The genomics of plant-virus co-evolution in wild Brassica species

The genomics of plant-virus
co-evolution in wild Brassica
species
Mike Kearsey
Agriculture and
Agri-Food Canada
Environmental Genomics Grant Number NER/T/S/2002/00902
1

Questions.
• Do wild plants show a different genetic response
to local virus pathotypes than to foreign ones
• Is there competition between different virus
pathotypes replicating within a single plant and
does the outcome depend on plant genotype
• Do these effects vary with plant species
2

Transcriptomics Approach.
2. To develop and optimise an oligonucleotide
microarray to study Brassica genomics in
order to…..
4. … identify genes involved in differential plant
host responses to challenge from different viral
genotypes.
3

Experimental System:
Plant communities & virus isolate origin
Turnip Mosaic Virus
(TuMV)
Llandudno, Wales
Brassica oleracea
& TuMV GBR83
• Potyvirus, singlestranded
+ sense RNA
genome (10K bp)
Winspit, Dorset
Brassica oleracea
& TuMV GBR98
Culham, Oxon
Brassica rapa
Sturminster Newton, Dorset
Brassica rapa
4

Genetic variation – TuMV coat
100
100
100
protein nucleotide sequence
2 - 4 major phylogenetic
groups supported by high
bootstrap values
The genetic diversity of
Winspit, Dorset
sub-populations Chapman‘s Pool, Dorset is partially
Kimmeridge, Dorset
defined Great by Orme, the Walesgeographic
Staithes,
location
North Yorkshire
GBR 48(S)
GBR 47(S)
100
GBR 98(W)
GBR 44(W)
GBR 43(W) GBR 98
GBR 38(W) Pathotype 1
GBR 36(W)
GBR 37(W)
GBR 23(C)
GBR 31(C)
GBR 18(K)
GBR 21(W)
GBR 29(C)
GBR 19(C)
GBR 35(W)
GBR 32(C)
GBR 28(C)
UK 1
UK2
1
GBR 56(GO)
GBR 26(K)
GBR 27(K)
GBR 25(K)
GBR 24(K)
GBR 20(K)
GBR 30(K)
GBR 91(GO)
GBR 22(C)
GBR 46(S)
GBR 49(S)
GBR 50(S)
GBR 45(S)
GBR 57(GO)
GBR 53(GO) 2
GBR 52(GO)
GBR 51(S)
GBR 55(GO)
GBR 54(GO)
GBR 83(GO)
GBR 83
Pathotype 3
3
5
4

Results from 1 st competition experiment
Wild cabbage from Llandudno (LL3);
local isolate is GBR 83
number of plants
8
7
6
5
4
3
2
1
0
B. napus – susceptible control
Inoculation 1st round 2nd round 3rd round 4th round 5th round
mixed infected
Infected with GBR 98 from Winspit
Infected with GBR 83 from Llandudno
number of plants
Wild cabbage from Winspit (WS15);
local isolate is GBR 98
number of plants
8
7
6
5
4
3
2
1
0
8
7
6
5
4
3
2
1
0
Inoculation 1st round 2nd round 3rd round 4th round 5th round
Inoculation 1st round 2nd round 3rd round 4th round 5th round
6

Brassica Array / Sub-array Layout - Schematic
15,000 oligos spotted in duplicate
-14,996 Brassica napus
- 4 TuMV
26
12
26
4
Landing light
Stratagene Alien Oligo Controls
676
Oligos duplicated within a subarray
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Why create a Brassica array
Brassicas closely related to Arabidopsis,
BUT:
• Brassicas are ancient polyploid species
so need to distinguish between multiple
members of related gene families.
• B.napus chip has both A and C genomes.
• Many Brassica genes would not be
represented on an Arabidopsis array.
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Gene Families Arrayed.
Enriched for:
• Stress (biotic and abiotic)
• Meiosis and reproduction
• Genes recognisable in Arabidopsis
• Genes with no Arabidopsis equivalent.
Avoided:
• Housekeeping and ribosomal genes
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Experimental design
for each population/species.
Mock
(n plants)
X r slides
X r slides
TuMVGBR83
(n plants)
X r slides
TuMV GBR98
(n plants)
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Results – Quantitative analysis
Genes Differentially Expressed
Mock vs. 83
Mock vs. 98
83 vs. 98
B. rapa - Culham
1107
1199
656
B. rapa – Stur. Newt
762
615
362
B. oleracea - Llandudno
141
103
1
B. oleracea - Winspit
30
29
1
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Results – Qualitative analysis
• GO analysis:
1. Biological Processes - describes what it changes
2. Molecular Function – describes what it does
3. Cellular Component – describes where it acts
• GOSSIP: uses GO annotations to test whether terms
are enriched within a group of genes compared to a
reference set.
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GO Analysis - Highlights
Significant
GO Terms
Photosystem II
/Thylakoid
Proteolysis
Biological significance
Known to be down-regulated in
several plant-virus systems
Fungal attack causes degradation
of unnecessary proteins
Oxidative
stress
Water
deprivation
Known plant-resistance related pathways
Useful markers for programmed
cell death (PCD)
Link to environmental conditions
and possible explanation for
TuMV’s absence in wild B.rapa
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GO Analysis
Significant
GO Terms
Carbon
utilisation
Nucleosome
&
Nucleosome
assembly
Biological significance
Culham plants down-reg these
genes in response to both
isolates. Not significant in
Sturminster Newton
Both are related terms in two
separate ontologies. Significant
difference between isolates:
83 up-regulated w.r.t 98;
detected in both plant pops.
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Summary
• We developed a 15K Brassica oligo array
to analyse gene expression of three related
Brassica species.
• We detected ~10-fold greater expression in
wild B. rapa than B.oleracea in response to
two TuMV isolates.
• GO analysis suggests that many genes
detected are involved in failing B. rapa
resistance pathways and PCD.
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CEH Oxford:
Acknowledgements
Jono Reeves, Denise Pallett, Hui Wang, Ian
Cooper, Bela Tiwari, Delia McCall.
HRI Warwick:
John Walsh, Christian Obermeier.
University Of Birmingham:
Rachel Machado, Zewei Luo, Elaine Howell.
Agriculture and Agri-food Canada:
Andrew Sharpe, Derek Lydiate, Branimir
Gretvaj, Chris Lewis
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