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

植物与微生物相互作用的结果是由微生物毒力库与植物免疫系统的相互作用决定的。在玉米中，致病真菌麦氏黑穗病菌调节宿主免疫，建立生物营养相互作用，引起黑穗病。协调植物防御的重要枢纽是木瓜样半胱氨酸蛋白酶（PLCPs）。我们之前的工作表明，这些酶对于激活玉米叶片中水杨酸（SA）依赖性防御至关重要。在真菌感染期间，玉米PLCPs的活性受到寄主植物通过胱抑素CC9和真菌效应蛋白Pit2的调节。在该项目的第一阶段，我们发现了一种新的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阻断plcp介导的防御机制，释放跨界保守抑制剂肽（cMIP）。我们观察到根中PLCPs的器官特异性，sa介导的激活，并发现根内生细菌分泌根PLCPs的抑制剂。基于这些发现，我们将重点研究两个主要方面：1)Zip1信号及其进化保护。这部分项目旨在了解Zip1激活sa信号的机制。我们将解决以下问题：PROZIP1是如何定位的，它是如何被处理以释放外胞体Zip1的？Zip1的受体是什么？Zip1信号是玉米特有的，还是这种机制在其他植物物种中是保守的？2) plcp诱导SA防御的器官特异性及其对微生物根相互作用的抑制。该项目的这一部分旨在了解器官特异性，plcp激活的SA信号的分子基础，以及表征plcp抑制剂在根微生物组中的作用。我们的主要目标是1)根特异性外胞体plcp及其抑制剂的生化和功能表征；ii)研究根特异性SA信号网络，重点研究器官特异性PLCP的作用；iii)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.