional</strong> Nudix hydrolase [24] . Details <strong>of</strong> the target <strong>and</strong> mode<br />
<strong>of</strong> action are known for P. <strong>in</strong>festans effector Avr3a, which stabilises the host<br />
E3 ligase CMPG1 <strong>in</strong> order to manipulate host immunity [25] . Host defences are<br />
also affected by P. <strong>in</strong>festans AVRBlb2, which associates with C14, a papa<strong>in</strong>-like<br />
cyste<strong>in</strong>e protease normally secreted by the host as a defence measure. This<br />
association occurs around haustoria <strong>and</strong> blocks secretion <strong>of</strong> C14 [26] . The targets <strong>and</strong><br />
functions <strong>of</strong> other <strong>effectors</strong> rema<strong>in</strong> to be elucidated, though there are <strong>in</strong>dications<br />
that multiple <strong>effectors</strong> <strong>of</strong> different species may share the same host targets.<br />
High-throughput yeast-2-hybrid <strong>analysis</strong> <strong>of</strong> H. arabidopsidis effector c<strong>and</strong>idates,<br />
P. syr<strong>in</strong>gae <strong>effectors</strong>, <strong>and</strong> A. thaliana prote<strong>in</strong>s revealed that, though separated by<br />
two billion years <strong>of</strong> evolution, 18 <strong>of</strong> 165 effector targets are shared between both<br />
pathogens [27] . Between the targets 139 prote<strong>in</strong>-prote<strong>in</strong> <strong>in</strong>teractions were identified,<br />
<strong>in</strong>dicat<strong>in</strong>g that the direct targets are highly <strong>in</strong>terconnected. In other words, both<br />
pathogens act on a limited <strong>and</strong> overlapp<strong>in</strong>g set <strong>of</strong> target modules <strong>in</strong> plant defence.<br />
Although the lettuce-Bremia <strong>in</strong>teraction has been studied extensively, details<br />
<strong>of</strong> Bremia <strong>effectors</strong> <strong>and</strong> their activities have only recently started to emerge.<br />
Previously, we identified potential RXLR <strong>and</strong> RXLR-like <strong>effectors</strong> <strong>of</strong> Bremia from<br />
the transcriptome <strong>of</strong> <strong>downy</strong> <strong>mildew</strong>-<strong>in</strong>fected lettuce plants [13] . Here we <strong>in</strong>vestigate<br />
the role <strong>of</strong> these potential <strong>effectors</strong> <strong>in</strong> modulat<strong>in</strong>g host susceptibility or suppress<strong>in</strong>g<br />
plant defence.<br />
Results<br />
A Bremia effector set for <strong>in</strong> planta expression<br />
Previously, we predicted 77 potential RXLR <strong>and</strong> RXLR-like <strong>effectors</strong> from our<br />
Bremia transcriptome data [13] . Based on these predictions we cloned a set <strong>of</strong> 16<br />
full-length effector c<strong>and</strong>idates that were verified by Sanger sequenc<strong>in</strong>g. We had<br />
previously verified <strong>and</strong> cloned 12 potential <strong>effectors</strong> that were predicted <strong>in</strong> previous<br />
prelim<strong>in</strong>ary assemblies. F<strong>in</strong>ally, we also determ<strong>in</strong>ed the full-length sequence <strong>of</strong> 6<br />
potential <strong>effectors</strong> that were not full-length <strong>in</strong> the assembled 454-transcript data by<br />
us<strong>in</strong>g additional data from short reads <strong>of</strong> spore-derived genomic DNA or 3’ RACE.<br />
The complete set therefore comprises 34 Bremia effector c<strong>and</strong>idates. An overview
102 Chapter 4<br />
Table 1: Overview <strong>of</strong> cloned Bremia effector c<strong>and</strong>idates. The first RXLR or RXLR-like motif <strong>and</strong><br />
position with<strong>in</strong> 100 am<strong>in</strong>o acids from the start codon are <strong>in</strong>dicated. The first EER or EER-like motif<br />
<strong>and</strong> position <strong>of</strong> which the first am<strong>in</strong>o acid is with<strong>in</strong> 20 am<strong>in</strong>o acids <strong>of</strong> the first am<strong>in</strong>o acid <strong>of</strong> the RXLR<br />
motif are shown. Source: M – ma<strong>in</strong> assembly, 3 – 3’ RACE, E – transcript extended with SOLiD data,<br />
P – prelim<strong>in</strong>ary assemblies.<br />
RXLR-like<br />
EER-like<br />
ID Source Contig ID length start motif start motif<br />
BLR01 P - 86 42 RKLR 52 EQK<br />
BLR02 P - 146 85 RLLR<br />
BLR03 P - 141 48 RFLR 59 EEER<br />
BLR04 P - 76 45 RELR 60 DIK<br />
BLR05 P - 97 32 RALR 58 DED<br />
BLR06 P - 281 46 RCLR<br />
BLR07 P - 253 47 RALR 68 EEER<br />
BLR08 P - 135 38 RLLR<br />
BLR09 P - 112 37 RRLR 81 EER<br />
BLR10 P - 112 37 RRLR 81 EER<br />
BLR11 P - 463 46 RRLR 57 DESER<br />
BLR12 P - 123 49 RYLR 61 ELEK<br />
BLR13 M 16131 363 44 RRLR 55 EER<br />
BLR14 M 29191 75 46 RKLR<br />
BLR15 M 50216 102 47 RSLR 60 DEER<br />
BLR16 M 32917-1 98 47 RSLR 60 NDER<br />
BLR17 M 18684 282 50 RRLR 64 DAEK<br />
BLR18 M 48006 92 46 RALR 55 NEDR<br />
BLR19 M 31910 160 45 RLLR 54 DNNEER<br />
BLR20 M 45396 130 53 RLLR 69 DEAD<br />
BLR21 M 33962 65 39 RILR<br />
BLR22 M 43968 185 33 RGLR<br />
BLR23 M 24965 107 57 RSLR 62 DENR<br />
BLR24 M 43687 91 55 RSLR 74 ELEQ<br />
BLR25 M 48013 82 55 RALR<br />
BLR26 E 16394 187 46 RRLR 59 QNDER<br />
BLR27 E 38529 434 40 RQLR<br />
BLR28 3 08983 279 49 RRLR<br />
BLR29 3 43449 311 35 RMLR 46 EES<br />
BLR30 3 50216 101 47 RSLR 60 DEER<br />
BLQ01 3 59265 79 49 QLLR 61 DEEQR<br />
BLG01 M 25695 336 44 GKLR 57 DER<br />
BLG02 M 31920 233 44 GRLR 57 DER<br />
BLG03 M 23857 243 42 GKLR 55 DER
Bremia <strong>effectors</strong> enhance host susceptibility<br />
103<br />
<strong>of</strong> the cloned effector c<strong>and</strong>idates is given <strong>in</strong> Table 1, <strong>and</strong> sequences can found <strong>in</strong><br />
Supplemental Information 1.<br />
Our set <strong>of</strong> potential <strong>effectors</strong> consists <strong>of</strong> 30 RXLR prote<strong>in</strong>s (BLR, for Bremia<br />
lactucae RXLR) <strong>and</strong> four c<strong>and</strong>idates with an RXLR-like motif. These Bremia<br />
RXLR-like effector c<strong>and</strong>idates were identified based on their similarity to <strong>effectors</strong><br />
<strong>of</strong> other oomycetes <strong>and</strong> by comparison to a hidden Markov model based on the<br />
am<strong>in</strong>o acid sequence surround<strong>in</strong>g the RXLR motifs <strong>of</strong> Bremia <strong>effectors</strong> with an<br />
RXLR motif. In three <strong>of</strong> these RXLR-like motifs the first arg<strong>in</strong><strong>in</strong>e is replaced by a<br />
glyc<strong>in</strong>e (BLG01, BLG02 <strong>and</strong> BLG03), <strong>and</strong> <strong>in</strong> the fourth this residue is a glutam<strong>in</strong>e<br />
(BLQ01). EER-like doma<strong>in</strong>s (am<strong>in</strong>o acid sequences rich <strong>in</strong> E,Q,D or N residues,<br />
preferably end<strong>in</strong>g <strong>in</strong> R or K) were found <strong>in</strong> the majority <strong>of</strong> effector c<strong>and</strong>idates. The<br />
length <strong>of</strong> the cloned <strong>effectors</strong> varies from 65 to 463 am<strong>in</strong>o acids, with an average<br />
length <strong>of</strong> just under 180 am<strong>in</strong>o acids.<br />
Constructs for Agrobacterium-mediated transient expression <strong>in</strong> planta were<br />
created by eng<strong>in</strong>eer<strong>in</strong>g a new start codon upstream <strong>of</strong> the cod<strong>in</strong>g sequence <strong>of</strong> the<br />
cleaved secreted prote<strong>in</strong>, so that the first am<strong>in</strong>o acid after the start codon <strong>in</strong> the<br />
constructs corresponds to the codon for the first am<strong>in</strong>o acid after the predicted<br />
signal peptide cleavage site. In planta expression <strong>of</strong> the constructs is driven by<br />
a 35S CaMV promoter. Because <strong>of</strong> the deletion <strong>of</strong> the signal peptide sequence,<br />
the effector prote<strong>in</strong> will be translated <strong>in</strong> the host cell cytoplasm, where it is also<br />
expected to be present dur<strong>in</strong>g <strong>in</strong>fection by Bremia.<br />
Bremia <strong>effectors</strong> modulate plant susceptibility<br />
Modulation <strong>of</strong> host susceptibility by the Bremia RXLR <strong>effectors</strong> was tested <strong>in</strong><br />
a leaf-disc assay based on the idea that transient expression <strong>of</strong> <strong>effectors</strong> <strong>in</strong> leaf<br />
tissue could enhance the susceptibility to Bremia <strong>in</strong>fection, result<strong>in</strong>g <strong>in</strong> higher<br />
colonisation <strong>and</strong> sporulation levels. To test our system we transiently expressed<br />
the lettuce orthologue <strong>of</strong> a gene encod<strong>in</strong>g a negative regulator <strong>of</strong> plant immunity,<br />
DMR6 [28] , as positive control. A T-DNA GUS-<strong>in</strong>tron construct (hereafter: GUS)<br />
was used as a control for T-DNA transfer <strong>and</strong> transgene expression, <strong>and</strong> was used<br />
to determ<strong>in</strong>e the effect <strong>of</strong> Agrobacterium <strong>in</strong>filtration on susceptibility to Bremia<br />
<strong>in</strong> our assay. Figure 1 shows the amount <strong>of</strong> spores that could be recovered from<br />
mock-<strong>in</strong>filtrated <strong>and</strong> DMR6 express<strong>in</strong>g leaf discs, relative to the amount <strong>of</strong> spores<br />
recovered from leaf discs express<strong>in</strong>g GUS. Leaf discs that are <strong>in</strong>filtrated with only<br />
<strong>in</strong>filtration medium are more susceptible to Bremia than those <strong>in</strong>filtrated with Agrobacterium,<br />
probably because <strong>of</strong> the <strong>in</strong>duction <strong>of</strong> plant defence. The Agrobacterium<br />
sta<strong>in</strong> carry<strong>in</strong>g a GUS construct is therefore a more relevant control to <strong>in</strong>vestigate<br />
the effect <strong>of</strong> the Bremia effector c<strong>and</strong>idates on host susceptibility. DMR6 <strong>in</strong>creases<br />
host susceptibility to Bremia, as is evident from the <strong>in</strong>creased numbers <strong>of</strong> spores
104 Chapter 4<br />
Figure 1: Median relative amount <strong>of</strong> Bremia spores harvested from lettuce discs <strong>in</strong>filtrated with<br />
<strong>in</strong>filtration medium (IM), GUS-Agrobacterium or DMR6-Agrobacterium. Based on 23 observations per<br />
treatment. Error bars <strong>in</strong>dicate 95% confidence <strong>in</strong>terval.<br />
that can be harvested from leaf discs transiently over-express<strong>in</strong>g DMR6. This is<br />
<strong>in</strong> accordance with observations <strong>of</strong> enhanced susceptibility <strong>of</strong> A. thaliana DMR6<br />
over-expression l<strong>in</strong>es to the <strong>downy</strong> <strong>mildew</strong> H. arabidopsidis <strong>and</strong> the bacterial pathogen<br />
P. syr<strong>in</strong>gae pv tomato DC3000 [28] . We verified that Bremia can be negatively<br />
affected by effector-<strong>in</strong>duced responses us<strong>in</strong>g the P. sojae effector PsojNIP, which<br />
<strong>in</strong>duces cell death <strong>in</strong> lettuce [29] . PsojNIP-express<strong>in</strong>g leaf discs underwent extensive<br />
cell-death <strong>and</strong> no Bremia spores could be recovered from them as a result. This<br />
leaf-disc assay is therefore capable <strong>of</strong> uncover<strong>in</strong>g changes <strong>in</strong> susceptibility <strong>and</strong> can<br />
be used to <strong>in</strong>vestigate the effect <strong>of</strong> Bremia <strong>effectors</strong> on host immunity.<br />
We used the leaf-disc assay to test the 34 cloned Bremia <strong>effectors</strong>. The effect<br />
<strong>of</strong> transient over-expression <strong>of</strong> Bremia <strong>effectors</strong> <strong>in</strong> advance <strong>of</strong> <strong>in</strong>oculation with<br />
Bremia differs between tested <strong>effectors</strong> (Figure 2). Transient expression <strong>of</strong> all<br />
but three <strong>effectors</strong> appeared to allow recovery <strong>of</strong> more Bremia spores from leaf<br />
discs compared to GUS controls, though variation between replicates is high.<br />
Expression <strong>of</strong> six <strong>effectors</strong> even led to sporulation levels higher then those on leaf<br />
discs <strong>in</strong>filtrated with only medium. Sample sizes were small (4-23 observations)<br />
<strong>and</strong> the assumption <strong>of</strong> homogeneity <strong>of</strong> variances is violated (p < 0.05), rul<strong>in</strong>g out<br />
the use <strong>of</strong> an ANOVA. All treatment groups were compared <strong>in</strong>dividually to GUS<br />
to determ<strong>in</strong>e which Bremia <strong>effectors</strong> <strong>and</strong> controls significantly affect spore counts<br />
us<strong>in</strong>g the Mann-Whitney U test, <strong>and</strong> correction for multiple test<strong>in</strong>g was performed<br />
us<strong>in</strong>g the Bonferroni method. Significantly enhanced susceptibility was observed<br />
only for host tissue express<strong>in</strong>g BLR16 <strong>and</strong> BLR27. Leaf tissue <strong>in</strong> which either <strong>of</strong>
Bremia <strong>effectors</strong> enhance host susceptibility<br />
105<br />
Figure 2: Median relative amount <strong>of</strong> Bremia spores harvested from lettuce leaf-discs <strong>in</strong>filtrated with<br />
Agrobacterium carry<strong>in</strong>g <strong>in</strong>dicated Bremia RXLR effector c<strong>and</strong>idates, or control genes for GUS <strong>and</strong><br />
DMR6, or <strong>in</strong>filtration medium (IM). Striped bars <strong>in</strong>dicate the treatments that did not <strong>in</strong>clude Bremia<br />
<strong>effectors</strong>. Spores amounts are <strong>in</strong>dicated relative to the number <strong>of</strong> spores harvested from Agrobacterium-<br />
GUS <strong>in</strong>filtrated leaf discs. Values are based on m<strong>in</strong>imally 4 observations. Error bars <strong>in</strong>dicate 95%<br />
confidence <strong>in</strong>terval. Asterisks <strong>in</strong>dicate treatments that differ significantly from Agrobacterium-GUS<br />
treatment (p < 0.05) after Bonferroni correction.<br />
these <strong>effectors</strong> is expressed supports spore production levels that lie closer to those<br />
on DMR6-express<strong>in</strong>g tissue than those on GUS-express<strong>in</strong>g tissue. Conversely,<br />
expression <strong>of</strong> BLR03 has a significant negative impact on host susceptibility<br />
result<strong>in</strong>g <strong>in</strong> lower sporulation levels <strong>of</strong> Bremia. The number <strong>of</strong> spores harvested<br />
from BLR03-express<strong>in</strong>g host tissue was generally around half <strong>of</strong> that harvested<br />
from GUS-express<strong>in</strong>g control tissue.<br />
To test if the presumed translocation doma<strong>in</strong> is dispensable for the effects on<br />
host susceptibility, we also expressed the effector doma<strong>in</strong> only <strong>of</strong> BLR03 <strong>and</strong><br />
BLR16 <strong>in</strong> lettuce-leaf discs. Prelim<strong>in</strong>ary results <strong>in</strong>dicate that reduction <strong>of</strong> host<br />
susceptibility <strong>in</strong> leaf discs express<strong>in</strong>g BLR03 without translocation doma<strong>in</strong> was<br />
comparable to that found <strong>in</strong> leaf discs express<strong>in</strong>g BLR03 with translocation doma<strong>in</strong><br />
(not shown). Likewise, susceptibility was comparably enhanced <strong>in</strong> leaf discs<br />
express<strong>in</strong>g BLR16 without its translocation doma<strong>in</strong> <strong>and</strong> similar to that <strong>of</strong> BLR16<br />
with its translocation doma<strong>in</strong>. These prelim<strong>in</strong>ary results suggest that the translocation<br />
doma<strong>in</strong> is not required for the <strong>in</strong> planta susceptibility-modulat<strong>in</strong>g activities <strong>of</strong><br />
these <strong>effectors</strong>, confirm<strong>in</strong>g that the effector doma<strong>in</strong> starts after the EER-like motif.<br />
Effectors are expressed at different stages <strong>of</strong> <strong>in</strong>fection<br />
To <strong>in</strong>vestigate at which stage <strong>of</strong> the <strong>in</strong>fection process the potential <strong>effectors</strong><br />
are expressed, we determ<strong>in</strong>ed the relative mRNA levels <strong>of</strong> selected effector genes.<br />
The selection <strong>of</strong> effector genes was based on promis<strong>in</strong>g prelim<strong>in</strong>ary results <strong>in</strong> our
106 Chapter 4<br />
leaf disc assay <strong>and</strong> <strong>in</strong>cludes the <strong>effectors</strong> that significantly affected susceptibility<br />
to Bremia. All <strong>effectors</strong> were cloned from cDNA <strong>and</strong> were therefore already<br />
known to be expressed <strong>in</strong> Bremia. A more detailed look at expression levels <strong>and</strong><br />
differential expression throughout the <strong>in</strong>fection process is given <strong>in</strong> Figure 3.<br />
Bremia Act<strong>in</strong> expression levels were compared to L. sativa Act<strong>in</strong> expression at<br />
each time po<strong>in</strong>t by subtract<strong>in</strong>g the number <strong>of</strong> qPCR cycles required for the L.<br />
sativa Act<strong>in</strong> amplicon to reach a threshold, from the number <strong>of</strong> qPCR cycles <strong>of</strong><br />
the Bremia Act<strong>in</strong> amplicon to reach the same threshold (ΔC T<br />
). Similarly, effector<br />
gene expression was normalised to Bremia Act<strong>in</strong>. Relative Bremia Act<strong>in</strong> levels<br />
<strong>in</strong>dicate substantial relative Bremia growth dur<strong>in</strong>g the entire time course (Figure 3,<br />
Act<strong>in</strong>). Based on our transcriptome data, i.e. relative abundance <strong>of</strong> non-normalised<br />
5’ reads <strong>in</strong> the spore-derived <strong>and</strong> <strong>in</strong>oculated-lettuce-derived pools, we predicted<br />
BLR22 to be ma<strong>in</strong>ly present <strong>in</strong> spores, <strong>and</strong> BLR23 to be more highly expressed<br />
dur<strong>in</strong>g <strong>in</strong> planta growth [11] . Indeed, expression <strong>of</strong> BLR22 strongly decreased after<br />
the first two days post <strong>in</strong>oculation (dpi), whilst the expression <strong>of</strong> BLR23 <strong>in</strong>creased<br />
Figure 3: Temporal expression <strong>of</strong> <strong>in</strong>dicated Bremia genes. Bremia Act<strong>in</strong> expression was compared to<br />
lettuce Act<strong>in</strong> expression. All other Bremia genes were normalised to Bremia Act<strong>in</strong>. As lower C T<br />
values<br />
<strong>in</strong>dicate higher expression, the Y-axis scale has been reversed to ease <strong>in</strong>terpretation. Error bars <strong>in</strong>dicate<br />
st<strong>and</strong>ard deviation from the mean <strong>of</strong> three technical replicates.
Bremia <strong>effectors</strong> enhance host susceptibility<br />
107<br />
from two dpi onwards (Figure 3). BLR05, BLR09 <strong>and</strong> BLR27 are more actively<br />
transcribed dur<strong>in</strong>g the spore stage <strong>and</strong> at 1 dpi, with expression slightly decl<strong>in</strong><strong>in</strong>g<br />
until 3 dpi. BLG02 expression levels are much higher at 1 dpi compared to spores,<br />
but expression decreases steadily from then on. BLR16, which shows a significant<br />
positive effect on host-susceptibility, appears to be slightly up-regulated at 1 dpi,<br />
with expression be<strong>in</strong>g lower at 2 dpi <strong>and</strong> steadily <strong>in</strong>creas<strong>in</strong>g aga<strong>in</strong> to higher levels<br />
at the end <strong>of</strong> the time course, similar to that <strong>of</strong> BLR23. The expression <strong>of</strong> BLR03,<br />
on the other h<strong>and</strong>, drops rapidly dur<strong>in</strong>g early <strong>in</strong> planta stages, similar to that <strong>of</strong><br />
BLR22, though the low expression levels <strong>of</strong> BLR03 are reached at 3 dpi, whereas<br />
expression <strong>of</strong> BLR22 is already low at 2 dpi. This suggests an early role for BLR03,<br />
<strong>and</strong> could expla<strong>in</strong> that when expressed outside <strong>of</strong> its normal w<strong>in</strong>dow <strong>of</strong> expression<br />
BLR03 can have a negative <strong>in</strong>fluence on host susceptibility, as we observed <strong>in</strong> the<br />
leaf disc assay. The expression pr<strong>of</strong>iles <strong>of</strong> the Bremia <strong>effectors</strong> were found to be<br />
similar <strong>in</strong> a second experiment on an <strong>in</strong>dependent time course (not shown).<br />
Modulation <strong>of</strong> biotic stress-associated gene expression <strong>in</strong> lettuce<br />
To address the question whether <strong>effectors</strong> change the expression <strong>of</strong> genes that are<br />
responsive to biotic stress we set out to identify immunity-related marker genes <strong>in</strong><br />
lettuce. We identified lettuce sequences that are highly abundant <strong>in</strong> the transcriptome<br />
<strong>of</strong> Bremia-<strong>in</strong>fected lettuce plants. A non-normalised sequenc<strong>in</strong>g pool derived<br />
from full-length mRNA <strong>of</strong> lettuce <strong>in</strong>fected with Bremia was previously sequenced<br />
from the 5’ end <strong>and</strong> used for de novo assembly <strong>of</strong> Bremia <strong>and</strong> lettuce transcripts [13] .<br />
The number <strong>of</strong> reads derived from this pool that is assembled <strong>in</strong>to each contig<br />
allows an estimate <strong>of</strong> the relative abundance <strong>of</strong> the transcript represented by the<br />
contig. Our transcript data did not <strong>in</strong>clude transcripts derived from un<strong>in</strong>fected<br />
lettuce that could be used to identify Bremia-<strong>in</strong>duced transcripts based on relative<br />
abundance, so highly abundant transcripts were searched for biotic stress-associated<br />
transcripts as follows. First, transcripts were compared with annotated<br />
Arabidopsis gene models to identify homologous sequences. Potential markers<br />
were then selected by two methods. Homologues <strong>of</strong> Arabidopsis genes annotated<br />
as biotic stress-responsive were selected. Furthermore, Arabidopsis homologues <strong>of</strong><br />
the most highly present lettuce transcripts were <strong>in</strong>vestigated <strong>in</strong> over 6000 publicly<br />
available Arabidopsis microarray experiments via Genevestigator [30] to determ<strong>in</strong>e<br />
whether they were strongly responsive to biotic stresses. The selected genes, listed<br />
<strong>in</strong> Table 2, were further <strong>in</strong>vestigated for their usefulness as markers.<br />
We determ<strong>in</strong>ed the response <strong>of</strong> the selected lettuce genes to Bremia <strong>in</strong>fection<br />
<strong>and</strong> Agrobacterium <strong>in</strong>oculation. Induction by Bremia was tested to determ<strong>in</strong>e<br />
the relevance <strong>of</strong> the markers dur<strong>in</strong>g Bremia <strong>in</strong>fection <strong>of</strong> lettuce (Figure 4A). The<br />
expression <strong>of</strong> all <strong>of</strong> the eleven tested marker c<strong>and</strong>idates was <strong>in</strong>duced dur<strong>in</strong>g Bremia
108 Chapter 4<br />
Table 2: Overview <strong>of</strong> lettuce biotic stress-associated marker c<strong>and</strong>idates <strong>and</strong> their Arabidopsis<br />
homologues.<br />
ID<br />
(contig)<br />
5’<br />
reads<br />
Arabidopsis<br />
gene ID<br />
Blast<br />
e-value<br />
Symbols Description<br />
00337 44 AT5G58600.2 2e-95 PMR5<br />
Plant prote<strong>in</strong> <strong>of</strong> unknown<br />
function (DUF828)<br />
00744 25 AT4G30210.2 0 ATR2, AR2 P450 reductase 2<br />
03859 32 AT5G64120.1 1e-67 Peroxidase superfamily prote<strong>in</strong><br />
04174 21 AT3G45640.1<br />
ATMPK3, MPK3, Mitogen-activated prote<strong>in</strong><br />
7e-64<br />
ATMAPK3 k<strong>in</strong>ase 3<br />
06346 51 AT4G16260.1 2e-95<br />
Glycosyl hydrolase superfamily<br />
prote<strong>in</strong><br />
08313 23 AT4G33430.1<br />
BAK1, RKS10,<br />
SERK3, ELG, BRI1-associated receptor k<strong>in</strong>ase<br />
ATSERK3, ATBAK1<br />
07164 37 AT3G10985.1<br />
SAG20, WI12,<br />
2e-23<br />
ATWI-12<br />
Senescence associated gene 20<br />
15736 37 AT1G70170.1 4e-42 MMP Matrix metalloprote<strong>in</strong>ase<br />
15964 22 AT2G39210.1 0<br />
Major facilitator superfamily<br />
prote<strong>in</strong><br />
33411 99 AT2G36690.1 3e-119<br />
2-oxoglutarate (2OG) <strong>and</strong><br />
Fe(II)-dependent oxygenase<br />
superfamily prote<strong>in</strong><br />
37274 22 AT2G25000.1 3e-39 WRKY60,<br />
ATWRKY60<br />
WRKY DNA-b<strong>in</strong>d<strong>in</strong>g prote<strong>in</strong> 60<br />
<strong>in</strong>fection. Induction <strong>of</strong> two marker genes, 3859 <strong>and</strong> 4174, was higher than 3 C T<br />
<strong>and</strong><br />
three other markers, 15964, 33411 <strong>and</strong> 37274, showed a higher than 6 C T<br />
<strong>in</strong>duction.<br />
The Bremia effector genes that we planned to <strong>in</strong>vestigate were to be tested by<br />
Agrobacterium-mediated transient transformation. We therefore <strong>in</strong>vestigated<br />
the lettuce response to Agrobacterium <strong>in</strong>filtration (Figure 4B) for marker gene<br />
c<strong>and</strong>idates that showed a greater than 3 C T<br />
<strong>in</strong>duction <strong>in</strong> Bremia-treated compared<br />
to mock-treated leaf material. Induction was similar between Bremia <strong>and</strong> Agrobacterium<br />
<strong>in</strong>oculation, with the largest differences <strong>in</strong> the level <strong>of</strong> <strong>in</strong>duction <strong>of</strong> markers<br />
3859 (stronger <strong>in</strong>duction by Agrobacterium) <strong>and</strong> 37274 (stronger <strong>in</strong>duction by<br />
Bremia). The Agrobacterium <strong>in</strong>filtration <strong>of</strong> lettuce can therefore serve a dual<br />
purpose: i, to <strong>in</strong>duce biotic stress, <strong>and</strong> ii, for transient effector gene expression.<br />
We <strong>in</strong>vestigated whether transient expression <strong>of</strong> any <strong>of</strong> the six <strong>effectors</strong> that<br />
most strongly enhanced susceptibility <strong>in</strong> leaf discs, <strong>and</strong> BLR03, which reduces host<br />
susceptibility (Figure 2), modulates expression <strong>of</strong> lettuce biotic stress-markers.<br />
Background expression <strong>of</strong> the biotic stress-markers was determ<strong>in</strong>ed <strong>in</strong> mocktreated<br />
plants. All marker gene expression values were normalised to L. sativa<br />
Act<strong>in</strong> (ΔC T<br />
) <strong>and</strong> normalised values were compared between different treatments
Bremia <strong>effectors</strong> enhance host susceptibility<br />
109<br />
Figure 4: (A) Induction <strong>of</strong> expression <strong>of</strong> biotic-stress-responsive genes <strong>in</strong> Bremia <strong>in</strong>oculated lettuce.<br />
All expression values were normalised towards L. sativa Act<strong>in</strong> <strong>and</strong> compared to normalised mocktreated<br />
values, <strong>and</strong> are given as ΔΔC T<br />
. Error bars <strong>in</strong>dicate the st<strong>and</strong>ard deviation <strong>of</strong> the average <strong>of</strong><br />
three technical replicates. The experiment was repeated with similar results. The scale <strong>of</strong> the Y-axis<br />
has been <strong>in</strong>verted to ease <strong>in</strong>terpretation. (B) Induction <strong>of</strong> expression <strong>of</strong> biotic-stress-responsive genes<br />
<strong>in</strong> Agrobacterium <strong>in</strong>filtrated lettuce. Values are calculated similar to those <strong>in</strong> (A). Error bars <strong>in</strong>dicate<br />
the st<strong>and</strong>ard deviation <strong>of</strong> the average <strong>of</strong> three technical replicates. The experiment was repeated with<br />
similar results. The scale <strong>of</strong> the Y-axis has been <strong>in</strong>verted to ease <strong>in</strong>terpretation.<br />
<strong>and</strong> mock (ΔΔC T<br />
). The <strong>in</strong>duction level <strong>of</strong> biotic stress-markers <strong>in</strong> lettuce leaf<br />
material transiently express<strong>in</strong>g <strong>in</strong>dividual <strong>effectors</strong> is shown <strong>in</strong> Figure 5. Basel<strong>in</strong>e<br />
Agrobacterium-<strong>in</strong>duced marker expression can be determ<strong>in</strong>ed <strong>in</strong> leaf material<br />
transiently express<strong>in</strong>g GUS, as the encoded β-glucuronidase is not expected to<br />
modulate host processes. Induction <strong>of</strong> biotic stress-markers is affected to a limited<br />
degree by transient expression <strong>of</strong> DMR6, a negative regulator <strong>of</strong> host defence, with<br />
a 1-1.5 C T<br />
difference <strong>in</strong> <strong>in</strong>duction <strong>of</strong> both 33411 <strong>and</strong> 37274 compared to expression<br />
<strong>of</strong> GUS. No large differences <strong>in</strong> the expression <strong>of</strong> markers 3859 <strong>and</strong> 4174 were<br />
found between GUS <strong>and</strong> <strong>in</strong>dividual Bremia <strong>effectors</strong>. A trend towards higher<br />
expression <strong>of</strong> 15964 was found <strong>in</strong> response to the majority <strong>of</strong> <strong>effectors</strong>, whilst<br />
expression <strong>of</strong> 33411 is reduced <strong>in</strong> response to nearly half <strong>of</strong> the <strong>effectors</strong>. Expression<br />
<strong>of</strong> 37274 is also <strong>in</strong>duced by a number <strong>of</strong> <strong>effectors</strong>, though it is suppressed by<br />
expression <strong>of</strong> DMR6. Response <strong>of</strong> the markers to different Bremia <strong>effectors</strong> turned<br />
out to be variable, when tested <strong>in</strong> an <strong>in</strong>dependent second experiment (not shown).<br />
The biological variation <strong>in</strong> these experiments was too large to rule out m<strong>in</strong>or<br />
effects. We conclude that there is no strong major effect <strong>of</strong> any <strong>of</strong> the tested Bremia<br />
<strong>effectors</strong> on the expression <strong>of</strong> the selected markers <strong>of</strong> biotic stress.
110 Chapter 4<br />
Figure 5: Relative transcript abundance <strong>of</strong> L. sativa biotic stress-markers <strong>in</strong> leaf sections that were<br />
<strong>in</strong>filtrated with Agrobacterium <strong>and</strong> transiently expressed <strong>in</strong>dicated Bremia <strong>effectors</strong>, GUS, or L. sativa<br />
DMR6. All expression values were normalised towards L. sativa Act<strong>in</strong> <strong>and</strong> compared to normalised<br />
mock-treated values, <strong>and</strong> are given as ΔΔC T<br />
. Error bars <strong>in</strong>dicate the st<strong>and</strong>ard deviation <strong>of</strong> the average <strong>of</strong><br />
three technical replicates. The scale <strong>of</strong> the Y-axis has been <strong>in</strong>verted to ease <strong>in</strong>terpretation (so that higher<br />
bars <strong>in</strong>dicate higher <strong>in</strong>duction levels).<br />
Discussion<br />
A set <strong>of</strong> 34 predicted Bremia <strong>effectors</strong> was analysed for their contribution to<br />
the modulation <strong>of</strong> host susceptibility. Effects <strong>of</strong> over-expression <strong>of</strong> <strong>effectors</strong> <strong>in</strong><br />
the pathogen itself have been reported for other species. For example, P. sojae<br />
transformants constitutively express<strong>in</strong>g high levels <strong>of</strong> Avr1b-1 are more virulent on<br />
soybean than wild-type isolates [31] . Silenc<strong>in</strong>g <strong>of</strong> <strong>effectors</strong> <strong>in</strong> Phytophthora species<br />
has also revealed contributions to pathogenicity, e.g. by P. <strong>in</strong>festans AVR3a [25] <strong>and</strong><br />
P. sojae AVH238 [21] . Unfortunately, attempts at transformation <strong>of</strong> Bremia have been<br />
unsuccessful so far, rul<strong>in</strong>g out the possibility <strong>of</strong> manipulat<strong>in</strong>g expression <strong>of</strong> <strong>effectors</strong><br />
<strong>in</strong> the oomycete itself. We therefore transiently expressed Bremia <strong>effectors</strong> <strong>in</strong><br />
lettuce leaf material us<strong>in</strong>g Agrobacterium.<br />
Most <strong>of</strong> the <strong>in</strong>vestigated Bremia <strong>effectors</strong> enhance susceptibility <strong>in</strong> the host.<br />
The experimental setup we used measures the level <strong>of</strong> susceptibility by quantify<strong>in</strong>g<br />
spore-production <strong>of</strong> Bremia. Susceptibility is likely a comb<strong>in</strong>ation <strong>of</strong> multiple<br />
factors (see <strong>in</strong>troduction) <strong>and</strong> by quantify<strong>in</strong>g <strong>in</strong>fection levels based on a late stage<br />
<strong>of</strong> the pathogen life cycle, any manipulation <strong>of</strong> host processes that impacts both<br />
early <strong>and</strong> late pathogen development can potentially be identified. The system is<br />
potentially limited by its dependence on three different organisms (i.e. L. sativa,<br />
Agrobacterium <strong>and</strong> Bremia) that each <strong>in</strong>troduce their own variation. By us<strong>in</strong>g<br />
appropriate controls <strong>and</strong> us<strong>in</strong>g fixed leaf area <strong>and</strong> <strong>in</strong>oculum size, we m<strong>in</strong>imised
Bremia <strong>effectors</strong> enhance host susceptibility<br />
111<br />
variation.<br />
Increased susceptibility by suppression <strong>of</strong> plant immunity has been more<br />
extensively studied <strong>and</strong> appears to be a common function <strong>of</strong> <strong>effectors</strong>. In a P.<br />
sojae effector screen, Wang <strong>and</strong> co-workers assayed for suppression <strong>of</strong> cell death<br />
<strong>in</strong>duced by BAX, other <strong>effectors</strong>, or INF-1 <strong>in</strong> N. benthamiana [21] . When expressed<br />
24 hours before challenge, 107 <strong>of</strong> 169 <strong>effectors</strong> were able to suppress cell death,<br />
suggest<strong>in</strong>g that cell-death suppression is a common function <strong>of</strong> P. sojae <strong>effectors</strong>.<br />
Hemibiotrophs such as P. sojae may use cell death suppress<strong>in</strong>g <strong>effectors</strong> to tightly<br />
regulate the onset <strong>of</strong> the necrotrophic phase [17] . A screen <strong>of</strong> <strong>effectors</strong> <strong>in</strong> the <strong>downy</strong><br />
<strong>mildew</strong> H. arabidopsidis by Fabro et al. [23] focused on the manipulation <strong>of</strong> immune<br />
responses triggered by Ps. syr<strong>in</strong>gae pv tomato DC3000, which was also used as<br />
effector delivery system. The authors conclude from the screen that many <strong>of</strong> the<br />
tested <strong>effectors</strong> suppress immunity, but also that the effects <strong>of</strong> <strong>in</strong>dividual <strong>downy</strong><br />
<strong>mildew</strong> <strong>effectors</strong> are <strong>of</strong>ten weak <strong>and</strong> host accession-specific. Similar host-dependent<br />
differences between Bremia <strong>effectors</strong> may await discovery.<br />
Effector screens allow study<strong>in</strong>g the effect <strong>of</strong> <strong>in</strong>dividual effector prote<strong>in</strong>s on<br />
a particular host process <strong>of</strong> <strong>in</strong>terest. Currently these processes are still broadly<br />
def<strong>in</strong>ed, e.g. ‘host susceptibility’. Effector activities can be assayed for <strong>in</strong> more<br />
specific screens, though this requires better underst<strong>and</strong><strong>in</strong>g <strong>of</strong> the specific components<br />
that underlie host susceptibility. Based on the knowledge <strong>of</strong> non-oomycete<br />
<strong>effectors</strong>, host susceptibility can be <strong>in</strong>fluenced at many levels. A previously<br />
mentioned example is PthX01-<strong>in</strong>duced OsSWEET11 expression that is thought<br />
to result <strong>in</strong> a sugar efflux that feeds the bacterial pathogen [9] . The corn-smut<br />
fungus Ustilago maydis directs a chorismate mutase to the host-cell cytoplasm to<br />
metabolically prime the cell for <strong>in</strong>fection, reduc<strong>in</strong>g the levels <strong>of</strong> salicylic acid <strong>and</strong><br />
thereby enhanc<strong>in</strong>g susceptibility [32] . Further <strong>in</strong>vestigation is required to elucidate<br />
the mechanisms by which Bremia <strong>effectors</strong> enhance susceptibility.<br />
We found one effector, BLR03, with a seem<strong>in</strong>gly negative contribution to<br />
virulence. This effector does not <strong>in</strong>duce a hypersensitive response <strong>in</strong> host plants,<br />
but does decrease susceptibility to Bremia when tested on its own. The effector<br />
may fulfil a function dur<strong>in</strong>g normal <strong>in</strong>fection that is not detected <strong>in</strong> our assay. The<br />
negative effect observed <strong>in</strong> our assay can be the result <strong>of</strong> the tim<strong>in</strong>g or high-level<br />
<strong>of</strong> effector expression. Tight regulation <strong>of</strong> effector expression has been shown to be<br />
important for pathogen virulence. For example, misexpression <strong>of</strong> P. sojae effector<br />
genes Avh172 <strong>and</strong> Avh238 can disrupt <strong>in</strong>fection [21] . Interest<strong>in</strong>gly, both constitutive<br />
over-expression <strong>and</strong> transient silenc<strong>in</strong>g <strong>of</strong> Avh238 <strong>in</strong> P. sojae led to reduced<br />
virulence. The expression pr<strong>of</strong>ile <strong>of</strong> BLR03 dur<strong>in</strong>g <strong>in</strong>fection shows a strong decl<strong>in</strong>e<br />
<strong>of</strong> transcript levels dur<strong>in</strong>g the first days <strong>of</strong> <strong>in</strong>fection. The 35S-driven <strong>in</strong> planta<br />
transient expression level <strong>of</strong> BLR03 is likely far higher than expression levels<br />
<strong>in</strong> Bremia. The negative effect on host susceptibility <strong>in</strong> our leaf-disc assay may
112 Chapter 4<br />
therefore be the effect <strong>of</strong> misexpression <strong>of</strong> the effector.<br />
Us<strong>in</strong>g lettuce biotic stress-associated markers we show that both Bremia <strong>and</strong><br />
Agrobacterium <strong>in</strong>duce strong transcriptional responses <strong>in</strong> lettuce. Over-expression<br />
<strong>of</strong> DMR6, which encodes a negative regulator <strong>of</strong> plant immunity, appeared to<br />
have a limited effect on marker expression levels. Variation <strong>in</strong> marker expression<br />
levels <strong>in</strong> leaf material express<strong>in</strong>g different Bremia <strong>effectors</strong> between <strong>in</strong>dependent<br />
experiments was too large to detect suppression by the <strong>effectors</strong> on marker gene<br />
expression. The observations do, however, suggest that it is unlikely that the tested<br />
Bremia <strong>effectors</strong> <strong>in</strong>duce strong major changes <strong>in</strong> expression <strong>of</strong> biotic stress-markers.<br />
Enhancement <strong>of</strong> host susceptibility by these Bremia <strong>effectors</strong> therefore is not<br />
likely to act by general repression <strong>of</strong> biotic stress responses, but is more likely the<br />
result <strong>of</strong> very specific <strong>and</strong> limited modulation <strong>of</strong> the host. Also, modulation <strong>of</strong> host<br />
processes may not lead to detectable transcriptional changes; e.g. block<strong>in</strong>g <strong>of</strong> C14<br />
secretion by AVRBlb2 [26] is not likely to <strong>in</strong>duce a strong transcriptional response.<br />
The basis <strong>of</strong> the susceptibility enhanc<strong>in</strong>g effects <strong>of</strong> Bremia <strong>effectors</strong> thus rema<strong>in</strong>s<br />
to be determ<strong>in</strong>ed. The identification <strong>of</strong> disease susceptibility-enhanc<strong>in</strong>g <strong>effectors</strong> <strong>of</strong><br />
Bremia now opens the way to <strong>in</strong>vestigate their activity <strong>and</strong> host targets.<br />
Materials <strong>and</strong> methods<br />
Clon<strong>in</strong>g <strong>and</strong> sequenc<strong>in</strong>g<br />
Effector predictions were sequence-verified from PCR product us<strong>in</strong>g flank<strong>in</strong>g<br />
primers <strong>and</strong> sequenced by Macrogen Inc. For primer sequences used <strong>in</strong> this study,<br />
see Supplemental Table 1. Verified effector c<strong>and</strong>idates were TOPO-cloned <strong>in</strong>to<br />
the pENTR vector us<strong>in</strong>g the pENTR/D-TOPO Clon<strong>in</strong>g kit (Invitrogen) accord<strong>in</strong>g<br />
to manufacturer’s <strong>in</strong>structions start<strong>in</strong>g from the sequence after the predicted<br />
signal peptide cleavage site <strong>of</strong> the <strong>effectors</strong>, unless otherwise <strong>in</strong>dicated, <strong>and</strong> were<br />
preceded by a newly <strong>in</strong>troduced start codon. Constructs were then recomb<strong>in</strong>ed <strong>in</strong>to<br />
the pK2GW7 vector (Plant-Systems Biology VIB, Ghent). The PsojNIP construct<br />
was previously described [33] . Clones were electro-transformed <strong>in</strong>to Agrobacterium<br />
tumefaciens stra<strong>in</strong> C58C1 (pGV2260).<br />
Leaf disc assays<br />
Lactuca sativa cv. Ol<strong>of</strong> was sown <strong>and</strong> kept at high humidity (closed tray with<br />
transparent lid) at 17°C, 9 h <strong>of</strong> light (100 μE/m 2 /s) to germ<strong>in</strong>ate for one week.<br />
Seedl<strong>in</strong>gs were then grown for 2 weeks at 21°C, 16 h <strong>of</strong> light (200 μE/m 2 /s), 70%<br />
RH. Per experiment, a number <strong>of</strong> leaf discs (14 mm diameter) <strong>in</strong> excess <strong>of</strong> that<br />
required for the experiment were punched from Lactuca sativa cv. Ol<strong>of</strong>, mixed, <strong>and</strong>
Bremia <strong>effectors</strong> enhance host susceptibility<br />
113<br />
r<strong>and</strong>omly divided over treatment conditions. Agrobacterium tumefaciens stra<strong>in</strong>s<br />
conta<strong>in</strong><strong>in</strong>g the 35S-effector T-DNA were grown overnight <strong>in</strong> selective media at<br />
28°C, 220 RPM. Cells were spun down at 2500 g for 10 m<strong>in</strong>utes <strong>and</strong> re-suspended<br />
<strong>in</strong> <strong>in</strong>duction medium (1x M9 salts, 1% glucose, 50μM acetosyr<strong>in</strong>gone) with appropriate<br />
antibiotics. After growth for 4 h at 28°C, 220 RPM, cells were spun down<br />
at 2500 g for 10 m<strong>in</strong> <strong>and</strong> re-suspended <strong>in</strong> <strong>in</strong>filtration medium (0.5x Murashige &<br />
Skoog salts, 10mM MES, 0.5% fructose, 0.5% sucrose, 150μM acetosyr<strong>in</strong>gone).<br />
Leaf discs were vacuum <strong>in</strong>filtrated with Agrobacterium suspension until fully suspension-logged.<br />
Infiltrated leaf-discs were placed on 4 layers <strong>of</strong> 260x410mm filter<br />
paper wet with 65ml dH 2<br />
O <strong>and</strong> were kept at 21°C for 24 h. Drop-<strong>in</strong>oculation <strong>of</strong> 20<br />
μl <strong>of</strong> 150 spores μl -1 spore suspension was performed the next day. Leaf discs were<br />
from then on kept at 17°C <strong>and</strong> high humidity. Seven days post-<strong>in</strong>oculation lids<br />
were sprayed with dH 2<br />
O. Spores were harvested at 8 dpi by pool<strong>in</strong>g 9 discs <strong>in</strong> 4 ml<br />
H 2<br />
O. Each observation is the average <strong>of</strong> the spore count <strong>in</strong> 5 x 1μl. All statistical<br />
analyses were performed us<strong>in</strong>g SPSS 19 (IBM). The assumption <strong>of</strong> homogeneity<br />
<strong>of</strong> variances was tested by Levene’s test. An <strong>in</strong>dependent samples median test was<br />
performed to determ<strong>in</strong>e whether <strong>in</strong>filtration with different Agrobacterium stra<strong>in</strong>s<br />
had a significant effect on spore counts. Spore counts <strong>of</strong> leaf discs <strong>in</strong>filtrated with<br />
<strong>in</strong>dividual Agrobacterium stra<strong>in</strong>s were then compared to those <strong>of</strong> the discs <strong>in</strong>filtrated<br />
with Agrobacterium-GUS (Mann-Whitney U test). Correction for multiple<br />
test<strong>in</strong>g was performed us<strong>in</strong>g the Bonferroni method.<br />
qPCR <strong>analysis</strong><br />
For time-course experiments one-week old L. sativa cv. Ol<strong>of</strong> seedl<strong>in</strong>gs were<br />
spray-<strong>in</strong>oculated with 150 spores μl -1 until run<strong>of</strong>f was imm<strong>in</strong>ent. Seedl<strong>in</strong>gs were<br />
grown under high humidity (closed tray with transparent lid) at 17°C with 9 h <strong>of</strong><br />
light (100 μE/m 2 /s) <strong>and</strong> kept under these conditions for the duration <strong>of</strong> the experiment.<br />
Samples were taken <strong>and</strong> snap-frozen immediately after spray<strong>in</strong>g <strong>and</strong> every<br />
24 h until 5 days post-<strong>in</strong>oculation.<br />
For the <strong>analysis</strong> <strong>of</strong> expression changes <strong>of</strong> biotic stress-responsive genes<br />
Agrobacterium stra<strong>in</strong>s were prepared as described <strong>and</strong> pressure <strong>in</strong>filtrated with<br />
a needleless syr<strong>in</strong>ge <strong>in</strong>to three-week old lettuce leaves. Lettuce was grown as<br />
described for leaf disc assays <strong>and</strong> kept at 21°C, 16 h <strong>of</strong> light (200 μE/m 2 /s), 70%<br />
RH after <strong>in</strong>filtration. Infiltration sites were excised <strong>and</strong> snap-frozen 65 h post-<strong>in</strong>filtration.<br />
Total RNA was extracted us<strong>in</strong>g the Spectrum Plant Total RNA Kit (Sigma<br />
Life Science) <strong>and</strong> treated with DNAse (Fermentas). cDNA was synthesised us<strong>in</strong>g<br />
RevertAid H m<strong>in</strong>us Reverse Transcriptase (Fermentas) <strong>and</strong> Oligo(dT)15. Cycle<br />
thresholds were determ<strong>in</strong>ed <strong>in</strong> triplicate per transcript us<strong>in</strong>g the ABI PRISM 7900<br />
HT or the Life technologies ViiA7 system us<strong>in</strong>g SYBR Green as reporter dye.
114 Chapter 4<br />
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sequenc<strong>in</strong>g <strong>of</strong> Pseudoperonospora cubensis reveals effector prote<strong>in</strong>s with a putative QXLR<br />
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19 Kale SD, Gu B, Capelluto DGS, Dou D, Feldman E, Rumore A, Arredondo FD, Hanlon R,<br />
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<strong>in</strong>to plant <strong>and</strong> animal host cells. Cell 142, 284-95.<br />
20 Oh S-K, Young C, Lee M, Oliva R, Bozkurt TO, Cano LM, W<strong>in</strong> J, Bos JIB, Liu H-Y, Van<br />
Damme M, Morgan W, Choi D, Van der Vossen EAG, Vleeshouwers VGAA & Kamoun S (2009)<br />
In planta expression screens <strong>of</strong> Phytophthora <strong>in</strong>festans RXLR <strong>effectors</strong> reveal diverse phenotypes,<br />
<strong>in</strong>clud<strong>in</strong>g activation <strong>of</strong> the Solanum bulbocastanum disease resistance prote<strong>in</strong> Rpi-blb2. The Plant<br />
Cell 21, 2928-47.<br />
21 Wang Q, Han C, Ferreira AO, Yu X, Ye W, Tripathy S, Kale SD, Gu B, Sheng Y, Sui Y,<br />
Wang X, Zhang Z, Cheng B, Dong S, Shan W, Zheng X, Dou D, Tyler BM & Wang Y (2011)<br />
Transcriptional programm<strong>in</strong>g <strong>and</strong> <strong>functional</strong> <strong>in</strong>teractions with<strong>in</strong> the Phytophthora sojae RXLR<br />
effector repertoire. The Plant Cell 23, 2064-86.<br />
22 Sohn KH, Lei R, Nemri A & Jones JDG (2007) The <strong>downy</strong> <strong>mildew</strong> effector prote<strong>in</strong>s ATR1 <strong>and</strong><br />
ATR13 promote disease susceptibility <strong>in</strong> Arabidopsis thaliana. The Plant Cell 19, 4077-90.<br />
23 Fabro G, Ste<strong>in</strong>brenner J, Coates ME, Ishaque N, Baxter L, Studholme DJ, Körner E, Allen RL,<br />
Piquerez SJM, Rougon-Cardoso A, Greenshields D, Lei R, Badel JL, Caillaud M-C, Sohn KH,<br />
Van den Ackerveken G, Parker JE, Beynon J & Jones JDG (2011) Multiple c<strong>and</strong>idate <strong>effectors</strong><br />
from the oomycete pathogen Hyaloperonospora arabidopsidis suppress host plant immunity. PLoS<br />
pathogens 7, e1002348.
116 Chapter 4<br />
24 Dong S, Y<strong>in</strong> W, Kong G, Yang X, Qutob D, Chen Q, Kale SD, Sui Y, Zhang Z, Dou D, Zheng X,<br />
Gijzen M, M Tyler B & Wang Y (2011) Phytophthora sojae avirulence effector Avr3b is a<br />
secreted NADH <strong>and</strong> ADP-ribose pyrophosphorylase that modulates plant immunity. PLoS<br />
pathogens 7, e1002353.<br />
25 Bos JIB, Armstrong MR, Gilroy EM, Boev<strong>in</strong>k PC, He<strong>in</strong> I, Taylor RM, Zhendong T, Engelhardt S,<br />
Vetukuri RR, Harrower B, Dixelius C, Bryan G, Sadan<strong>and</strong>om A, Whisson SC, Kamoun S<br />
& Birch PRJ (2010) Phytophthora <strong>in</strong>festans effector AVR3a is essential for virulence <strong>and</strong><br />
manipulates plant immunity by stabiliz<strong>in</strong>g host E3 ligase CMPG1. Proceed<strong>in</strong>gs <strong>of</strong> the National<br />
Academy <strong>of</strong> Sciences <strong>of</strong> the United States <strong>of</strong> America 107, 9909-14.<br />
26 Bozkurt TO, Schornack S, W<strong>in</strong> J, Sh<strong>in</strong>do T, Ilyas M, Oliva R, Cano LM, Jones AME, Huitema E,<br />
Van der Hoorn RAL & Kamoun S (2011) Phytophthora <strong>in</strong>festans effector AVRblb2 prevents<br />
secretion <strong>of</strong> a plant immune protease at the haustorial <strong>in</strong>terface. Proceed<strong>in</strong>gs <strong>of</strong> the National<br />
Academy <strong>of</strong> Sciences <strong>of</strong> the United States <strong>of</strong> America 108, 20832-7.<br />
27 Mukhtar MS, Carvunis A-R, Dreze M, Epple P, Ste<strong>in</strong>brenner J, Moore J, Tasan M, Galli M,<br />
Hao T, Nishimura MT, Pevzner SJ, Donovan SE, Ghamsari L, Santhanam B, Romero V,<br />
Poul<strong>in</strong> MM, Gebreab F, Gutierrez BJ, Tam S, Monachello D, Boxem M, Harbort CJ,<br />
McDonald N, Gai L, Chen H, He Y, V<strong>and</strong>enhaute J, Roth FP, Hill DE, Ecker JR, Vidal M,<br />
Beynon J, Braun P & Dangl JL (2011) Independently evolved virulence <strong>effectors</strong> converge onto<br />
hubs <strong>in</strong> a plant immune system network. Science 333, 596-601.<br />
28 Zeilmaker T (2012) Functional <strong>and</strong> applied aspects <strong>of</strong> the DOWNY MILDEW RESISTANT 1 <strong>and</strong><br />
6 genes <strong>in</strong> Arabidopsis. Thesis, Utrecht University, 1-147.<br />
29 Jeuken MJW, Zhang NW, McHale LK, Pelgrom K, den Boer E, L<strong>in</strong>dhout P, Michelmore RW,<br />
Visser RGF & Niks RE (2009) R<strong>in</strong>4 causes hybrid necrosis <strong>and</strong> race-specific resistance <strong>in</strong> an<br />
<strong>in</strong>terspecific lettuce hybrid. The Plant Cell 21, 3368-78.<br />
30 Zimmermann P, Hirsch-H<strong>of</strong>fmann M, Hennig L & Gruissem W (2004) GENEVESTIGATOR.<br />
Arabidopsis microarray database <strong>and</strong> <strong>analysis</strong> toolbox. Plant Physiology 136, 2621-32.<br />
31 Dou D, Kale SD, Wang X, Chen Y, Wang Q, Wang X, Jiang RHY, Arredondo FD, Anderson RG,<br />
Thakur PB, McDowell JM, Wang Y & Tyler BM (2008) Conserved C-term<strong>in</strong>al motifs required for<br />
avirulence <strong>and</strong> suppression <strong>of</strong> cell death by Phytophthora sojae effector Avr1b. The Plant Cell 20,<br />
1118-33.<br />
32 Djamei A, Schipper K, Rabe F, Ghosh A, V<strong>in</strong>con V, Kahnt J, Osorio S, Tohge T, Fernie AR,<br />
Feussner I, Feussner K, Me<strong>in</strong>icke P, Stierh<strong>of</strong> Y-D, Schwarz H, Macek B, Mann M & Kahmann R<br />
(2011) Metabolic prim<strong>in</strong>g by a secreted fungal effector. Nature 478, 395-8.<br />
33 Cabral A, Oome S, S<strong>and</strong>er N, Küfner I, Nürnberger T & Van den Ackerveken G (2012) Nontoxic<br />
Nep1-like prote<strong>in</strong>s <strong>of</strong> the <strong>downy</strong> <strong>mildew</strong> pathogen Hyaloperonospora arabidopsidis: repression <strong>of</strong><br />
necrosis-<strong>in</strong>duc<strong>in</strong>g activity by a surface-exposed region. Molecular Plant-Microbe Interactions 25,<br />
697-708.
Bremia <strong>effectors</strong> enhance host susceptibility<br />
117<br />
Supplemental Information<br />
Supplemental Table 1 (cont<strong>in</strong>ued next page): Overview <strong>of</strong> primers used <strong>in</strong> this study. Types are<br />
Flank<strong>in</strong>g, Clon<strong>in</strong>g, qPCR, 3’ RACE, <strong>and</strong> Clon<strong>in</strong>g Effector Doma<strong>in</strong>. Reverse primers start<strong>in</strong>g with TCM<br />
yield products that <strong>in</strong>clude a stop codon (TGA) or a codon for Glyc<strong>in</strong>e (GGA) <strong>in</strong>stead. Products without<br />
stop codon can be used de generate constructs with a C-term<strong>in</strong>al tag (not used <strong>in</strong> this study).<br />
Effector Type Forward Reverse<br />
BLG01 F AAACGCGATAAGGTCTCAAAA TGACTTCGGTCCTCATTAAAC<br />
BLG01 C CACCATGACTTTACAGTTGACGTCGGTA TCAATTTCGTTTGGCGTTGGG<br />
BLG02 F AATTTCTATTGCGAGCACAGC GTTCGCATGACTCACACCTC<br />
BLG02 C CACCATGACTTCACCACTGACGCTGG TCAAGTAGGTCGCAATTTGTTC<br />
BLG02 q GACGAAGCAAGCGTGTACTG TCGGTTACGTCGAACGTTTT<br />
BLG03 F AAGATGTCGCAACGCAGAG TCCACTTGGCAAAGAGCAC<br />
BLG03 C CACCATGTCATCGGTAACTACTGAAGAAGGTG TCACATCTCAACGTCGCTAGGTG<br />
BLQ01 3 CTCCAACACTTTCCGCTCTC -<br />
BLQ01 C CACCATGTTTCCAATGGAGAAGATTTCGT TCAAAATASTTTTGGCAACCAAC<br />
BLR01 F AGTGTGACTGATGAGGTTGCT TGTCTTTCACATCGTTGTCTTC<br />
BLR01 C CACCATGCGAAACAACAGCCTGGTGAC TCMAAGAATTGCTTGAGCTTCTT<br />
BLR02 F AGCACATGCGTTTAAATTTTCG ACACGACCATCACTCCTCCATT<br />
BLR02 C CACCATGCAGCTGGACACTCACGAAA TCMCTCCTCCATTGAGCAAAC<br />
BLR03 F GCTTTGACCTCGAGCAATAAAATAGC TTAGAGGCGCCCAAATGCTATGAAGC<br />
BLR03 C CACCATGGCGGATGCAGCTAAAGTCAA TCMGTCTGGTCCTCTAATTATAAA<br />
BLR03 E CACCATGTTAAGCATTTGGACCTCTTTTAAAG TCMGTCTGGTCCTCTAATTATAAA<br />
BLR03 q TCAAAATGCTTACCCAGTCG TCTTCCTCTGCTTCGTCAATG<br />
BLR04 F ACCATGGGATCGCTGCTCTTCAATCT GCAACTGTAGCCCGTTACAATACGCA<br />
BLR04 C CACCATGCGCTGGATCGCTGAAC TCMGCAGCGAGAAAAAACATGC<br />
BLR05 F TGGTTGATGGCGCTAACATTTGCCA TTGCGGTGATCCCTAAGACAGTTGA<br />
BLR05 C CACCATGTATTACGAAATAGACGATGATTC TCMCAGGCAAAACGGAAGACAG<br />
BLR05 q ATCTCTTGGCGTTGGTTGTC ACGGAAGGACGTAAGGCTCT<br />
BLR06 F TGACGGTAGTTGCTAATCAAGGGGCA ACGTGAAAATGCAGGTCTACCAGTTCC<br />
BLR06 C CACCATGGCTGAGAACGGATCAT TCMGGTTTGCCCAACATCAGA<br />
BLR07 F CAACAACTTTTGCAATGGCGTA AACCACGTTGCAGCCATCTT<br />
BLR07 C CACCATGACTATAGAAGGGTCCAAGCG TCAATTCGCTTTTTTCCCAGG<br />
BLR08 F TGTCGATTGGTCAAATCTCTAAAA AGGCATGAAAGCAATTATTATTTAGG<br />
BLR08 C CACCATGAACGCCGACCTCACTCC TCMGCTCGAGCGATACTTGA<br />
BLR09 F AGTTTGCCGTCCAGCTATTATCCCGA TCGCATTGGTCCATGTCGCCAGTTTA<br />
BLR09 C CACCATGACCAAGAACGTCACCCAAGT TCMAGTGATGCCGTAGCAAG<br />
BLR09 q GTGAATTGGAAGAGCGAGGA ATGCCGTAGCAAGCAGAGAT<br />
BLR10 F TAAATTTGTCGCATCGGACATT CCACGAGCAAATATGATTAGCAA<br />
BLR10 C CACCATGAAGAACGTCACCCAAGTG TCMAGTGATGCCGTAGCAAGC<br />
BLR11 F GCAGCGAAAACCTAAAACAATCC TTCTAATCTTTACCGAGTCTTTATTCACT<br />
BLR11 C CACCATGGCCTTCCAACAAGTGCCAGC TCMATACTTGAGACCAGAGCTA<br />
BLR12 F AATGGAAGGGAGGCGATTCGGTACAA ATGCACTGGAGCAGTCATTGTGAAGC<br />
BLR12 C CCACATGGCTGTATTGAGCTCAACGC TCMCCGATTACAGACGTAGAA<br />
BLR13 F CTCAGCTGTCTCTGTCGTTTTAGCA TGCATGTTATCAAAAGCGCCCCAGT<br />
BLR13 C CACCATGGCACTGGTGGCTAGGGAG TCAAGATCCAGTTTTCTGCTTT<br />
BLR14 F CCAACTTTCATCATTCCACCA GCTACCGGCTCCCTATCTTT<br />
BLR14 C CACCATGGCGATGAATAGCGCTGCTC TCMACTGAAAACCTTGGACAATA<br />
BLR15 F TCCACAAATGACTAGTCGCAAGCGT TTTATTGATCTCACGCAGCCAGCGA<br />
BLR15 C CACCATGAAATTTAGCTTGGGCACTGCA TCAAGGATCTGGAGGAAAGAA<br />
BLR16 F TTCGTGGATTAACGAAGCTTTT AAGCACCATTCACAGCTATCACA
118 Chapter 4<br />
Effector Type Forward Reverse<br />
BLR16 C CACCATGGAATCCAGCTTGGTCCGTGC TCMGATAGGAAGCGATCTGTAT<br />
BLR16 E CACCATGATCAGTTTGAAAGATGTCGTGG TCMGATAGGAAGCGATCTGTAT<br />
BLR16 q ATGGAGAGCCTCGAAGTCAA CTTTCCCACGACATCTTTCAA<br />
BLR17 F ATCGTAATTTTCCCGCCAACCACCA CACGCCGTAGATGAAACATACAGCA<br />
BLR17 C CACCATGCACAGTRTGTCCATATTTACGC TCMCGATTTTAGTAAGGCCTGA<br />
BLR18 F TCCATAGACCACAAGAGCCTTT CGGGTTATGGTGTGAGAGCTT<br />
BLR18 C CACCATGATTGAAACTCTAGAGGGTC TCMTTCGTTGAGAGCTTTCGC<br />
BLR19 F AGCGACTTACCTGCTGACTTTG AAGCTGCGACATGCTTCTTTC<br />
BLR19 C CACCATGCAAGACTCGGAAGCAGATC TCMCCTGCTGACTTTGTCCC<br />
BLR20 F ACACCGCATGTGAACGACCAAAACT TGCCAAATCAAACTTGTCCTTAGCCCG<br />
BLR20 C CACCATGAGTTGGGAGACCATTTCGGT TCMATGATGATTATTGTAGTATCGT<br />
BLR21 F TATTCCTTGATTGCCGACTTCT CTGGAAGGTGGGGCAGAT<br />
BLR21 C CACCATGTCCAACCCTGAAGACGACG TCMTTGCCTAGGGTCATTATACAG<br />
BLR22 F GGCAAACAACAGTCAAAAGCAG CGAACAACAACACTCACGATGG<br />
BLR22 C CACCATGGAAGTAGAATCCAGCATCGC TCMAACGGAAAGCAAATCAACTC<br />
BLR22 q CGTCGCTAAACTTGAACTTGC TGACTCCCCGTTCTGATGTT<br />
BLR23 F CTCCACATCATTGGCAAAAGC GAGCCCTTTGCCTGATTTCAA<br />
BLR23 C CACCATGGATTTCCAAGCGGCTTCC TCMATTAATATGTTTGGCTGGATA<br />
BLR23 q GGAAGTGATGCGAAAACGAT TGGCTGGATAAAATGGATTG<br />
BLR24 F TGCAGCAATGAAGAATTGACGGCAC ACGCCTACCACCACGTCACTAACATT<br />
BLR24 C CACCATGAAACATGGTAAAGCTGATAAGGA TCMTGTYAACTTAAAGTGAAGCA<br />
BLR25 F TTTTATTTCTACCAAAGTATCAATTCG CGGAGAGAGTCTGCCTATGG<br />
BLR25 C CACCATGAGTCCTAACACTATGACTCTTA TCAGAATGCCAACTTCCAGC<br />
BLR26 F TTGTTGTACGCCTTGGCTGTCGATT ATTGGGTAGCGTTGCACAAAAGCCA<br />
BLR26 C CACCATGGCAACAACAGCACCAAACGC TCMAAGGCTCAGCATGCATCGA<br />
BLR27 F AAAATACCTTTTGTCTCATCTCATC TCGCATTGGTCCATGTCGCCAGTTTA<br />
BLR27 C CACCATGATCGAGAACGTCGCGCTGA TCAAGCTATTTTGACCCCC<br />
BLR27 q AGCACTCGTGACCAGACTTG ATAGCAAGGCCAACCACTTG<br />
BLR28 3 ACCAGCTTCAAGACACCAAGAT -<br />
BLR28 C CACCATGCATACTGTGGCGCTATCCAC TCATATTCCGAACTTTCGACAGAC<br />
BLR29 3 CATCTCATAACTCCAACACTTT -<br />
BLR29 C CACCATGTACACGGGTGCCGCATCCT TCAAGGCGGGGGGGATACG<br />
BLR30 3 TTCACTTCTTCGTTCGCATC -<br />
BLR30 C CACCATGGAGTCCAGCTTGGGCACTG TCAACGTTCTGGTAAGTGCTC<br />
Gene Type Forward Reverse<br />
337 q TCGCATTGGATACAGGCTTA TGGGTAGGGGACACTGATTG<br />
744 q CGGATCTATGGAATTTGCTTTC ACAATGGTGTGGAGGGTTCT<br />
3859 q ATGGACCCGACCCGTCTATT CCAGTATCAAGAGCCACACG<br />
4174 q AATTACATGGATGCTAAGAGAACC CAGGTGGAGGAACTACATCTTTT<br />
8313 q CCGATGTTTTTGGATATGGTG AGCAACATGACATCATCATCATT<br />
6346 q CAGAGGGACAACAGGGACAC CTCCGACTCTTCACCAGGTT<br />
7164 q GGCATAATCACTCAGGTACGG TAATGGAGCGGGATTTTGAA<br />
15736 q TGATGCGGATGAAAATTGG ATGACCAAGCCCAAGAAGGT<br />
15964 q AAAGCAGGGTGAGGATCTGA AAGCGAAATAAATGCACCAAA<br />
33411 q CGTCACCAAAGCTCATTGAC TTGCACTTGAATTCCGAAGA<br />
37274 q TTCAAATCTCCAAAGGAGGTG CAGCGAGCTCTTCAATGAAAT<br />
Lettuce<br />
Act<strong>in</strong><br />
q CTATCCAGGCTGTGCTTTCC ACCCTTCGTAGATCGGGACT<br />
Bremia<br />
Act<strong>in</strong><br />
q AGGCCGTGCTGTCTCTCTAC GCGCATAGCCTTCGTAGATT
Bremia <strong>effectors</strong> enhance host susceptibility<br />
119<br />
Supplemental Information 1: Am<strong>in</strong>o acid <strong>and</strong> nucleotide sequences <strong>of</strong> cloned <strong>effectors</strong>.<br />
Indicated <strong>in</strong> bold <strong>and</strong> underl<strong>in</strong>ed is the first codon after the predicted signal peptide cleavage site. All<br />
<strong>effectors</strong> were cloned start<strong>in</strong>g from this codon <strong>and</strong> preceded by a newly <strong>in</strong>troduced start codon.<br />
BLR01<br />
1 M R L L H A A L K R L A D L L A Q I Y L<br />
1 ATGAGGTTGCTGCATGCAGCGCTGAAAAGGCTTGCGGACTTGCTAGCGCAGATATACTTA<br />
21 A T L N V A R S R N N S L V T R Y P I E<br />
61 GCTACATTAAACGTAGCAAGATCTCGAAACAACAGCCTGGTGACGAGGTATCCGATCGAA<br />
41 L R K L R R H S C E R E Q K M R N V G G<br />
121 TTGCGCAAATTACGGAGGCATTCCTGCGAGCGCGAACAAAAAATGAGAAATGTAGGAGGA<br />
61 S N A A S P Q A S E T N L Q E A Q A I L<br />
181 AGCAATGCAGCATCACCGCAAGCAAGTGAAACAAATTTACAAGAAGCTCAAGCAATTCTT<br />
81 -<br />
241 TGA<br />
BLR02<br />
1 M R L N F R S F S D M M V V L L V F V S<br />
1 ATGCGTTTAAATTTTCGTTCTTTTTCCGACATGATGGTTGTTCTGCTGGTCTTCGTATCG<br />
21 G I V L H K A L A Q L D T H E S S A R V<br />
61 GGGATTGTGCTTCACAAAGCTCTCGCCCAGCTGGACACTCACGAAAGTAGCGCAAGAGTC<br />
41 N V W E R S F S A F R D T P A S I V E S<br />
121 AACGTATGGGAAAGATCCTTTTCGGCTTTCAGGGACACTCCTGCTTCGATCGTCGAAAGC<br />
61 M S F E V L Q Y N L F G R P Y E V S K D<br />
181 ATGAGCTTTGAGGTGCTTCAGTACAATTTATTCGGCCGGCCGTATGAAGTGTCCAAGGAC<br />
81 G Q R E R L L R V P E S L H R I S E T I<br />
241 GGACAGAGAGAGCGGCTGCTGCGAGTACCGGAATCACTGCATCGGATCTCCGAAACAATT<br />
101 D I V T F A E A D I Q T Q R D E M L T N<br />
301 GACATTGTGACGTTTGCTGAGGCAGATATTCAAACTCAACGCGATGAAATGCTGACGAAT<br />
121 F N N L D F I F G R R F C M T Q T R L R<br />
361 TTCAACAATTTGGATTTCATTTTTGGACGACGATTTTGCATGACCCAGACCCGTTTACGA<br />
141 V C S M E E -<br />
421 GTTTGCTCAATGGAGGAGTGA<br />
BLR03<br />
1 M P R C L A V L S F A L F T C C I D A S<br />
1 ATGCCCAGGTGCCTTGCTGTGCTCAGTTTTGCATTATTTACTTGCTGTATCGACGCTTCG<br />
21 T A A D A A K V K M L T Q S R Y P G T V<br />
61 ACAGCTGCGGATGCAGCTAAAGTCAAAATGCTTACCCAGTCGCGCTACCCCGGAACTGTT<br />
41 D R I E K V A R F L R S H S I D E A E E<br />
121 GACAGAATTGAGAAAGTCGCTAGATTCTTGCGTTCTCATAGCATTGACGAAGCAGAGGAA<br />
61 E R L S I W T S F K E L L F G G P F S P<br />
181 GAGAGATTAAGCATTTGGACCTCTTTTAAAGAGCTACTCTTTGGCGGTCCCTTTTCGCCG<br />
81 T R L R A M T A D N A V V Q F T F S A W<br />
241 ACTCGCTTGCGGGCCATGACGGCAGATAACGCAGTCGTTCAATTTACGTTCTCTGCGTGG<br />
101 N K F S H V D I R K L L A E G M K D M D<br />
301 AATAAATTTTCTCACGTCGACATTCGCAAGTTGCTTGCCGAAGGCATGAAGGACATGGAT<br />
121 K Q E I E K L N S I I E L Y F I I R G P<br />
361 AAACAAGAGATAGAGAAGTTGAACAGCATCATCGAGCTTTATTTTATAATTAGAGGACCA<br />
141 D -<br />
421 GACTAA
120 Chapter 4<br />
BLR04<br />
1 M A A L V A L L L A L Y V V G V L L P M<br />
1 ATGGCGGCACTGGTGGCACTATTACTAGCGCTGTACGTCGTTGGTGTTCTATTGCCTATG<br />
21 R W I A E Q E A G K A S A N R K Q P E T<br />
61 CGCTGGATCGCTGAACAGGAGGCAGGTAAAGCTTCCGCAAATAGAAAGCAGCCAGAAACG<br />
41 T S F R R E L R R R G S F S M S F V T D<br />
121 ACGTCATTCCGTAGAGAGCTGCGGCGTCGGGGTAGCTTCAGCATGAGCTTTGTGACCGAT<br />
61 I K A H V F S R C -<br />
181 ATTAAGGCGCATGTTTTTTCTCGCTGCTAA<br />
BLR05<br />
1 M G P Q H L L A L V V V S I L V A A G N<br />
1 ATGGGTCCTCAACATCTCTTGGCGTTGGTTGTCGTGTCCATACTAGTAGCTGCTGGAAAT<br />
21 A Y Y E I D D D S V T R A L R P S V I A<br />
61 GCGTATTACGAAATAGACGATGATTCTGTGACGAGAGCCTTACGTCCTTCCGTCATAGCC<br />
41 D Q E H A V H A I P A T N F I S K D E D<br />
121 GACCAAGAGCACGCAGTTCATGCTATTCCAGCCACAAATTTTATCTCAAAGGACGAAGAT<br />
61 H S K N E K K E I E I I R I A I F S L L<br />
181 CACAGCAAGAACGAGAAAAAGGAAATTGAAATTATAAGAATTGCAATATTTTCACTTCTC<br />
81 V V G V F A I M A L R C L P F C L -<br />
241 GTAGTGGGAGTTTTTGCAATTATGGCGCTTCGCTGTCTTCCGTTTTGCCTGTAG<br />
BLR06<br />
1 M H R X N A S T I A A L L T L L I C L T<br />
1 ATGCACCGTCRAAACGCTTCCACCATCGCTGCGTTATTGACATTGCTCATCTGCCTTACC<br />
21 S V T A A E N G S C S S H E G C C L L C<br />
61 TCCGTCACAGCGGCTGAGAACGGATCATGCTCGTCACACGAGGGGTGCTGCTTGCTCTGC<br />
41 G D E L E R C L R G P C S M H W I T R S<br />
121 GGCGATGAGCTAGAGCGCTGTTTACGCGGTCCATGCAGCATGCATTGGATTACACGCTCT<br />
61 F F S I S A P V I D V G F W D V A F S F<br />
181 TTCTTCAGCATTAGCGCGCCCGTTATTGACGTGGGCTTCTGGGATGTCGCTTTTTCCTTT<br />
81 Y G T V P Y L V P V A I L L D F I F Y C<br />
241 TATGGCACAGTGCCATACCTCGTGCCAGTAGCGATACTGCTCGACTTTATATTTTACTGC<br />
101 R S W T R L F A F L F I P I V G L L N S<br />
301 CGTAGCTGGACGCGGCTTTTTGCATTTCTCTTCATTCCGATCGTAGGTTTATTAAATTCC<br />
121 G I L V T I L G D C S D C P R P C G S C<br />
361 GGCATTCTAGTAACGATTCTCGGTGACTGCAGCGATTGTCCTCGTCCGTGTGGTAGCTGC<br />
141 V S S N G M P S G H A T T A I G L F M W<br />
421 GTTTCCAGCAACGGAATGCCGTCGGGGCATGCCACTACTGCTATTGGATTATTCATGTGG<br />
161 I L L E S L L G V G Y Q W K V V K K V A<br />
481 ATACTACTTGAGTCGCTGCTGGGCGTTGGTTATCAGTGGAAGGTGGTAAAAAAGGTTGCT<br />
181 V C V G I T L L F V P V P Y S R I Y L G<br />
541 GTTTGTGTGGGCATCACCCTGCTTTTTGTACCTGTGCCATACAGCCGCATTTATCTGGGC<br />
201 D H T R L Q V V I G C I N G V L F G L F<br />
601 GATCACACAAGATTGCAAGTCGTCATTGGATGTATAAATGGCGTCTTGTTTGGCCTTTTC<br />
221 Y F F V L R Y G L G R R L S V A T K R I<br />
661 TATTTCTTTGTGCTTCGTTACGGATTAGGAAGACGGCTGTCCGTGGCCACGAAGCGCATT<br />
241 N E G R F H F L H M V N D F H V D R R Y<br />
721 AATGAAGGGCGATTTCATTTTCTGCACATGGTTAACGACTTTCACGTGGATCGTAGATAC<br />
261 L L D S Q F N V E D Q Q F V R S D V G Q<br />
781 TTGCTTGATTCACAATTTAATGTTGAAGACCAGCAGTTCGTCCGATCTGATGTTGGGCAA<br />
281 T -<br />
841 ACCTAA
Bremia <strong>effectors</strong> enhance host susceptibility<br />
121<br />
BLR07<br />
1 M H L N H V A A I F L V G V V T C F T E<br />
1 ATGCATCTCAACCACGTTGCAGCCATCTTCCTTGTTGGAGTCGTGACGTGCTTCACGGAG<br />
21 T I E G S K R R S E I I R E H L A Y P V<br />
61 ACTATAGAAGGGTCCAAGCGTCGAAGTGAAATCATTCGTGAACATCTTGCATATCCTGTA<br />
41 D V A I G S R A L R H T V A S E V K D K<br />
121 GATGTGGCCATCGGTTCACGAGCTCTACGGCATACGGTAGCCTCTGAGGTAAAAGACAAG<br />
61 G D T G P D L E E E R A W W E N A V Q E<br />
181 GGGGACACTGGACCAGATCTAGAGGAAGAGCGAGCTTGGTGGGAGAATGCTGTACAGGAG<br />
81 L R W R L K G P G D F F K S L D A V R K<br />
241 CTGCGGTGGCGACTAAAAGGGCCTGGCGACTTCTTTAAAAGCTTAGATGCCGTACGCAAA<br />
101 G K D L H T D R S F Q W W L H R A A Q F<br />
301 GGGAAAGATTTGCATACTGATCGATCTTTTCAATGGTGGCTGCATCGCGCAGCCCAGTTC<br />
121 R N A H N G F D T S F L K V L Q Q E T S<br />
361 AGGAATGCCCATAACGGGTTTGATACTTCTTTTTTAAAGGTCCTTCAGCAAGAAACATCG<br />
141 Y E Q Q A K L F V W L Q Y S N F Y R D M<br />
421 TACGAGCAACAGGCAAAGCTTTTTGTATGGCTTCAGTATTCCAATTTCTATCGTGATATG<br />
161 N A F A K D Q L L I M E A N P A S R H A<br />
481 AATGCATTTGCGAAAGATCAGCTACTGATTATGGAAGCCAACCCTGCTTCTCGTCACGCC<br />
181 V Y E A W L Q D G K T P G D I C D S S R<br />
541 GTGTATGAAGCATGGCTACAGGATGGAAAAACACCTGGAGATATCTGCGATTCCAGTCGA<br />
201 I G L N F E S W L G Y V V Y Y R S K G N<br />
601 ATCGGCCTTAACTTTGAGAGTTGGCTTGGTTACGTTGTTTATTACCGATCCAAAGGAAAT<br />
221 Q F T N K D I L K T L T S Q D L I K E A<br />
661 CAATTTACCAATAAAGACATTCTCAAGACGCTAACGAGTCAAGATTTGATTAAAGAGGCT<br />
241 V K K L I L L P G K K A N -<br />
721 GTGAAGAAATTGATTTTACTACCTGGGAAAAAAGCGAATTGA<br />
BLR08<br />
1 M A E F R F R P T T L L F L I V A L I F<br />
1 ATGGCCGAGTTTCGGTTCCGCCCCACGACATTGCTATTCCTGATTGTTGCTTTGATCTTC<br />
21 R C A I D P V S A N A D L T P S P R L L<br />
61 AGGTGCGCCATCGACCCTGTCTCCGCCAACGCCGACCTCACTCCCAGCCCGCGATTACTG<br />
41 R D L V Q F D A T Q T V A H T L S N S P<br />
121 CGCGACCTAGTGCAATTCGACGCGACACAGACTGTTGCCCACACCCTATCCAACTCTCCG<br />
61 S A S S S S A E A T V H P A A S A E A S<br />
181 AGCGCCAGCAGCTCCAGTGCAGAAGCGACNGTTCACCCCGCTGCCTCTGCTGAAGCATCG<br />
81 A I S N H T T D N A A S A E S S A E S K<br />
241 GCGATATCGAATCACACTACCGACAATGCCGCCTCGGCTGAGTCCTCGGCTGAATCGAAG<br />
101 H E L P S I M S F I G P A A A G V L A I<br />
301 CACGAATTACCGTCCATCATGTCGTTCATCGGCCCAGCTGCTGCTGGCGTGCTAGCCATC<br />
121 V L I G A V I A F K Y R S S K -<br />
361 GTGCTTATTGGGGCCGTCATCGCTTTCAAGTATCGCTCGAGCAAGTAA<br />
BLR09<br />
1 M R H K C L L A M A V V A S M A F Y S V<br />
1 ATGCGTCACAAGTGCCTCCTAGCTATGGCTGTGGTCGCCAGTATGGCTTTCTACAGCGTC<br />
21 I S T K N V T Q V D T V Q Q E N R R L R<br />
61 ATCAGTACCAAGAACGTCACCCAAGTGGATACGGTGCAACAAGAAAACCGCCGTTTGAGA<br />
41 P R V E P T A N E L D K Q S D V D T K L<br />
121 CCCCGCGTAGAGCCGACAGCTAATGAGTTGGATAAGCAGAGTGATGTCGATACGAAGCTA<br />
61 E A D R R L G Y P G E S G F M L E G E L<br />
181 GAAGCAGATCGACGTTTGGGATACCCTGGAGAGTCGGGTTTCATGCTGGAAGGTGAATTG
122 Chapter 4<br />
81 E E R G G F P W R T F F L G L F A S V I<br />
241 GAAGAGCGAGGAGGATTTCCTTGGAGAACATTTTTTCTTGGTTTATTCGCNTCTGTAATT<br />
101 G V S I I S A C Y G I T -<br />
301 GGTGTCAGCATCATCTCTGCTTGCTACGGCATCACTTAA<br />
BLR10<br />
1 M R H K C L L A M A V V A S M A F Y S V<br />
1 ATGCGTCACAAGTGCCTCCTAGCTATGGCTGTGGTCGCCAGTATGGCTTTCTACAGCGTC<br />
21 I S T K N V T Q V D T V Q Q E N R R L R<br />
61 ATCAGTACCAAGAACGTCACCCAAGTGGATACGGTGCAACAAGAAAACCGCCGTTTGAGA<br />
41 P R V E P T A N E L D K Q S D V D T K L<br />
121 CCCCGCGTAGAGCCGACAGCTAATGAGTTGGATAAGCAGAGTGATGTCGATACGAAGCTA<br />
61 E A D R R L G Y P G E S G F M L E G E L<br />
181 GAAGCAGATCGACGTTTGGGATACCCTGGAGAGTCGGGTTTCATGCTGGAAGGTGAATTG<br />
81 E E R G G F P W R T F F L G L F A S V I<br />
241 GAAGAGCGAGGAGGATTTCCTTGGAGAACATTTTTTCTTGGTTTATTCGCTTCTGTAATT<br />
101 G V S I I S A C Y G I T -<br />
301 GGTGTCAGCATCATCTCTGCTTGCTACGGCATCACTTAA<br />
BLR11<br />
1 M V R L Y L A V L A T F L A Q G N S P L<br />
1 ATGGTTCGATTGTATCTCGCAGTGCTGGCAACTTTCCTCGCTCAAGGCAACAGCCCACTA<br />
21 T L A A F Q Q V P A D A E L L A P D Q E<br />
61 ACTTTAGCTGCCTTCCAACAAGTGCCAGCTGATGCCGAATTGTTAGCTCCTGACCAGGAA<br />
41 G G S L S R R L R V Y D E T S A D E S E<br />
121 GGTGGTTCTTTATCGAGAAGATTACGAGTGTACGATGAAACAAGTGCTGACGAAAGTGAG<br />
61 R G G V S S L L A K L N A S S K P K L E<br />
181 CGAGGAGGAGTCAGCAGTTTGCTAGCAAAACTAAACGCATCTAGTAAGCCAAAACTGGAA<br />
81 D I L A S A K L K V G L D H I L K F Y L<br />
241 GACATATTGGCGTCTGCAAAGTTAAAGGTAGGTCTCGATCATATACTAAAATTTTATCTC<br />
101 F R L E D L E K Q I V K Y N K Y L P K D<br />
301 TTCAGGTTAGAAGATCTGGAGAAACAGATTGTAAAATACAACAAATACTTGCCGAAAGAC<br />
121 K Q L S L Y K L L R E H Q S I P N I A T<br />
361 AAGCAATTATCATTGTATAAGCTACTGAGAGAACATCAAAGCATCCCCAACATAGCAACG<br />
141 A L Y A A K V H I G D E L P P L I A R L<br />
421 GCGCTATACGCCGCTAAAGTGCACATTGGCGACGAGTTGCCACCACTAATTGCTCGTCTA<br />
161 W E D F R N E W K G I D G G I Q K L A N<br />
481 TGGGAGGATTTTCGGAACGAATGGAAAGGAATCGACGGAGGCATACAGAAGCTTGCAAAC<br />
181 E L D M V A D G K E A I L N G K V A V L<br />
541 GAACTTGACATGGTCGCTGATGGTAAAGAAGCCATTCTTAATGGAAAGGTTGCGGTATTG<br />
201 R D Y I A S F S K E N E V D T I L R E Q<br />
601 CGGGACTATATTGCGTCATTCTCGAAAGAGAATGAAGTCGATACCATATTGCGCGAGCAA<br />
221 L T I V F G G E K N L V S I L A K L Y S<br />
661 TTGACGATCGTGTTTGGTGGTGAGAAGAACTTGGTGTCTATTCTTGCGAAACTTTATTCA<br />
241 S A L G N R S A F L E I K P A L P I I E<br />
721 TCGGCACTTGGGAATCGCTCTGCATTCCTTGAAATTAAACCGGCCTTGCCAATTATTGAA<br />
261 S I Q L S V F D K W L K E G L S L N Q V<br />
781 AGCATTCAATTATCTGTCTTTGACAAGTGGCTGAAGGAGGGCCTGTCGCTGAACCAAGTT<br />
281 W S L I V S G R N K G Q D L S I G E Y S<br />
841 TGGTCCCTTATAGTGTCCGGTAGAAACAAGGGCCAAGATCTTTCGATTGGGGAATATTCG<br />
301 V F K A Y A N A F L D R Q M T L K S D K<br />
901 GTGTTTAAAGCGTACGCAAACGCCTTTTTAGATCGTCAAATGACTCTCAAATCAGACAAA
Bremia <strong>effectors</strong> enhance host susceptibility<br />
123<br />
321 F V L Y S V K D I S A A P E A T F L D Y<br />
961 TTTGTGCTTTATAGCGTAAAAGACATCTCGGCGGCACCCGAAGCGACTTTTCTCGATTAT<br />
341 Y D L V L D M S L L D G E R L F F L L D<br />
1021 TACGATTTGGTTTTGGATATGAGCCTTTTGGATGGCGAACGTTTATTTTTTCTATTGGAT<br />
361 R V G A S S F V F E P D F Y R K L Q G F<br />
1081 CGGGTCGGAGCTTCCTCATTTGTATTTGAGCCCGATTTCTATCGAAAACTTCAAGGATTT<br />
381 D K T G D L P G L I T E A I A A K E K T<br />
1141 GATAAAACCGGTGACCTTCCTGGTTTGATTACTGAAGCTATTGCGGCAAAAGAGAAAACG<br />
401 F K L L N T N G E A I K T A K K R G T F<br />
1201 TTCAAGTTACTTAATACAAACGGTGAGGCCATCAAGACTGCAAAAAAACGTGGAACTTTC<br />
421 D V V T P E A K K I N H L Q N K W K Q D<br />
1261 GATGTAGTTACGCCGGAAGCTAAGAAAATCAACCATCTTCAAAATAAGTGGAAGCAAGAC<br />
441 M S F L L K A K H V A S S A K A R S S G<br />
1321 ATGTCGTTTTTACTAAAAGCCAAGCACGTGGCAAGCAGCGCTAAGGCGCGTAGCTCTGGT<br />
461 L K Y -<br />
1381 CTCAAGTATTGA<br />
BLR12<br />
1 M G P G F S F T L L I F T L L T Y Y N E<br />
1 ATGGGTCCTGGCTTCTCCTTCACTCTGCTCATCTTTACTCTACTTACGTACTACAATGAG<br />
21 L S S A A V L S S T H S T S T D I A S S<br />
61 CTTTCAAGTGCGGCTGTATTGAGCTCAACGCATTCAACGTCCACCGATATTGCTAGTAGC<br />
41 R S I V R I A R R Y L R E T A A M E G Q<br />
121 CGCAGCATCGTCCGTATCGCCCGTCGGTACCTTCGGGAGACCGCAGCTATGGAAGGACAG<br />
61 E L E K N L G F D Q T I K N V K K P S L<br />
181 GAGCTGGAAAAGAATCTTGGTTTTGACCAGACTATCAAGAACGTGAAGAAGCCCTCACTT<br />
81 P Q K V Y E N V L E Y L G R M N G K S K<br />
241 CCTCAGAAAGTCTATGAAAACGTACTCGAGTACCTTGGAAGAATGAATGGTAAATCGAAA<br />
101 R D K F F V I A T L L L L P F G I F Y V<br />
301 AGAGATAAGTTTTTCGTCATCGCGACTCTCTTGCTGCTTCCGTTCGGCATCTTCTACGTC<br />
121 C N R -<br />
361 TGTAATCGGTAG<br />
BLR13<br />
1 M K F V F V F G A L F A L F I R L N C A<br />
1 ATGAAGTTTGTTTTTGTGTTTGGAGCGCTTTTTGCTCTCTTCATACGCTTGAATTGTGCC<br />
21 A L V A R E A N S L E L S H D R A N F R<br />
61 GCACTGGTGGCTAGGGAGGCAAATTCACTTGAATTGAGCCACGACCGAGCGAATTTTCGT<br />
41 T I T R R L R I D T L N A S E E R N I F<br />
121 ACAATCACGAGGAGGCTAAGAATTGACACTTTGAACGCTAGTGAAGAGAGAAACATCTTT<br />
61 N F W R R K K H A N W E G L L N V D D V<br />
181 AATTTCTGGCGTCGGAAAAAGCATGCAAACTGGGAGGGTCTTTTGAACGTTGATGATGTG<br />
81 P Q F T P R Q I Q E N I K W S K K D L K<br />
241 CCCCAATTCACTCCTCGTCAAATTCAAGAAAATATCAAGTGGTCGAAGAAGGACTTGAAA<br />
101 E V L T A L N F R K V T S P E V T R Q K<br />
301 GAAGTCTTGACGGCGCTGAACTTTCGCAAAGTGACAAGCCCAGAAGTTACGAGACAGAAG<br />
121 M F Q L D E Y L D S L D L K T E V K T M<br />
361 ATGTTCCAACTCGACGAGTATCTCGATTCGCTGGACCTAAAGACAGAGGTTAAGACGATG<br />
141 D E I V A K R L K V L G M L K G N D W E<br />
421 GACGAAATTGTAGCGAAAAGGTTGAAGGTCTTAGGCATGCTTAAAGGAAATGATTGGGAG<br />
161 H T K E L K R H L A H V W V I K R I P V<br />
481 CACACGAAGGAATTGAAGAGACATTTGGCTCATGTTTGGGTTATTAAAAGGATTCCTGTG
124 Chapter 4<br />
181 R N V Y R G L K M N E E E S L E N L F S<br />
541 AGAAACGTCTACAGGGGCCTAAAAATGAATGAAGAAGAGTCTTTGGAAAACCTTTTCTCG<br />
201 I R S G E E T L D F F L D F F Y A T R K<br />
601 ATTCGAAGCGGCGAAGAGACGCTTGACTTTTTCCTTGATTTCTTTTACGCCACTCGTAAA<br />
221 Y H N D K E E L A E L F L E T Y D V A T<br />
661 TACCACAATGATAAGGAAGAGTTGGCTGAATTGTTTCTCGAGACGTACGATGTAGCAACT<br />
241 V L H L I N V G S R L D S A Q V F V A Q<br />
721 GTCTTGCATTTAATAAATGTCGGATCGCGACTAGACTCAGCACAAGTATTTGTAGCGCAG<br />
261 L E H S L H R K W S S M S H N K V F F D<br />
781 TTGGAGCATAGTCTTCACCGCAAGTGGAGCTCAATGAGCCACAATAAGGTGTTTTTCGAT<br />
281 I L H L D K K G S Q V F N T F E M Q T W<br />
841 ATTCTTCACCTCGACAAAAAGGGATCTCAAGTCTTTAATACGTTTGAGATGCAGACGTGG<br />
301 Y R F I K G T D P D E A E A K T L I F L<br />
901 TACCGATTTATTAAAGGCACCGATCCTGACGAGGCAGAAGCCAAAACATTAATTTTTTTG<br />
321 I H S Y G T A F L S D E L I R G Q G Q S<br />
961 ATCCATAGCTATGGCACCGCTTTTCTCTCGGACGAGTTGATTCGTGGACAAGGACAATCG<br />
341 I S S L R P I F R K A L L D H W A K Q K<br />
1021 ATCTCATCGCTACGTCCGATTTTCCGAAAAGCGTTGCTTGATCATTGGGCAAAGCAGAAA<br />
361 T G S -<br />
1081 ACTGGATCTTAG<br />
BLR14<br />
1 M R L L D V L V A F V I L A A T S S A M<br />
1 ATGCGTCTGCTTGACGTGCTCGTTGCTTTCGTTATCCTCGCTGCTACATCTTCGGCGATG<br />
21 N S A A Q L T P S V D T Q L A D R K H S<br />
61 AATAGCGCTGCTCAGCTTACGCCATCAGTCGACACACAATTGGCCGACAGAAAACATTCT<br />
41 T E D V R R K L R G F N P I T Y L S L K<br />
121 ACCGAGGATGTACGACGAAAATTACGAGGTTTCAATCCGATAACGTATCTAAGTCTCAAA<br />
61 I K E N V A K K V L S K V F S -<br />
181 ATCAAGGAGAACGTTGCCAAGAAGGTATTGTCCAAGGTTTTCAGTTGA<br />
BLR15<br />
1 M Q V C F H V T A L V M I N A L S I S S<br />
1 ATGCAGGTCTGTTTCCACGTTACTGCGTTAGTCATGATCAATGCGCTGTCGATATCAAGT<br />
21 L T S A K F S L G T A A F A L E P S H M<br />
61 CTCACCTCGGCAAAATTTAGCTTGGGCACTGCAGCATTTGCACTTGAACCATCTCACATG<br />
41 E S H E A M R S L R A Q Q T S T L D V D<br />
121 GAGAGCCACGAAGCCATGCGATCGCTGCGGGCCCAACAAACTTCAACTCTCGACGTTGAC<br />
61 E E R L R F K L T R L V G N I C E K I V<br />
181 GAGGAGCGCTTACGCTTCAAACTTACACGTCTTGTGGGAAATATTTGTGAAAAGATTGTT<br />
81 D A F Y R V K N R T K V F Q G E F F P P<br />
241 GATGCTTTCTATAGAGTGAAGAACCGTACTAAGGTTTTCCAGGGTGAGTTCTTTCCTCCA<br />
101 D P -<br />
301 GATCCTTGA<br />
BLR16<br />
1 M Q L P F H V L A L V T T Y A L S T I S<br />
1 ATGCAGCTTCCTTTCCACGTTTTAGCGTTAGTCACGACCTATGCGCTGTCAACGATTAGT<br />
21 Q T S A E S S L V R A T F A H E R Y H M<br />
61 CAAACCTCTGCAGAATCCAGCTTGGTCCGTGCAACATTCGCGCATGAGCGATATCATATG<br />
41 E S L E V K R S L R G Q Q T S P L K D D<br />
121 GAGAGCCTCGAAGTCAAGCGATCGCTAAGAGGCCAGCAGACTTCGCCTCTCAAAGATGAC<br />
61 D E R I S L K D V V G K I K G I I R K I<br />
181 GACGAGCGCATCAGTTTGAAAGATGTCGTGGGAAAGATTAAAGGCATAATCCGAAAAATT
Bremia <strong>effectors</strong> enhance host susceptibility<br />
125<br />
81 M P Q R K G T L R K T K T Y R S L P I -<br />
241 ATGCCTCAAAGAAAAGGCACCTTACGAAAAACCAAAACATACAGATCGCTTCCTATCTAG<br />
BLR17<br />
1 M F S T V L F L V A A C A K S S Y G H S<br />
1 ATGTTTAGCACGGTGCTTTTTCTTGTGGCTGCGTGCGCCAAGTCGTCGTATGGCCACAGT<br />
21 V S I F T R D S T K Y I A S N F D E Y S<br />
61 GTGTCCATATTTACGCGTGACTCTACTAAGTACATTGCGTCAAACTTCGACGAGTATTCG<br />
41 T I P E D I D A K R R L R E A V G V D G<br />
121 ACGATCCCAGAGGATATTGATGCAAAGCGCAGGCTTCGAGAAGCTGTTGGCGTGGATGGG<br />
61 I A R D A E K T F A D I R H S L D R L N<br />
181 ATTGCGCGCGATGCTGAAAAGACTTTTGCAGACATTAGACACTCACTCGATAGACTGAAT<br />
81 K D F V R S N S F K N I N P L I T A E A<br />
241 AAGGACTTTGTGCGTTCAAATTCATTTAAGAACATTAATCCATTAATAACGGCAGAAGCT<br />
101 L V K Q P S F Y Q E A F L P L I T A R G<br />
301 TTGGTCAAACAACCATCTTTTTATCAAGAAGCTTTCTTACCGCTCATAACAGCTCGTGGG<br />
121 I K C S K D F E F A T M A L L G V S P G<br />
361 ATAAAATGTAGCAAAGATTTTGAATTTGCCACAATGGCTTTGCTCGGTGTATCTCCGGGT<br />
141 T L R Q K I Q V A A Q Q P S N I W S Y Q<br />
421 ACTTTGAGACAGAAGATACAGGTAGCAGCACAACAACCCTCCAATATATGGAGTTACCAG<br />
161 T S E Y N E H F V A S Y K K Y L D V V F<br />
481 ACCTCGGAATACAATGAACATTTTGTCGCCAGCTACAAGAAGTATCTCGACGTTGTTTTT<br />
181 M A P T I S E S S A F V K K H I S M S I<br />
541 ATGGCACCGACCATATCAGAATCGTCCGCCTTTGTCAAGAAGCACATCTCAATGTCTATA<br />
201 P E P S E M T Y K L L N A A V I Q L L Q<br />
601 CCTGAGCCGTCCGAGATGACTTATAAGCTTCTCAACGCTGCCGTTATTCAACTGTTGCAG<br />
221 L A I R N V D D V N E L A K L A T S V T<br />
661 TTGGCCATTCGAAATGTAGATGATGTCAACGAGTTGGCGAAATTGGCGACATCGGTTACG<br />
241 F K Y A L E H D E E F S T I M M F G N L<br />
721 TTCAAGTACGCGCTAGAGCACGATGAAGAGTTTTCTACCATTATGATGTTTGGAAACCTG<br />
261 E A W I S N P V L N K L L M V H Q V L L<br />
781 GAAGCTTGGATTTCCAATCCCGTTTTGAACAAACTTCTTATGGTCCATCAGGTCTTACTA<br />
281 K S -<br />
841 AAATCGTAG<br />
BLR18<br />
1 M R T T F F L T V V I A M F C A C I T A<br />
1 ATGCGCACGACATTTTTCCTGACCGTCGTGATAGCAATGTTCTGTGCTTGTATCACTGCG<br />
21 I E T L E G P T T T G R E P N I A D A T<br />
61 ATTGAAACTCTAGAGGGTCCAACTACAACGGGACGTGAGCCGAACATTGCGGACGCAACC<br />
41 P T V V A R A L R G A V A F N E D R L D<br />
121 CCTACTGTCGTTGCAAGGGCGTTGCGAGGTGCCGTGGCGTTCAATGAAGATCGACTTGAT<br />
61 L E G L T N S V A T L F D G A L H E I N<br />
181 CTCGAAGGTCTTACTAATTCCGTTGCTACTTTGTTTGATGGCGCGCTTCATGAAATAAAT<br />
81 P E L V R T A K A L N E -<br />
241 CCGGAGCTTGTGCGAACTGCGAAAGCTCTCAACGAATAA<br />
BLR19<br />
1 M L L S R A I S V V A L L A C I X C G A<br />
1 ATGCTTCTTTCCCGCGCNATATCTGTCGTCGCCCTTCTCGCATGCATTCNTTGTGGGGCG<br />
21 H A Q D S E V D L G T L L T T L D S S M<br />
61 CACGCTCAAGACTCGGAAGTAGATCTCGGGACTCTATTGACCACCCTTGACAGTTCGATG<br />
41 V T S Q R L L R T S V D L D N N E E R V<br />
121 GTCACTTCGCAGCGGCTTCTCAGAACGAGCGTGGACTTGGACAACAACGAAGAACGCGTC
126 Chapter 4<br />
61 K W P F Q N L V T D Y L N Q K A I R K S<br />
181 AAATGGCCATTTCAAAATTTGGTCACTGATTATCTTAATCAGAAAGCAATCAGGAAAAGT<br />
81 L V N Q A K K T V N A H D E N V L E E A<br />
241 CTCGTAAATCAGGCCAAAAAAACAGTTAACGCTCACGATGAGAACGTATTGGAGGAAGCG<br />
101 V K K E I N A G R V K N V K Q A L S K L<br />
301 GTGAAGAAGGAGATAAATGCAGGTCGCGTGAAAAATGTCAAGCAAGCGTTAAGCAAACTG<br />
121 K N G D P A K A K L Q R L Y N A E I L R<br />
361 AAAAATGGCGATCCCGCAAAAGCTAAACTACAACGACTCTACAATGCTGAAATTTTAAGG<br />
141 N L P K T H N S G Q V R I S R D K V S R<br />
421 AATTTACCAAAAACACATAATTCAGGTCAAGTTAGGATCTCGAGGGACAAAGTCAGCAGG<br />
161 -<br />
481 TAA<br />
BLR20<br />
1 M R F I L V I F M T A A T I C A S W E T<br />
1 ATGCGCTTTATTCTCGTCATTTTTATGACTGCAGCCACAATATGTGCAAGTTGGGAGACC<br />
21 I S V E K D G E H D K L L V I T S T T R<br />
61 ATTTCGGTTGAAAAGGATGGCGAACATGATAAACTTTTGGTAATAACTTCAACAACTCGC<br />
41 Y L K E D Y G A V S N S R L L R V S V G<br />
121 TACCTCAAGGAAGACTATGGTGCCGTCTCCAATTCGCGGCTTTTGCGCGTCAGCGTCGGC<br />
61 S N Q S G T M Q D E A D I G E D S Y M D<br />
181 TCCAACCAAAGTGGAACCATGCAAGACGAAGCGGACATTGGCGAAGACTCCTACATGGAT<br />
81 T V Y F K L W R L I G K T P G E V Y T D<br />
241 ACTGTGTACTTTAAGCTCTGGCGTTTGATTGGCAAGACGCCTGGTGAGGTATACACCGAC<br />
101 S F G T M D P A S A A Q N P S Y N R Y I<br />
301 TCATTCGGTACCATGGACCCAGCCTCTGCTGCCCAGAACCCAAGCTATAATAGGTATATT<br />
121 R Y K R Y Y N N H H -<br />
361 CGATACAAACGATACTACAATAATCATCATTAG<br />
BLR21<br />
1 M R T C F L I F V A G A T I L L S T K A<br />
1 ATGCGTACTTGTTTCTTGATCTTTGTGGCTGGGGCAACGATTTTACTCAGCACTAAAGCT<br />
21 S N P E D D V E D R G S A I G Q V R R I<br />
61 TCCAACCCTGAAGACGACGTCGAAGACCGCGGATCAGCAATTGGTCAAGTGAGAAGAATT<br />
41 L R P K T I N Y G A L E P G R S N P L Y<br />
121 TTACGGCCTAAAACGATCAACTACGGGGCGTTGGAGCCTGGTCGATCAAATCCTCTGTAT<br />
61 N D P R Q -<br />
181 AATGACCCTAGGCAATAG<br />
BLR22<br />
1 M L S C K K T F A L C A T A A L I M S G<br />
1 ATGCTTTCCTGCAAAAAAACTTTCGCGCTATGCGCAACTGCGGCTTTGATCATGTCTGGC<br />
21 F S E V E S S I A S Y R R G L R E G A V<br />
61 TTTTCAGAAGTAGAATCCAGCATCGCAAGTTACAGACGCGGCCTGAGGGAGGGCGCTGTA<br />
41 S G E G Y S D K M D Y K R K H Y E H T D<br />
121 AGCGGTGAAGGCTACAGCGACAAGATGGATTACAAGAGAAAACACTATGAACACACCGAT<br />
61 S D G D V G H N G D D T V L Y Q D S Q L<br />
181 TCGGATGGAGACGTTGGCCACAACGGCGATGATACGGTTTTGTATCAGGACTCGCAGCTA<br />
81 H D G L I D I D L V A K L E L A L G L L<br />
241 CATGACGGGCTCATTGATATCGACCTCGTCGCTAAACTTGAACTTGCACTTGGGCTATTG<br />
101 R E D G V D L L S H H N G A L Q H Q N G<br />
301 CGCGAAGATGGAGTCGACCTTTTGAGCCACCACAACGGCGCCTTACAACATCAGAACGGG<br />
121 E S P L H L D D L L E I G V G L E I A I<br />
361 GAGTCACCATTGCACTTGGATGACCTTCTCGAAATCGGCGTCGGTCTTGAAATTGCAATT
Bremia <strong>effectors</strong> enhance host susceptibility<br />
127<br />
141 D L L R E K G V G L L N H H G A E Q H Q<br />
421 GATCTGTTGCGCGAAAAAGGAGTCGGCCTTTTGAACCACCACGGTGCTGAACAGCATCAA<br />
161 N A E T P L H L D N I V D I D A D V G V<br />
481 AACGCGGAGACGCCATTGCACTTGGATAACATTGTTGACATCGATGCCGATGTAGGAGTT<br />
181 D L L S V -<br />
541 GATTTGCTTTCCGTTTAA<br />
BLR23<br />
1 M R L R G S F V T L V A I I A V F Y Q G<br />
1 ATGCGTCTTCGTGGTAGCTTCGTTACATTGGTCGCAATCATCGCGGTTTTCTACCAAGGT<br />
21 E G M D F Q A A S A T F K H V R S P I Q<br />
61 GAAGGTATGGATTTCCAAGCGGCTTCCGCCACCTTCAAGCATGTACGGTCTCCTATACAG<br />
41 N P P N F E M E S V P S A Q M T R S L R<br />
121 AACCCCCCAAACTTCGAAATGGAGAGTGTACCAAGCGCCCAGATGACTCGATCTCTAAGG<br />
61 V D E N R N G G Q L G S D A K T I I T K<br />
181 GTTGACGAAAATCGCAATGGAGGGCAGCTCGGAAGTGATGCGAAAACGATCATAACTAAG<br />
81 V K F P K K A F E N L T M K I K H N P F<br />
241 GTCAAGTTTCCAAAGAAGGCATTTGAAAACCTAACGATGAAGATTAAGCACAATCCATTT<br />
101 Y P A K H I N -<br />
301 TATCCAGCCAAACATATTAATTAA<br />
BLR24<br />
1 M T F A A V M E M S T V K I T L A L A V<br />
1 ATGACATTTGCCGCGGTTATGGAGATGAGTACTGTGAAAATAACTTTAGCTCTGGCTGTT<br />
21 A W G V L A K H G K A D K E E H L E I Y<br />
61 GCATGGGGCGTACTCGCCAAACATGGTAAAGCTGATAAGGAAGAGCATTTGGAAATATAT<br />
41 L C A P I G I E I G E K I L R S L R V Q<br />
121 TTATGCGCTCCGATTGGAATAGAGATTGGCGAAAAAATATTGCGGTCGCTCCGCGTGCAG<br />
61 S P R I S F S G C K K H Y E L E Q A G R<br />
181 AGCCCAAGAATCTCGTTCTCCGGCTGCAAAAAACACTATGAATTAGAGCAGGCGGGGCGA<br />
81 L W S Q L L H F K L T -<br />
241 CTTTGGAGCCAATTGCTTCACTTTAAGTTRACATAA<br />
BLR25<br />
1 M V R S T S K G L T S C T V R R L M L L<br />
1 ATGGTGCGCTCCACGTCCAAGGGTTTAACGTCTTGTACCGTGCGCAGACTCATGCTGCTG<br />
21 A V F A L S A C V A T L S T S P N T M T<br />
61 GCAGTTTTTGCTCTATCAGCGTGCGTGGCAACCCTCTCGACGAGTCCTAACACTATGACT<br />
41 L S S Y R H L K E L P D T R R A L R F P<br />
121 CTTAGCTCTTATCGACACTTGAAAGAGCTGCCTGACACACGACGAGCTCTGCGCTTTCCG<br />
61 K T G S I R T T T C S K Q W L N S W K L<br />
181 AAAACAGGCTCAATTCGTACTACGACTTGCTCAAAGCAATGGCTCAACAGCTGGAAGTTG<br />
81 A F -<br />
241 GCATTCTGA<br />
BLR26<br />
1 M M P V P S E L V I T Q H R R I L L L F<br />
1 ATGATGCCTGTACCATCCGAGCTTGTAATAACTCAGCACAGGCGTATATTACTGCTATTT<br />
21 L A L L A G V T I V M A A T T A P N A V<br />
61 TTGGCTCTGCTGGCGGGCGTGACAATTGTCATGGCGGCAACAACAGCACCAAACGCTGTC<br />
41 A G V F R R R L R E V P N A L V D G Q N<br />
121 GCCGGTGTCTTCCGTCGACGATTAAGAGAGGTGCCAAACGCTCTTGTCGACGGGCAGAAC<br />
61 D E R N S S P S I A V R Q I A E K S L A<br />
181 GATGAAAGGAACAGCTCTCCAAGTATTGCCGTCCGGCAAATTGCGGAAAAGTCGTTGGCA
128 Chapter 4<br />
81 S A K T K G I L P K N F E E F A E N F S<br />
241 TCGGCGAAAACGAAAGGCATTTTGCCGAAGAACTTTGAAGAATTCGCCGAAAACTTTTCA<br />
101 K N N Y S G N D D L D H R M Y A F L N A<br />
301 AAGAACAATTACTCAGGGAATGACGACTTGGATCATCGCATGTACGCATTCCTCAATGCT<br />
121 L T K N K R E A L E Y V F S P N W L Q K<br />
361 CTTACAAAGAATAAAAGAGAAGCTTTGGAGTATGTATTTTCGCCAAATTGGTTGCAGAAA<br />
141 P H A L R F Y N F L V N R W S K Q K L G<br />
421 CCCCACGCGCTGAGATTTTACAATTTCTTAGTAAATCGGTGGTCTAAACAAAAATTAGGC<br />
161 Q N L H L D G I S R E D L N A L I G L K<br />
481 CAAAATTTGCACTTGGACGGGATAAGCCGAGAGGACCTAAATGCTTTGATTGGATTAAAA<br />
181 T R L E K T L -<br />
541 ACGCGACTCGAAAAAACATTATAA<br />
BLR27<br />
1 M T K Y K C S S L I C S L L L L S C Y H<br />
1 ATGACCAAGTACAAGTGCTCGTCGTTGATCTGCTCCCTCCTCCTGCTATCCTGCTACCAC<br />
21 V V T S I E N V A L I E A P V L G K S R<br />
61 GTCGTGACAAGCATCGAGAACGTCGCGCTGATTGAGGCGCCAGTCCTCGGCAAGTCTCGA<br />
41 Q L R S S V E L V E G L L L T S D K L A<br />
121 CAATTACGCTCGAGTGTCGAGCTGGTGGAAGGATTATTGCTTACCTCAGATAAATTGGCC<br />
61 K V I G T L P K N L A K E W L A T F K W<br />
181 AAGGTTATAGGTACACTGCCAAAAAACCTGGCCAAGGAATGGCTCGCGACCTTCAAATGG<br />
81 G I N H F K I L L A E D K V A A A G K I<br />
241 GGTATCAATCATTTCAAGATCCTGCTCGCAGAGGACAAGGTCGCGGCCGCTGGTAAAATT<br />
101 F P D D S A N P I M T K L F E S D A F L<br />
301 TTCCCCGATGATTCGGCCAATCCCATAATGACAAAGCTCTTTGAAAGTGATGCCTTTCTA<br />
121 W W E K L I R R T V D D T Q E E V D T I<br />
361 TGGTGGGAAAAACTCATTCGACGAACTGTAGACGATACCCAAGAGGAAGTGGACACGATC<br />
141 I T S A L V T R L G K G R A I K L F T E<br />
421 ATTACTTCAGCACTCGTGACCAGACTTGGCAAAGGCAGGGCCATCAAACTCTTTACGGAA<br />
161 A G F E A D A I N A K W L A L L Y H A L<br />
481 GCAGGGTTCGAGGCCGATGCGATCAATGCCAAGTGGTTGGCCTTGCTATATCATGCATTG<br />
181 D D E D M P K L F K E I V S S G G F D K<br />
541 GACGATGAAGATATGCCGAAGTTATTTAAAGAGATCGTTTCGAGTGGAGGTTTCGATAAG<br />
201 N S V E F F I K L C A R E N N K D A A Y<br />
601 AACAGCGTCGAGTTTTTCATTAAGTTATGTGCAAGGGAGAACAATAAAGATGCCGCGTAT<br />
221 E I I N S L L Q D K K E Y R E V Y Q K F<br />
661 GAAATTATCAATAGTCTGCTGCAAGACAAGAAAGAATATCGGGAGGTTTATCAGAAATTC<br />
241 D G V V E T Y R T K W D S L K S V Y F K<br />
721 GATGGAGTTGTCGAAACGTACCGTACCAAATGGGATTCTCTCAAAAGCGTGTATTTCAAG<br />
261 A V S D P K L D K S V S P M Q R L R N L<br />
781 GCAGTATCCGATCCGAAACTTGATAAAAGTGTTTCACCAATGCAGAGGCTTCGGAATTTA<br />
281 Y Q T E E L D P V S V A S K I A D I Y A<br />
841 TACCAAACTGAAGAGCTCGATCCTGTGTCCGTGGCATCAAAAATTGCCGACATTTATGCC<br />
301 K T S E G L H L L Q I V K Q T L H D K A<br />
901 AAAACGTCGGAAGGTCTTCATTTACTTCAGATCGTAAAGCAGACGTTGCATGATAAGGCA<br />
321 A T E A D K A F A G L V L D K V H V K W<br />
961 GCAACGGAAGCTGATAAAGCGTTTGCTGGACTAGTCTTGGACAAAGTACATGTGAAGTGG<br />
341 I Q T N Y D V G K M L K S L T R K I K P<br />
1021 ATTCAAACTAATTATGACGTGGGCAAGATGCTCAAGTCTCTTACGCGCAAAATTAAACCA<br />
361 V E T L L N T S E G V A F V F A I R E M<br />
1081 GTCGAAACTCTTCTTAATACGTCAGAGGGTGTGGCATTTGTATTTGCCATAAGAGAAATG
Bremia <strong>effectors</strong> enhance host susceptibility<br />
129<br />
381 K A T E S E G Y S E A V G V F R K T F E<br />
1141 AAGGCGACCGAGTCGGAAGGATATAGTGAGGCGGTCGGTGTGTTTCGAAAGACTTTTGAG<br />
401 A D V L E K M M K N A N S D D T I V A G<br />
1201 GCCGACGTTCTCGAAAAGATGATGAAAAACGCAAATTCAGATGACACTATTGTCGCGGGC<br />
421 F I D A Y I E L M G V K I A -<br />
1261 TTCATTGATGCCTATATAGAGCTTATGGGGGTCAAAATAGCTTAA<br />
BLR28<br />
1 M L R V V L F L V A A C A K T S Y S H T<br />
1 ATGCTCCGTGTGGTGCTTTTTCTTGTGGCTGCGTGTGCCAAGACTTCGTACAGCCATACT<br />
21 V A L S T R N S Q Y I A S K A N E H A T<br />
61 GTGGCGCTATCCACGCGTAACTCGCAGTACATTGCGTCGAAAGCCAACGAGCATGCTACG<br />
41 I P E D I N L N R R L R K A A V I T E V<br />
121 ATTCCAGAGGACATCAACTTAAACCGCAGGCTTCGCAAAGCTGCTGTGATTACAGAGGTA<br />
61 A E T L E S I I E A F N P L R T L R S D<br />
181 GCCGAGACTCTTGAATCTATAATTGAAGCGTTCAATCCACTACGAACTTTAAGAAGTGAT<br />
81 V R S E M S S K T K L E Q D A M L K E P<br />
241 GTCAGATCGGAGATGTCATCGAAAACTAAACTCGAACAGGATGCAATGCTTAAGGAACCA<br />
101 S F Y F R M L K P F S E F R I R A C F E<br />
301 TCGTTTTATTTCAGAATGTTGAAACCTTTCTCTGAATTTCGAATTCGCGCATGTTTTGAG<br />
121 V Y E I D T L I L F G T S P H L L K Q Y<br />
361 GTTTATGAAATCGATACGTTGATTCTGTTTGGTACGTCTCCTCATTTATTGAAACAATAT<br />
141 I Q N G I P R G I L P E S V T V L A T T<br />
421 ATTCAAAATGGAATACCTCGAGGTATCCTCCCCGAAAGCGTAACTGTACTTGCAACTACT<br />
161 G E K L K R F Q R Q F D I F F N P P T G<br />
481 GGAGAAAAACTGAAGCGGTTTCAAAGGCAGTTCGATATATTTTTTAATCCGCCAACAGGG<br />
181 S K P S K P S R A W P Y A R G P Q V Q A<br />
541 TCAAAACCGAGTAAACCCAGTCGCGCTTGGCCTTACGCCCGGGGCCCTCAAGTTCAAGCG<br />
201 N F K K I Y S S D H I K F L A Y A F H H<br />
601 AATTTTAAAAAGATCTATAGTTCCGACCATATTAAATTTTTGGCATACGCCTTTCACCAT<br />
221 L D D V N I L A K L S S S I I Y R F V L<br />
661 CTTGATGATGTGAACATCTTGGCGAAATTGTCGTCATCAATTATTTACAGGTTCGTTCTC<br />
241 D N F K E C R A T I R Y G T V E D W Y K<br />
721 GACAATTTTAAAGAGTGCCGAGCTACTATTCGTTATGGAACGGTGGAAGACTGGTACAAA<br />
261 H P M L N K L L R V H E V C R K F G I -<br />
781 CATCCCATGCTGAACAAACTTCTTCGAGTGCATGAGGTCTGTCGAAAGTTCGGAATATAG<br />
BLR29<br />
1 M I G N F K K G L F V A A V A V A L A T<br />
1 ATGATTGGAAATTTTAAGAAAGGTTTGTTCGTGGCCGCCGTGGCAGTGGCCCTGGCCACA<br />
21 S V E G Y T G A A S Y E S K R M L R Q Q<br />
61 AGTGTCGAAGGGTACACGGGTGCCGCATCCTATGAATCAAAGCGCATGCTTCGACAGCAG<br />
41 T Q A I A E E S V A D D P E C G S L E M<br />
121 ACGCAAGCAATTGCAGAGGAATCAGTTGCGGACGATCCCGAGTGCGGTTCGCTAGAAATG<br />
61 A E I D D P E C G S L E M A E E N N D N<br />
181 GCCGAGATTGACGACCCCGAGTGCGGATCGCTCGAAATGGCCGAAGAGAACAATGACAAC<br />
81 N D N N D N T N D N N N N Y S S G N N G<br />
241 AATGACAACAATGACAATACTAACGACAATAACAACAATTACTCAAGTGGCAACAATGGC<br />
101 N N G N F W T P P D N G N N G N S G N Y<br />
301 AACAATGGCAACTTTTGGACGCCGCCAGACAACGGCAACAACGGCAACAGTGGCAATTAT<br />
121 W T P P D G K K N L H T G Q E G T P S G<br />
361 TGGACGCCACCCGATGGCAAGAAGAATTTACATACGGGCCAAGAAGGAACGCCTTCTGGT
130 Chapter 4<br />
141 Q D T K G N T V I Q T D D T V S N E A E<br />
421 CAGGATACCAAGGGGAATACTGTAATCCAAACGGACGATACCGTGAGCAATGAGGCAGAG<br />
161 T I D P E C G S L D M A E E D N G N E G<br />
481 ACGATAGACCCCGAGTGTGGGTCTTTGGATATGGCCGAGGAGGACAATGGCAATGAAGGA<br />
181 G N Q K S E N P S I K S N L N G G G N A<br />
541 GGCAATCAGAAGTCTGAAAATCCGTCAATAAAGAGTAATTTGAACGGCGGCGGAAACGCT<br />
201 G E K Q E S N K D E K K D G N T N T K E<br />
601 GGCGAAAAACAAGAGTCTAACAAGGATGAGAAGAAAGATGGCAACACGAATACCAAGGAA<br />
221 E N K E S N N G D K N T L Q Q D I G G D<br />
661 GAGAACAAGGAATCCAACAATGGCGACAAGAATACGCTTCAACAAGACATTGGAGGCGAT<br />
241 E V G K A G P Y G D G V E P E C G S L D<br />
721 GAAGTAGGTAAAGCTGGACCCTACGGAGATGGCGTTGAGCCCGAGTGTGGGTCGCTTGAT<br />
261 M A E D N D E C D S L E M A E I G Q E G<br />
781 ATGGCCGAAGACAACGACGAGTGTGATTCGCTTGAAATGGCGGAAATTGGGCAAGAGGGC<br />
281 K S D A V F T G S K S N L S F S P Y Q G<br />
841 AAGAGCGATGCTGTGTTTACTGGATCAAAGTCGAACCTTTCCTTTTCGCCGTACCAAGGT<br />
301 E V N V G T V S P P P -<br />
901 GAGGTCAACGTTGGCACCGTATCCCCCCCGCCTTAA<br />
BLR30<br />
1 M Q V C F K I T A L V M I N A L S I S S<br />
1 ATGCAGGTCTGTTTTAAGATTACTGCGTTAGTCATGATCAATGCGCTGTCGATATCAAGT<br />
21 L I S A E S S L G T A A F A L K T P H M<br />
61 CTCATCTCGGCAGAGTCCAGCTTGGGCACTGCAGCATTTGCGCTTAAAACACCTCACATG<br />
41 E S H E A M R S L R A R H T S T L N V D<br />
121 GAGAGCCACGAAGCCATGCGATCGCTGCGGGCCCGACATACTTCTACTCTCAACGTTGAC<br />
61 E E R L R L K L L R R V E Y I C E K I V<br />
181 GAGGAGCGTTTACGCCTCAAACTTTTACGTCGTGTGGAATATATTTGTGAAAAGATTGTT<br />
81 E A Y N R V K N R N K V F H V E H L P E<br />
241 GAAGCTTATAATAGAGTGAAGAACCGTAATAAGGTTTTCCATGTTGAGCACTTACCAGAA<br />
101 R -<br />
301 CGTTGA<br />
BLQ01<br />
1 M L A L S K I I E A V V I A S I L I G S<br />
1 ATGCTTGCTCTATCCAAGATCATCGAAGCGGTGGTCATCGCATCAATACTCATTGGTAGT<br />
21 S S S F P M E K I S S A T S N D Q T R H<br />
61 AGCAGTTCTTTTCCAATGGAGAAGATTTCGTCAGCAACTTCGAATGACCAAACTCGTCAC<br />
41 Y G F E Q G N G Q L L R G A E K T R L K<br />
121 TACGGATTTGAACAAGGAAACGGACAGCTTTTACGTGGAGCCGAAAAGACGCGTCTCAAG<br />
61 D E E Q R F F K F G L P S W L P K L F -<br />
181 GACGAAGAACAAAGATTTTTTAAATTCGGTCTGCCAAGTTGGTTGCCAAAACTATTTTGA<br />
BLG01<br />
1 M V R V Y V A A L T G F L A L S A S A S<br />
1 ATGGTTCGTGTCTACGTCGCCGCATTGACAGGTTTCCTCGCGCTTAGCGCGTCGGCCTCT<br />
21 A T L Q L T S V N E S L A D A Y D S T A<br />
61 GCTACTTTACAGTTGACGTCGGTAAACGAATCATTGGCTGATGCTTATGACAGTACCGCT<br />
41 P A R G K L R A Y A A T N V E S D E R A<br />
121 CCTGCACGAGGGAAATTACGCGCTTACGCAGCAACAAATGTCGAGAGCGACGAAAGAGCT<br />
61 F S N L L E H L K T L N V L F S P L S K<br />
181 TTCAGTAATTTACTTGAACATCTCAAAACCCTTAATGTTTTGTTCTCGCCGCTTTCAAAG
Bremia <strong>effectors</strong> enhance host susceptibility<br />
131<br />
81 E R M E A A I S K S D S S V V K H L S E<br />
241 GAGCGCATGGAAGCAGCAATATCTAAAAGCGACAGCAGCGTGGTCAAGCATCTCTCGGAG<br />
101 D G A V N E K K L G I G R D G V K A F E<br />
301 GATGGAGCGGTCAACGAGAAGAAACTTGGAATTGGAAGAGATGGTGTAAAAGCTTTTGAA<br />
121 P A K V S K V K M I L E D I F R K P Y N<br />
361 CCTGCAAAAGTTTCGAAGGTGAAGATGATTCTAGAAGACATCTTTCGAAAGCCTTACAAT<br />
141 K I L L K V L S K A N G G E R N L V R N<br />
421 AAAATTTTGTTGAAAGTGCTTTCGAAAGCGAATGGGGGTGAGCGAAATCTAGTGCGCAAC<br />
161 L A Q A E M L G Y K V F F L K S T L A S<br />
481 TTAGCGCAAGCGGAAATGCTCGGTTATAAAGTGTTTTTTCTTAAGTCAACTCTCGCTTCC<br />
181 K W E R D G V S L M D V W S Y I C K D V<br />
541 AAGTGGGAGCGAGACGGTGTGTCGCTGATGGATGTCTGGTCATATATCTGCAAAGATGTG<br />
201 K A P T E E E M K I F S H W C G Y A F V<br />
601 AAAGCCCCAACTGAAGAGGAAATGAAGATTTTCTCGCACTGGTGTGGATATGCTTTTGTT<br />
221 L S A K G K F S A E E T V I E K T L L K<br />
661 CTATCGGCGAAAGGGAAATTCTCGGCAGAGGAGACTGTTATTGAAAAGACGCTCCTCAAA<br />
241 V H N D N K N K K G K L A I N M L A Q I<br />
721 GTTCATAATGACAACAAGAACAAGAAAGGCAAGCTTGCAATCAACATGCTGGCTCAGATT<br />
261 H L S R K F K V D A N F F E L Y D P S P<br />
781 CACCTGTCGCGCAAGTTTAAGGTCGATGCCAATTTCTTTGAATTGTATGATCCGTCACCT<br />
281 S S L K K L L L E L E K S P R A C D L D<br />
841 TCTTCACTCAAGAAATTATTGCTTGAACTTGAGAAGAGTCCGCGTGCTTGCGATCTCGAT<br />
301 V E L Y R L L K Q A V T D D K F M K G I<br />
901 GTAGAGTTATATCGACTTCTCAAACAGGCTGTAACCGACGATAAGTTCATGAAAGGCATT<br />
321 W D R S T K K P E Q P N A K R N -<br />
961 TGGGACAGATCGACCAAGAAGCCTGAACAGCCCAACGCCAAACGAAATTAG<br />
BLG02<br />
1 M V R I Y V A A L T V V L A F S A S C S<br />
1 ATGGTTCGTATCTACGTAGCCGCATTGACCGTCGTCCTCGCGTTTAGTGCCTCGTGCTCT<br />
21 A T S P L T L A K A T P V T A N G S G G<br />
61 GCTACTTCACCACTGACGCTGGCAAAAGCTACACCAGTTACTGCTAATGGAAGTGGCGGT<br />
41 R A Q G R L R A H T T T N V E I D E R S<br />
121 CGTGCTCAAGGGAGATTACGCGCTCACACGACAACAAATGTCGAAATCGACGAGAGAAGC<br />
61 I S D V I Y A I A R Q V A D H R S N L V<br />
181 ATTTCTGACGTTATATATGCTATAGCTCGTCAAGTTGCAGACCACCGTTCTAATTTGGTA<br />
81 Q A P K R T Y T T Y L A T L D L S L D K<br />
241 CAGGCTCCAAAAAGAACATATACTACGTATTTAGCTACACTGGACTTATCCTTAGATAAA<br />
101 V L E Q N W S E L R L L Y V K Q T K R K<br />
301 GTTCTCGAACAGAATTGGAGTGAGCTTCGATTGTTGTACGTGAAGCAAACTAAAAGAAAG<br />
121 R P K D F V S M Y E L L V K K Y G L L K<br />
361 CGTCCAAAAGACTTTGTCTCAATGTATGAGCTGTTGGTAAAAAAGTATGGCCTCTTGAAA<br />
141 L T D M M K K P D Y I S L S E T D N F K<br />
421 TTGACTGACATGATGAAGAAGCCAGATTATATCTCATTAAGCGAAACGGACAATTTTAAG<br />
161 Y L Y D E A S V Y W R K N S V L K Q Y A<br />
481 TATCTGTACGACGAAGCAAGCGTGTACTGGAGAAAAAACAGCGTTTTAAAGCAGTATGCG<br />
181 S E L Y D S K G N S K T F D V T D F E S<br />
541 TCTGAACTATATGATAGCAAAGGGAATAGCAAAACGTTCGACGTAACCGACTTTGAAAGC<br />
201 L Q P F F E R I G R R G D Y L E I T A A<br />
601 CTGCAACCTTTTTTCGAACGCATCGGCCGTCGCGGTGATTATTTGGAGATTACTGCTGCC<br />
221 E H E K W K T N K L R P T -<br />
661 GAGCATGAGAAATGGAAAACGAACAAATTGCGACCTACTTAG
132 Chapter 4<br />
BLG03<br />
1 M V R V Y V A A L A A I F A L S A S A T<br />
1 ATGGTTCGTGTCTACGTCGCCGCATTGGCAGCCATTTTCGCATTAAGTGCCTCTGCTACT<br />
21 L H L T L A N A S S V T T E E G D G R P<br />
61 TTACATTTGACGCTCGCAAACGCTTCATCGGTAACTACTGAAGAAGGTGACGGTCGTCCT<br />
41 Q G K L R V N A A T N V E S D E R F L D<br />
121 CAAGGGAAGTTACGCGTCAATGCAGCAACAAATGTCGAAAGCGACGAGAGATTTCTAGAC<br />
61 G L K A L V R G F Y D L T P F A P R F Q<br />
181 GGATTAAAAGCTCTTGTCCGTGGTTTCTATGACCTTACGCCTTTCGCACCACGCTTTCAG<br />
81 K D T Y S N L L L K L N L S F E Q F S G<br />
241 AAGGACACATATTCCAACCTTTTATTGAAACTGAATTTGTCTTTCGAACAGTTTTCTGGG<br />
101 K D T L Q L R R F Y D A A R R H N K V N<br />
301 AAGGACACCTTGCAGCTTCGGCGATTTTACGACGCCGCTAGGAGGCATAACAAGGTGAAT<br />
121 P T N P V S V Y T G L V E K Y G E F E I<br />
361 CCAACAAACCCTGTCTCGGTATATACAGGATTGGTAGAAAAGTATGGTGAGTTTGAAATA<br />
141 V N M V Y S L K H A Q S S K S R D V L K<br />
421 GTAAACATGGTGTATTCGCTAAAACATGCACAATCAAGCAAATCCCGTGATGTGCTCAAG<br />
161 R L G K E E K W Y W K D K K D A G K M Y<br />
481 CGACTCGGCAAAGAGGAAAAATGGTATTGGAAGGATAAGAAGGATGCGGGGAAAATGTAC<br />
181 A E A L D L G N E F T V K N M V S K L S<br />
541 GCAGAGGCGCTCGACCTGGGCAACGAGTTTACAGTGAAGAATATGGTTTCGAAATTATCG<br />
201 K L E Q F L N R I D Q K A S E E Q L K A<br />
601 AAGCTAGAACAGTTTTTAAATCGCATCGACCAAAAGGCTAGCGAGGAACAATTGAAAGCT<br />
221 L V V A D I E R V K T K K E S V S P S D<br />
661 TTGGTTGTTGCCGATATTGAGAGGGTGAAAACGAAAAAAGAAAGCGTTTCACCTAGCGAC<br />
241 V E M -<br />
721 GTTGAGATGTGA
133<br />
Chapter 5:<br />
Two GKLR prote<strong>in</strong>s <strong>of</strong> the lettuce <strong>downy</strong><br />
<strong>mildew</strong> Bremia lactucae <strong>in</strong>duce effectortriggered<br />
immunity<br />
Joost H.M. Stassen 1 , Erik den Boer 2 , Pim W. J. Vergeer 1 ,<br />
Ursula Ellendorff 4 , Koen Pelgrom 2 , Mathieu Pel 3 ,<br />
Johan Schut 4 , Olaf Zonneveld 5 , Marieke J.W. Jeuken 2 ,<br />
<strong>and</strong> Guido Van den Ackerveken 1,6 .<br />
1<br />
Plant-Microbe Interactions, Department <strong>of</strong> Biology, Utrecht University,<br />
Utrecht, 3584 CH, The Netherl<strong>and</strong>s<br />
2<br />
Laboratory <strong>of</strong> Plant Breed<strong>in</strong>g, Wagen<strong>in</strong>gen University,<br />
Wagen<strong>in</strong>gen, 6700 AJ, The Netherl<strong>and</strong>s<br />
3<br />
Enza zaden, Enkhuizen, 1600 AA, The Netherl<strong>and</strong>s<br />
4<br />
Rijk Zwaan, De Lier, 2678 ZG, The Netherl<strong>and</strong>s<br />
5<br />
Syngenta, Enkhuizen, 1601 BK, The Netherl<strong>and</strong>s<br />
6<br />
Centre for BioSystems Genomics, Wagen<strong>in</strong>gen, 6700 AB, The Netherl<strong>and</strong>s
134 Chapter 5<br />
Abstract<br />
Bremia lactucae, the <strong>downy</strong> <strong>mildew</strong> pathogen <strong>of</strong> lettuce (Lactuca sativa), causes<br />
large losses <strong>in</strong> susceptible crops. Dom<strong>in</strong>ant <strong>downy</strong> <strong>mildew</strong> (Dm) resistance genes<br />
have been used extensively by breeders to generate Bremia-resistant lettuce<br />
cultivars. However, turnover <strong>of</strong> Dm genes has been rapid as Bremia is quick to<br />
adapt. Underst<strong>and</strong><strong>in</strong>g the mechanism <strong>of</strong> resistance gene-mediated recognition <strong>of</strong><br />
<strong>downy</strong> <strong>mildew</strong> <strong>effectors</strong> <strong>and</strong> result<strong>in</strong>g effector-triggered immunity (ETI), as well as<br />
how Dm genes are rendered <strong>in</strong>effective, is essential for the development <strong>of</strong> novel<br />
resistance breed<strong>in</strong>g approaches.<br />
We have previously cloned the cod<strong>in</strong>g sequence <strong>of</strong> 34 potential <strong>effectors</strong><br />
<strong>of</strong> Bremia, based on transcriptome sequenc<strong>in</strong>g, that are RXLR <strong>and</strong> RXLR-like<br />
prote<strong>in</strong>s predicted to be translocated <strong>in</strong>to the host cell, where they may <strong>in</strong>duce<br />
ETI. We screened a collection <strong>of</strong> 129 Lactuca l<strong>in</strong>es, belong<strong>in</strong>g to different species<br />
<strong>and</strong> harbour<strong>in</strong>g putative novel resistance traits, for specific effector recognition.<br />
Two <strong>effectors</strong> <strong>in</strong>duced ETI, visible as a hypersensitive response (cell death). The<br />
first, BLG01 triggered a response <strong>in</strong> 52 l<strong>in</strong>es, predom<strong>in</strong>antly <strong>of</strong> L. saligna, that are<br />
non-hosts <strong>of</strong> Bremia. The second, BLG03 was recognised <strong>in</strong> two L. sativa l<strong>in</strong>es<br />
conta<strong>in</strong><strong>in</strong>g the resistance gene Dm2. BLG01 <strong>and</strong> BLG03 have similar N-term<strong>in</strong>al<br />
sequences conta<strong>in</strong><strong>in</strong>g a signal peptide <strong>and</strong> a GKLR variant <strong>of</strong> the RXLR translocation<br />
motif, but have different C-term<strong>in</strong>al effector doma<strong>in</strong>s that are polymorphic<br />
between Bremia isolates. BLG03 triggered ETI <strong>in</strong> l<strong>in</strong>es that were susceptible to the<br />
Bremia isolate from which it was cloned. Nevertheless, BLG03 ETI co-segregated<br />
with resistance to a Dm2–<strong>in</strong>compatible Bremia isolate, <strong>in</strong>dicat<strong>in</strong>g the locus<br />
required for BLG03 ETI maps to the RGC2 cluster <strong>of</strong> resistance genes. The L.<br />
saligna locus required for BLG01 ETI maps to the bottom <strong>of</strong> chromosome 9 <strong>in</strong> <strong>of</strong>fspr<strong>in</strong>g<br />
<strong>of</strong> a L. saligna x L. sativa cross. However, <strong>of</strong>fspr<strong>in</strong>g that show BLG01 ETI<br />
were susceptible to Bremia isolates that express BLG01. This suggests that these<br />
virulent Bremia races can suppress the recognition <strong>of</strong> BLG <strong>effectors</strong>, possibly by<br />
means <strong>of</strong> other host-translocated <strong>effectors</strong> that have not been identified so far. We<br />
are explor<strong>in</strong>g the Lactuca loci underly<strong>in</strong>g the ETI responses for their application <strong>in</strong><br />
Bremia-resistance breed<strong>in</strong>g <strong>of</strong> lettuce.
Bremia GKLR prote<strong>in</strong>s <strong>in</strong>duce ETI<br />
135<br />
Introduction<br />
The lettuce <strong>downy</strong> <strong>mildew</strong> pathogen, Bremia lactucae (hereafter: Bremia), causes<br />
large losses <strong>in</strong> susceptible host plants <strong>and</strong> has been classified as a pathogen with a<br />
high risk <strong>of</strong> quick adaptation to lettuce resistance traits <strong>and</strong> chemical control [1,2] .<br />
Its large effective population size, high gene flow <strong>and</strong> mixed (both sexual <strong>and</strong><br />
asexual) reproductive system contribute greatly to this risk. Bremia belongs to<br />
the peronosporales, an order <strong>of</strong> the oomycetes that <strong>in</strong>cludes <strong>downy</strong> <strong>mildew</strong>- <strong>and</strong><br />
Phytophthora species. The <strong>downy</strong> <strong>mildew</strong>s are obligate biotrophs that are found<br />
on many plant species, <strong>in</strong>clud<strong>in</strong>g Arabidopsis (Hyaloperonospora arabidopsidis),<br />
cucurbits (Pseudoperonospora cubensis), grapev<strong>in</strong>e (Plasmopara viticola), <strong>and</strong><br />
sunflower (Plasmopara halstedii). Obligate biotrophs depend on the liv<strong>in</strong>g host for<br />
their growth <strong>and</strong> reproduction [3] . For successful <strong>in</strong>fection it is therefore <strong>of</strong> prime<br />
importance that biotrophs cope with <strong>in</strong>ducible defences <strong>of</strong> the host, which can be<br />
described as consist<strong>in</strong>g <strong>of</strong> two overlapp<strong>in</strong>g layers <strong>of</strong> plant immunity [4–6] . The first is<br />
triggered by the recognition <strong>of</strong> pathogen-derived molecules termed pathogen-associated<br />
molecular patterns (PAMPs) by transmembrane pattern recognition receptors<br />
(PRRs), <strong>and</strong> is referred to as PAMP-triggered immunity (PTI). Though pathogens<br />
can avoid <strong>in</strong>duc<strong>in</strong>g PTI, e.g. by mask<strong>in</strong>g PAMPs, a more common mechanism<br />
is the suppression <strong>of</strong> PTI with<strong>in</strong> the host cell [5,7] . Pathogens can achieve this by<br />
translocat<strong>in</strong>g prote<strong>in</strong>s (<strong>effectors</strong>) <strong>in</strong>to host <strong>in</strong>tracellular compartments where they<br />
can manipulate cellular processes, e.g. the suppression <strong>of</strong> plant defence responses,<br />
lead<strong>in</strong>g to effector-triggered susceptibility (ETS). Gram-negative bacterial pathogens<br />
deploy a type III secretion system to br<strong>in</strong>g <strong>effectors</strong> <strong>in</strong>to the host cytoplasm<br />
by means <strong>of</strong> a pilus-like structure (reviewed by Büttner <strong>and</strong> He [8] ). Oomycetes get<br />
<strong>in</strong> close contact with host cells by penetrat<strong>in</strong>g the plant-cell wall <strong>and</strong> <strong>in</strong>vag<strong>in</strong>at<strong>in</strong>g<br />
the plant cell membrane to form haustoria, feed<strong>in</strong>g structures that are thought to<br />
contribute to pathogenicity [3,9,10] . From the haustoria <strong>effectors</strong> are secreted from the<br />
pathogen before they cross the host membrane [10] . In the case <strong>of</strong> the peronosporales<br />
two ma<strong>in</strong> classes <strong>of</strong> <strong>effectors</strong> that enter host cells have been def<strong>in</strong>ed: Cr<strong>in</strong>klers <strong>and</strong><br />
RXLR prote<strong>in</strong>s (reviewed by Stassen <strong>and</strong> Van den Ackerveken [11] ). The canonical<br />
RXLR effector conta<strong>in</strong>s an N-term<strong>in</strong>al signal peptide, <strong>and</strong> a translocation doma<strong>in</strong><br />
that conta<strong>in</strong>s an RXLR am<strong>in</strong>o acid motif <strong>and</strong>, optionally, a dEER motif. The<br />
part C-term<strong>in</strong>al <strong>of</strong> the translocation doma<strong>in</strong> is referred to as the effector doma<strong>in</strong>.<br />
Variation <strong>in</strong> the presence <strong>and</strong> exact sequence <strong>of</strong> motifs <strong>in</strong> the translocation doma<strong>in</strong><br />
have been observed, e.g. QXLR motifs <strong>in</strong> P. cubensis <strong>effectors</strong> [12] <strong>and</strong> the presence<br />
<strong>of</strong> an EER motif but no RXLR motif <strong>in</strong> H. arabidopsis ATR5 [13] .<br />
A second layer <strong>of</strong> plant defence is triggered when host cells recognise pathogen<br />
<strong>effectors</strong>. This effector-triggered immunity (ETI) is mediated by resistance (R)<br />
prote<strong>in</strong>s that can detect <strong>effectors</strong> or their activity on host targets. Most known
136 Chapter 5<br />
R-prote<strong>in</strong>s belong to the family <strong>of</strong> cytoplasmic nucleotide b<strong>in</strong>d<strong>in</strong>g (NB) <strong>and</strong> leuc<strong>in</strong>e-rich<br />
repeat (LRR) prote<strong>in</strong>s [5,14,15] . Defence triggered by recognition <strong>of</strong> <strong>effectors</strong><br />
by R-prote<strong>in</strong>s is <strong>of</strong>ten associated with the hypersensitive response (HR) that is<br />
visible as programmed cell death <strong>of</strong> host tissue. Effectors that are recognised by<br />
host R-prote<strong>in</strong>s <strong>and</strong> trigger HR are termed avirulence prote<strong>in</strong>s (AVRs). All AVRs<br />
cloned from oomycete pathogens so far are RXLR <strong>and</strong> RXLR-like <strong>effectors</strong> [11,16] ,<br />
with the exception <strong>of</strong> ATR5 [13] . RXLR <strong>effectors</strong> are predicted to be present <strong>in</strong> large<br />
numbers <strong>in</strong> the genomes <strong>of</strong> oomycetes belong<strong>in</strong>g to the peronosporales, from 134<br />
<strong>in</strong> H. arabidopsidis [17] to 563 <strong>in</strong> P. <strong>in</strong>festans [18] . RXLR <strong>effectors</strong> are highly diverse<br />
between species [19] <strong>and</strong> can also be differentially present with<strong>in</strong> different isolates<br />
<strong>of</strong> a pathogen species [16,20] . Oomycete pathogens rapidly evolve to overcome R<br />
prote<strong>in</strong>-mediated recognition or ETI by (i) am<strong>in</strong>o acid substitutions <strong>in</strong> the effector<br />
prote<strong>in</strong> [21,22] , (ii) by down-regulation, loss, or silenc<strong>in</strong>g <strong>of</strong> the effector gene [23] , or<br />
(iii) by suppression <strong>of</strong> ETI by other <strong>effectors</strong> [16,24,25] . Resistance is therefore the<br />
outcome <strong>of</strong> a complex network <strong>of</strong> <strong>in</strong>teractions between <strong>effectors</strong> <strong>and</strong> components<br />
<strong>of</strong> the host’s defence mach<strong>in</strong>ery. Unravell<strong>in</strong>g such a network requires knowledge<br />
about the <strong>in</strong>dividual <strong>in</strong>teractions between <strong>effectors</strong>, R-prote<strong>in</strong>s <strong>and</strong> host targets.<br />
The <strong>in</strong>teraction between lettuce <strong>and</strong> Bremia has been extensively studied as a<br />
host-pathogen model for gene-for-gene <strong>in</strong>teractions (reviewed by Michelmore <strong>and</strong><br />
Wong, [26] ). More than 40 major <strong>downy</strong> <strong>mildew</strong>-resistance (Dm) genes are known,<br />
as well as m<strong>in</strong>or-effect resistance genes that may confer partial or field resistance.<br />
A s<strong>in</strong>gle Dm gene, Dm3, has been cloned <strong>and</strong> is part <strong>of</strong> a large locus <strong>of</strong> several<br />
megabases known as Resistance Gene C<strong>and</strong>idate 2 locus (RGC2 locus) [27] that<br />
conta<strong>in</strong>s at least 30 other NB-LRR genes [28] .<br />
Cultivated lettuce (Lactuca sativa) can be crossed with some difficulty with<br />
wild lettuce species that <strong>in</strong>clude Lactuca species that are considered Bremia<br />
non-hosts (e.g. L. saligna). These wild lettuce species provide a pool <strong>of</strong> genetic<br />
material from which new Dm genes <strong>and</strong> resistance QTLs have been identified [29–31] .<br />
Dom<strong>in</strong>ant resistance genes have been used extensively by breeders to generate<br />
Bremia-resistant lettuce cultivars. Turnover <strong>of</strong> Dm genes has, however, been rapid,<br />
as Bremia is quick to adapt to newly <strong>in</strong>troduced resistance genes. To underst<strong>and</strong><br />
the molecular basis <strong>of</strong> ETI <strong>in</strong> the lettuce-Bremia <strong>in</strong>teraction <strong>and</strong> to identify new<br />
R‐genes for resistance breed<strong>in</strong>g we deployed c<strong>and</strong>idate RXLR <strong>and</strong> RXLR-like<br />
<strong>effectors</strong> that were previously identified by transcriptome sequenc<strong>in</strong>g (Chapter 3<br />
<strong>and</strong> 4, [32] ). Here, we describe the screen<strong>in</strong>g <strong>of</strong> a large collection <strong>of</strong> lettuce breed<strong>in</strong>g<br />
l<strong>in</strong>es for the ability to recognise a selection <strong>of</strong> 34 Bremia effector c<strong>and</strong>idates. We<br />
discovered two GKLR prote<strong>in</strong>s that are recognised <strong>in</strong> planta, one <strong>of</strong> which is<br />
recognised by L. sativa cultivars conta<strong>in</strong><strong>in</strong>g the Dm2 resistance gene.
Bremia GKLR prote<strong>in</strong>s <strong>in</strong>duce ETI<br />
137<br />
Results<br />
Bremia <strong>effectors</strong> <strong>and</strong> Lactuca accessions<br />
Of 34 c<strong>and</strong>idate <strong>effectors</strong> <strong>of</strong> Bremia isolate Bl:24 we cloned the cod<strong>in</strong>g sequence,<br />
start<strong>in</strong>g directly after the signal peptide-encod<strong>in</strong>g sequence, <strong>in</strong>to a T-DNA expression<br />
vector under control <strong>of</strong> the 35S promoter. A new start codon was <strong>in</strong>serted<br />
before the cloned sequence to construct a gene that encodes the translocation<br />
doma<strong>in</strong> <strong>and</strong> effector doma<strong>in</strong> <strong>of</strong> each predicted effector. In our assay the <strong>effectors</strong><br />
are expressed <strong>in</strong> the host cell cytoplasm, where they are reta<strong>in</strong>ed as no signal<br />
peptide is encoded <strong>in</strong> our constructs. Intracellular recognition <strong>of</strong> <strong>effectors</strong> by<br />
R-prote<strong>in</strong>s is expected to <strong>in</strong>duce a clearly visible cell death response.<br />
Lactuca accessions were selected represent<strong>in</strong>g a large range <strong>of</strong> Bremia<br />
resistance genes, both dom<strong>in</strong>ant Dm genes as well as partial resistance genes, (See<br />
Supplemental Table 1) <strong>and</strong> other unknown sources <strong>of</strong> resistance to Bremia. They<br />
were chosen on the basis <strong>of</strong> the differential sets EU-A <strong>and</strong> EU-B proposed by the<br />
International Bremia Evaluation Board (IBEB, http://www.worldseed.org/isf/ibeb.<br />
html), a set <strong>of</strong> resistant accessions proposed by Michelmore et al. [33] , parents <strong>of</strong><br />
RIL-populations segregat<strong>in</strong>g for Bremia-resistance [34–37] , <strong>and</strong> other known sources<br />
<strong>of</strong> Bremia-resistance [31,38,39] . Also one resistant L. aculeata <strong>and</strong> two resistant L.<br />
altaica accessions [31] were <strong>in</strong>cluded to widen genetic diversity. To test whether any<br />
<strong>of</strong> the 34 <strong>effectors</strong> are recognised <strong>in</strong> planta, we pressure-<strong>in</strong>filtrated suspensions<br />
<strong>of</strong> the Agrobacterium tumefaciens (hereafter: Agrobacterium) stra<strong>in</strong>s carry<strong>in</strong>g<br />
the effector constructs <strong>in</strong>to leaves <strong>of</strong> the selected lettuce l<strong>in</strong>es. Visual responses<br />
to transient expression <strong>of</strong> effector c<strong>and</strong>idates were scored 5-8 days after Agrobacterium<br />
<strong>in</strong>filtration. We <strong>in</strong>cluded stra<strong>in</strong>s carry<strong>in</strong>g a YFP-conta<strong>in</strong><strong>in</strong>g vector as a<br />
negative control, <strong>and</strong> a Necrosis-<strong>in</strong>duc<strong>in</strong>g prote<strong>in</strong> (NIP) gene-conta<strong>in</strong><strong>in</strong>g vector as<br />
a positive control. Expression <strong>of</strong> YFP is not expected to elicit visible cell death, so<br />
any response seen after <strong>in</strong>filtration with Agrobacterium carry<strong>in</strong>g the YFP construct<br />
is considered background. The NIP gene encod<strong>in</strong>g PsojNIP that is derived from<br />
Phytophthora sojae <strong>in</strong>duces a cell death response <strong>in</strong> lettuce that is visible 1-2 days<br />
after <strong>in</strong>filtration <strong>of</strong> the Agrobacterium stra<strong>in</strong> <strong>and</strong> develops <strong>in</strong>to a dark necrotic<br />
lesion after 48 hours. Hence, the PsojNIP response serves as control for successful<br />
T-DNA transfer <strong>and</strong> transient transgene expression.<br />
Screen<strong>in</strong>g <strong>of</strong> Lactuca accessions reveals recognition <strong>of</strong> two <strong>effectors</strong><br />
Screen<strong>in</strong>g <strong>of</strong> the responses <strong>of</strong> 129 lettuce l<strong>in</strong>es to each <strong>of</strong> the 34 <strong>effectors</strong> was performed.<br />
To identify robust ETI observations were scored <strong>and</strong> averaged per lettuce/<br />
effector comb<strong>in</strong>ation. Scor<strong>in</strong>g was based on the presence <strong>of</strong> no or few visible
138 Chapter 5<br />
Table 1: Details <strong>of</strong> 34 Bremia <strong>effectors</strong> <strong>and</strong> the number <strong>of</strong> Lactuca l<strong>in</strong>es <strong>in</strong> which robust ETI (average<br />
score <strong>of</strong> ≥ 1.3 from ≥ 2 replicates) was observed.<br />
RXLR-like EER-like<br />
ID length start motif start motif Robust ETI<br />
BLR01 86 42 RKLR 52 EQK 0<br />
BLR02 146 85 RLLR 0<br />
BLR03 141 48 RFLR 59 EEER 0<br />
BLR04 76 45 RELR 60 DIK 0<br />
BLR05 97 32 RALR 58 DED 0<br />
BLR06 281 46 RCLR 0<br />
BLR07 253 47 RALR 68 EEER 0<br />
BLR08 135 38 RLLR 0<br />
BLR09 112 37 RRLR 81 EER 0<br />
BLR10 112 37 RRLR 81 EER 0<br />
BLR11 463 46 RRLR 57 DESER 0<br />
BLR12 123 49 RYLR 61 ELEK 0<br />
BLR13 363 44 RRLR 55 EER 0<br />
BLR14 75 46 RKLR 0<br />
BLR15 102 47 RSLR 60 DEER 0<br />
BLR16 98 47 RSLR 60 NDER 0<br />
BLR17 282 50 RRLR 64 DAEK 0<br />
BLR18 92 46 RALR 55 NEDR 0<br />
BLR19 160 45 RLLR 54 DNNEER 0<br />
BLR20 130 53 RLLR 69 DEAD 0<br />
BLR21 65 39 RILR 0<br />
BLR22 185 33 RGLR 0<br />
BLR23 107 57 RSLR 62 DENR 0<br />
BLR24 91 55 RSLR 74 ELEQ 0<br />
BLR25 82 55 RALR 0<br />
BLR26 187 46 RRLR 59 QNDER 0<br />
BLR27 434 40 RQLR 0<br />
BLR28 279 49 RRLR 0<br />
BLR29 311 35 RMLR 46 EES 0<br />
BLR30 101 47 RSLR 60 DEER 0<br />
BLQ01 79 49 QLLR 61 DEEQR 0<br />
BLG01 A 336 44 GKLR 57 DER 23<br />
BLG01 E variant <strong>of</strong> BLG01 A 41<br />
BLG02 233 44 GRLR 57 DER 0<br />
BLG03 243 42 GKLR 55 DER 2
Bremia GKLR prote<strong>in</strong>s <strong>in</strong>duce ETI<br />
139<br />
symptoms (0), strong yellow<strong>in</strong>g <strong>of</strong> the leaf (1) or cell death (2). The average scores<br />
<strong>of</strong> all replicates per lettuce/effector comb<strong>in</strong>ation are given <strong>in</strong> Supplemental Table<br />
2. Details <strong>of</strong> the 34 <strong>effectors</strong>, as well as the number <strong>of</strong> l<strong>in</strong>es show<strong>in</strong>g robust ETI,<br />
def<strong>in</strong>ed as an average score from at least two replicates ≥ 1.3, are given <strong>in</strong> Table 1.<br />
Agrobacterium-mediated transient expression was robust <strong>in</strong> nearly all tested l<strong>in</strong>es,<br />
as can been seen from the cell death response that is <strong>in</strong>duced by PsojNIP expression<br />
(Supplemental Table 2). None <strong>of</strong> the tested l<strong>in</strong>es showed a strong response to<br />
the YFP-conta<strong>in</strong><strong>in</strong>g Agrobacterium stra<strong>in</strong>.<br />
Effector BLG01 was recognised <strong>in</strong> many <strong>of</strong> the tested wild lettuce l<strong>in</strong>es. Of 129<br />
l<strong>in</strong>es, 23 showed a strong reproducible cell death response to BLG01. A variant <strong>of</strong><br />
the mature BLG01 prote<strong>in</strong> (without signal peptide), BLG01 E - based on an allele<br />
from Bremia isolates NL519 <strong>and</strong> F703, was found to <strong>in</strong>duce an even stronger<br />
response than the prote<strong>in</strong> encoded by our reference stra<strong>in</strong> Bl:24 (BLG01 A ). Of the<br />
129 tested l<strong>in</strong>es 41 gave a strong cell death response to BLG01 E (Table 1). Weak,<br />
<strong>in</strong>consistent or unverified responses to BLG01 A <strong>and</strong> BLG01 E were observed for 17<br />
<strong>and</strong> 11 Lactuca l<strong>in</strong>es, respectively (see Supplemental Figure 1 for a full overview).<br />
The higher number <strong>of</strong> BLG01 E responsive l<strong>in</strong>es is likely due to a stronger ETI<br />
response that is more easily detected, as almost all l<strong>in</strong>es that showed a weak<br />
response to BLG01 A showed a strong response to BLG01 E . Additionally, 16 l<strong>in</strong>es<br />
respond only to BLG01 E . Most <strong>of</strong> the BLG01-responsive Lactuca l<strong>in</strong>es <strong>in</strong> our<br />
collection <strong>of</strong> Lactuca l<strong>in</strong>es are <strong>of</strong> the L. saligna species.<br />
A second effector, BLG03, was specifically recognised <strong>in</strong> two L. sativa l<strong>in</strong>es,<br />
Amplus <strong>and</strong> UCDM2 (Table 1, Figure 1). In other l<strong>in</strong>es BLG03 did not <strong>in</strong>duce<br />
responses that could be dist<strong>in</strong>guished from the GUS negative control (e.g. Ol<strong>of</strong>,<br />
Figure 4). The set <strong>of</strong> cultivated lettuce l<strong>in</strong>es (L. sativa) <strong>in</strong>cluded <strong>in</strong> the screen conta<strong>in</strong>s<br />
differential l<strong>in</strong>es that provide a wide range <strong>of</strong> genetically known R-genes <strong>and</strong><br />
can be used to determ<strong>in</strong>e R‐gene specificities. Both Amplus <strong>and</strong> UCDM2 conta<strong>in</strong><br />
the Dm2 resistance specificity that is absent <strong>in</strong> all other L. sativa l<strong>in</strong>es tested. The<br />
Figure 1: ETI triggered by BLG03 <strong>in</strong> L. sativa cv. Amplus <strong>and</strong> L. sativa cv. UCDM2. NIP: GUS<br />
<strong>and</strong> PsojNIP (NIP) serve as controls for responses to Agrobacterium <strong>and</strong> successful T-DNA transfer,<br />
respectively.
140 Chapter 5<br />
recognition <strong>of</strong> BLG03 could therefore be mediated by Dm2.<br />
Strik<strong>in</strong>gly, BLG01 <strong>and</strong> BLG03, which are recognized <strong>in</strong> specific Lactuca l<strong>in</strong>es,<br />
both conta<strong>in</strong> the RXLR-like motif GKLR. In addition, the signal peptide- <strong>and</strong><br />
GKLR-conta<strong>in</strong><strong>in</strong>g N-term<strong>in</strong>i <strong>of</strong> BLG01 <strong>and</strong> BLG03 are highly similar (Figure 2).<br />
The N-term<strong>in</strong>us <strong>of</strong> a third Bremia prote<strong>in</strong>, BLG02, also shows homology to these<br />
<strong>effectors</strong>. However, BLG02 is not recognised <strong>in</strong> any <strong>of</strong> the lettuce l<strong>in</strong>es tested. The<br />
GKLR <strong>and</strong> DER motifs are identical <strong>in</strong> BLG01 <strong>and</strong> BLG03, whilst BLG02 has a<br />
GRLR variant <strong>of</strong> the RXLR motif. The effector doma<strong>in</strong>s do not share the high level<br />
<strong>of</strong> similarity that is seen for the signal peptides <strong>and</strong> G K / R<br />
LR-conta<strong>in</strong><strong>in</strong>g N-term<strong>in</strong>i.<br />
<br />
BLG01 MVRVYVAALTGFLALSASASATLQLTSVNESLADAYDSTAPARGKLRAYAATNVESDERA 60<br />
BLG02 MVRIYVAALTVVLAFSASCSATSPLTLAKATPVTANGSGGRAQGRLRAHTTTNVEIDERS 60<br />
BLG03 MVRVYVAALAAIFALSA--SATLHLTLANASSVTTEEGDGRPQGKLRVNAATNVESDER- 57<br />
***:*****: .:*:** *** ** .: : . : . . .:*:**. ::**** ***<br />
BLG01 FSNLLEHLKTLNVLFSPLSKERMEAAISKSDSSVVKHLSEDGAVNEKKLGIGRDGVKAFE 120<br />
BLG02 ISDVIYAIARQVADHRSNLVQAPKRTYTTYLATLDLSLDKVLEQNWSELRLLY--VKQTK 118<br />
BLG03 FLDGLKALVRGFYDLTPFAPRFQKDTYSNLLLKLNLSFEQFSGKDTLQLRRLYDAARRHN 117<br />
: : : : . . : : :. .: :.: : :* .: :<br />
BLG01 PAKVSKVKMILEDIFRKPYNKILLKVLSKANGGERNLVRNLAQAEMLGYKVFFLKSTLAS 180<br />
BLG02 RKRPKDFVSMYELLVKK-------------------------------YGLLKLTDMMKK 147<br />
BLG03 KVNPTNPVSVYTGLVEK-------------------------------YGEFEIVNMVYS 146<br />
. .. : :..* * : : . : .<br />
BLG01 KWERDGVSLMDVWSYICKDVKAPTEEEMKIFSHWCGYAFVLSAKGKFSAEETVIEKTLLK 240<br />
BLG02 PDYISLSET-DNFKYLYDEASVYWR-KNSVLK---QYASELYDSKGNSKTFDVTDFESLQ 202<br />
BLG03 LKHAQSSKSRDVLKRLGKEEKWYWKDKKDAGK---MYAEALDLGNEFTVKNMVSKLSKLE 203<br />
. . * . : .: . . : . . ** * : * . *:<br />
BLG01 VHNDNKNKKGKLAINMLAQIHLSRKFKVDANFFELYDPSPSSLKKLLLELEKSPRACDLD 300<br />
BLG02 PFFERIGRRGD-----YLEITAAEHEKWKT---NKLRPT--------------------- 233<br />
BLG03 QFLNRIDQKASEEQLKALVVADIERVKTKK---ESVSPSDVEM----------------- 243<br />
. :. .::.. : .: * . : *:<br />
BLG01 VELYRLLKQAVTDDKFMKGIWDRSTKKPEQPNAKRN- 336<br />
BLG02 -------------------------------------<br />
BLG03 -------------------------------------<br />
Figure 2: Alignment <strong>of</strong> the am<strong>in</strong>o acid sequence <strong>of</strong> c<strong>and</strong>idate <strong>effectors</strong> BLG01, BLG02 <strong>and</strong> BLG03.<br />
Signal peptide <strong>and</strong> translocation doma<strong>in</strong>s are <strong>in</strong>dicated above the alignment, the GXLR <strong>and</strong> DER motifs<br />
are underl<strong>in</strong>ed <strong>in</strong> the alignment. Symbols under the alignment <strong>in</strong>dicate the degree <strong>of</strong> conservation <strong>of</strong> the<br />
above residues <strong>and</strong> <strong>in</strong>dicate identical (*) residues, the presence <strong>of</strong> conserved substitutions (:) or semiconserved<br />
substitutions (.). Numbers to the right <strong>of</strong> the alignment <strong>in</strong>dicate the residue number <strong>of</strong> the last<br />
residue <strong>in</strong> the column counted from the start <strong>of</strong> the prote<strong>in</strong>, skipp<strong>in</strong>g gaps.<br />
BLG01, BLG02 <strong>and</strong> BLG03 do not have significant homology to any sequences<br />
<strong>in</strong> the NCBI non-redundant prote<strong>in</strong> database (e-value < 1e-3), nor are there any<br />
significant matches to Pfam [40] doma<strong>in</strong>s. The best BLAST matches <strong>in</strong> a comb<strong>in</strong>ed<br />
database <strong>of</strong> oomycete prote<strong>in</strong>s (H. arabidopsidis, P. <strong>in</strong>festans, P. ramorum, P.<br />
sojae, Pythium ultimum <strong>and</strong> Saprolegnia parasitica) were to a putative P. <strong>in</strong>festans<br />
RXLR effector (PITG_15128, e-value 0.037) for BLG01, a P. sojae RXLR<br />
effector (Ps_133875, e-value 0.81) for BLG03, <strong>and</strong> a P. ramorum RXLR effector<br />
(Pr_97351, e-value 0.032) for BLG02 (Supplemental Information 2).
Bremia GKLR prote<strong>in</strong>s <strong>in</strong>duce ETI<br />
141<br />
Recognition <strong>of</strong> the BLG01 <strong>and</strong> BLG03 <strong>effectors</strong> is specific to a subset <strong>of</strong><br />
lettuce l<strong>in</strong>es, mak<strong>in</strong>g cell death responses as a result <strong>of</strong> over-load<strong>in</strong>g the translation<br />
mach<strong>in</strong>ery or recognition <strong>of</strong> the translocation doma<strong>in</strong> unlikely. To verify whether<br />
the translocation doma<strong>in</strong> is <strong>in</strong>deed dispensable for the recognition we <strong>in</strong>vestigated<br />
whether the effector doma<strong>in</strong> <strong>of</strong> BLG01 is sufficient for recognition <strong>in</strong> lettuce.<br />
Us<strong>in</strong>g the same Agrobacterium-mediated transient transformation system as used<br />
to screen the collection <strong>of</strong> lettuce l<strong>in</strong>es, we expressed BLG01 60-336 <strong>in</strong> L. saligna<br />
l<strong>in</strong>e CGN05271, which showed a cell death response towards BLG01 with the<br />
translocation doma<strong>in</strong> (Figure 3). Cell death responses <strong>in</strong> the zones <strong>in</strong>filtrated with<br />
BLG01 60-336 did not differ from those <strong>in</strong>filtrated with BLG01 with translocation<br />
doma<strong>in</strong> (BLG01 22-336 ), <strong>in</strong>dicat<strong>in</strong>g that the translocation doma<strong>in</strong> is not required for <strong>in</strong><br />
planta recognition <strong>in</strong> CGN05271. The same approach with effector BLG03 (with<br />
translocation doma<strong>in</strong> BLG01 20-243 , without BLG01 58-243 ) <strong>in</strong> Amplus <strong>and</strong> UCDM2<br />
revealed that recognition <strong>of</strong> BLG03 is also <strong>in</strong>dependent <strong>of</strong> the translocation doma<strong>in</strong><br />
(Shown for UCDM2 <strong>in</strong> Figure 4).<br />
Figure 3: Recognition <strong>of</strong> BLG01 without signal<br />
peptide (BLG01 22-336 ) <strong>and</strong> without signal peptide<br />
<strong>and</strong> translocation doma<strong>in</strong> (BLG01 60-336 ) <strong>in</strong> L.<br />
saligna CGN05271. GUS <strong>and</strong> PsojNIP serve<br />
as controls for responses to Agrobacterium <strong>and</strong><br />
successful T-DNA transfer, respectively. Pictures<br />
were taken 8 dpi.<br />
Figure 4: Recognition <strong>of</strong> BLG03 with (+TD;<br />
BLG03 20-243 ) <strong>and</strong> without translocation doma<strong>in</strong><br />
(-TD; BLG03 58-243 ) <strong>in</strong> L. sativa cv. UCDM2.<br />
Neither is recognised <strong>in</strong> L. sativa cv. Ol<strong>of</strong>.<br />
GUS (G) <strong>and</strong> PsojNIP (N) serve as controls<br />
for responses to Agrobacterium <strong>and</strong> successful<br />
T-DNA transfer, respectively. Pictures were<br />
taken 6 dpi.
142 Chapter 5<br />
BLG gene expression <strong>and</strong> genetic variation<br />
In order to be recognized <strong>in</strong> planta these G K / R<br />
LR <strong>effectors</strong> need to be expressed<br />
dur<strong>in</strong>g the <strong>in</strong>fection process. Expression <strong>in</strong> planta was already observed, as the<br />
effector transcripts were identified by transcriptome sequenc<strong>in</strong>g <strong>of</strong> <strong>in</strong>fected lettuce<br />
leaves. To determ<strong>in</strong>e the changes <strong>in</strong> expression dur<strong>in</strong>g the different stages <strong>of</strong> <strong>in</strong>fection<br />
we analysed a time-series by quantitative PCR (qPCR) (Figure 5). L. sativa<br />
cv. Ol<strong>of</strong> seedl<strong>in</strong>gs were spray-<strong>in</strong>oculated with spores <strong>of</strong> Bremia isolate Bl:24 after<br />
which samples were taken every 24 hours start<strong>in</strong>g immediately after spray<strong>in</strong>g.<br />
With<strong>in</strong> the first 24 hours, the majority <strong>of</strong> spores germ<strong>in</strong>ated <strong>and</strong> Bremia had penetrated<br />
the epidermis. Substantial Bremia hyphal growth <strong>and</strong> formation <strong>of</strong> haustoria<br />
<strong>in</strong> mesophyll cells occurred over the next four days. Conidiophores formed by six<br />
days post <strong>in</strong>oculation (dpi), at which time sampl<strong>in</strong>g was stopped. Expression levels<br />
were determ<strong>in</strong>ed as the number <strong>of</strong> qPCR cycles required for the abundance <strong>of</strong> each<br />
amplicon to reach threshold level (C T<br />
), <strong>and</strong> were normalised to L. sativa Act<strong>in</strong> or<br />
Bremia Act<strong>in</strong> (result<strong>in</strong>g <strong>in</strong> ΔC T<br />
values). The expression <strong>of</strong> Bremia Act<strong>in</strong> relative<br />
to L. sativa Act<strong>in</strong> shows the substantial relative growth <strong>of</strong> Bremia throughout the<br />
entire time course (Figure 5). Expression <strong>of</strong> BLG03 <strong>in</strong>creases immediately after<br />
<strong>in</strong>oculation <strong>and</strong> appears to be stable from one dpi onwards. BLG01 gene expression<br />
decreases slightly dur<strong>in</strong>g the course <strong>of</strong> <strong>in</strong>fection, with expression levels comparable<br />
to those <strong>of</strong> BLG03 at the later stages <strong>of</strong> <strong>in</strong>fection. BLG02 shows an <strong>in</strong>crease <strong>of</strong><br />
expression at the first day after <strong>in</strong>oculation similar to that <strong>of</strong> BLG03. After one dpi<br />
expression levels <strong>of</strong> BLG02 decl<strong>in</strong>e, approach<strong>in</strong>g the level seen immediately after<br />
<strong>in</strong>oculation. Similar expression patterns were detected <strong>in</strong> an <strong>in</strong>dependent biological<br />
replicate <strong>of</strong> the time series.<br />
Figure 5: Bremia isolate Bl:24 growth <strong>and</strong> effector gene expression dur<strong>in</strong>g <strong>in</strong>fection <strong>of</strong> L. sativa<br />
cv. Ol<strong>of</strong>. Growth is <strong>in</strong>ferred by the <strong>in</strong>crease <strong>of</strong> Bremia Act<strong>in</strong> relative to lettuce Act<strong>in</strong> throughout the<br />
time course calculated as (ΔC T<br />
). Effector gene expression is determ<strong>in</strong>ed relative to Bremia Act<strong>in</strong>. The<br />
difference <strong>in</strong> C T<br />
required to reach threshold is given: as lower values <strong>in</strong>dicate higher expression, the<br />
Y-axis has been reversed to ease <strong>in</strong>terpretation.
Bremia GKLR prote<strong>in</strong>s <strong>in</strong>duce ETI<br />
143<br />
Allelic diversity <strong>of</strong> the three G K / R<br />
LR <strong>effectors</strong> <strong>in</strong> a selection <strong>of</strong> Bremia<br />
isolates was <strong>in</strong>vestigated to test if the prote<strong>in</strong>s show signs <strong>of</strong> selection. A set <strong>of</strong><br />
eight Bremia isolates was chosen as a group with high diversity based on their<br />
R‐gene-specificities. As Bremia is not haploid isolates can be heterozygous result<strong>in</strong>g<br />
<strong>in</strong> mixed peaks when sequenc<strong>in</strong>g PCR products. Therefore PCR products were<br />
cloned <strong>and</strong> from each isolate a m<strong>in</strong>imum <strong>of</strong> eight clones per gene were sequenced.<br />
Seven different alleles were found for BLG01 <strong>and</strong> BLG03, <strong>and</strong> six alleles for<br />
BLG02. The distribution <strong>of</strong> alleles is represented <strong>in</strong> Table 2, the prote<strong>in</strong> translations<br />
<strong>of</strong> the different alleles can be found <strong>in</strong> Supplemental Information 1. Two strik<strong>in</strong>g<br />
observations can be made regard<strong>in</strong>g BLG01. First, as can be seen from the allele<br />
distribution, isolate Bl:5 appears to possess 4 alleles <strong>of</strong> this effector. Rather than<br />
alleles, these may represent duplicated genes. Secondly, the allele sequences reveal<br />
that no <strong>functional</strong> BLG01 prote<strong>in</strong>s are encoded <strong>in</strong> isolates NL519 <strong>and</strong> F703 due to<br />
a nonsense mutation <strong>in</strong> the fifth codon (TAC→TAA, Y→stop). The same stop is<br />
found <strong>in</strong> one <strong>of</strong> the alleles <strong>of</strong> Bl:5, whereas two other Bl:5 alleles have premature<br />
stops at other positions. In one <strong>of</strong> these alleles the stop (CAG→TAG, Q→stop) is<br />
at am<strong>in</strong>o acid position 24, the third residue after the predicted signal peptide cleavage<br />
site (SA|TL), <strong>in</strong> the other case a two nucleotide deletion <strong>in</strong> the effector doma<strong>in</strong><br />
(am<strong>in</strong>o acid position 286) <strong>in</strong>duces a frame shift that reads <strong>in</strong>to a stop after two<br />
am<strong>in</strong>o acids. In contrast to NL519 <strong>and</strong> F703, a full-length copy <strong>of</strong> the effector is<br />
present <strong>in</strong> Bl:5. For most isolates two different alleles <strong>of</strong> BLG02 <strong>and</strong> BLG03 were<br />
found. BLG02 A <strong>and</strong> BLG02 B encode the same prote<strong>in</strong> sequence <strong>and</strong> only have synonymous<br />
nucleotide variants. All am<strong>in</strong>o acid differences encoded <strong>in</strong> BLG02 alleles<br />
were found <strong>in</strong> the effector doma<strong>in</strong>. This is <strong>in</strong> contrast to BLG03, <strong>in</strong> which am<strong>in</strong>o<br />
acid polymorphisms are also found <strong>in</strong> the signal peptide <strong>and</strong> translocation doma<strong>in</strong>.<br />
Table 2: Distribution <strong>of</strong> alleles <strong>of</strong> BLG01, BLG02 <strong>and</strong> BLG03 over eight Bremia isolates. na: not<br />
amplified.<br />
Race BLG01 BLG02 BLG03<br />
Bl:5 C,D 1 ,F 1 ,G 2 A,E B,G<br />
Bl:16 A,B A,B 3 A<br />
Bl:17 na A,C D,F 2<br />
Bl:24 A A,B 3 A,C<br />
NL519 E 1 B 3 ,C A,D<br />
F703 E 1 C,D na<br />
CA3 C A,F A<br />
CA6 C A A,E<br />
1<br />
Premature stop codon before translocation doma<strong>in</strong><br />
2<br />
Premature stop codon after translocation doma<strong>in</strong><br />
3<br />
Encodes same prote<strong>in</strong> sequence as A.
144 Chapter 5<br />
Although one allele has an <strong>in</strong>sertion <strong>of</strong> two am<strong>in</strong>o acids <strong>in</strong> the signal peptide, all<br />
BLG03 alleles are predicted to encode a signal peptide. BLG03 F , only present <strong>in</strong><br />
Bl:17, has a premature stop codon <strong>in</strong> the effector doma<strong>in</strong>. Furthermore, BLG03 G ,<br />
found <strong>in</strong> Bl:5, is more sequence divergent from the Bl:24 reference sequence than<br />
all other BLG03 alleles. None <strong>of</strong> our <strong>effectors</strong> are predicted to be under positive<br />
selection to ma<strong>in</strong>ta<strong>in</strong> am<strong>in</strong>o acid diversity based on the ratio <strong>of</strong> synonymous to<br />
non-synonymous substitutions (codon-based test <strong>of</strong> positive selection averag<strong>in</strong>g<br />
over all sequence pairs; BLG01: Z-score = 0.05790, p = 0.47696; BLG02: Z-score<br />
= 0.15217, p = 0.43965; BLG03: Z-score = -1.31461, p = 1.0). However, the many<br />
different alleles <strong>and</strong> <strong>in</strong> particular the nonsense alleles suggest that BLG01 <strong>and</strong><br />
BLG03 have been under selective pressure.<br />
The different BLG01 alleles were next used to make constructs <strong>of</strong> the effector<br />
without signal peptide for <strong>in</strong> planta expression. Constructs with BGL01 alleles A,<br />
B, C <strong>and</strong> E (see Supplemental Information 1) were tested <strong>in</strong> L. saligna CGN5271<br />
to determ<strong>in</strong>e their potential to trigger cell death. As shown <strong>in</strong> Figure 6, the visible<br />
symptoms differ <strong>in</strong> response to the alleles <strong>of</strong> the various isolates. Responses to<br />
BLG01 A <strong>and</strong> BLG01 C appear comparable, whilst the response to BLG01 B appears<br />
to be slightly stronger. Strik<strong>in</strong>gly, BLG01 E triggered the strongest responses when<br />
expressed <strong>in</strong> L. saligna CGN05271. However, as there is a premature stop <strong>in</strong> the<br />
signal peptide <strong>of</strong> BLG01 E this prote<strong>in</strong> will not be produced by the Bremia isolates<br />
F703 <strong>and</strong> NL519.<br />
Figure 6: Response to transient expression <strong>of</strong> <strong>in</strong>dicated alleles <strong>of</strong> BLG01 (see also Table 2 <strong>and</strong><br />
Supplemental Figure 2) <strong>in</strong> four separate leaves <strong>of</strong> L. saligna CGN05271 at 6 dpi.
Bremia GKLR prote<strong>in</strong>s <strong>in</strong>duce ETI<br />
145<br />
Lactuca loci confer BLG recognition but not Bremia resistance<br />
To genetically map the locus responsible for the response to BLG01, Backcross<br />
Inbred L<strong>in</strong>es (BILs), which cover 96% <strong>of</strong> the L. saligna CGN05271 genome <strong>in</strong> a<br />
L. sativa Ol<strong>of</strong> background [41] , were tested by transient Agrobacterium-mediated<br />
expression <strong>of</strong> the BLG01 alleles A <strong>and</strong> E. Unexpectedly, none <strong>of</strong> the BILs showed a<br />
cell death response to BLG01 (Table 3). As 4% <strong>of</strong> the L. saligna genome is absent<br />
<strong>in</strong> the set <strong>of</strong> 28 BILs the locus could be located <strong>in</strong> one <strong>of</strong> the four chromosomal<br />
regions (bottom chr 3, top chr 5, top chr 7, bottom chr 9) that are not represented<br />
<strong>in</strong> the BILs [41,42] . From the orig<strong>in</strong>al L. saligna CGN05271 x L. sativa cv. Ol<strong>of</strong><br />
mapp<strong>in</strong>g population [34] viable F3, F4 <strong>and</strong> BC1S1 families were obta<strong>in</strong>ed from<br />
selfed parental l<strong>in</strong>es that were heterozygous or homozygous L. saligna at one <strong>of</strong> the<br />
four regions that were not represented <strong>in</strong> the BILs. Several families were found to<br />
segregate for cell death <strong>in</strong> response to both allele BLG01 A <strong>and</strong> allele BLG01 E (Table<br />
3). Most F3 plants that were responsive to the stronger BLG01 E allele were also<br />
responsive to the BLG01 A allele. Only six plants responsive to BLG01 E were not<br />
visually scored as responsive to BLG01 A probably because <strong>of</strong> the weaker response<br />
to BLG01 A . This segregation was also observed <strong>in</strong> F3 plants from a cross between<br />
the responsive L. saligna parent CGN11341 <strong>and</strong> the non-responsive L. sativa<br />
parent cv. Norden (Table 3). Compar<strong>in</strong>g the parental genotypes from the families<br />
that showed cell-death, revealed that the response to BLG01 was l<strong>in</strong>ked to a region<br />
at the bottom <strong>of</strong> Chromosome 9 that was always homozygous or heterozygous L.<br />
saligna, <strong>in</strong>dicat<strong>in</strong>g that the trait is dom<strong>in</strong>ant. This was confirmed <strong>in</strong> four F1 plants<br />
obta<strong>in</strong>ed from a cross between CGN05271 <strong>and</strong> Ol<strong>of</strong> that were all responsive to the<br />
effector (Table 3). The position <strong>of</strong> the locus on chromosome 9 was further con-<br />
Table 3: Response to BLG01 transient expression <strong>in</strong> L. saligna <strong>and</strong> L. sativa parental l<strong>in</strong>es <strong>and</strong> progeny.<br />
Numbers between brackets <strong>in</strong>dicate the number <strong>of</strong> families.<br />
Assay 1 <strong>and</strong> 2 Assay 3<br />
L<strong>in</strong>es/Populations<br />
# plants BLG01 E BLG01 A # plants BLG01 E<br />
tested # cell death # cell death tested # cell death remark *<br />
L. sativa cv. Ol<strong>of</strong> 6 0 0 3 0 NRP<br />
L. saligna CGN05271 6 6 6 3 3 RP<br />
Set <strong>of</strong> 28 BILs 84 0 0<br />
BC1S1 5 2 (1) 1 (1)<br />
F3 48 15 (11) 13 (9) 74 19 (8)<br />
F4 3 3 (1) 2 (1)<br />
F1 4 4<br />
L. sativa cv. Norden 4 0 0 2 0 NRP<br />
L. saligna CGN11341 7 7 7 3 3 RP<br />
F3 14 7 (4) 5 (4) 3 3 (2)<br />
*<br />
NRP: Non-responsive parent, RP: Responsive parent
146 Chapter 5<br />
CLSM5902<br />
cM<br />
98.2<br />
QGC18F20<br />
CLS4696<br />
CLSX3110<br />
CLS4656<br />
CLSX3880<br />
CLS3349<br />
QGF21K05<br />
LE9038<br />
LE0456<br />
LE3016<br />
F3 Plant Cell death<br />
10_2 NO<br />
30_3 YES<br />
130_2 YES<br />
91_3 YES<br />
103.2<br />
104.3<br />
106.0<br />
110.4<br />
112.1<br />
113.2<br />
113.8<br />
114.3<br />
115.1<br />
115.4<br />
Figure 7: Locus <strong>of</strong> the BLG01 cell-death<br />
response <strong>in</strong> L. saligna CGN05271 at the bottom<br />
<strong>of</strong> Chromosome 9. Genotype graphs for four F3<br />
plants with the closest recomb<strong>in</strong>ation are shown.<br />
Blue is homozygous L. sativa cv. Ol<strong>of</strong>, yellow<br />
is heterozygous, grey represent <strong>in</strong>tervals with a<br />
recomb<strong>in</strong>ation event, <strong>and</strong> white means unknown<br />
genotype. Green <strong>in</strong>dicates the smallest region <strong>in</strong><br />
which the cell-death response to BLG01 is f<strong>in</strong>e<br />
mapped.<br />
Table 4: Detached leaf assay at adult plant stage, ADTG with B. lactucae Bl:24 on genotyped BC1S2<br />
populations from two BC1S1 plants that showed a cell-death response to BLG01 E .<br />
Plant genotype bottom C9 # plants tested # leaf segments Race Bl:24 ISL<br />
BC1S2 90_2<br />
Hom L. sativa 3 18 84<br />
Heterozygous 6 36 75<br />
BC1S2 90_6<br />
Hom L. sativa 3 18 75<br />
Heterozygous 6 36 92<br />
L. sativa cv. Ol<strong>of</strong> Hom L. sativa 3 27 84<br />
Figure 8: Response to BLG03 <strong>in</strong> Dm2-<br />
conta<strong>in</strong><strong>in</strong>g lettuce cultivars. GUS <strong>and</strong> PsojNIP<br />
(NIP) serve as controls for responses to<br />
Agrobacterium <strong>and</strong> successful T-DNA transfer,<br />
respectively.
Bremia GKLR prote<strong>in</strong>s <strong>in</strong>duce ETI<br />
147<br />
firmed <strong>and</strong> more precisely positioned us<strong>in</strong>g F3 families <strong>of</strong> which the F2 parent had<br />
a recomb<strong>in</strong>ation event near the c<strong>and</strong>idate region. The locus could thus be mapped<br />
to a region <strong>of</strong> 4.4 cM between markers CLSX3110 <strong>and</strong> CLSX4656 as shown by the<br />
genotype <strong>of</strong> four <strong>in</strong>formative F3 plants (Figure 7).<br />
The BLG01-triggered response resembles ETI <strong>and</strong> was therefore expected to be<br />
causally l<strong>in</strong>ked to Bremia resistance. As the responsive L. saligna l<strong>in</strong>e CGN05271<br />
is a non-host for Bremia, because <strong>of</strong> multiple quantitative resistance loci [30,43] , we<br />
tested segregat<strong>in</strong>g families for l<strong>in</strong>kage <strong>of</strong> the response to BLG01 <strong>and</strong> the Bremia<br />
resistance phenotype. Surpris<strong>in</strong>gly, both BC1S2 families obta<strong>in</strong>ed from BC1S1<br />
plants 90_2 <strong>and</strong> 90_6 that were responsive to BLG01 were as susceptible to<br />
isolate Bl:24 as the L. sativa cv. Ol<strong>of</strong> parent (Table 4) <strong>in</strong>dicat<strong>in</strong>g that the locus on<br />
chromosome 9 does not confer disease resistance. We conclude that the locus for<br />
responsiveness to BLG01 does not confer resistance to Bremia isolate Bl:24 that<br />
expresses the recognized effector prote<strong>in</strong>.<br />
BLG03 is recognised by lettuce l<strong>in</strong>es Amplus <strong>and</strong> UCDM2 (Supplemental Table<br />
2 <strong>and</strong> Figure 1), which carry known resistance loci to Bremia. Amplus conta<strong>in</strong>s<br />
two R‐genes, Dm2 <strong>and</strong> Dm4, whereas UCDM2 conta<strong>in</strong>s a s<strong>in</strong>gle locus, Dm2. These<br />
two l<strong>in</strong>es therefore have the Dm2 locus <strong>in</strong> common, which is absent from the other<br />
tested l<strong>in</strong>es. This raises the possibility that BLG03 recognition is mediated by the<br />
Dm2-encoded R-prote<strong>in</strong>. We <strong>in</strong>vestigated whether resistance to Bremia isolate Bl:5,<br />
to which the lettuce Dm2 confers resistance, co-segregates with the ability to recognise<br />
BLG03 <strong>in</strong> an F2 population <strong>of</strong> a UCDM2 x Cobham Green cross, <strong>in</strong> which the<br />
Dm2 resistance locus segregates. The response to BLG03 was determ<strong>in</strong>ed by Agrobacterium<br />
<strong>in</strong>filtration <strong>in</strong> leaves, <strong>and</strong> resistance to Bremia Bl:5 was determ<strong>in</strong>ed <strong>in</strong><br />
a leaf disc assay. Of 143 tested F2 plants all but 28 recognised BLG03, <strong>in</strong>dicat<strong>in</strong>g<br />
that the ability to recognise BLG03 is a dom<strong>in</strong>ant trait. All plants that recognised<br />
BLG03 were resistant to Bl:5. Conversely, all plants that did not recognise BLG03<br />
were susceptible to Bl:5. This <strong>in</strong>dicates that Dm2 or a closely l<strong>in</strong>ked gene from<br />
the Dm2 background is required for recognition <strong>of</strong> BLG03. To <strong>in</strong>vestigate this<br />
further we tested BLG03 on additional Dm2-conta<strong>in</strong><strong>in</strong>g l<strong>in</strong>es; Claret (Dm2+3+11),<br />
Maurice (Dm2+16), Mir<strong>and</strong>a (Dm2+7), <strong>and</strong> Parker (Dm2+11). Expression <strong>of</strong><br />
BLG03 triggered a cell death response <strong>in</strong> both Mir<strong>and</strong>a <strong>and</strong> Parker, but only a very<br />
weak response <strong>in</strong> Claret <strong>and</strong> no response <strong>in</strong> Maurice (Figure 8).<br />
As Amplus <strong>and</strong> UCDM2 are susceptible to Bremia isolate Bl:24, from which<br />
we cloned BLG03, we set out to <strong>in</strong>vestigate whether any <strong>of</strong> the other <strong>effectors</strong><br />
cloned from Bl:24 could suppress the cell death-response <strong>in</strong>duced by BLG03. We<br />
<strong>in</strong>oculated a mixture (total OD600 = 0.8) <strong>of</strong> BLG03 <strong>and</strong> <strong>in</strong>dividual other effector<br />
genes at ratios <strong>of</strong> 1:2 (UCDM2) or 1:3 (Amplus). These ratios were determ<strong>in</strong>ed as<br />
the lowest Agrobacterium-BLG03:Agrobacterium-GUS ratio at which a consistent<br />
cell death response could be seen. Us<strong>in</strong>g this setup we did not f<strong>in</strong>d any reduction <strong>of</strong>
148 Chapter 5<br />
the cell death responses <strong>in</strong>duced by BLG03 <strong>in</strong> comb<strong>in</strong>ation with any <strong>of</strong> the 33 other<br />
<strong>effectors</strong> compared to the comb<strong>in</strong>ation <strong>of</strong> BLG03 <strong>and</strong> GUS (not shown). A similar<br />
setup us<strong>in</strong>g BLG01 <strong>in</strong> L. saligna CGN05271 also did not reveal any reduction <strong>of</strong><br />
BLG01-<strong>in</strong>duced cell death responses by other <strong>effectors</strong> (not shown).<br />
Discussion<br />
In planta effector recognition<br />
Two Bremia RXLR-like <strong>effectors</strong> were found to be specifically recognised <strong>in</strong><br />
Lactuca breed<strong>in</strong>g material. A large set <strong>of</strong> wild lettuce species were capable <strong>of</strong><br />
recognis<strong>in</strong>g BLG01 <strong>and</strong> mount<strong>in</strong>g a cell death response. BLG03, <strong>in</strong> contrast, is<br />
recognised specifically <strong>in</strong> two cultivated lettuce l<strong>in</strong>es that share the Dm2 resistance<br />
specificity. Our Bremia effector screen <strong>of</strong> <strong>effectors</strong> for <strong>in</strong> planta recognition has<br />
uncovered potential gene-for-gene <strong>in</strong>teractions. The screen<strong>in</strong>g <strong>of</strong> 54 <strong>effectors</strong> <strong>of</strong> the<br />
oomycete P. <strong>in</strong>festans <strong>in</strong> 10 wild Solanum genotypes by transformation with potato<br />
virus X (PVX) uncovered 36 specific <strong>in</strong>teractions [44] . In contrast, screen<strong>in</strong>g <strong>of</strong> 60<br />
<strong>effectors</strong> <strong>of</strong> the <strong>downy</strong> <strong>mildew</strong> H. arabidopsidis <strong>in</strong> 12 A. thaliana accessions us<strong>in</strong>g<br />
Pseudomonas syr<strong>in</strong>gae pv. DC3000 for delivery did not uncover any hypersensitive<br />
response (cell death) upon effector delivery [25] .<br />
Wild Lactuca species that are Bremia non-hosts can be used to <strong>in</strong>trogress resistance<br />
genes or QTLs <strong>in</strong>to lettuce cultivars. For example, the Dm3 resistance gene<br />
orig<strong>in</strong>ates from a L. serriola accession, but is very rare <strong>in</strong> natural populations, with<br />
only a s<strong>in</strong>gle accession <strong>of</strong> 1033 tested from 49 natural populations hav<strong>in</strong>g an <strong>in</strong>tact<br />
Dm3 gene [45] . Dm3 is a fast evolv<strong>in</strong>g resistance gene from the RGC2 locus. This<br />
locus encodes two types <strong>of</strong> resistance gene c<strong>and</strong>idates, a fast evolv<strong>in</strong>g type <strong>and</strong><br />
a type that evolves at a much slower rate <strong>and</strong> is more conserved among different<br />
accessions [28] . Recognition <strong>of</strong> the Bremia effector BLG01 appears to be relatively<br />
common <strong>in</strong> L. saligna species <strong>and</strong> to only occur sporadically <strong>in</strong> other species. The<br />
wide recognition <strong>of</strong> BLG01 may <strong>in</strong>dicate recognition by a slowly evolv<strong>in</strong>g or more<br />
ancient resistance gene. The exact nature <strong>of</strong> the recognition <strong>of</strong> BLG01 <strong>in</strong> L. saligna<br />
<strong>and</strong> whether this recognition is dependent on the same gene <strong>in</strong> all l<strong>in</strong>es rema<strong>in</strong>s to<br />
be determ<strong>in</strong>ed.<br />
The recognition <strong>of</strong> BLG01 <strong>in</strong> L. saligna CGN05271 was mapped at the bottom<br />
<strong>of</strong> chromosome 9, where no R‐gene clusters to Bremia <strong>in</strong> L. serriola <strong>and</strong> L. sativa<br />
are known so far [37,46] . None <strong>of</strong> the 23 lettuce EST sequences from the Lettuce<br />
SFP Chip Project (http://chiplett.ucdavis.edu) that are mapped with<strong>in</strong> the 4.4 cM<br />
region between our flank<strong>in</strong>g markers (CLSX3110 & CLS4656) show homology<br />
to NB-LRR-like resistance prote<strong>in</strong>s. We are aware <strong>of</strong> only three R‐genes from L.<br />
saligna that have been <strong>in</strong>trogressed <strong>in</strong>to L. sativa, none <strong>of</strong> which are located on
Bremia GKLR prote<strong>in</strong>s <strong>in</strong>duce ETI<br />
149<br />
chromosome 9 [47, 57, 58] . Interest<strong>in</strong>gly, the cell death response to the P. syr<strong>in</strong>gae<br />
effector AvrPto did map <strong>in</strong> the same region on chromosome 9 <strong>in</strong> a L. sativa x L.<br />
serriola RIL population [48] .<br />
BLG03 is recognised <strong>in</strong> only two lettuce breed<strong>in</strong>g l<strong>in</strong>es <strong>of</strong> the <strong>in</strong>itial screen.<br />
These l<strong>in</strong>es share the Dm2 resistance locus, which maps <strong>in</strong> or near the RGC2<br />
locus [27] . The Dm2 locus provides resistance to Bl:5, <strong>and</strong> recognition <strong>of</strong> BLG03<br />
correlated with resistance to Bl:5 <strong>in</strong> 143 F2 plants <strong>of</strong> a UCDM2 x Cobham Green<br />
cross, <strong>in</strong> which Dm2 segregates. This <strong>in</strong>dicates that the response to BLG03 maps<br />
to the RGC2 locus <strong>in</strong> UCDM2. Of four additional l<strong>in</strong>es that are reported to conta<strong>in</strong><br />
Dm2, two also showed a response towards BLG03. The two l<strong>in</strong>es that did not show<br />
a response to BLG03, Claret <strong>and</strong> Maurice, also have other resistance specificities<br />
that map to the RGC2 locus. As we have not yet tested the response to BLG03<br />
from Bl:5, it is too early to conclude that the effector is not recognised by the<br />
Dm2-encoded prote<strong>in</strong>.<br />
Sequenc<strong>in</strong>g <strong>of</strong> BLG03 <strong>in</strong> eight different isolates revealed 7 different alleles.<br />
Of the sequenced Bremia isolates, Bl:5 <strong>and</strong> F703 are unable to successfully <strong>in</strong>fect<br />
Dm2-conta<strong>in</strong><strong>in</strong>g hosts. Both Bl:5 BLG03 alleles were not found <strong>in</strong> other sequenced<br />
Bremia isolates. We were not able to amplify BLG03 from Bremia isolate F703,<br />
although the quality <strong>of</strong> DNA was not a problem as we could amplify BLG01 <strong>and</strong><br />
BLG02. The two Bl:5 BLG03 alleles rema<strong>in</strong> to be cloned <strong>and</strong> tested <strong>in</strong> the different<br />
Dm2-conta<strong>in</strong><strong>in</strong>g l<strong>in</strong>es to determ<strong>in</strong>e whether responses to these alleles differ from<br />
those to the Bl:24 reference allele.<br />
Effector recognition versus resistance<br />
In most reported cases effector recognition is l<strong>in</strong>ked to resistance. Recognition <strong>of</strong><br />
BLG01, however, was not l<strong>in</strong>ked to resistance to Bl:24 <strong>in</strong> laboratory assays, <strong>and</strong><br />
BLG03 was cloned from a Bremia isolate that can successfully <strong>in</strong>fect the lettuce<br />
l<strong>in</strong>es <strong>in</strong> which BLG03 is recognised. Examples <strong>of</strong> a lack <strong>of</strong> correlation between<br />
recognition <strong>of</strong> an effector <strong>and</strong> resistance to pathogen isolates that express the<br />
effector have previously been reported. Screen<strong>in</strong>g <strong>of</strong> 54 P. <strong>in</strong>festans <strong>effectors</strong> <strong>in</strong><br />
Solanum species by Potato Virus X (PVX) expression uncovered two <strong>in</strong>teractions<br />
that were not correlated to resistance. In an F2 population <strong>of</strong> a cross between a<br />
resistant <strong>and</strong> susceptible Solanum species no correlation between the ability to<br />
recognise certa<strong>in</strong> P. <strong>in</strong>festans <strong>effectors</strong> <strong>and</strong> resistance to P. <strong>in</strong>festans was found,<br />
despite perfect correlation between recognition <strong>of</strong> other P. <strong>in</strong>festans <strong>effectors</strong> <strong>and</strong><br />
resistance [44] . Likewise, not all A. thaliana accessions that recognise ATR39-1 or<br />
ATR1Emco5 are resistant to isolates that encode these alleles [49–51] .<br />
An explanation for the lack <strong>of</strong> resistance <strong>in</strong> plants that can recognise <strong>in</strong>dividual<br />
Bremia <strong>effectors</strong> is that Bremia uses additional <strong>effectors</strong> to suppress ETI. A study
150 Chapter 5<br />
that <strong>in</strong>vestigated crosses <strong>of</strong> virulent <strong>and</strong> avirulent Bremia isolates revealed a<br />
possible locus that <strong>in</strong>hibits avirulence triggered by Avr5/8, but no evidence for<br />
other <strong>in</strong>hibitors <strong>of</strong> avirulence <strong>in</strong> Bremia [52] . The study highlights that <strong>in</strong>hibitor<br />
loci exist, but that they are polymorphic. S<strong>in</strong>ce this study was performed over 20<br />
years <strong>of</strong> selective pressure on Bremia has given rise to new isolates that break<br />
various resistances. However, none <strong>of</strong> our 34 tested <strong>effectors</strong> were able to suppress<br />
BLG01- or BLG03-<strong>in</strong>duced cell death. We cannot rule out the existence <strong>of</strong> suppressors<br />
<strong>of</strong> cell death as our selection <strong>of</strong> <strong>effectors</strong> is non-exhaustive <strong>and</strong> suppressors<br />
may even be non-RXLR <strong>effectors</strong>, which we did not <strong>in</strong>vestigate.<br />
F<strong>in</strong>d<strong>in</strong>gs described for the P. <strong>in</strong>festans effector AvrSmira2 [53] provide an alternative<br />
explanation for an apparent lack <strong>of</strong> correlation between effector recognition<br />
<strong>and</strong> resistance. Agrobacterium-mediated transient transformation <strong>of</strong> potato cultivar<br />
‘Sarpo Mira’ with AvrSmira2 <strong>in</strong>duced a cell death response <strong>and</strong> <strong>in</strong>dicates the presence<br />
<strong>of</strong> a resistance factor named Rpi-Smira2. Cell death responses to AvrSmira2<br />
were found to segregate <strong>in</strong> <strong>of</strong>fspr<strong>in</strong>g <strong>of</strong> a cross between an Rpi-Smira2-conta<strong>in</strong><strong>in</strong>g<br />
parent <strong>and</strong> a universally susceptible parent. As with our Bremia effector BLG03,<br />
no resistance to P. <strong>in</strong>festans stra<strong>in</strong>s conta<strong>in</strong><strong>in</strong>g AvrSmira2 could be scored <strong>in</strong> plants<br />
that show a cell death response towards AvrSmira2 <strong>in</strong> laboratory assays. Field<br />
trials, however, revealed a partial resistance phenotype correlated with the ability<br />
to recognise AvrSmira2. Therefore, field trials <strong>in</strong> plants conta<strong>in</strong><strong>in</strong>g the L. saligna<br />
locus for recognition <strong>of</strong> BLG01 are important to determ<strong>in</strong>e whether the locus<br />
confers a partial resistance phenotype <strong>and</strong> if the locus is <strong>of</strong> value for breed<strong>in</strong>g<br />
Bremia-resistant lettuce.
Bremia GKLR prote<strong>in</strong>s <strong>in</strong>duce ETI<br />
151<br />
Materials <strong>and</strong> Methods<br />
Clon<strong>in</strong>g, sequenc<strong>in</strong>g <strong>and</strong> qPCR experiments<br />
Effectors were TOPO-cloned as described <strong>in</strong> Chapter 4. The PsojNIP <strong>and</strong> YFP<br />
constructs were k<strong>in</strong>dly provided by A. Cabral [54] . Clones were electro-transformed<br />
<strong>in</strong>to Agrobacterium tumefaciens stra<strong>in</strong> C58C1_pGV2260. For sequenc<strong>in</strong>g <strong>of</strong> effector<br />
alleles, DNA was PCR amplified us<strong>in</strong>g primers flank<strong>in</strong>g the cod<strong>in</strong>g sequence,<br />
blunt-end ligated <strong>in</strong>to pJET1.2 (Fermentas), <strong>and</strong> transformed <strong>in</strong>to E. coli DH5α<br />
by heat-shock. Plasmid isolation <strong>and</strong> sequenc<strong>in</strong>g was carried out by Macrogen<br />
Inc. Time-course sampl<strong>in</strong>g <strong>and</strong> qPCR <strong>analysis</strong> were performed as described <strong>in</strong><br />
chapter 4. All primers used are listed <strong>in</strong> Supplementary Table 3.<br />
Agrobacterium-mediated transient transformation assay<br />
Agrobacterium stra<strong>in</strong>s were prepared for <strong>in</strong>filtration as described <strong>in</strong> chapter 4.<br />
Stra<strong>in</strong>s were then pressure <strong>in</strong>filtrated <strong>in</strong>to leaves us<strong>in</strong>g a needleless syr<strong>in</strong>ge.<br />
Responses <strong>in</strong> lettuce l<strong>in</strong>es were scored 8 dpi, unless otherwise <strong>in</strong>dicated.<br />
Codon-based test for positive selection<br />
Insertions <strong>and</strong> deletions were removed from sequences <strong>and</strong> sequences with <strong>in</strong>ternal<br />
stop codons were removed. Substitution rates were calculated us<strong>in</strong>g Nei <strong>and</strong><br />
Gojobori’s method [55] , us<strong>in</strong>g MEGA4 [56] . St<strong>and</strong>ard error was determ<strong>in</strong>ed by 500<br />
bootstrap replications. The null hypothesis <strong>of</strong> no selection (dN = dS) versus the<br />
positive selection hypothesis (dN > dS) were determ<strong>in</strong>ed us<strong>in</strong>g the Z-test: Z = (dNdS)/<br />
√(Var(dS)+Var(dN)).<br />
Materials for mapp<strong>in</strong>g the BLG01 response<br />
Two crosses <strong>of</strong> L. saligna <strong>and</strong> L. sativa were previously made: Cross 1: L. saligna<br />
CGN05271 x L. sativa cv. Ol<strong>of</strong> <strong>and</strong> Cross 2: L. saligna CGN11341 x L. sativa cv.<br />
Norden [34] . Materials for Assays 1, 2, <strong>and</strong> 3 were as follows. Assay 1: three replicates<br />
<strong>of</strong> 28 BILs derived from Cross 1 that together cover 96% <strong>of</strong> the L. saligna<br />
CGN5271 genome [41] , <strong>and</strong> the parental l<strong>in</strong>es <strong>of</strong> Cross 1 <strong>and</strong> Cross 2. Assay 2: F3,<br />
F4 <strong>and</strong> BC1S1 plants derived from Cross 1 <strong>and</strong> Cross 2. Assay 3: parental l<strong>in</strong>es <strong>of</strong><br />
Cross 1 <strong>and</strong> Cross 2, F1 <strong>of</strong>fspr<strong>in</strong>g <strong>of</strong> Cross 1, <strong>and</strong> F3 <strong>of</strong>fspr<strong>in</strong>g <strong>of</strong> F2 plants <strong>of</strong> both<br />
Cross 1 <strong>and</strong> Cross 2 with a recomb<strong>in</strong>ation near the C9 locus.
152 Chapter 5<br />
Marker development <strong>and</strong> genotyp<strong>in</strong>g<br />
For f<strong>in</strong>e mapp<strong>in</strong>g new markers were developed <strong>and</strong> selected to saturate the region.<br />
Based on alignment <strong>of</strong> our F2 map (improved version <strong>of</strong> Jeuken 2001) with the<br />
L. sativa x L. serriola RIL map by the Lettuce SFP Chip Project (http://chiplett.<br />
ucdavis.edu) <strong>and</strong> the Compositae Genome Project Database (CGPDB) (http://<br />
compgenomics.ucdavis.edu) we selected c<strong>and</strong>idate EST sequences <strong>and</strong> markers.<br />
Primers were developed, tested <strong>and</strong> <strong>in</strong> case <strong>of</strong> polymorphism run on the segregat<strong>in</strong>g<br />
populations for mapp<strong>in</strong>g. Polymorphisms between L. sativa <strong>and</strong> L. saligna PCR<br />
products <strong>of</strong> the EST markers were visualized by high-resolution melt<strong>in</strong>g curve<br />
differences on a LightScanner System (Idaho Technology). All plants with a cell<br />
death response <strong>in</strong> transient assay 2 <strong>and</strong> all the plants from assay 3 were genotyped<br />
us<strong>in</strong>g the new markers.<br />
Disease test on adult plants<br />
A detached leaf assay was conducted on adult plants accord<strong>in</strong>g the protocol <strong>of</strong><br />
ADTG as previously described [42] . Two genotyped BC1S2 populations from<br />
BC1S1 plant that showed a cell death response towards BLG01 were tested with<br />
Bremia Bl:24. From each plant at least six leaf squares were collected (2.5 x 2.5<br />
cm). Leaf squares were <strong>in</strong>oculated with <strong>in</strong>oculum from Bremia isolate Bl:24 at<br />
37 days post sow<strong>in</strong>g conta<strong>in</strong><strong>in</strong>g 4 x 10 5 spores per ml. At 9 dpi the percentage <strong>of</strong><br />
the area <strong>of</strong> each leaf square that showed Bremia sporulation was determ<strong>in</strong>ed. One<br />
way Analysis <strong>of</strong> Variances was used to analyse the data (with as fixed factor: l<strong>in</strong>e<br />
(<strong>of</strong>fspr<strong>in</strong>g from one parent with genotype at C9 locus) <strong>and</strong> as block factor: each<br />
different plant). For comparison between the heterozygous <strong>and</strong> homozygous L.<br />
sativa genotypes from each BC1S1 parent with each other, a Duncan’s multiple<br />
range test (α = 0.05) was performed with GenStat (14 th Edition) s<strong>of</strong>tware.
Bremia GKLR prote<strong>in</strong>s <strong>in</strong>duce ETI<br />
153<br />
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Supplemental Information<br />
Supplemental Table 1: List <strong>of</strong> Lactuca l<strong>in</strong>es used for screen<strong>in</strong>g Bremia <strong>effectors</strong>.<br />
The universal name, as well as known synonyms are <strong>in</strong>dicated. The species <strong>of</strong> Lactuca to which each<br />
l<strong>in</strong>e belongs is <strong>in</strong>dicated (Lactuca sp.). The ‘Gene(s)’ column <strong>in</strong>dicates current knowledge <strong>of</strong> resistance<br />
genes, if any, for each l<strong>in</strong>e. The use <strong>of</strong> the l<strong>in</strong>e as a parent for BIL crosses, or as a part <strong>of</strong> a differential<br />
set is <strong>in</strong>dicated under comments. Additional references (Ref.) are as follows:<br />
A - http://calgreens.org/control/uploads/Michelmore_<strong>and</strong>_Ochoa_-_Breed<strong>in</strong>g_Crisphead_Lettuce.pdf,<br />
B - http://compgenomics.ucdavis.edu/morphodb/<strong>analysis</strong>/viewTrait.php,<br />
C - http://www.worldseed.org/isf/ibeb.html,<br />
D - http://documents.plant.wur.nl/cgn/website/downloads/download/Cnr06Trait406.zip,<br />
Universal Name Synonym Lactuca sp. Gene(s) Comment Ref.<br />
CGN15692 aculeata<br />
[31]<br />
CGN04664 altaica<br />
[31]<br />
CGN15711 altaica<br />
[31]<br />
CGN04662 saligna<br />
[33]<br />
CGN05147 saligna<br />
[33]<br />
CGN05157 saligna<br />
[33]<br />
CGN05265 saligna<br />
[33]<br />
CGN05267 saligna<br />
[33]<br />
CGN05271 saligna BIL-parent<br />
[33,34]<br />
CGN05282 saligna<br />
[33]<br />
CGN05301 saligna<br />
[33]<br />
CGN05304 saligna<br />
[33]<br />
CGN05306 saligna<br />
[33]<br />
CGN05308 PIVT1267 saligna<br />
[33]<br />
CGN05309 saligna<br />
[33]<br />
CGN05310 saligna<br />
[33]<br />
CGN05311 saligna<br />
[33]<br />
CGN05313 saligna<br />
[33]<br />
CGN05314 saligna<br />
[33]<br />
CGN05315 saligna<br />
[33]<br />
CGN05317 saligna<br />
[33]<br />
CGN05318 saligna<br />
[33]<br />
CGN05320 saligna<br />
[33]<br />
CGN05321 PIVT1346 saligna<br />
[33]<br />
CGN05322 saligna<br />
[33]<br />
CGN05323 saligna<br />
[33]<br />
CGN05324 saligna<br />
[33]<br />
CGN05325 saligna<br />
[33]<br />
CGN05327 saligna<br />
[33]<br />
CGN05329 saligna<br />
[33]<br />
CGN05330 saligna<br />
[33]<br />
CGN05796 saligna<br />
[33]<br />
CGN05882 saligna<br />
[33]<br />
CGN05895 saligna<br />
[33]<br />
CGN05947 saligna<br />
[33]<br />
CGN09311 saligna<br />
[33]<br />
CGN09313 saligna<br />
[33]
Bremia GKLR prote<strong>in</strong>s <strong>in</strong>duce ETI<br />
159<br />
Universal Name Synonym Lactuca sp. Gene(s) Comment Ref.<br />
CGN09314 saligna<br />
[33]<br />
CGN10888 saligna<br />
[33]<br />
CGN11341 UC83US1 saligna BIL-parent<br />
[34]<br />
CGN13326 PI491206 saligna<br />
[33]<br />
CGN13327 PI491204 saligna<br />
[33]<br />
CGN13330 saligna<br />
[33]<br />
CGN14263 saligna<br />
[33]<br />
HRI11140 saligna<br />
[33]<br />
LJ85314 saligna R33<br />
[33],A<br />
PI491000 saligna<br />
[33],B<br />
PI491207 saligna<br />
[33]<br />
PI491208 saligna<br />
[33]<br />
PI491226 saligna<br />
[33],B<br />
PI503623 saligna<br />
[33]<br />
PI509523 saligna<br />
[33],B<br />
PI509524 saligna<br />
[33],B<br />
UC94ISR1 saligna<br />
[33]<br />
UC94US104 saligna<br />
[33]<br />
AMPLUS sativa Dm2,Dm4 new DM-resistance<br />
ARGELES sativa Dm24/38 EU-A/B differential set<br />
C<br />
BALESTA sativa R EU-B differential set<br />
C<br />
BEDFORD sativa R EU-B differential set<br />
C<br />
BELLISSIMO sativa R EU-B differential set<br />
C<br />
CAPITAN sativa Dm11 EU-A/B differential set<br />
C<br />
CG DM16 sativa Dm16 EU-B differential set<br />
C<br />
COBHAM<br />
no<br />
sativa<br />
GREEN<br />
Dm genes<br />
EU-A differential set<br />
C<br />
COLORADO sativa Dm18 EU-A/B differential set<br />
C<br />
DANDIE sativa Dm3 EU-A/B differential set<br />
C<br />
DESIGN sativa R c<strong>and</strong>idate differential set IBEB C<br />
DISCOVERY<br />
sativa<br />
Dm7,<br />
Dm36/37<br />
EU-A/B differential set<br />
C<br />
GRAND RAPIDS sativa<br />
[36]<br />
GREEN TOWERS<br />
sativa<br />
no<br />
Dm genes<br />
EU-B differential set<br />
C<br />
HILDE sativa Dm12 EU-A/B differential set<br />
C<br />
ICEBERG sativa<br />
[36,35]<br />
KIGALIE sativa R c<strong>and</strong>idate differential set IBEB C<br />
LEDNICKY sativa Dm1 EU-A/B differential set<br />
C<br />
LJ85289<br />
NINJA<br />
sativa<br />
(virosa)<br />
sativa<br />
R35<br />
Dm3,<br />
Dm11,<br />
Dm36/37<br />
R-gene orig<strong>in</strong>ally from L. virosa [33],A<br />
EU-A/B differential set<br />
NORDEN sativa BIL-parent<br />
[34]<br />
NUN Dm15 sativa Dm15 EU-B differential set<br />
C<br />
NUN DM17 sativa Dm17 EU-B differential set<br />
C<br />
OLOF sativa BIL-parent<br />
[34]<br />
PENNLAKE sativa Dm13 EU-A/B differential set<br />
C<br />
PRIMUS sativa<br />
[38]<br />
R4T57D sativa Dm4 EU-A/B differential set<br />
C<br />
C
160 Chapter 5<br />
Universal Name Synonym Lactuca sp. Gene(s) Comment Ref.<br />
RYZ2164 sativa Dm25 EU-B differential set<br />
C<br />
RYZ910457 sativa R EU-B differential set<br />
C<br />
SABINE sativa Dm6 EU-A/B differential set<br />
C<br />
SALADIN sativa<br />
[35]<br />
UC2202 sativa R33<br />
A<br />
UC2203 sativa R34<br />
A<br />
UC2204 sativa R35<br />
A<br />
UC2205 sativa R28<br />
A<br />
UC2206 sativa R29<br />
A<br />
UCDM 10 sativa Dm10 EU-A/B differential set<br />
C<br />
UCDM 14 sativa Dm14 EU-A/B differential set<br />
C<br />
UCDM 2 sativa Dm2 EU-A/B differential set<br />
C<br />
VALMAINE sativa Dm5/8 EU-A/B differential set<br />
C<br />
CGN05913 serriola<br />
[39]<br />
CGN05916 serriola<br />
[33]<br />
CGN10887 serriola<br />
D<br />
CGN14255 serriola<br />
[33]<br />
CGN14271 serriola<br />
[33]<br />
CGN14278 serriola<br />
[33]<br />
LJ85292 serriola R34<br />
[33],A<br />
LS 102 serriola Dm17/25 EU-A differential set<br />
C<br />
LSE 57/15 serriola Dm7 EU-A differential set<br />
C<br />
LSE/18 serriola Dm16 EU-A differential set<br />
C<br />
PI491108 serriola<br />
[33]<br />
PIVT 1309 serriola Dm15 EU-A/B differential set<br />
C<br />
UC96US23 serriola<br />
[37]<br />
W66331A serriola R29<br />
[33]<br />
W66336A serriola R28<br />
[33]<br />
CGN04683 PIVT280 virosa<br />
[33]<br />
CGN05148 virosa<br />
[33]<br />
CGN05332 virosa<br />
[33]<br />
CGN05333 virosa<br />
[33]<br />
CGN05816 virosa<br />
[31]<br />
CGN05941 virosa<br />
[31]<br />
CGN05978 virosa<br />
[31]<br />
CGN09364 virosa<br />
[31]<br />
CGN09365 PIVT1398 virosa<br />
[39]<br />
CGN13302 virosa<br />
[31]<br />
CGN13339 virosa<br />
[31]<br />
CGN13356 virosa<br />
[31]<br />
CGN13357 virosa<br />
[31]<br />
CGN17436 virosa<br />
[31]<br />
CGN18635 virosa<br />
[31]<br />
CGN19040 virosa<br />
[31]<br />
CGN22690 virosa<br />
[31]<br />
CGN23887 virosa<br />
[31]
Bremia GKLR prote<strong>in</strong>s <strong>in</strong>duce ETI<br />
161<br />
Supplemental Table 2 (next page <strong>and</strong> page thereafter): Recognition <strong>of</strong> <strong>effectors</strong> <strong>in</strong> lettuce breed<strong>in</strong>g<br />
l<strong>in</strong>es.<br />
The Lactuca species <strong>of</strong> each l<strong>in</strong>e is <strong>in</strong>dicated under ‘Sp.’, acu: aculeata, alt: altaica, sal: saligna, sat:<br />
sativa, ser: serriola, vir: virosa. More details <strong>of</strong> the Lactuca l<strong>in</strong>es can be found <strong>in</strong> Supplemental Table 1.<br />
Colours represent three scor<strong>in</strong>g levels, green for a low average score (no effects observed, score ≤0.7),<br />
yellow for an <strong>in</strong>termediate score (mild/<strong>in</strong>consistent effects observed) <strong>and</strong> red for a high average score<br />
(strong response observed, score ≥1.3). The number <strong>of</strong> biological replicates for each lettuce/effector<br />
comb<strong>in</strong>ation is <strong>in</strong>dicated by the <strong>in</strong>tensity <strong>of</strong> the colour. Lettuce/effector comb<strong>in</strong>ations for which no data<br />
is available are <strong>in</strong>dicated <strong>in</strong> grey.<br />
Supplemental Table 3: Primers <strong>and</strong> markers used <strong>in</strong> this study<br />
Gene Type Forward primer Reverse primer<br />
BLG01 Flank<strong>in</strong>g AAACGCGATAAGGTCTCAAAA TGACTTCGGTCCTCATTAAAC<br />
BLG01 Clon<strong>in</strong>g ED CACCATGGCTTTCAGTAATTTACTTGAACATCT TCAATTTCGTTTGGCGTTGGG<br />
BLG01 qPCR GTGCTTGCGATCTCGATGTA GGCTTCTTGGTCGATCTGTC<br />
BLG02 Flank<strong>in</strong>g AATTTCTATTGCGAGCACAGC GTTCGCATGACTCACACCTC<br />
BLG02 qPCR GACGAAGCAAGCGTGTACTG TCGGTTACGTCGAACGTTTT<br />
BLG03 Flank<strong>in</strong>g AAGATGTCGCAACGCAGAG TCCACTTGGCAAAGAGCAC<br />
BLG03 Clon<strong>in</strong>g ED CACCATGTTTCTAGACGGATTAAAAGCTCTTG TCACATCTCAACGTCGCTAGGTG<br />
BLG03 qPCR AAAAGGCTAGCGAGGAACAA CACATCTCAACGTCGCTAGG<br />
Lettuce<br />
Act<strong>in</strong>2<br />
qPCR CTATCCAGGCTGTGCTTTCC ACCCTTCGTAGATCGGGACT<br />
Bremia<br />
Act<strong>in</strong>2<br />
qPCR AGGCCGTGCTGTCTCTCTAC GCGCATAGCCTTCGTAGATT<br />
Marker name A Forward primer Reverse primer<br />
CLSM5902 AGGTACGCGAATTTGACCAC CACAACGGAAGAAACCGAAT<br />
QGC18F20 AGGTGACAGATGGGACCAAG GTATTCCGCCCACAGTTGAT<br />
CLS4696 AATCTCCAGCTTCGGGTTTT ACTACGAAACGACCCATTGC<br />
CLSX3110 CCACGGCTAGGGTCAGATTA TGAAGACATGGGAGACGACA<br />
CLS4656 CCGTATGCCGTTCATCTTCT GCACTCCAATTGAATGATCG<br />
CLSX3880 TTGCGAGATCCGTATCAGTG CTCCCACAGTTCCGCTAATC<br />
CLS3349 CTTTTTGGAAGGCAATCTGG TCCAGGGAAAACCATCTTTG<br />
QGF21K05 CAAAATGTTGCAGCGTCTGT GGGGGAGGGTCATAACAGTAA<br />
LE9038 GATGGAGCGTCCGATCAGTGTCTG GGATCACCATCATAGTCAGCTTGT<br />
LE0456 TTTGCTTGCTTGTTGGTTCA TCTGTGAAATCACCCTTGCC<br />
LE3016 ACCACCGCTTTCTCACAGTT TGGCCATTCGACCTTTACTC<br />
A<br />
Markers that start their name with LE were derived from the Compositae Genome Project Database<br />
(CGPDB)(http://compgenomics.ucdavis.edu). The orig<strong>in</strong>al EST contigs from which those markers<br />
were designed were QGD7H11.yd.ab1 for LE9038, QGG19L12.yg.ab1 for LE0456, <strong>and</strong> QG_CA_<br />
Contig2163 for LE3016. Markers that start with C or Q were designed based on EST contigs <strong>in</strong> the<br />
Lettuce SFP Chip Project (http://chiplett.ucdavis.edu).
162 Chapter 5
Bremia GKLR prote<strong>in</strong>s <strong>in</strong>duce ETI<br />
163
164 Chapter 5<br />
BLG01 E<br />
Strong<br />
10 0<br />
23<br />
BLG01 A<br />
Strong<br />
11<br />
1<br />
BLG01 A<br />
Weak<br />
4<br />
1<br />
6<br />
BLG01 E<br />
Weak<br />
Supplemental Figure 1: Overlap <strong>in</strong> responses <strong>of</strong> Lactuca l<strong>in</strong>es to BLG01 A <strong>and</strong> BLG01 E .<br />
Based on data <strong>in</strong> Supplemental Table 2. Strong responses are those with a score > 1.3 (red colours <strong>in</strong><br />
Supplemental Table 2), <strong>and</strong> weak responses those > 0.7 but ≤ 1.3 (yellow colours <strong>in</strong> Supplemental<br />
Table 2). For determ<strong>in</strong><strong>in</strong>g the overlap <strong>in</strong> responses to the effector alleles all scores were taken <strong>in</strong>to<br />
account, irrespective <strong>of</strong> the number <strong>of</strong> replicates on which the score is based (<strong>in</strong> contrast to the data <strong>in</strong><br />
Table 1, which only <strong>in</strong>cludes positive scores based on at least two replicates). Weak scores are more<br />
likely to be the result <strong>of</strong> falsely scor<strong>in</strong>g <strong>of</strong> <strong>in</strong>filtration damage as a response towards the effector, e.g.<br />
all four l<strong>in</strong>es that were scored as show<strong>in</strong>g a weak response only to BLG01 A each have an average<br />
score <strong>of</strong> 1 based on two replicates <strong>of</strong> which one was scored as 2 (strong <strong>in</strong>teraction), but the other as 0<br />
(no <strong>in</strong>teraction).<br />
Supplemental Information 2: Alignment <strong>of</strong> prote<strong>in</strong> translations <strong>of</strong> different alleles <strong>of</strong> BLG01, BLG02<br />
<strong>and</strong> BLG03.<br />
BLG01<br />
A MVRVYVAALTGFLALSASASATLQLTSVNESLADAYDSTAPARGKLRAYAATNVESDERA<br />
B ...--.......................................................<br />
C ............................................................<br />
D .......................*....................................<br />
E ....*.......................................................<br />
F ....*...............................A.......................<br />
G ............................................................<br />
A FSNLLEHLKTLNVLFSPLSKERMEAAISKSDSSVVKHLSEDGAVNEKKLGIGRDGVKAFE<br />
B ............................................................<br />
C ............................................................<br />
D ............................................................<br />
E ........................................................E...<br />
F ............................................................<br />
G ............................................................<br />
A PAKVSKVKMILEDIFRKPYNKILLKVLSKANGGERNLVRNLAQAEMLGYKVFFLKSTLAS<br />
B .............................................I..F...........<br />
C ............................................................<br />
D ................................................F...........<br />
E ............................................................<br />
F ................................................F...........<br />
G ................................................F...........
Bremia GKLR prote<strong>in</strong>s <strong>in</strong>duce ETI<br />
165<br />
A KWERDGVSLMDVWSYICKDVKAPTEEEMKIFSHWCGYAFVLSAKGKFSAEETVIEKTLLK<br />
B ....................N.......................................<br />
C ....................N.........................L.............<br />
D ....................N.......................................<br />
E ....................N.......................................<br />
F ....................N.......................................<br />
G ....................N.......................................<br />
A VHNDNKNKKGKLAINMLAQIHLSRKFKVDANFFELYDPSPSSLKKLLLELEKSPRACDLD<br />
B .........D..............................................Y...<br />
C ............................................................<br />
D ........................................................Y...<br />
E .........D..............................F.T.............Y...<br />
F ........................................................Y...<br />
G .............................................IA*------------<br />
A VELYRLLKQAVTDDKFMKGIWDRSTKKPEQPNAKRN<br />
B ....................................<br />
C ....................................<br />
D ....................................<br />
E ..........................T-........<br />
F ......................SRPRSLNSPTPNEIX<br />
G ------------------------------------<br />
BLG02<br />
A MVRIYVAALTVVLAFSASCSATSPLTLAKATPVTANGSGGRAQGRLRAHTTTNVEIDERS<br />
B ............................................................<br />
C ............................................................<br />
D ............................................................<br />
E ............................................................<br />
F ............................................................<br />
A ISDVIYAIARQVADHRSNLVQAPKRTYTTYLATLDLSLDKVLEQNWSELRLLYVKQTKRK<br />
B ............................................................<br />
C ............................................................<br />
D ............................................................<br />
E ..........................C.................................<br />
F .L..V............G..........................................<br />
A RPKDFVSMYELLVKKYGLLKLTDMMKKPDYISLSETDNFKYLYDEASVYWRKNSVLKQYA<br />
B ............................................................<br />
C ............................................................<br />
D ............................................................<br />
E ............................................................<br />
F ............................................................<br />
A SELYDSKGNSKTFDVTDFESLQPFFERIGRRGDYLEITAAEHEKWKTNKLRPT<br />
B .....................................................<br />
C .........G.K.........K....C..........................<br />
D .........G.K.........K..............N................<br />
E .....................................................<br />
F .....................................................<br />
BLG03<br />
A MVRVYVAALAAIFALS--ASATLHLTLANASSVTTEEGDGRPQGKLRVNAATNVESDERF<br />
B ................--..........................................<br />
C ................--..........................................<br />
D ................--..........................................<br />
E ..............F.AS..........................................<br />
F ..............F.--............P...A........................S<br />
G ..............F.--............P...A.......H.................<br />
A LDGLKALVRGFYDLTPFAPRFQKDTYSNLLLKLNLSFEQFSGKDTLQLRRFYDAARRHNK<br />
B ............................................................<br />
C ............................................................<br />
D ............................................................<br />
E .................................................Q..........<br />
F ............................*....................Q..........<br />
G ......F..............K....P......D..........................
166 Chapter 5<br />
A VNPTNPVSVYTGLVEKYGEFEIVNMVYSLKHAQSSKSRDVLKRLGKEEKWYWKDKKDAGK<br />
B ..............................D.............................<br />
C ............................................................<br />
D ............................................................<br />
E ............................................................<br />
F ............................................................<br />
G L..RT....D.........................E........................<br />
A MYAEALDLGNEFTVKNMVSKLSKLEQFLNRIDQKASEEQLKALVVADIERVKTKKESVSP<br />
B ............................................................<br />
C .....I......................................................<br />
D ....................................................M.......<br />
E .................I..................................M.......<br />
F ....................................................M.......<br />
G R...........................................................<br />
A SDVEM<br />
B .....<br />
C .....<br />
D ...A.<br />
E ...A.<br />
F ...A.<br />
G .....<br />
Supplemental Information 1: Best BLAST hit <strong>of</strong> BLG01, BLG02, <strong>and</strong> BLG03 to an oomycete prote<strong>in</strong>.<br />
BLG01<br />
>PITG_15128T0 | PITG_15128 | Phytophthora <strong>in</strong>festans T30-4 RXLR effector family,<br />
putative (332 aa)<br />
Length = 331<br />
Score = 37.4 bits (85), Expect = 0.037, Method: Compositional matrix adjust<br />
Identities = 31/96 (32%), Positives = 46/96 (47%), Gaps = 12/96 (12%)<br />
Query: 109 LGIGRDGVKAFEPAKVSKVKMILEDIFRK-----PYNKILLKVLSKANGGERNLVRNLAQ 163<br />
L + DG+ K +LED K ++ L+KVL+K GE NLVR L Q<br />
Sbjct: 34 LKLNDDGLNVLRSRKFE----VLEDYVTKLNHGQSVDETLVKVLTKHMDGEDNLVRLLDQ 89<br />
Query: 164 AEMLGY---KVFFLKSTLASKWERDGVSLMDVWSYI 196<br />
A+ K L++ L +KW+ + + M VWS +<br />
Sbjct: 90 AKWSTRSLDKANHLETALLTKWQNEKLLPMSVWSRL 125<br />
BLG02<br />
>Pr_97351T0 | Pr_97351 | Phytophthora ramorum RxLR effector (533 aa)<br />
Length = 532<br />
Score = 37.0 bits (84), Expect = 0.032, Method: Compositional matrix adjust.<br />
Identities = 50/171 (29%), Positives = 74/171 (43%), Gaps = 18/171 (10%)<br />
Query: 1 MVRIYVAALTVVLAFSASCSATSPLTLAKA---TPVTANGSGGRAQGRLRAHTTTNVEID 57<br />
M YV L +++A + + + TSP T+A A T A G G A+GR+ +T D<br />
Sbjct: 1 MRYCYVMMLVLIVALAINAAQTSPATIANARVPTRWVAAGLNG-ARGRVLTSDSTAETTD 59<br />
Query: 58 ERSISDVIYAIARQVADHRSNLVQAPKRTYTTYL---ATLD-----LSL----DKVLEQN 105<br />
E I A A + A V+AP +T +L ++D LSL DK+L+<br />
Sbjct: 60 EERAG--ISASAVEKAKALFTPVKAPTKTLQRWLNNGISVDDIFTHLSLAKAGDKLLDDT 117<br />
Query: 106 WSELRLLYVKQTKRKRPKDFVSMYELLVKKYGLLKLTDMMKKPDYISLSET 156<br />
L YV+ K P S L YG L M++ +S +E+<br />
Sbjct: 118 QFMTWLQYVELFNFKNPAQTKSTITTLTAHYGDKGLFQMLEAAKRVSSTES 168<br />
BLG03<br />
>Ps_133875T0 | Ps_133875 | Phytophthora sojae RxLR effector (225 aa)<br />
Length = 224<br />
Score = 32.3 bits (72), Expect = 0.81, Method: Compositional matrix adjust.<br />
Identities = 15/57 (26%), Positives = 31/57 (54%), Gaps = 2/57 (3%)<br />
Query: 88 LLKLNLSFEQFSGKDTLQLRRLYDAARRHNKVNPTNPVSVYTGLVEKYGEFEIVNMV 144<br />
LL+L+ E+ + L + + + N+ NP N V++ + ++ KYGE ++ M+<br />
Sbjct: 98 LLQLDDGLEKLLSRKNLDTWKTF--VNKLNQQNPENQVTMMSAIINKYGEEQVARMI 152
167<br />
Chapter 6:<br />
Discussion
168 Chapter 6
Discussion<br />
169<br />
Identify<strong>in</strong>g <strong>effectors</strong><br />
Microbial pathogens <strong>of</strong> plants <strong>and</strong> animals have evolved sophisticated ways to<br />
<strong>in</strong>terfere with host cell processes. To render the host susceptible to <strong>in</strong>fection pathogens<br />
deploy small molecules <strong>and</strong> prote<strong>in</strong>s, also called <strong>effectors</strong>, which <strong>in</strong>terfere<br />
with host processes [1,2] . An important process to perturb is detection by the host<br />
through recognition <strong>of</strong> molecular patterns that are present on the microbe’s surface<br />
or are part <strong>of</strong> other exposed molecules (microbe-associated molecular patterns,<br />
MAMPs) [3] . Successful pathogens use their <strong>effectors</strong> to suppress immune responses<br />
that are triggered after recognition or avoid detection altogether [1,2,4] . The <strong>in</strong>itial<br />
<strong>in</strong>teraction occurs mostly outside <strong>of</strong> host cells <strong>and</strong> pathogens deploy extracellular<br />
<strong>effectors</strong> to <strong>in</strong>terfere with MAMP recognition or other extracellular defences,<br />
e.g. <strong>in</strong>hibition <strong>of</strong> hydrolytic host enzymes [5,6] . Interest<strong>in</strong>gly, pathogens have also<br />
evolved different ways to get <strong>effectors</strong> <strong>in</strong>to the host cell. A well-studied mechanism<br />
by which <strong>effectors</strong> are translocated from pathogens <strong>in</strong>to host cells is the Type Three<br />
Secretion System used by various Gram-negative bacterial pathogens (reviewed by<br />
Büttner <strong>and</strong> He [7] ). Effectors conta<strong>in</strong><strong>in</strong>g a specific signal are targeted <strong>in</strong>to the host<br />
cell via a pilus-like structure that penetrates the host cell. Once <strong>in</strong>side, <strong>effectors</strong> act<br />
on the host cellular mach<strong>in</strong>ery to suppress immunity <strong>and</strong> to establish a successful<br />
<strong>in</strong>fection. Eukaryotic pathogens such as the oomycetes do not appear to use a direct<br />
<strong>in</strong>jection-like mechanism, but rather secrete prote<strong>in</strong>s that are then taken up by the<br />
host.<br />
Oomycetes are a remarkable <strong>and</strong> diverse group <strong>of</strong> fungal-like pathogens. Their<br />
hyphal growth <strong>and</strong> spore production give them the appearance <strong>of</strong> fungi, though<br />
they belong to the k<strong>in</strong>gdom Stramenopila <strong>and</strong> are more closely related to brown<br />
algae. Oomycete pathogens affect both animals <strong>and</strong> plants <strong>and</strong> <strong>in</strong>clude a number<br />
<strong>of</strong> notorious pathogens <strong>of</strong> crops, such as Phytophthora <strong>in</strong>festans, the potato late<br />
blight pathogen that is best known as the cause <strong>of</strong> the Irish potato fam<strong>in</strong>e. Like<br />
other successful pathogens, oomycetes use an arsenal <strong>of</strong> <strong>effectors</strong> to establish<br />
<strong>in</strong>fection [8,9] . Bio<strong>in</strong>formatics <strong>analysis</strong> showed that oomycetes have evolved many<br />
novel prote<strong>in</strong> doma<strong>in</strong> comb<strong>in</strong>ations compared to other species with a similar s<strong>in</strong>gle<br />
doma<strong>in</strong> repertoire [10] . These novel comb<strong>in</strong>ations are enriched <strong>in</strong> secreted prote<strong>in</strong>s,<br />
suggest<strong>in</strong>g they contribute to a diverse <strong>and</strong> unique effector repertoire. Analysis <strong>of</strong><br />
gene content has been <strong>in</strong>strumental <strong>in</strong> identify<strong>in</strong>g the vast repertoire <strong>of</strong> <strong>effectors</strong><br />
<strong>and</strong> has revealed extensive effector diversity between oomycete species [8,9] .
170 Chapter 6<br />
Genomes <strong>and</strong> transcriptomes<br />
The genomes <strong>of</strong> Albugo c<strong>and</strong>ida, Pythium ultimum, the Phytophthora species<br />
<strong>in</strong>festans, ramorum, sojae <strong>and</strong> capsici, <strong>and</strong> the <strong>downy</strong> <strong>mildew</strong> species Hyaloperonospora<br />
arabidopsidis <strong>and</strong> Pseudoperonospora cubensis have been sequenced<br />
<strong>and</strong> annotated [11–17] . Whilst these genome sequences have had great use <strong>in</strong> the<br />
identification <strong>of</strong> potential <strong>effectors</strong> <strong>in</strong> these species, obta<strong>in</strong><strong>in</strong>g <strong>and</strong> analys<strong>in</strong>g the<br />
genome sequences has been a large comb<strong>in</strong>ed effort, <strong>of</strong>ten <strong>in</strong>volv<strong>in</strong>g many collaborators<br />
with an <strong>in</strong>terest <strong>in</strong> these particular pathogens. At the moment technologies<br />
are still not sufficiently advanced <strong>and</strong> automated to <strong>in</strong>vestigate the genomes <strong>of</strong> all<br />
possible pathogens <strong>in</strong> an efficient <strong>and</strong> rapid way. However, the gene content <strong>of</strong><br />
organisms can also be obta<strong>in</strong>ed by transcriptomics from which <strong>effectors</strong> can then<br />
be discovered.<br />
In this thesis the identification <strong>and</strong> <strong>functional</strong> <strong>analysis</strong> <strong>of</strong> Bremia lactucae<br />
<strong>effectors</strong> from transcriptome-sequenc<strong>in</strong>g data is described. Sequenc<strong>in</strong>g the<br />
transcriptome rather than the entire genome <strong>of</strong> Bremia has several advantages. The<br />
result<strong>in</strong>g dataset consists <strong>of</strong> transcripts <strong>of</strong> genes that are expressed dur<strong>in</strong>g <strong>in</strong>fection,<br />
whereas a genome-derived set <strong>of</strong> predicted genes is more likely to also conta<strong>in</strong><br />
genes that are not expressed dur<strong>in</strong>g <strong>in</strong>fection or are even not expressed at all<br />
(be<strong>in</strong>g pseudogenes). Careful plann<strong>in</strong>g <strong>of</strong> the sampl<strong>in</strong>g strategy is, however, more<br />
important when sequenc<strong>in</strong>g a transcriptome, as sequenc<strong>in</strong>g will yield a snapshot <strong>of</strong><br />
the mRNA molecules present at the time <strong>of</strong> sampl<strong>in</strong>g. By <strong>in</strong>oculat<strong>in</strong>g leaf-material<br />
twice, <strong>in</strong>terspaced by three days, both earlier <strong>and</strong> later stages <strong>of</strong> pathogen growth<br />
are <strong>in</strong>cluded <strong>in</strong> the sample. This allows for a broad sampl<strong>in</strong>g <strong>of</strong> <strong>in</strong>fection-related<br />
transcripts, <strong>and</strong> enables identification <strong>of</strong> both effector c<strong>and</strong>idates that are expressed<br />
early (e.g. BLR03 <strong>and</strong> BLR22, Chapter 4) <strong>and</strong> those that are expressed late dur<strong>in</strong>g<br />
<strong>in</strong>fection (e.g. BLR23, Chapter 4). Prediction <strong>of</strong> prote<strong>in</strong> cod<strong>in</strong>g sequences from<br />
transcripts is more straight-forward as <strong>in</strong>trons do not disrupt the read<strong>in</strong>g frames <strong>and</strong><br />
the largest open read<strong>in</strong>g frame <strong>in</strong> each transcript is therefore most likely encod<strong>in</strong>g<br />
the correctly predicted prote<strong>in</strong>. Furthermore, the transcriptome conta<strong>in</strong>s fewer<br />
sequence repeats than a genome, which simplifies the assembly process. Another<br />
issue that has less impact on the assembly <strong>of</strong> transcriptome sequenc<strong>in</strong>g data than<br />
it has on the assembly <strong>of</strong> an entire genome is heterozygosity <strong>of</strong> the genome,<br />
e.g. that <strong>of</strong> Bremia. Overall, less sequenc<strong>in</strong>g data is required to provide good<br />
coverage <strong>of</strong> the transcriptome to allow robust assembly <strong>of</strong> full-length transcripts.<br />
The advantages <strong>of</strong> sequenc<strong>in</strong>g the transcriptome also mean that computational<br />
requirements to store, h<strong>and</strong>le, <strong>and</strong> analyse data are lower, allow<strong>in</strong>g such a project to<br />
be performed with modest comput<strong>in</strong>g hardware.
Discussion<br />
171<br />
Host-translocated <strong>effectors</strong><br />
The focus <strong>of</strong> the research described <strong>in</strong> this thesis was on RXLR <strong>and</strong> RXLR-like<br />
<strong>effectors</strong>. The RXLR motif was found to be common to all cloned avirulence<br />
prote<strong>in</strong>s <strong>of</strong> oomycetes known <strong>in</strong> 2005 [18] . Avirulence prote<strong>in</strong>s, or their effects on<br />
host prote<strong>in</strong>s, are recognised by <strong>in</strong>tracellular Resistance prote<strong>in</strong>s belong<strong>in</strong>g to the<br />
NB-LRR class <strong>and</strong> were therefore suspected to be present <strong>in</strong> the host cytoplasm.<br />
The fact that these prote<strong>in</strong>s shared the RXLR motif suggested the motif was<br />
<strong>in</strong>volved <strong>in</strong> translocation <strong>of</strong> the effector <strong>in</strong>to the host cell. Experiments us<strong>in</strong>g<br />
<strong>effectors</strong> with mutated RXLR motifs <strong>in</strong>deed revealed the motif to be required for<br />
translocation [19] . Translocation was shown to be <strong>in</strong>dependent <strong>of</strong> pathogen-encoded<br />
mach<strong>in</strong>ery as <strong>effectors</strong> are taken up <strong>in</strong>to host cells <strong>in</strong> the absence <strong>of</strong> the pathogen<br />
[20,21]<br />
. The mechanism by which translocation occurs, however, is still under debate.<br />
One study suggests that the RXLR motif b<strong>in</strong>ds PIP3 <strong>and</strong> that <strong>effectors</strong> are subsequently<br />
translocated <strong>in</strong> a lipid-raft-dependent manner [22] . This was also shown for<br />
fungal <strong>effectors</strong>, <strong>and</strong> allowed entry <strong>in</strong>to plant <strong>and</strong> human cells [22] . The lipid b<strong>in</strong>d<strong>in</strong>g<br />
properties <strong>of</strong> one <strong>of</strong> the fungal <strong>effectors</strong> could, however, not be confirmed <strong>in</strong> a<br />
second study, which was also able to separate lipid b<strong>in</strong>d<strong>in</strong>g properties <strong>of</strong> another<br />
fungal effector from the sequence required for translocation [23] . Likewise, <strong>in</strong>vestigation<br />
<strong>of</strong> P. <strong>in</strong>festans effector Avr3a revealed that lipid b<strong>in</strong>d<strong>in</strong>g properties could be<br />
separated from translocation, <strong>and</strong> that lipid b<strong>in</strong>d<strong>in</strong>g was required for stabilisation<br />
<strong>of</strong> Avr3a, but not for translocation [24] . Translocation <strong>of</strong> RXLR-motif-conta<strong>in</strong><strong>in</strong>g<br />
SpHtp1 <strong>of</strong> the oomycete fish pathogen Saprolegnia parasitica has been reported<br />
to be dependent on tyros<strong>in</strong>e-O-sulphate [21] . SpHtp1 did not, however, translocate<br />
<strong>in</strong>to plant <strong>and</strong> human cells, <strong>and</strong> <strong>in</strong> contrast to the secretomes <strong>of</strong> Peronosporales<br />
there is no enrichment <strong>of</strong> the RXLR motif <strong>in</strong> the S. parasitica secretome. The exact<br />
mechanism <strong>of</strong> RXLR-effector translocation thus rema<strong>in</strong>s to be determ<strong>in</strong>ed.<br />
The RXLR motif was <strong>in</strong>strumental <strong>in</strong> the identification <strong>of</strong> several hundreds <strong>of</strong><br />
potential <strong>effectors</strong> <strong>in</strong> the genomes <strong>of</strong> <strong>in</strong>dividual Phytophthora species. In <strong>downy</strong><br />
<strong>mildew</strong>s the numbers <strong>of</strong> identified RXLR <strong>effectors</strong> <strong>and</strong> other pathogenicity genes<br />
appeared to be lower [14,25] . Though am<strong>in</strong>o acid motifs provide a useful tool to<br />
identify oomycete <strong>effectors</strong>, the def<strong>in</strong>ition <strong>of</strong> the motifs may be too strict to detect<br />
all related <strong>effectors</strong>. Recently the host-translocated effector ATR5 <strong>of</strong> H. arabidopsidis<br />
was identified <strong>and</strong> found to lack a RXLR motif, though an RXLR-associated<br />
EER doma<strong>in</strong> was present. In Pseudoperonospora cubensis a variant <strong>of</strong> the RXLR<br />
motif, QXLR is common <strong>and</strong> enables translocation <strong>in</strong>to the host cell [25] . This<br />
QXLR motif was previously thought to be <strong>in</strong>capable <strong>of</strong> enabl<strong>in</strong>g translocation, as<br />
the am<strong>in</strong>o acid substitution RXLR → QXLR <strong>in</strong> the P. sojae RXLR effector Avr1b<br />
abolished the effector’s capability to re-enter soybean root cells <strong>in</strong> the absence <strong>of</strong><br />
a pathogen [22] . Mutated forms <strong>of</strong> Avr1b were expressed <strong>in</strong> <strong>and</strong> then secreted from
172 Chapter 6<br />
soybean root cells after delivery <strong>of</strong> constructs by particle bombardment. The ability<br />
to re-enter the cells was abolished <strong>in</strong> some <strong>of</strong> the tested mutants, but a wide range<br />
<strong>of</strong> mutations did not appear to impact re-entry <strong>and</strong> suggested a highly redundant<br />
motif ([RHK]X[LMIFYW]). In addition, mutations <strong>in</strong> EER-like motifs also had a<br />
negative effect on effector re-entry. The ability <strong>of</strong> RXLR <strong>effectors</strong> to translocate<br />
<strong>in</strong>to host cells is therefore more likely to be dependent on the context <strong>in</strong> which the<br />
motif occurs than the exact motif itself. Such f<strong>in</strong>d<strong>in</strong>gs warrant the <strong>in</strong>vestigation <strong>of</strong><br />
potential <strong>effectors</strong> that do not conform to the canonical RXLR effector with strict<br />
RXLR- <strong>and</strong> EER motifs. In this regard the presence <strong>of</strong> the RXLR motif can be<br />
seen as an <strong>in</strong>dication <strong>of</strong> whether a prote<strong>in</strong> may be a host-translocated effector, but<br />
its absence should not be taken as a sign that the prote<strong>in</strong> is not translocated. In this<br />
thesis GKLR <strong>effectors</strong> are described that are recognised <strong>in</strong> specific Lactuca hosts<br />
(Chapter 5). These <strong>effectors</strong> were identified based on similarity to other oomycete<br />
<strong>effectors</strong> <strong>and</strong> were selected for further <strong>in</strong>vestigation despite the variant RXLR<br />
motif. Effector c<strong>and</strong>idates without a clear variant <strong>of</strong> the RXLR motif that do have<br />
an EER motif (Chapter 3) were also identified, but these are yet to be <strong>in</strong>vestigated.<br />
Other classes <strong>of</strong> non-RXLR host-translocated oomycete effector have been<br />
identified, e.g. the Cr<strong>in</strong>klers, which are thought to have a more ancient orig<strong>in</strong><br />
based on their occurrence <strong>in</strong> phylogenetically more distant oomycete species such<br />
as Albugo <strong>and</strong> Aphanomyces [26] . Downy <strong>mildew</strong>s express relatively few Cr<strong>in</strong>kler<br />
prote<strong>in</strong>s (Chapter 3, [27–29] ). RXLR <strong>and</strong> Cr<strong>in</strong>kler effector sequences can be m<strong>in</strong>ed<br />
from sequence databases by search<strong>in</strong>g for the occurrence <strong>of</strong> the motifs that characterise<br />
these <strong>effectors</strong>. Additional classes <strong>of</strong> <strong>effectors</strong> can be identified as clusters<br />
<strong>of</strong> related sequences that are specific for the pathogen species. For example, <strong>in</strong><br />
the predicted secretome <strong>of</strong> the white rust Albugo laibachii an additional group<br />
<strong>of</strong> 31 <strong>effectors</strong> that share a CHXC motif was identified [30] . The N-term<strong>in</strong>us <strong>of</strong><br />
one such effector, CHXC9, was shown to be as efficient as the N-term<strong>in</strong>us <strong>of</strong> a<br />
Cr<strong>in</strong>kler effector <strong>in</strong> translocat<strong>in</strong>g the AVR3a effector doma<strong>in</strong> from Phytophthora<br />
capsici <strong>in</strong>to N. benthamiana cells, <strong>in</strong>dicat<strong>in</strong>g that these CHXC prote<strong>in</strong>s are genu<strong>in</strong>e<br />
host-translocated <strong>effectors</strong>. Search<strong>in</strong>g the Bremia secretome for clusters <strong>of</strong> related<br />
sequences identified additional RXLR-like effector c<strong>and</strong>idates. It did not, however,<br />
uncover novel types <strong>of</strong> <strong>effectors</strong> such as the Albugo CHXC <strong>effectors</strong>.<br />
Targets <strong>and</strong> effector variation<br />
A next challenge for effector research is the identification <strong>of</strong> the targets <strong>of</strong> <strong>effectors</strong>.<br />
The network <strong>of</strong> <strong>in</strong>teractions between H. arabidopsidis <strong>and</strong> P. syr<strong>in</strong>gae <strong>effectors</strong><br />
<strong>and</strong> Arabidopsis prote<strong>in</strong>s based on a recent high-throughput yeast-two-hybrid study<br />
<strong>in</strong>dicate that different pathogens target a limited number <strong>of</strong> highly connected cellular<br />
hubs [31] . The shared host targets are <strong>of</strong>ten <strong>in</strong>volved <strong>in</strong> immunity <strong>and</strong> are highly
Discussion<br />
173<br />
<strong>in</strong>terconnected. Some <strong>of</strong> the host targets are targeted by more than 20 different<br />
<strong>effectors</strong>.<br />
If a relatively small set <strong>of</strong> defence hubs are the ma<strong>in</strong> target <strong>of</strong> <strong>effectors</strong>, the<br />
question rema<strong>in</strong>s why some pathogens encode many hundreds <strong>of</strong> <strong>effectors</strong>. A<br />
study on <strong>downy</strong> <strong>mildew</strong>s <strong>effectors</strong> <strong>in</strong> Arabidopsis revealed that many <strong>effectors</strong><br />
enhance disease susceptibility, although many with subtle effects <strong>and</strong> not on all <strong>of</strong><br />
the 12 accessions tested [32] . These accession dependent effects reflect the variation<br />
<strong>in</strong> <strong>effectors</strong> <strong>and</strong> host targets. Variation <strong>in</strong> <strong>effectors</strong> between different isolates <strong>of</strong> a<br />
species has been shown to <strong>in</strong>clude mutations result<strong>in</strong>g <strong>in</strong> am<strong>in</strong>o acid substitutions,<br />
but also loss or ga<strong>in</strong> <strong>of</strong> entire <strong>effectors</strong>, differences <strong>in</strong> copy number <strong>and</strong> transcriptional<br />
polymorphism [28,33–35] . The complete arsenal <strong>of</strong> <strong>effectors</strong> encoded by a s<strong>in</strong>gle<br />
species is therefore likely to be much larger than the reference sequence <strong>of</strong> the<br />
species suggests. These different <strong>effectors</strong> may target subtly different variants <strong>of</strong><br />
the host target prote<strong>in</strong>s, or provide <strong>functional</strong> redundancy <strong>and</strong> <strong>in</strong>creased flexibility<br />
<strong>in</strong> adapt<strong>in</strong>g to hostile environments.<br />
Us<strong>in</strong>g <strong>effectors</strong> <strong>in</strong> resistance breed<strong>in</strong>g<br />
Identify<strong>in</strong>g effector c<strong>and</strong>idates by genome or transcriptome sequenc<strong>in</strong>g is becom<strong>in</strong>g<br />
<strong>in</strong>creas<strong>in</strong>gly affordable <strong>and</strong> achievable. The total number <strong>of</strong> c<strong>and</strong>idate <strong>effectors</strong><br />
identified from genome sequences has already surpassed 1000 <strong>in</strong> Phytophthora<br />
species alone. More pathogen species are be<strong>in</strong>g sequenced to learn more about<br />
the basis <strong>of</strong> their pathogenicity. The ultimate goal is to apply knowledge about the<br />
factors <strong>in</strong>volved <strong>in</strong> pathogenicity to breed pathogen-resistant crop plants. Effectors<br />
<strong>of</strong> pathogens can be used as tools to breed for novel resistances aga<strong>in</strong>st pathogens,<br />
an approach also referred to as effector-assisted breed<strong>in</strong>g. Knowledge <strong>of</strong> the P.<br />
<strong>in</strong>festans genome <strong>and</strong> the <strong>effectors</strong> encoded there<strong>in</strong>, has already provided useful<br />
<strong>in</strong>sights for breed<strong>in</strong>g <strong>of</strong> late blight resistance <strong>in</strong> potato (reviewed by Vleeshouwers<br />
et al. [36] )<br />
Detect<strong>in</strong>g R‐genes<br />
The most direct way <strong>in</strong> which <strong>effectors</strong> can currently aid resistance breed<strong>in</strong>g is by<br />
us<strong>in</strong>g them to identify <strong>and</strong> genetically follow resistance (R) genes. As has been<br />
done <strong>in</strong> the research described <strong>in</strong> this thesis <strong>and</strong> elsewhere [37–39] , <strong>effectors</strong> can be<br />
used to screen for the presence <strong>of</strong> R-prote<strong>in</strong>s that mediate their recognition. In<br />
this thesis <strong>effectors</strong> BLG01 <strong>and</strong> BLG03 are examples <strong>of</strong> such <strong>effectors</strong>. Effector<br />
BLG03 is recognised by either Dm2 or a gene closely l<strong>in</strong>ked to Dm2. In a<br />
segregat<strong>in</strong>g population the response to BLG03 can be used as a marker for Dm2<br />
without the need for DNA isolation <strong>and</strong> <strong>analysis</strong>. Effectors can therefore also act
174 Chapter 6<br />
as useful markers to identify <strong>and</strong> track resistance genes <strong>in</strong> breed<strong>in</strong>g l<strong>in</strong>es. Recently,<br />
the genetic basis <strong>of</strong> resistance <strong>of</strong> potato cultivar ‘Sarpo Mira’ to P. <strong>in</strong>festans was<br />
dissected us<strong>in</strong>g <strong>effectors</strong> <strong>of</strong> P. <strong>in</strong>festans to screen <strong>of</strong>fspr<strong>in</strong>g <strong>of</strong> a cross between<br />
‘Sarpo Mira’ <strong>and</strong> a universally susceptible cultivar [40] . Resistance was found to be<br />
based on recognition <strong>of</strong> at least five <strong>effectors</strong>. Recognition <strong>of</strong> one <strong>of</strong> these <strong>effectors</strong>,<br />
AvrSmira2, correlated with field resistance, but not with resistance <strong>in</strong> laboratory<br />
assays. The dissection <strong>of</strong> the basis <strong>of</strong> resistance <strong>in</strong> ‘Sarpo Mira’ would be too<br />
complex us<strong>in</strong>g only different isolates <strong>of</strong> P. <strong>in</strong>festans <strong>and</strong> would likely have missed<br />
the field resistance. Us<strong>in</strong>g <strong>effectors</strong> this resistance was uncovered, <strong>and</strong> a marker for<br />
the presence <strong>of</strong> the trait, namely the effector itself, is immediately available. Furthermore,<br />
R‐genes that are detected by effector screens can potentially be rapidly<br />
identified, if the genome sequence <strong>of</strong> the host plant species that carries the R‐gene<br />
is available. A library <strong>of</strong> NB-LRR resistance genes can be cloned <strong>and</strong> transiently<br />
co-expressed with the effector. Co-expression sites that show a cell death response<br />
express the R‐gene correspond<strong>in</strong>g to the effector. The cloned R‐gene can then be<br />
used for transgenic approaches, or its sequence can be used <strong>in</strong> marker-assisted<br />
breed<strong>in</strong>g.<br />
Work<strong>in</strong>g with host targets<br />
There is a relatively small number <strong>of</strong> pathogens whose effector arsenal is be<strong>in</strong>g<br />
extensively studied, <strong>in</strong> the case <strong>of</strong> the oomycetes these are predom<strong>in</strong>antly Phytophthora<br />
species <strong>and</strong> H. arabidopsidis. Sequenc<strong>in</strong>g <strong>of</strong> multiple oomycete species has<br />
revealed there is very little conservation <strong>of</strong> effector sequences between species.<br />
This makes extrapolat<strong>in</strong>g knowledge <strong>of</strong> effector-target <strong>in</strong>teractions uncovered <strong>in</strong>,<br />
e.g., P. <strong>in</strong>festans to other pathogens difficult. Effectors identified <strong>in</strong> one model<br />
system will <strong>of</strong>ten be unrecognisable <strong>in</strong> other related pathogen species. This is<br />
also shown <strong>in</strong> chapter 3, where very little overlap is found between the predicted<br />
<strong>effectors</strong> <strong>of</strong> the three <strong>downy</strong> <strong>mildew</strong> species, H. arabidopsidis, Bremia, <strong>and</strong> P.<br />
cubensis. The strength <strong>of</strong> us<strong>in</strong>g the more extensively studied <strong>in</strong>teraction between<br />
H. arabidopsidis <strong>and</strong> its host Arabidopsis thaliana as a model pathosystem for the<br />
Bremia-lettuce <strong>in</strong>teraction lies not <strong>in</strong> the identification <strong>of</strong> the sequences <strong>of</strong> <strong>effectors</strong>,<br />
but more <strong>in</strong> their utility <strong>in</strong> identify<strong>in</strong>g host targets. H. arabidopsidis <strong>effectors</strong><br />
have been used to identify <strong>in</strong> planta <strong>in</strong>teractors, which are likely the targets <strong>of</strong> these<br />
<strong>effectors</strong> [31] . These <strong>in</strong>teractors are highly <strong>in</strong>terconnected <strong>and</strong> can be seen as hubs <strong>in</strong><br />
the plant immune mach<strong>in</strong>ery. Comparison <strong>of</strong> the non-receptor immune <strong>in</strong>teractors<br />
<strong>of</strong> pathogen <strong>effectors</strong> <strong>in</strong> A. thaliana <strong>and</strong> Papaya revealed a slow overall rate <strong>of</strong><br />
evolution, <strong>in</strong>dicat<strong>in</strong>g that, <strong>in</strong> contrast to <strong>effectors</strong> <strong>of</strong> different species, host targets<br />
are likely conserved between species. Indeed, H. arabidopsidis effector HaRxL96<br />
was found to be able to suppress immunity <strong>in</strong> soybean, whilst a homologous P.
Discussion<br />
175<br />
sojae effector was found to suppress immunity <strong>in</strong> Arabidopsis, suggest<strong>in</strong>g the<br />
<strong>effectors</strong> are effective aga<strong>in</strong>st conserved targets <strong>in</strong> divergent hosts, despite limited<br />
homology between both <strong>effectors</strong> (26% identity, 43% similarity) [41] . When the<br />
genome sequence <strong>of</strong> the host plant is available (expected <strong>in</strong> 2012/2013 for lettuce)<br />
effector-target <strong>in</strong>teractions can be <strong>in</strong>vestigated start<strong>in</strong>g from the conserved host<br />
targets <strong>and</strong> effector arsenal <strong>of</strong> the pathogen. Effectors modulate host targets to<br />
enhance susceptibility or suppress defence. Changes <strong>in</strong> the host target may break<br />
down the <strong>in</strong>teraction between effector <strong>and</strong> target, lead<strong>in</strong>g to <strong>in</strong>sensitivity to the<br />
effector. Further underst<strong>and</strong><strong>in</strong>g <strong>of</strong> the modifications <strong>of</strong> host targets by <strong>effectors</strong><br />
will allow for <strong>functional</strong> screen<strong>in</strong>g for effector-<strong>in</strong>sensitive host-targets <strong>in</strong> breed<strong>in</strong>g<br />
material. Effector-<strong>in</strong>sensitive host targets may provide a novel source <strong>of</strong> resistance<br />
that can be used <strong>in</strong> addition to dom<strong>in</strong>ant R‐genes.<br />
Gene-for-gene <strong>in</strong>teractions <strong>and</strong> resistance<br />
Pathogen species are <strong>of</strong>ten capable <strong>of</strong> rapidly adapt<strong>in</strong>g to new resistance traits<br />
<strong>in</strong>troduced by breeders. Given sufficient knowledge <strong>of</strong> the effector arsenal <strong>of</strong><br />
a pathogen species, screen<strong>in</strong>g <strong>of</strong> the <strong>effectors</strong> <strong>of</strong> resistance-break<strong>in</strong>g isolates<br />
will allow for better underst<strong>and</strong><strong>in</strong>g <strong>of</strong> the mechanisms that underlie this rapid<br />
adaptation. In this thesis it is shown that plants from a Dm2 background are able<br />
to recognise an allele <strong>of</strong> Bremia effector BLG03 <strong>of</strong> an isolate to which these plants<br />
are not resistant. This suggests that host defence does not fail because <strong>of</strong> changes<br />
to the effector that abolish recognition by Dm2 or a gene <strong>in</strong> proximity, but by other<br />
changes <strong>in</strong> the effector complement that either block recognition <strong>of</strong> the effector<br />
or signall<strong>in</strong>g downstream <strong>of</strong> recognition. Similarly, <strong>in</strong>troduction <strong>of</strong> the L. saligna<br />
locus required for recognition <strong>of</strong> the Bremia effector BLG01 <strong>in</strong> L. sativa does not<br />
appear to confer resistance. These observations are not unique to Bremia. In the<br />
<strong>downy</strong> <strong>mildew</strong> H. arabidopsidis both ATR39-1 <strong>and</strong> ATR1Emco5 are recognised <strong>in</strong><br />
hosts that are susceptible to isolates that carry these <strong>effectors</strong> [18,39,42] . Observations<br />
also <strong>in</strong>dicate that <strong>in</strong>termediate resistance to H. arabidopsidis is relatively common<br />
<strong>and</strong> plays an important role <strong>in</strong> the H. arabidopsidis - A. thaliana <strong>in</strong>teraction. The<br />
aforementioned field resistance towards AvrSmira2‐conta<strong>in</strong><strong>in</strong>g P. <strong>in</strong>festans isolates<br />
<strong>in</strong> potato cultivar ‘Sarpo Mira’ is an illustration that such <strong>in</strong>termediate resistance<br />
can also be relevant for resistance breed<strong>in</strong>g. Krasileva et al. [42] po<strong>in</strong>t out that the<br />
outcome <strong>of</strong> the <strong>in</strong>teraction between host <strong>and</strong> pathogen cannot always be expla<strong>in</strong>ed<br />
by s<strong>in</strong>gle gene-for-gene <strong>in</strong>teractions, but is rather the result <strong>of</strong> genome-for-genome<br />
<strong>in</strong>teractions. Screen<strong>in</strong>g <strong>of</strong> resistance-break<strong>in</strong>g pathogens can assist <strong>in</strong> p<strong>in</strong>po<strong>in</strong>t<strong>in</strong>g<br />
the components that allow the break<strong>in</strong>g <strong>of</strong> resistance. It also will allow assessment<br />
<strong>of</strong> the level <strong>of</strong> allelic diversity <strong>of</strong> a recognised effector, which can provide an<br />
<strong>in</strong>dication <strong>of</strong> how durable a correspond<strong>in</strong>g R‐gene will be.
176 Chapter 6<br />
The zigzag model that describes the plant immune system [1,43] provides a model<br />
for the lack <strong>of</strong> correlation between the outcome <strong>of</strong> a gene-for-gene <strong>in</strong>teraction<br />
<strong>and</strong> resistance to an <strong>in</strong>vad<strong>in</strong>g pathogen. The model dist<strong>in</strong>guishes different layers<br />
<strong>of</strong> pathogen <strong>of</strong>fence <strong>and</strong> plant defence. Hosts are normally resistant to potential<br />
pathogens as they recognise MAMPs. Pathogens then use <strong>effectors</strong> to <strong>in</strong>duce<br />
susceptibility <strong>in</strong> hosts, so called effector-triggered susceptibility. Host species, <strong>in</strong><br />
return, evolved means <strong>of</strong> directly or <strong>in</strong>directly recognis<strong>in</strong>g these <strong>effectors</strong>, restor<strong>in</strong>g<br />
an effective defence response, termed effector-triggered-immunity. The plant-pathogen<br />
prote<strong>in</strong>-prote<strong>in</strong>-<strong>in</strong>teractome network <strong>of</strong> Mukhtar et al. [31] <strong>in</strong>dicates that many<br />
<strong>in</strong>teractors <strong>of</strong> the resistance prote<strong>in</strong>s that recognise <strong>effectors</strong> are targeted by <strong>effectors</strong>,<br />
rather than the resistance prote<strong>in</strong>s themselves. In other words, pathogens use<br />
<strong>effectors</strong> to suppress the defence responses triggered by other <strong>effectors</strong> to restore<br />
effector-triggered susceptibility. Screen<strong>in</strong>g <strong>of</strong> <strong>effectors</strong> <strong>in</strong> soybean has <strong>in</strong>deed<br />
revealed that many are capable <strong>of</strong> suppress<strong>in</strong>g effector-<strong>in</strong>duced cell death [34] .<br />
Unravell<strong>in</strong>g this web <strong>of</strong> <strong>in</strong>teract<strong>in</strong>g prote<strong>in</strong>s is required to make predictions on how<br />
to use the available pool <strong>of</strong> defence strategies most effectively for each different<br />
crop species.<br />
Future prospects: Break<strong>in</strong>g the cycle<br />
Bremia is among the plant pathogens with the highest risk <strong>of</strong> evolv<strong>in</strong>g adaptations<br />
to measures <strong>in</strong>troduced to control disease [44] . Factors that contribute to this risk<br />
<strong>in</strong>clude the large effective population size <strong>and</strong> mixed reproductive system. This risk<br />
is not only dependent on the biology <strong>of</strong> the pathogen, as breed<strong>in</strong>g efforts <strong>in</strong> lettuce<br />
impose a strong directional selection on Bremia, which contributes to this risk.<br />
Resistant cultivars <strong>of</strong>ten conta<strong>in</strong> dom<strong>in</strong>ant resistant genes <strong>and</strong> are grown <strong>in</strong> large<br />
monocultures <strong>in</strong> relatively large regions. With sequenc<strong>in</strong>g costs decreas<strong>in</strong>g <strong>and</strong><br />
knowledge <strong>of</strong> pathogen <strong>effectors</strong> <strong>in</strong>creas<strong>in</strong>g, genetic screen<strong>in</strong>g <strong>of</strong> field isolates will<br />
become a useful tool to more accurately type isolates <strong>and</strong> underst<strong>and</strong> why resistance<br />
breaks. Such screen<strong>in</strong>g is also <strong>in</strong>valuable for a localised rather than global<br />
approach to deployment <strong>of</strong> resistant cultivars. By comb<strong>in</strong><strong>in</strong>g novel, broad range<br />
resistance genes <strong>and</strong> alleles that are <strong>in</strong>sensitive to specific effector action, more<br />
durable forms <strong>of</strong> resistance can potentially be bred. This reduces the directional<br />
selection on Bremia, <strong>and</strong> together with a more localised approach to resistance<br />
can serve to reduce the evolutionary risk so that resistance is not easily broken or<br />
rendered <strong>in</strong>effective.
Discussion<br />
177<br />
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44 McDonald BA & L<strong>in</strong>de C (2002) Pathogen population genetics, evolutionary potential, <strong>and</strong><br />
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182
183<br />
Summary<br />
Oomycete pathogens cause large losses <strong>in</strong> many crop plants. Chemical control<br />
measures becom<strong>in</strong>g less effective <strong>and</strong> are be<strong>in</strong>g phased out. Resistance breed<strong>in</strong>g<br />
has traditionally made use <strong>of</strong> dom<strong>in</strong>ant resistance genes to which oomycete pathogens<br />
have <strong>of</strong>ten been quick to adapt. Novel approaches towards resistance breed<strong>in</strong>g<br />
are, therefore, required to <strong>in</strong>troduce new <strong>and</strong> more durable forms <strong>of</strong> resistance <strong>in</strong><br />
crops. A better underst<strong>and</strong><strong>in</strong>g <strong>of</strong> host-pathogen <strong>in</strong>teractions at the molecular level<br />
is required to design new breed<strong>in</strong>g approaches. Important molecular players, that<br />
are the ma<strong>in</strong> subject <strong>of</strong> this study, are the effector prote<strong>in</strong>s that <strong>downy</strong> <strong>mildew</strong>s<br />
use to manipulate their host <strong>and</strong> cause disease. Sequenc<strong>in</strong>g <strong>of</strong> Expressed Sequence<br />
Tags <strong>of</strong> Hyaloperonospora arabidopsidis proved a powerful method <strong>of</strong> identify<strong>in</strong>g<br />
<strong>effectors</strong> that are active dur<strong>in</strong>g <strong>in</strong>fection. This revealed isolate-specific <strong>effectors</strong><br />
that <strong>in</strong>crease susceptibility <strong>of</strong> the host. Scal<strong>in</strong>g up this approach us<strong>in</strong>g massively<br />
parallel transcriptome sequenc<strong>in</strong>g allowed for an extensive overview <strong>of</strong> genes that<br />
are active dur<strong>in</strong>g <strong>downy</strong> <strong>mildew</strong> <strong>in</strong>fection <strong>of</strong> lettuce, caused by Bremia lactucae.<br />
Bremia transcript sequences were identified from the mixed set <strong>of</strong> plant <strong>and</strong> pathogen<br />
transcripts based on mapp<strong>in</strong>g <strong>of</strong> spore-derived short reads <strong>of</strong> Bremia genomic<br />
DNA. Effector c<strong>and</strong>idates were then identified based on sequence characteristics<br />
known from <strong>effectors</strong> <strong>of</strong> other oomycetes. A set <strong>of</strong> 34 potential host-translocated<br />
<strong>effectors</strong> with an RXLR or RXLR-like motif was cloned for further <strong>analysis</strong>. The<br />
role <strong>of</strong> these <strong>effectors</strong> <strong>in</strong> promot<strong>in</strong>g disease susceptibility on the lettuce host was<br />
<strong>in</strong>vestigated, reveal<strong>in</strong>g two <strong>effectors</strong>, BLR16 <strong>and</strong> BLR27 that show a strong <strong>and</strong><br />
robust susceptibility-enhanc<strong>in</strong>g effect when transiently expressed. Many other<br />
tested <strong>effectors</strong> showed a trend towards enhanc<strong>in</strong>g susceptibility, though a s<strong>in</strong>gle<br />
effector, BLR03, consistently reduced susceptibility. BLR16 <strong>and</strong> BLR27 were found<br />
to be expressed throughout Bremia <strong>in</strong>fection <strong>of</strong> lettuce, whilst a strong reduction <strong>in</strong><br />
the expression <strong>of</strong> BLR03 was observed already at one day post <strong>in</strong>oculation. Lettuce<br />
markers <strong>of</strong> biotic stress-responses were developed based on high abundance <strong>and</strong><br />
homology to Arabidopsis biotic stress-<strong>in</strong>duced genes. However, no evidence<br />
was found that susceptibility is enhanced by a general repression <strong>of</strong> biotic-stress<br />
responses <strong>of</strong> the host. F<strong>in</strong>ally, 129 lettuce breed<strong>in</strong>g l<strong>in</strong>es were tested for their<br />
response to the set <strong>of</strong> cloned <strong>effectors</strong> by Agrobacterium-mediated transient transformation<br />
assays to reveal new resistance specificities. Two <strong>effectors</strong> conta<strong>in</strong><strong>in</strong>g<br />
a GKLR-variant <strong>of</strong> the RXLR motif were found to be recognised result<strong>in</strong>g <strong>in</strong> the
184<br />
<strong>in</strong>duction <strong>of</strong> cell death; BLG01 <strong>in</strong> wild lettuce l<strong>in</strong>es, <strong>and</strong> BLG03 <strong>in</strong> cultivated<br />
lettuce l<strong>in</strong>es that conta<strong>in</strong> the known resistance locus Dm2. Recognition <strong>of</strong> BLG01<br />
is dependent on a region on the short arm <strong>of</strong> chromosome 9 <strong>in</strong> L. saligna, <strong>and</strong><br />
recognition <strong>of</strong> BLG03 is l<strong>in</strong>ked to the Dm2 resistance locus. Bremia isolates carry<strong>in</strong>g<br />
these <strong>effectors</strong> were still able to cause disease, suggest<strong>in</strong>g that recognition or<br />
subsequent defence is suppressed by other <strong>effectors</strong>. The generation <strong>of</strong> an overview<br />
<strong>of</strong> the Bremia effector arsenal, identification <strong>of</strong> susceptibility-enhanc<strong>in</strong>g activities<br />
<strong>of</strong> Bremia <strong>effectors</strong>, <strong>and</strong> uncover<strong>in</strong>g <strong>of</strong> recognition specificities are important steps<br />
forward towards underst<strong>and</strong><strong>in</strong>g the molecular mechanisms underly<strong>in</strong>g disease<br />
outcome.
185<br />
Samenvatt<strong>in</strong>g<br />
Ziekteverwekkers behorende tot de oömyceten veroorzaken grote schade <strong>in</strong><br />
vele gewassen. Chemische gewasbescherm<strong>in</strong>gsmethoden zijn <strong>in</strong> afnemende<br />
mate effectief en worden steeds meer <strong>in</strong>geperkt. Bij de veredel<strong>in</strong>g van gewassen<br />
op ziekteresistentie wordt traditionelerwijze gebruik gemaakt van dom<strong>in</strong>ante<br />
resistentiegenen. Die worden echter snel door oomyceetpathogenen omzeild.<br />
Nieuwe methoden voor resistentieveredel<strong>in</strong>g zijn dan ook gewenst om nieuwe,<br />
duurzame vormen van resistentie <strong>in</strong> gewassen te verkrijgen. Een beter begrip<br />
van waard-plantpathogeen<strong>in</strong>teracties op moleculair niveau is nodig voor nieuwe<br />
strategieën <strong>in</strong> de resistentieveredel<strong>in</strong>g. Belangrijke moleculaire spelers en het<br />
ho<strong>of</strong>donderwerp van dit proefschrift zijn de effectoreiwitten, die valse meeldauwsoorten<br />
gebruiken om hun waardplanten te manipuleren en ziek te maken. Het<br />
bepalen van de nucleotidesequenties van de valse meeldauw Hyaloperonospora<br />
arabidopsidis ‘expressed sequence tags’ bleek een krachtige methode om<br />
effectoreiwitten die actief zijn tijdens het <strong>in</strong>fectieproces te voorspellen. Deze<br />
aanpak onthulde isolaat-specifieke effectoreiwitten die de vatbaarheid van de<br />
waardplant verhoogden. Door schaalvergrot<strong>in</strong>g door middel van ‘massively parallel<br />
transcriptome sequenc<strong>in</strong>g’ kon een breed overzicht van de genen van de valse<br />
meeldauw Bremia lactucae die actief zijn bij de <strong>in</strong>fectie van sla worden verkregen.<br />
Gebaseerd op vergelijk<strong>in</strong>gen met korte genomische DNA-sequenties afkomstig uit<br />
Bremia-sporen zijn de Bremia-eiwitten voorspeld. Effectork<strong>and</strong>idaten zijn vervolgens<br />
geïdentificeerd gebaseerd op de sequentiekenmerken die bekend zijn van<br />
effectoreiwitten van <strong>and</strong>ere oömyceten. Een set van 34 effectork<strong>and</strong>idaten met een<br />
RXLR- <strong>of</strong> RXLR-achtig am<strong>in</strong>ozuurmotief is vervolgens gekloneerd voor verdere<br />
analyse. De rol van deze k<strong>and</strong>idaten <strong>in</strong> het bevorderen van de vatbaarheid van sla<br />
is onderzocht, wat onthulde dat twee effectoreiwitten, BLR16 en BLR27, een sterk<br />
robuust vatbaarheidverhogende activiteit <strong>in</strong> de waardplant hebben. Veel van de<br />
<strong>and</strong>ere k<strong>and</strong>idaten bleken de vatbaarheid <strong>in</strong> m<strong>in</strong>dere mate te verhogen, terwijl één<br />
effector, BLR03, stelselmatig de vatbaarheid verlaagde. BLR16 en BLR27 bleken<br />
gedurende de hele <strong>in</strong>fectiecyclus tot expressie te komen, terwijl de expressie van<br />
BLR03 al na één dag sterk verm<strong>in</strong>derd was. Er werden merkers voor biotische<br />
stress <strong>in</strong> sla ontwikkeld, gebaseerd op hoge relatieve aanwezigheid van transcripten<br />
en gelijkenis met biotische stress-geïnduceerde Arabidopsisgenen. Deze genen<br />
werden echter niet sterk onderdrukt door de geteste Bremia-effectoren, wat er
186<br />
op duidt dat de vatbaarheidverhogende activiteit niet het gevolg is van een brede<br />
onderdrukk<strong>in</strong>g van de biotische stress-respons. In een tweede groot experiment<br />
werden 129 slalijnen getest op herkenn<strong>in</strong>g van de set gekloneerde effectoreiwitk<strong>and</strong>idaten<br />
met behulp van Agrobacterium-gemedieerde transiënte expressie, om<br />
nieuwe herkenn<strong>in</strong>gsspecificiteiten te onthullen. Twee effectoreiwitten met een<br />
GKLR-variant van het RXLR-am<strong>in</strong>ozuurmotief bleken herkend te worden, met<br />
<strong>in</strong>ductie van celdood tot gevolg. BLG01 en BLG03 werden herkend, <strong>in</strong> een aantal<br />
wilde slalijnen, respectievelijk gecultiveerde slalijnen die de gekarakteriseerde<br />
resistentielocus Dm2 bevatten. Een locus op de korte arm van chromosoom 9 van<br />
L. saligna bleek verantwoordelijk voor herkenn<strong>in</strong>g van BLG01, terwijl herkenn<strong>in</strong>g<br />
van BLG03 gekoppeld bleek te zijn aan de Dm2-locus. Bremia-isolaten die deze<br />
effectoreiwitten gebruiken, bleken echter <strong>in</strong> staat ziekte te veroorzaken. Dit duidt<br />
erop dat de herkenn<strong>in</strong>g van de effectoreiwitten <strong>of</strong> de daaropvolgende <strong>in</strong>ductie<br />
van de afweer mogelijk wordt onderdrukt door <strong>and</strong>ere effectoreiwitten van deze<br />
isolaten. Het <strong>in</strong> kaart brengen van het arsenaal van Bremia-effectoreiwitten, het<br />
identificeren van hun vatbaarheidverhogende activiteiten en het onthullen van<br />
nieuwe herkenn<strong>in</strong>gsspecificiteiten zijn belangrijke stappen <strong>in</strong> het begrijpen van de<br />
moleculaire mechanismen die bepalend zijn voor de <strong>in</strong>fectie van sla door Bremia.
187<br />
Acknowledgements<br />
I would like to use this place to thank everyone who contributed pr<strong>of</strong>essionally to<br />
my doctoral studies. Most importantly, I would like to thank the follow<strong>in</strong>g people<br />
who were especially closely <strong>in</strong>volved:<br />
First <strong>of</strong> all, thank you to all those <strong>in</strong>volved <strong>in</strong> the project at the seed companies<br />
<strong>and</strong> Wagen<strong>in</strong>gen University for the stimulat<strong>in</strong>g discussions dur<strong>in</strong>g the project meet<strong>in</strong>gs<br />
<strong>and</strong> the contribution <strong>of</strong> the lettuce l<strong>in</strong>es <strong>and</strong> Bremia isolates used throughout<br />
the project. I’m especially grateful for the screen<strong>in</strong>g <strong>of</strong> the <strong>effectors</strong> <strong>in</strong> the lettuce<br />
l<strong>in</strong>es. Com<strong>in</strong>g from a molecular background it was <strong>in</strong>terest<strong>in</strong>g for me to get an<br />
<strong>in</strong>sight <strong>in</strong>to the world <strong>of</strong> plant breed<strong>in</strong>g. I wish you the best <strong>of</strong> luck <strong>in</strong> your efforts<br />
to breed pest- <strong>and</strong> disease-resistant, easy to grow, nice-look<strong>in</strong>g, tasty lettuce with a<br />
long shelf life <strong>and</strong> all other required traits.<br />
Guido van den Ackerveken <strong>and</strong> Marieke Jeuken, thanks for sett<strong>in</strong>g up this <strong>in</strong>terest<strong>in</strong>g<br />
project with all those <strong>in</strong>volved. I hope the proposal for additional Bremia<br />
research will be funded. Guido, thanks for your supervision, first dur<strong>in</strong>g my f<strong>in</strong>al<br />
<strong>in</strong>ternship <strong>and</strong> later on as co-promoter. I hope your near-limitless optimism will<br />
prove to have had its effect on me. Thanks for the opportunity to dig deeper <strong>in</strong>to<br />
the life <strong>of</strong> an oomycete, as well as for all the patience you have shown <strong>in</strong> deal<strong>in</strong>g<br />
with my occasional scepticism.<br />
I am also grateful for the supervision by the rest <strong>of</strong> the Plant-Microbe Interactions<br />
group: my promoter Corné Pieterse (for tell<strong>in</strong>g me to be more worried), ‘role<br />
model’ Saskia van Wees (for tell<strong>in</strong>g me to be less worried) <strong>and</strong> Peter Bakker (for<br />
the nuance).<br />
Thanks Miek Andel, for the supervision dur<strong>in</strong>g my <strong>in</strong>ternship, the clones for the<br />
project <strong>and</strong> for be<strong>in</strong>g a paranymph. Pim Vergeer, my other paranymph <strong>and</strong> lettuce<br />
tra<strong>in</strong>er, I thank you for grow<strong>in</strong>g all the lettuce, tam<strong>in</strong>g the Agrobacterium stra<strong>in</strong>s,<br />
<strong>and</strong> not gett<strong>in</strong>g too fed up with spore count<strong>in</strong>g. Thank you Michael Seidl for all the<br />
discussions <strong>and</strong> the use <strong>of</strong> your bio<strong>in</strong>formatics expertise. Hans van Pelt, thanks for<br />
the excellent photographic support.<br />
S<strong>in</strong>ce this is also my goodbye from PMI, I would like to thank all PMI-people<br />
past <strong>and</strong> present for mak<strong>in</strong>g my time at PMI all the more fun <strong>and</strong> <strong>in</strong>terest<strong>in</strong>g.<br />
Keep up the good work,<br />
Joost
188
189<br />
Curriculum vitae<br />
Johannes (Joost) H.M. Stassen was born on 1 November, 1984 <strong>in</strong> Woerden, The<br />
Netherl<strong>and</strong>s. He f<strong>in</strong>ished his secondary education at M<strong>in</strong>kema College, Woerden<br />
<strong>in</strong> 2002 <strong>and</strong> cont<strong>in</strong>ued his education at University College Utrecht. In 2005 he<br />
obta<strong>in</strong>ed his BSc degree <strong>and</strong> registered for the Master’s programme Biomolecular<br />
Sciences. As part <strong>of</strong> this programme he performed a research project entitled<br />
‘A Search for the Role <strong>of</strong> BAIAP3’ <strong>in</strong> the group <strong>of</strong> Dr Peter van der Sluis at the<br />
Department <strong>of</strong> Cell Biology at the University Medical Centre, Utrecht. He worked<br />
on a second research project, ‘Genetic Mapp<strong>in</strong>g <strong>of</strong> the Downy Mildew Resistance<br />
Loci dmr4 <strong>and</strong> dmr2 <strong>in</strong> Arabidopsis thaliana’, <strong>in</strong> the group <strong>of</strong> Dr Guido van den<br />
Ackerveken, which was <strong>in</strong>itially part <strong>of</strong> the Molecular Genetics group at Utrecht<br />
University, but fused with the Phytopathology group to form the Plant-Microbe<br />
Interactions group. In 2007, he received his MSc degree <strong>and</strong> started work on the<br />
project described <strong>in</strong> this thesis under supervision <strong>of</strong> Dr Guido van den Ackerveken<br />
<strong>and</strong> Pr<strong>of</strong>essor Corné Pieterse at the Plant-Microbe Interactions group at Utrecht<br />
University.
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