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Jianping Cui Naomi Pierce Fred Ausubel

Coronatine-Dependent Systemic Induced Susceptibility (SIS) in Virulent Pseudomonas syringae Infection. Jianping Cui Naomi Pierce Fred Ausubel. Overview : Pathogens Get Perspective, Too (Successful pathogen vs.Successful plant).

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Jianping Cui Naomi Pierce Fred Ausubel

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  1. Coronatine-Dependent Systemic Induced Susceptibility (SIS) in Virulent Pseudomonas syringae Infection Jianping Cui Naomi Pierce Fred Ausubel

  2. Overview : Pathogens Get Perspective, Too (Successful pathogen vs.Successful plant) Systemic acquired resistance (SAR) In response to avirulent pathogens, plants activate long-lasting resistance responses systemically to defend against a broad spectrum microbial pathogens. Systemic induced susceptibility (SIS)Virulent pathogens suppress basal and induced host defenses systemically to facilitate colonization. Mechanisms of SIS Coronatine, phytotoxin produced by Pseudomonas syringae in some virulent infections, suppresses SA-dependent basal and induced host responses probably through SA/JA antagonism. Coronatine is a structural and functional JA mimic.

  3. avrB avrB avrB pLAFR pLAFR pLAFR MgSO4 MgSO4 MgSO4 avrRpt2 avrRpt2 avrRpt2 Col rps2 rpm1 Virulent P. syringae Induces Systemic Susceptibility to Insects Virulent pathogen has been shown to induce similar transcriptional profile as avirulent pathogen, but at lower magnitude and later stage. However… 2 ns * * * * Tricoplusia ni (T. ni) 1.5 Relative Larval Weight Gain 1 ** ** ** ** Virulent Psm 0.5 0 Virulent infection Avirulent infection

  4. Rationale and History INPUT (Local) PLANT OUTPUT (Systemic) Pathogen growth () Avirulent Psm Insect performance () Pathogen growth (?) Virulent Psm Insect performance () SAR Unknown Changes Virulent pathogen induces similar transcriptional profile as avirulent pathogen

  5. Virulent Psm makes upper part of plant more susceptible 5.6 Psm growth * 5.4 5.2 5 Psm growth 4 dpi (Log cfu/cm2) 4.8 Virulent Psm * 4.6 4.4 4.2 4 MgSO4 Psm(Rpt2) Psm(PLAFR)

  6. Virulent Pst also Induces Systemic Susceptibility 7 * Pst DC3000 growth 6.5 6 Pst growth 4 dpi (log cfu/cm2) * 5.5 Virulent Pst DC3000 5 4.5 4 Pst DC3000 MgSO4 Pst(avrRpm1)

  7. Pathogens must overcome plant basal defenses to become virulent LOWER LEAF UPPER LEAF Type III secretion system (TTSS) Virulence factors (TTSS effectors, toxins) Virulent SIS Avirulence (avr) gene Resistance (R) gene Psm (avrRpt2) Avirulent SAR Hypersensitive response (HR) Counteracting target Non- pathogen Many others / SAR (SA, PR proteins) Basal defenses Psm Avr-R recognition Signaling molecules (Salicylic acid (SA))

  8. SIS Inducer Candidate : Phytotoxin Coronatine Rationale and previous knowledge Coronatine is primary virulence factor of several strains of Ps, including Psm and Pst. COR- mutants are less virulent. In Psm, rpoN is essential for COR production. A rpoN mutant does not multiply, does not elicit disease symptoms, does not induce PR genes. Coronatine is a structural and functional JA mimic (Bender et al). Coronatine induces Ps susceptibility when sprayed locally. 5. Genes involved in the biosynthesis of coronatine are up-regulated during Ps pathogenesis (Wang et al., 2002). 6. Arabidopsis coi1(coronatine-insensitive) mutants are more resistant to Ps than wild type plants (Kloek et al., 2001). 7. JA and SA are known to mediate systemic responses. Interference with these signal molecules might result in systemic changes. SIS is systemic. Hypothesis 1. Psm produces the phytotoxin, coronatine, which mimics JA. 2. Coronatine suppresses SA-mediated basal and induced defenses, not only locally but also systemically.

  9. Coronatine Induces SIS 6 * * 5.5 Coronatine 5 Psm growth 4 dpi (log cfu/cm2) 4.5 * Psm 4 3.5 Coronatine MgSO4 Psm (avrRpt2) Psm (pLAFR) Coronatine

  10. Construction of COR- mutant By Gustavo Guzman COR quantification 1. We have constructed a defined mutation in the COR gene cluster of Psm ES4326. The cfa6 mutant, Psm ES62, is COR-. 2. COR production was restored to Psm ES62 when is was complemented with pHLAFR3G4 (contains CFA gene cluster and regulatory region) and partially with pVRC4 (contains only CFA region). 40 30 µgCOR/ml culture 20 10 0 Psm ES 62 62(3G4) 62(VRC4)

  11. COR- Mutant Does Not Induce SIS 6 * 5.5 5 Psm growth 4 dpi (log cfu/cm2) 4.5 * 4 3.5 3 MgSO4 Psm (avrRpt2) Psm (pLAFR) ES62

  12. O H H NH C CO2H O Structure of coronatine and its components, coronafacic acid (CFA) and coronamic acid (CMA) From Carol Bender CORONATINE 1. CFA shows structural similarity to jasmonate 2. CMA is similar to ACC (aminocyclopropyl carboxylic acid), the immediate precursor to ethylene CFA CMA O NH H H CO2H C O CH3 ACC O ETHYLENE JASMONATE

  13. Complete Structure of Coronatine Is Necessary for SIS Induction 6 * 5.5 5 Psm growth 4 dpi (log cfu/cm2) 4.5 4 3.5 CFA CMA MgSO4 Coronatine

  14. Coronatine Induces SIS at Nanomolar Level, Suggesting It Acts as Signal Compound 6.5 ** ** * 6 * * 5.5 Psm growth 4 dpi (log cfu/cm2) 5 4.5 4 MgSO4 COR(1) COR(10) COR(0.1) COR(0.01) COR(0.001) Coronatine dose (µM)

  15. COR- Avirulent Psm Strain Induces Stronger SAR 5.5 5 * 4.5 Psm growth 4 dpi (log cfu/cm2) 4 ** 3.5 3 MgSO4 Psm (avrRpt2) ES62 (avrRpt2)

  16. Summary Ps SIS is a critical component of P. syringae pathogenesis Virulent Psm not only overcomes basal defense at the site of infection, but also disarms host defense systemically. Coronatine can induce SIS independent of Psm infection. This strongly suggests that coronatine, when produced by virulent Psm during growth in infected leaves, is able to induce SIS during the infection process. ES62, which is a COR- mutant, fails to induce SIS, which supports the hypothesis that virulent Ps induces SIS through coronatine. Induction of SIS requires both the CFA and CMA components of coronatine. Coronatine induces SIS by acting as signal compound. Avirulent Psm partiallysuppresses SAR by counteracting it with coronatine.

  17. Et SA JA JA JA Et JA SA SA Et Et JA SA Involvement of SA, JA and Et Signaling Pathways in SIS Rationale 1. SA, JA and Et mediate defense signaling pathways in Arabidopsis. These signaling cascades are interconnected, both antagonistically and synergistically. 2. Pathogens evolve counter-defense strategy by targeting various steps of the signaling cascades. 3. Signal antagonism is the most efficient way to suppress host defense system, because the suppression is systemic. Experiments Test SA, JA and Et mutants for loss of SIS. SA pathway transgenic plant and mutant: nahG, npr1 JA pathway mutant: jar1, coi1 Et pathway mutant: ein2

  18. Et SA JA JA JA Et SA JA SA Et Et JA SA Psm SIS is dependent on SA Col NahG 9 8 7 * 6 * 5 4 Psm growth 4 dpi (log cfu/cm2) 3 2 1 0 MgSO4 MgSO4 Untreated Psm(PLAFR) Untreated Psm(avrRpt2) Psm(PLAFR) Psm(avrRpt2)

  19. MgSO4 Psm(avrRpt2) Psm(PLAFR) Untreated SIS is npr1-and ein2-independent 8 * 7.5 7 6.5 * 6 Psm growth 4 dpi (log cfu/cm2) 5.5 * 5 * 4.5 4 3.5 Col jar1 ein2 npr1 jar1 is a weak JA mutant. The ambiguity of the data of JA mutant is typical

  20. SIS is coronatine (JA)-dependent 3.9 coi1 mutant 3.7 3.5 3.3 Psm growth 4 dpi (log cfu/cm2) 3.1 2.9 2.7 2.5 MgSO4 Psm(avrRpt2) Psm(pLAFR)

  21. Ps Summary • SIS is SA-dependent, but npr1-independent. 2. SIS is ethylene-independent. • SIS is JA-dependent. This agrees with our hypothesis that SIS is induced through SA/JA antagonism. Ps exploits plant signaling molecules (meant for fine-tuning defense responses) and uses them to suppress defense. 4. Natural selection gives Ps the capability to make coronatine and makes it successful phytopathogen among other microorganisms that lack equally exquisite arsenal to subvert plant surveillance system.

  22. Possible Molecular Mechanism of SIS: Some PR genes Are Suppressed by Virulent Psm and by Coronatine MgSO4 Psm (avrRpt2) Psm (pLAFR) 2 hr Gene expression PR2 ::GUS plant 72 hr Suppression of Bean Defense Responses by Pseudomonas syringae. Plant Cell. 1993 Jan;5(1):57-63

  23. coronatine Gene Expression Profiling Hypothesis: Genes involved in biochemical and physiological changes that lead to SIS are transcriptionally regulated by virulent infection and coronatine. Expression profiling will help identify the genes and reveal the mechanistic mechanisms. Mock Avirulent Psm Virulent Psm Expression of genes altered by virulent pathogen infection and coronatine infiltration; Do the alterations correlate? Do they correlate with SIS? Exp. Microarray assay of gene expression using “full”genome Affymetrix chips

  24. Acknowledgements Julia Dewdney Lisa Racki Gang Wu Carine Denoux Fred Ausubel Gustavo Guzman Carol L Bender Naomi Pierce

  25. SA and Npr1 Is separable in Resistance Responses NBS-LRR R-proteins coiled-coil (CC)-like domain (leucine zipper domain) TIR domain (Drosophila Toll and human interleukin-1 transmembrane receptors) EDR1dependent EDS1dependent Pst-specific RPM1, RPS2 (SA-dependent) (npr1-independent) RPS4 (SA-dependent) (npr1-independent) RPP8 (SA-dependent) (npr1-independent) P.parasitica-specific RPP5 (SA-dependent) (npr1-dependent) 1. The majority of Arabidopsis R-genes require SA accumulation for full resistance activity 2. Npr1/Nim1 may play a role only in resistance to P. parasitica mediated by TIR-class R genes

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