75 Integrating Membrane Transport with Male Gametophyte ... - TAIR

265 Translesion polymerases influence growth rate in the presence of DNA lesions
Marc Curtis, John Hays
Oregon State University
DNA damage induced by UV-B light, reactive oxygen species, or other agents threatens plant genomic integrity.
DNA lesions present during S-phase can block replication. Persistent collapsed replication forks can give rise to lethal
double-strand breaks. Specialized translesion polymerases (TLPs) with inherently low fidelity synthesize DNA past
template lesions, with relatively 'error-free' or 'error-prone' results. Although all TLPs lack proofreading exonucleases,
the extent of errors depends on the particular TLP or combination of TLPs involved, and the particular template lesion
being bypassed. Four TLPs are conserved across eukaryotic kingdoms. Pol κ, Pol η and Rev1 are members of the Y-family
of DNA polymerases. The fourth conserved eukaryotic TLP is Pol ζ, a member of the B-family of DNA polymerases,
which includes the major replicative (proofreading) polymerases. Unlike the high fidelity replicative polymerases, Pol
ζ is able to elongate distorted primer-template termini that arise at DNA lesions.
In root assays, mutant Arabidopsis lacking Pol η (AtRAD30-1/1) are sensitive to UV-B, exhibiting a transient inhibition
of growth rate. Plants lacking Pol ζ (AtREV3-2/2) are more sensitive to UV-B, perhaps due to a cell cycle arrest. Plants
lacking both polymerases show a prolonged UV-B-induced inhibition of growth, perhaps due to irreparable damage
to initial cells and some death of initials; recovery appears to occur by replacement of initial cells from the quiescent
center. In double mutants, sensitivity was more than additive, suggesting inter-dependent functions. The two major UV
photoproducts are cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6,4) pyrimidone dimers; Pol η is known to
bypass the former efficiently but not the latter. Bypass of the 6,4 photoproduct requires Pol ζ, which also may extend
primer termini across from CPDs that are in sequence contexts problematic for Pol η.
TLPs also influence the coordinated growth of ovule and gamete development (absent UVB): in AtREV3-2/+
heterozygotes, 50% of the female gametes are buried in the integuments. Moreover, progeny of selfed AtREV3-2/+
AtRAD30-1/+ double heterozygotes exhibit non-Mendelian segregation. Surprisingly, single heterozygotes, double
mutants, and wild-type progeny were all underrepresented, consistent with selective loss of wild-type and double-mutant
gametes. (Supported by NSF grant MCB-0345061 to J.B.H.)
266 Functional evaluation of plant defence signalling against Fusarium ear blight disease in
Arabidopsis
Alayne Cuzick, Kerry Maguire, Sarah Lee, Kim Hammond-Kosack
Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, UK
Fusarium ear blight (FEB) infections of cereal crops cause considerable losses to grain quality and safety. The
two main causative agents of this disease on wheat crops are the hemibiotrophic pathogens F. culmorum (Fc) and
F. graminearum (Fg) (sexual stage Gibberella zeae). Floral infections by Fc and Fg also cause the developing cereal
grains to become contaminated with various fungal mycotoxins, including the highly toxic trichothecene mycotoxin
deoxynivalenol (DON). The molecular basis of resistance to FEB in cereal species is poorly understood but it is QTL
based and Fusarium species non-specific.
We have previously demonstrated that Fc and Fg conidia can infect the floral tissues of Arabidopsis to cause disease
symptoms on flowers, siliques and upper stem tissue [1]. DON mycotoxin production was detected in infected flowers.
This novel Arabidopsis floral model provides a tractable system for elucidating fundamental aspects of this globally
important cereal-fungal interaction.
We are now undertaking a detailed analysis of the FEB infection phenotype in Arabidopsis genotypes with defined
gene mutations. Data will be presented on the effects of genes involved in basal and race-specific defence signalling,
including RAR1, SGT1 and EDS1. Infections are quantified by FAD values (Fusarium-Arabidopsis Disease) [1].
Forward genetics experiments are also being conducted to select EMS mutagenised Arabidopsis lines resistant to
DON mycotoxin.
We are in the process of obtaining / generating a selection of transgenic fusarium strains that contain different reporter
contructs. These include constitutive expression of GFP (green flurorescent protein) or GUS (ß-glucuronidase) as well as
TRI5 promoter mediated GUS expression to mark the onset of mycotoxin production. These strains will be used to aid the
visualisation and understanding of the Fusarium-Arabidopsis pathosystem in both wild-type and mutant genotypes.
This research is supported by the Biotechnology and Biological Sciences Research Council of the UK .
References
1. Urban et al., (2002) Plant Journal 32, 961-973