Apoplastic cysteine proteases as inducers of plant defense and their inhibition by microbial effectors

植物-微生物相互作用的结果取决于微生物毒力与植物免疫系统的相互作用。在玉米中，病原菌Ustilago maydis调节寄主免疫以建立生物营养相互作用并引起黑穗病。木瓜蛋白酶样半胱氨酸蛋白酶(PLCP)是协调植物防御的重要枢纽。我们以前的工作表明，这些酶对于激活玉米叶片中依赖水杨酸(SA)的防御系统至关重要。在玉米条斑病菌侵染过程中，玉米PLCP的活性既受寄主植物通过半胱氨酸氨基转移酶CC9的调节，也受真菌效应蛋白Pit2的调节。在这个项目的第一阶段，我们鉴定了一种新的plcp释放肽信号(ZIP1)，它可以激活SA介导的免疫。我们发现真菌效应蛋白(PIT2)通过作为分子模拟分子来阻断PLCP介导的防御机制，从而释放一种跨王国保守的抑制肽(CMIP)。我们观察到了根中PLCPs的器官特异性激活，并发现根内生细菌分泌根PLCPs的抑制物。基于这些发现，我们将主要集中在两个方面：1)ZIP1信号及其进化保守。这部分项目旨在从机制上理解ZIP1激活SA信号的机制。我们将解决以下问题：PROZIP1是如何本地化的，它是如何加工来释放质外体ZIP1的？ZIP1的受体是什么？ZIP1信号是玉米特有的，还是在其他植物中保守的？2)PLCP诱导SA防御的器官特异性及其在微生物根相互作用中的抑制。该项目的这一部分旨在了解器官特异的、plcp激活的SA信号的分子基础，以及plcp抑制物在根微生物群中的作用。我们的主要目标是：1)根特异的质外体PLCP及其抑制剂的生化和功能特征；2)研究根特异的SA信号网络，重点是器官特异的PLCP的作用；3)研究细菌PLCP抑制剂在建立根-微生物相互作用中的作用。

The outcome of plant-microbe interactions is determined by the interplay of the microbial virulence repertoire with the plant immune system. In maize, the pathogenic fungus Ustilago maydis modulates host immunity to establish a biotrophic interaction and cause smut disease. Important hubs for the coordination of plant defence are papain-like cysteine proteases (PLCPs). Our previous work showed that these enzmes are crucial for the activation of salicylic acid (SA)-dependent defenses in maize leaves. During U. maydis infection, the activity of maize PLCPs is modulated by both the host plant via the cystatin CC9, as well as by the fungal effector protein Pit2. During the first phase of this project, we identified a novel, PLCP-release peptide signal (Zip1) which activates SA-mediated immunity. We showed that a fungal effector protein (Pit2) blocks the PLCP-mediated defense mechanism by acting as a molecular mimicry molecule, which releases a cross-kingdom conserved inhibitor peptide (cMIP). We observed an organ-specific, SA-mediated activation of PLCPs in roots, and found that root endophytic bacteria secrete inhibitors of root PLCPs. Based on these findings, we will focus on two main aspects:1) Zip1 signaling and its evolutionary conservation.This part of the project aims to gain mechanistic understanding on the activation of SA-signaling by Zip1. We will address the following questions: How is PROZIP1 localized and how is it processed to release apoplastic Zip1? What is the receptor for Zip1? Is Zip1 signaling specific to maize, or is this mechanismconserved in other plant species? 2) Organ specificity in PLCP-induced SA defense and its suppression in microbial root interactions. This part of the project aims to understand the molecular basis of organ-specific, PLCP-activated SA signaling, as well as to characterize the role of PLCP-inhibitors in the root microbiome. Our major goals are i) the biochemical and functional characterization of root-specific apoplastic PLCPs and their inhibitors; ii) to investigate root-specific SA signaling networks with an emphasis on the role of organ-specific PLCPs, and iii) to study the role of bacterial PLCP inhibitors for the establishment of the root-microbe interactions.