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Phenotypic and genetic evidence for tolerance to bacterial wilt in Arabidopsis plants Dave Berger Plant Science Department Forestry and Agricultural Biotechnology.

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Presentation on theme: "Phenotypic and genetic evidence for tolerance to bacterial wilt in Arabidopsis plants Dave Berger Plant Science Department Forestry and Agricultural Biotechnology."— Presentation transcript:

1 Phenotypic and genetic evidence for tolerance to bacterial wilt in Arabidopsis plants Dave Berger Plant Science Department Forestry and Agricultural Biotechnology Institute (FABI) University of Pretoria

2 Ralstonia solanacearum Soil-borne vascular pathogen Wide host range Symptoms – wilting and necrosis Species complex (Fegan & Prior 2005) Bacterial wilt APSNET

3 Bacterial wilt on Eucalyptus trees APSNET AFRICA Coutinho 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: Roux J, Coutinho TA, Wingfield MJ, Bouillet J-P (2000) Diseases of plantation Eucalyptus in the Republic of Congo. S Afr J Sci 96: Roux J, Coutinho TA, Byabashaija DM, Wingfield MJ (2001) Diseases of plantation Eucalyptus in Uganda. S Afr J Sci 97: BRAZIL (2005) Susceptibility to wilt associated with Pseudomonas solanacearum among six species of Eucalyptus growing in equatorial Brazil. Austral Plant Pathol 19: CHINA (2009) Genetic diversity of Ralstonia solanacearum strains from China. European Journal of Plant Pathology 125: 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:

4 Identify mechanisms of plant resistance to bacterial wilt APSNET STRATEGY Screen 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:

5 Dogma in molecular plant pathology Resistance / : gene-for-gene interactions Immunity Tolerance :polygenic, QTLs of small effect

6 mock inoculated Be-0 Kil-0 Nd-1 BCCF402 + BCCF402 The pathosystem Ralstonia solanacearum BCCF 402 (from Eucalyptus) vs Arabidopsis thaliana

7 + BCCF402 A curious result Kil-0

8 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:

9 Kil-0 does not show significant reduction in yield/fecundity in response to R. solanacearum, in contrast to Be-0 Support for Tolerance hypothesis

10 What is the genetic basis of tolerance? Cross-fertilization of Kil-0 and Be-0

11 CAPS markers confirm cross-fertilization ie F1 progeny are hybrids LweI digestion of PCR products (CAPS = cleaved amplified polymorphic sequences) F 1 progeny were susceptible to R. solanacearum BCCF402 Tolerance is recessive

12 Be-0F 2 progeny Kil-0 Kil-0 tolerance to R. solanacearum conferred by a single recessive gene F 2 progeny segregate for tolerance:susceptibility in a 1: 3 ratio

13 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 Be-0

14 Tolerance in Kil-0 is allelic to resistance in Nd-1 mock inoculatedinoculated Be-0 Kil-0 Nd-1 F1 (Kil-0 X Nd-1) Bacterial numbers High Low High* Kil-0 tolerance conferred by RRS1 or tightly linked gene

15 SusceptibilityResistance 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

16 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

17 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

18 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?

19 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 GMI1000 Catalytic triad conserved Autoacetylated lysine conserved

20 RRS1 truncated in susceptible ecotypes Only 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) AA

21 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 requires RRS1 – popP2 interaction

22 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

23 Collaborators Yves Marco & Stephane Genin, CNRS/INRA, Toulouse, France Katherine Denby, University of Warwick, UK Sanushka Naidoo, Dept of Genetics, UP Students Liesl van der Linden Jane Bredenkamp Acknowledgements Funding NRF, South Africa CNRS & Agropolis -South Africa exchange programme

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26 Multiplication of R. solanacearum BCCF402 bacteria in A. thaliana accessions Be ‑ 0 and Kil-0 is hrp-dependent.  + BCCF402 hrp Be-0 Kil-0 Be-0 Kil-0 (Hrp cluster encodes type III secretion system)


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