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 induced susceptibility (SIS) Virulent pathogens suppress basal and induced host defenses systemically to facilitate colonization. Systemic acquired resistance (SAR) In response to avirulent pathogens, plants activate long-lasting resistance responses systemically to defend against a broad spectrum microbial pathogens. 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. Virulent P. syringae Induces Systemic Susceptibility to Insects Virulent Psm 0 0.5 1 1.5 2 Colrps2rpm1 Relative Larval Weight Gain MgSO4 pLAFR avrRpt2 avrB MgSO4 pLAFR avrRpt2 avrB MgSO4 pLAFR avrRpt2 avrB ** * * * ns ** * Virulent infectionAvirulent infection Tricoplusia ni (T. ni) Virulent pathogen has been shown to induce similar transcriptional profile as avirulent pathogen, but at lower magnitude and later stage. However…

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

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

6. Virulent Pst also Induces Systemic Susceptibility Virulent Pst DC3000 growth MgSO4Pst(avrRpm1) Pst DC3000 4 4.5 5 5.5 6 6.5 7 Pst growth 4 dpi (log cfu/cm2) * *

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

8. SIS Inducer Candidate : Phytotoxin Coronatine Rationale and previous knowledge 1.Coronatine is primary virulence factor of several strains of Ps, including Psm and Pst. COR- mutants are less virulent. 2.In Psm, rpoN is essential for COR production. A rpoN mutant does not multiply, does not elicit disease symptoms, does not induce PR genes. 3.Coronatine is a structural and functional JA mimic (Bender et al). 4.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 Psm Coronatine Psm growth 4 dpi (log cfu/cm2) 3.5 4 4.5 5 5.5 6 MgSO4Psm (avrRpt2) Psm (pLAFR) Coronatine * * *

10. 0 10 20 30 40 Psm ES6262(3G4)62(VRC4) µgCOR/ml culture Construction of COR- mutant 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). By Gustavo Guzman

11. COR- Mutant Does Not Induce SIS 3 3.5 4 4.5 5 5.5 6 MgSO4Psm (avrRpt2) Psm (pLAFR) ES62 Psm growth 4 dpi (log cfu/cm2) * *

12. Structure of coronatine and its components, coronafacic acid (CFA) and coronamic acid (CMA) CORONATINE O C O HH OCH 3 NH CO 2 H C NH CO 2 H O HH O ETHYLENE JASMONATE CFA CMA ACC 1. CFA shows structural similarity to jasmonate 2. CMA is similar to ACC (aminocyclopropyl carboxylic acid), the immediate precursor to ethylene From Carol Bender

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

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

15. COR- Avirulent Psm Strain Induces Stronger SAR Psm growth 4 dpi (log cfu/cm2) 3 3.5 4 4.5 5 5.5 MgSO4Psm (avrRpt2) ES62 (avrRpt2) * **

16. Summary Ps 1.Virulent Psm not only overcomes basal defense at the site of infection, but also disarms host defense systemically. 2.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. 3.ES62, which is a COR- mutant, fails to induce SIS, which supports the hypothesis that virulent Ps induces SIS through coronatine. 4.Induction of SIS requires both the CFA and CMA components of coronatine. 5.Coronatine induces SIS by acting as signal compound. 6.Avirulent Psm partially suppresses SAR by counteracting it with coronatine. SIS is a critical component of P. syringae pathogenesis

17. 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 JA SA Et SA JA

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

19. SIS is npr1-and ein2-independent 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 Coljar1ein2npr1 MgSO4 Psm(avrRpt2) Psm(PLAFR) Untreated Psm growth 4 dpi (log cfu/cm2) * * * * jar1 is a weak JA mutant. The ambiguity of the data of JA mutant is typical

20. SIS is coronatine (JA)-dependent Psm growth 4 dpi (log cfu/cm2) 2.5 2.7 2.9 3.1 3.3 3.5 3.7 3.9 MgSO4Psm(avrRpt2)Psm(pLAFR) coi1 mutant

21. Summary Ps 1.SIS is SA-dependent, but npr1-independent. 2.SIS is ethylene-independent. 3.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 Gene expression PR2 ::GUS plant MgSO4 Psm (pLAFR) Psm (avrRpt2) 2 hr 72 hr Suppression of Bean Defense Responses by Pseudomonas syringae. Plant Cell. 1993 Jan;5(1):57-63

23. 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. Exp. Microarray assay of gene expression using “full”genome Affymetrix chips Avirulent Psm Virulent Psm Mock coronatine Expression of genes altered by virulent pathogen infection and coronatine infiltration; Do the alterations correlate? Do they correlate with SIS?

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) RPM1, RPS2 (SA-dependent) (npr1-independent) RPS4 (SA-dependent) (npr1-independent) P.parasitica- specific Pst-specific RPP8 (SA-dependent) (npr1-independent) 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 EDS1dependent EDR1dependent