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Phenotypic and genetic evidence for tolerance to bacterial wilt in Arabidopsis plants
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  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. Bacterial wilt • Ralstoniasolanacearum • Soil-borne vascular pathogen • Wide host range • Symptoms – wilting and necrosis • Species complex (Fegan & Prior 2005) APSNET

  3. Bacterial wilt on Eucalyptus trees AFRICA Coutinho TA, Roux J, Riedel KH, Terblanche J, Wingfield MJ (2000) First report of bacterial wilt caused by Ralstoniasolanacearum on eucalypts in South Africa. For Pathol 30: 205-210 Roux J, Coutinho TA, Wingfield MJ, Bouillet J-P (2000) Diseases of plantation Eucalyptus in the Republic of Congo. S Afr J Sci 96: 454-456 Roux 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 Ralstoniasolanacearum strains from China. European Journal of Plant Pathology 125: 641-653 Fouche-WeichJ, Berger D, Poussier S, Trigalet-Demery D, Coutinho T (2006) Molecular identification of some African strains of Ralstoniasolanacearum from eucalypt and potato. Journal of General Plant Pathology 72: 369-373 APSNET

  4. Identify mechanisms of plant resistance to bacterial wilt STRATEGY Screen Natural Diversity of Arabidopsis thaliana for resistance to bacterial wilt using Eucalyptus isolate of RalstoniasolanacearumBCCF 402* APSNET * Fouche-WeichJ, Berger D, Poussier S, Trigalet-Demery D, Coutinho T (2006) Molecular identification of some African strains of Ralstoniasolanacearum from eucalypt and potato. Journal of General Plant Pathology 72: 369-373

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

  6. The pathosystem BCCF402 RalstoniasolanacearumBCCF 402 (from Eucalyptus) vsArabidopsis thaliana Be-0 mock inoculated Kil-0 Nd-1 + BCCF402 + BCCF402 + BCCF402

  7. A curious result + BCCF402 + BCCF402 + BCCF402 Kil-0

  8. Tolerance:plant does not show a significant reduction in fitness despite high pathogen numbers in plantaResistance:plant does not show a significant reduction in fitness but severely restricts pathogen numbers in plantaSusceptibility:plant shows a significant reduction in fitness and has high pathogen numbers in planta Kover and Schaal (2002) PNAS 99:11270-11274

  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 LweIdigestion of PCR products (CAPS = cleaved amplified polymorphic sequences) F1 progeny were susceptible to R. solanacearumBCCF402 Tolerance is recessive

  12. Be-0 F2 progeny Kil-0 F2 progeny segregate for tolerance:susceptibility in a 1: 3 ratio Kil-0 tolerance to R. solanacearumconferred by a single recessive gene

  13. 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 Tolerant F2 progeny Kil-0 Be-0 Susceptible F2 progeny Be-0 Kil-0 • F3 Kil-0 tolerance to R. solanacearumlinked to RRS1

  14. Tolerance in Kil-0 is allelic to resistance in Nd-1 Bacterial numbers High High Low High* Kil-0 tolerance conferred by RRS1 or tightly linked gene

  15. Susceptibility Resistance Tolerance R. solanacearum R. solanacearum popP2 popP2 popP2 R. solanacearum RRS1-R RRS1-R Be-0 Nd-1 Kil-0 Col-5 Nd-1 Effector triggered susceptibility (ETS) Effector triggered immunity (ETI) Effector triggered tolerance (ETT) ( adapted from da Cunha et al. 2006)

  16. Susceptibity Tolerance Predict: popP2 mutant RRS1-R R. solanacearum R. solanacearum popP2 popP2 R. solanacearum RRS1-R Be-0 Kil-0 Col-5 Effector triggered susceptibility (ETS) ETT breaks down Effector triggered tolerance (ETT) ( adapted from da Cunha et al. 2006)

  17. Kil-0 tolerance requires R. solanacearum popP2 effector mock inoculated BCCF402 BCCF402 ΔpopP2 BCCF402 ΔpopP2 pLAFR6::popP2 Kil-0 Be-0 Supports hypothesis that Kil-0 tolerance conferred by RRS1 and not another linked gene

  18. Do AA sequences of RRS1 or popP2 explain difference between ETI and ETT? Tolerance Resistance R. solanacearum R. solanacearum popP2 popP2 RRS1-R RRS1-R Kil-0 Nd-1 Effector triggered tolerance (ETT) Effector triggered immunity (ETI)

  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. Do AA sequence differences in RRS1 explain difference between ETI and ETT? 1378 AA RRS1 truncated in susceptible ecotypes Only 8 AA difference between Nd-1 and Kil-0

  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. Model of Effector triggered tolerance (ETT) R. solanacearum R. solanacearum popP2 popP2 RRS1-R RRS1-R Bergelson lab Rpm1 – fitness benefit at high inoculum levels i.e. single gene tolerance (Genetics 2010) Rps5 – no fitness benefit (New Phytol 2009) Kil-0 Nd-1 Nd-1 Effector triggered immunity (ETI) Effector triggered tolerance (ETT) ( adapted from da Cunha et al. 2006)

  23. Acknowledgements Students • Liesl van der Linden • JaneBredenkamp Collaborators • Yves Marco & StephaneGenin,CNRS/INRA, Toulouse, France Katherine Denby, University of Warwick, UK SanushkaNaidoo, Dept of Genetics, UP Funding NRF, South Africa CNRS &Agropolis-South Africa exchangeprogramme

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