phenotypic and genetic evidence for tolerance to bacterial wilt in arabidopsis plants dave berger...
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Phenotypic and genetic evidence for tolerance to bacterial wilt in Arabidopsis plants
Dave BergerPlant Science Department
Forestry and Agricultural Biotechnology Institute (FABI)University of Pretoria
• Ralstonia solanacearum• Soil-borne vascular pathogen • Wide host range• Symptoms – wilting and necrosis• Species complex (Fegan & Prior 2005)
Bacterial wilt
APSNET
Bacterial wilt on Eucalyptus trees
APSNET
AFRICACoutinho TA, Roux J, Riedel KH, Terblanche J, Wingfield MJ (2000) First report of bacterial wilt caused by Ralstonia solanacearum on eucalypts in South Africa. For Pathol 30: 205-210Roux J, Coutinho TA, Wingfield MJ, Bouillet J-P (2000) Diseases of plantation Eucalyptus in the Republic of Congo. S Afr J Sci 96: 454-456Roux J, Coutinho TA, Byabashaija DM, Wingfield MJ (2001) Diseases of plantation Eucalyptus in Uganda. S Afr J Sci 97: 16-18
BRAZIL(2005) Susceptibility to wilt associated with Pseudomonas solanacearum among six species of Eucalyptus growing in equatorial Brazil. Austral Plant Pathol 19: 71-76
CHINA(2009) Genetic diversity of Ralstonia solanacearum strains from China. European Journal of Plant Pathology 125: 641-653
Fouche-Weich J, Berger D, Poussier S, Trigalet-Demery D, Coutinho T (2006) Molecular identification of some African strains of Ralstonia solanacearum from eucalypt and potato. Journal of General Plant Pathology 72: 369-373
Identify mechanisms of plant resistance to bacterial wilt
APSNET
STRATEGYScreen Natural Diversity of Arabidopsis thaliana for resistance to bacterial wilt using Eucalyptus isolate of Ralstonia solanacearum BCCF 402*
* Fouche-Weich J, Berger D, Poussier S, Trigalet-Demery D, Coutinho T (2006) Molecular identification of some African strains of Ralstonia solanacearum from eucalypt and potato. Journal of General Plant Pathology 72: 369-373
Dogma in molecular plant pathology
Resistance / : gene-for-gene interactionsImmunity
Tolerance : polygenic, QTLs of small effect
mock inoculated
Be-0
Kil-0
Nd-1
BCCF402
2 4 6 8 10 12 140.0
0.2
0.4
0.6
0.8
1.0Be-0
Kil0
Nd1
days post inoculation
Dis
ea
se
ind
ex
+ BCCF402
+ BCCF402
+ BCCF402
The pathosystemRalstonia solanacearum BCCF 402 (from Eucalyptus) vs Arabidopsis thaliana
2 4 6 8 10 12 140.0
0.2
0.4
0.6
0.8
1.0Be-0
Kil0
Nd1
days post inoculation
Dis
ea
se
ind
ex
+ BCCF402
+ BCCF402
+ BCCF402
A curious result
4 8 12 166
7
8
9
10
11
12
days post inoculation
log
(c
fu/g
fre
sh
we
igh
t)
Kil-0
Tolerance: plant does not show a significant reduction in fitness despite high pathogen numbers in planta
Resistance:plant does not show a significant reduction in fitness but severely restricts pathogen numbers in planta
Susceptibility:plant shows a significant reduction in fitness and has high pathogen numbers in planta
Kover and Schaal (2002) PNAS 99:11270-11274
Kil-0 does not show significant reduction in yield/fecundity in response to R. solanacearum,in contrast to Be-0
Support for Tolerance hypothesis
What is the genetic basis of tolerance?
Cross-fertilization of Kil-0 and Be-0
CAPS markers confirm cross-fertilization ie F1 progeny are hybrids
LweI digestion of PCR products(CAPS = cleaved amplified polymorphic sequences)
F1 progeny were susceptible to R. solanacearum BCCF402
Tolerance is recessive
Be-0 F2 progeny Kil-0
Kil-0 tolerance to R. solanacearum conferred by a single recessive gene
F2 progeny segregate for tolerance:susceptibility in a 1: 3 ratio
Table 2.2. Segregation analysis of R. solanacearum isolate BCCF 402 resistance in the F2 progeny from crosses between ecotypes Kil-0 (resistant) and Be-0 (susceptible).
Number of plants
Trial Cross Resistant Susceptible TotalExpected ratio (R:S)
Observed ratio (R:S) χ2a P
1 Kil-0 × Be-0 74 215 289 1:3 1:2.9 0.06 0.9>P>0.72 Kil-0 × Be-0 92 295 387 1:3 1:3.2 0.31 0.7>P>0.5
a χ2 values were calculated for a segregation ratio of 1 resistant : 3 susceptible plants.
Kil-0 tolerance to R. solanacearum linked to RRS1
Where in the Arabidopsis genome is the tolerance gene?Hypothesis: Tolerance conferred by allele of the RRS1 gene which confers R to a tomato isolate
F3
Tolerant F2 progeny
Susceptible F2 progeny
Kil-0
Kil-0
Be-0
Be-0
Tolerance in Kil-0 is allelic to resistance in Nd-1
mock inoculated inoculated
Be-0
Kil-0
Nd-1
F1 (Kil-0 X Nd-1)
Bacterial numbers
High
High
Low
High*
Kil-0 tolerance conferred by RRS1 or tightly linked gene
Susceptibility Resistance
Col-5 Nd-1
( adapted from da Cunha et al. 2006)
R. solanacearum
popP2
Be-0 Nd-1
Effector triggered susceptibility (ETS)
Effector triggered immunity
(ETI)
popP2
RRS1-R
R. solanacearum
Tolerance
Kil-0
Effector triggered tolerance
(ETT)
popP2
RRS1-R
R. solanacearum
Susceptibity
Col-5
( adapted from da Cunha et al. 2006)
R. solanacearum
popP2
Be-0
Effector triggered susceptibility
(ETS)
Tolerance
Kil-0
Effector triggered tolerance
(ETT)
popP2
RRS1-R
R. solanacearum
Predict: popP2 mutant
ETT breaks down
R. solanacearum
RRS1-R
mock inoculated
BCCF402
pLAFR6::popP2
Kil-0 Be-0
BCCF402 ΔpopP2
BCCF402 ΔpopP2
Kil-0 tolerance requires R. solanacearum popP2 effector
Supports hypothesis that Kil-0 tolerance conferred by RRS1 and not another linked gene
Tolerance
Kil-0
Effector triggered tolerance
(ETT)
popP2
RRS1-R
R. solanacearum
Resistance
Nd-1
Effector triggered immunity
(ETI)
popP2
RRS1-R
R. solanacearum
Do AA sequences of RRS1 or popP2 explain difference between ETI and ETT?
Do AA sequence differences in popP2 explain difference between ETI and ETT?
R.solanaceraum BCCF402 elicits ETI in Nd-1 and ETT in Kil-0.
R.solanaceraum GMI1000 elicits ETI in Nd-1.Only 4 AA difference between PoP2 of BCCF402 and GMI1000Catalytic triad conservedAutoacetylated lysine conserved
RRS1 truncated in susceptible ecotypesOnly 8 AA difference between Nd-1 and Kil-0
Do AA sequence differences in RRS1 explain difference between ETI and ETT?
Nd-1 (R )
% identity
Kil-0 (R ) 98.9
Be-0 (S) 97.3
Col-0 (S) 91.8
1378 AA
Conclusion: Gene-for-gene tolerance in Kil-0
R.solanacearum inoculation of Kil-0 plants:
• Kil-0 did not wilt but had high bacterial numbers in planta
• Plant biomass yield, seed number, germination not reduced
• Kil-0 response distinct from “resistant” ecotype Nd-1
Genetic evidence Kil-0 tolerance conferred by RRS1
Knockout/complementation evidence that Kil-0 requiresRRS1 – popP2 interaction
Nd-1
( adapted from da Cunha et al. 2006)
Kil-0
Effector triggered tolerance
(ETT)
popP2
RRS1-R
R. solanacearum
Model of Effector triggered tolerance (ETT)
Bergelson lab
Rpm1 – fitness benefit at high inoculum levels
i.e. single gene tolerance (Genetics 2010)
Rps5 – no fitness benefit (New Phytol 2009)
Nd-1
Effector triggered immunity
(ETI)
popP2
RRS1-R
R. solanacearum
CollaboratorsYves Marco & Stephane Genin, CNRS/INRA, Toulouse, FranceKatherine Denby, University of Warwick, UKSanushka Naidoo, Dept of Genetics, UP
StudentsLiesl van der LindenJane Bredenkamp
Acknowledgements
FundingNRF, South AfricaCNRS & Agropolis -South Africa exchange programme
0 2 6 8 12 143
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Days After Inoculation
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Multiplication of R. solanacearum BCCF402 bacteria in A. thaliana accessions Be‑0 and Kil-0 is hrp-dependent.
+ BCCF402
+ BCCF402
+ BCCF402
+ BCCF402 hrp
hrp
Be-0
Kil-0
Be-0
Kil-0
(Hrp cluster encodes type III secretion system)