Molecular Dissection of Tomato Resistance to Necrotrophic Fungi

Siming Xu, Purdue University

Abstract

Plant pathogens are classified into two categories based on their mode of nutrition. In contrast to biotrophs that feed on living plant tissues, necrotrophic pathogens actively destroy host tissues and feed on the dead and dying cells. In general, plant immune mechanisms to biotophic and necrotrophic pathogens are known to be different with some overlaps. However, the detailed molecular mechanisms of resistance to necrotrophic pathogens are generally poorly understood. Botrytis cinerea is a fungal necrotroph which causes severe pre- and post-harvest diseases in horticultural and agronomic crops resulting in significant economic losses. In this study, we examined the mechanisms of tomato resistance to B. cinerea starting from known genetic components of tomato defense. It was previously shown that TOMATO PROTEIN KINASE B (TPK1b), a receptor-like cytoplasmic kinase, mediates tomato defense responses to B. cinerea and feeding by larvae of tobacco hornworm. In an effort to identify TPK1b-interacting proteins, we found TPK1b associates with multiple receptor-like kinases (RLKs) including the tomato FLAGELLIN RECEPTOR 2 (FLS2), BRASSINOSTEROID-INSENSITIVE1 (BRI) and PEPR1 ORTHOLOGUE RECEPTOR LIKE KINASE1 (PORK1) suggesting it integrates and relays signals from different upstream RLKs. Particularly, TPK1b interacts with PORK1, identified as a tomato homolog of Arabidopsis PEPR1 and PEPR2 that was demonstrated to mediate DAMP-triggered immune responses to B. cinerea. We hypothesized that PORK1 regulates tomato resistance to B. cinerea, other microbial pathogens and pests that trigger the production of damage associated molecular patterns (DAMPs). To test this hypothesis, we characterized the biological function of PORK1 in disease resistance to B. cinerea and insect herbivores using virus induced gene silencing (VIGS) and PORK1 RNA interference (RNAi) plants suppressed for PORK1 gene expression. In addition, we determined the role of PORK1 in wounding/systemin-mediated responses. Finally, we examined the molecular and biochemical links between PORK1 and TPK1b. In general, suppression of PORK1 through VIGS caused increased susceptibility to B. cinerea accompanied by reduced expression of PROTEASE INHIBITOR II (PI-II), a downstream defense marker gene. Expression of PI-II in responses to both mechanical wounding and systemin was compromised in PORK1-silenced plants. Interestingly, silencing of PORK1 in tomato plants that overexpress the systemin precursor prosystemin severely compromised resistance to B. cinerea conferred by prosystemin overexpression. In addition, transgenic tomato PORK1 RNAi lines that show reduced PORK1 expression exhibited increased susceptibility to feeding by larvae of tobacco hornworm. Interestingly, PORK1 interacts with TPK1b in co-immunoprecipitation experiments suggesting their presence in the same protein complex. In addition, PORK1 is a functional kinase with autophosphorylation and transphosphorylation of TPK1b when supplied as a substrate. PORK1 and TPK1b kinase activities are also coregulated as suggested by the increased phosphorylation of both proteins in response to wounding and systemin. Interestingly, phosphorylation of TPK1b in response to wounding and systemin were dramatically compromised when PORK1 is suppressed, suggesting PORK1 is the upstream kinase of TPK1b in wounding/systemin-mediated signaling. Together we demonstrate that tomato PORK1 regulates responses to fungal pathogens and wounding through phosphorylation of TPK1b. In parallel with the above, we studied the role of TPK1b phosphomimic variants on tomato immunity to pathogens and other traits. Previous studies identified residues which are essential for TPK1b kinase activities. Transgenic tomato lines expressing phosphomimic TPK1b were generated by substituting Threonine (Thr) at positions 238 or 243 to Aspartate (Asp). Tomato plants expressing TPK1bT243D exhibit increased resistance to B. cinerea infection accompanied by increased basal and B. cinerea induced expression of PI-II. Interestingly, tomato plants expressing TPK1bT238D display hypersensitivity to ethylene responses but not disease resistance. In summary, our data suggests that phosphomimic variants of TPK1b are sufficient to activate resistance to to fungal infection and responses to ethylene. The data also suggest that specific phosphorylation sites are responsible for mediating different responses. Together, our data implicate PORK1 and TPK1b as central regulators of plant immunity to necrotrophic fungal pathogens and insect pest through a mechanism that involve systemin.

Degree

Ph.D.

Advisors

Mengiste, Purdue University.

Subject Area

Molecular biology|Plant Pathology

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