病原菌致病因子与磷脂互作的功能研究

Plant diseases caused by pathogenic microbes represent one of the most important threats to global food security. One important reason that plant diseases continue to threaten cop production despite tremendous efforts in breeding and pesticide input is that we are far from understanding the basic disease susceptibility mechanisms. New discoveries on disease susceptibility mechanisms can provide potentially transformative new strategies for improving disease resistance in crop plants. Type III effectors are key virulence weapons of Gram-negative phytopathogenic bacteria; these virulence proteins are translocated into the plant cell to promote infection. The AvrE-family of effectors is arguably the most widely-spread and conserved effector proteins found in diverse phytopathogenic bacterial species, and plays essential roles in pathogenesis. The principal investigator (PI) recently discovered that bacterial pathogens, such as Pseudomonas syringae, utilize AvrE effector to drive the formation of "water soaking" in the leaf apoplast (i.e., the intercellular space between leaf mesophyll cells) of infected plants, as a previously-unknown yet crucial virulence mechanism. However, the exact mechanism by which AvrE creates “water soaking” in the leaf remains elusive. We recently made a striking discovery that AvrE protein interacts with phosphatidylinositol phosphates (PIPs), particularly PI(3)P (phosphatidylinositol 3-monophosphate) and PI(3,5)P2 (phosphatidylinositol 3,5-biphosphates). This is in line with our previous study showing that AvrE is localized on the plant cell plasma membrane, in which PI(3)P and PI(3,5)P2 are important lipid components. This finding raises the exciting possibility that the primary virulence activity of AvrE may be mediated through interaction with phospholipid molecules in the plant plasma membrane. Accordingly, we hypothesize that, by interacting with PIPs, AvrE could disrupt the plasma membrane integrity of the plant cell, leading to leakage of the plant cell content and creating a localized high osmotic potential near the infected plant cells. We have preliminary evidence to show that localized high osmotic pressure is sufficient to attract water from surrounding tissues and drive “water soaking” formation. This project aims to test this exciting hypothesis and gain a deep understanding of the functional consequences of AvrE interaction with PIPs. Specifically, we will examine whether AvrE promotes liposome leakage and form pores on liposome. We will identify the domains and amino acid sites that mediate AvrE-lipid binding and examine the conservation of the lipid-interacting property of AvrE homologs from other pathogens. The long-term goal of this project is to discover the elusive mechanism of “water-soaking” formation during plant disease, and use this basic knowledge to engineer plants with an ability to counter “water soaking” formation as an innovative approach to improve plant resistance against pathogen infections.

病原微生物导致的植物病害仍是现代农业的一大危害。这其中的一个重要原因是我们对病原微生物的致病机制还未全面了解。AvrE III型效应子家族是一个在不同病原细菌中广泛存在并发挥重要致病作用的毒性蛋白家族。申请人近期研究表明病原细菌能够利用AvrE在植物叶片中诱导产生“水渍”，这是一个重要致病机制。但是AvrE介导“水渍”产生的机制并不清楚。本课题组近期研究表明AvrE蛋白可以与磷脂分子-磷脂酰肌醇磷酸(phosphatidylinositol phosphates)发生互作。这与AvrE在植物细胞中的细胞膜定位相符，表明AvrE可能通过与磷脂互作改变宿主细胞的膜结构而发挥功能。本项目将深入探索AvrE蛋白与植物磷脂互作的生物学特性及其如何介导AvrE的毒性功能和“水渍”形成，对阐明“水渍”这一广泛存在的致病机制及磷脂分子在生物胁迫中的作用具有重要意义，并为改良植物抗病性提供新的思路。

由病原微生物导致的植物病害严重威胁植物生长和作物产量。丁香假单胞杆菌是一类革兰氏阴性细菌，具有广泛的植物宿主，这一种属的不同菌株几乎可以侵染所有具重要经济意义的农作物。丁香假单胞杆菌利用保守的III型分泌系统向宿主细胞分泌多个效应蛋白，通过干扰植物免疫等机制促进侵染。其中两个III型效应子，AvrE和HopM1，在植物叶片中诱导产生“水渍”，但是它们诱导“水渍”形成的分子机制还不清楚。AvrE家族是一个在不同病原细菌中广泛存在并发挥重要致病作用的毒性蛋白家族。前期研究表明，AvrE与某些植物的磷脂酰肌醇磷酸互作。磷脂是构成生物膜的主要成分之一，已有研究表明磷脂分子在植物-微生物互作中发挥重要的调节作用，但是磷脂酰肌醇磷酸（PIPs）在植物-病原细菌互作中的功能还未见报道。本研究中，我们通过对AvrE家族不同成员的氨基酸序列比对和点突变体构建，发现了AvrE N端几个较保守的赖氨酸/精氨酸(K/R)对AvrE在植物中的活性是必需的，暗示了这些位点有可能通过电荷间相互作用介导AvrE与磷脂的互作。此外，为了深入揭示AvrE的生化活性和功能特征，我们与结构生物学实验室合作，试图通过冷冻电镜和X射线衍射晶体学方法解析AvrE、AvrE与互作蛋白复合体以及AvrE同源蛋白AvrEpsj和WtsE的三维结构。经过大量实验尝试和优化，利用冷冻电镜方法，我们初步获得了一个低分辨率的AvrE三维重构模型；后续还需优化实验条件，从而解析高分辨率的AvrE或其复合体结构。此外，我们在项目开展过程中发现了AvrE与拟南芥植物的I型磷酸酶TOPP互作，通过对TOPP突变体植物的表型分析发现TOPP对于介导AvrE的功能很重要。之后通过生物化学、细胞生物学等多种手段对AvrE-TOPP调控的机制进行解析，发现AvrE通过调节拟南芥中的TOPP-SnRK2模块上调叶片中的脱落酸激素通路，并诱导气孔关闭，从而导致叶片中水分积累、促进病害发生。此部分研究工作于2022年发表于Cell Host & Microbe。综上，我们的研究鉴定到了影响AvrE毒力功能的重要氨基酸位点、探索了利用冷冻电镜方法解析AvrE及复合体结构，为后续解析高分辨率的AvrE结构奠定了基础，并发现AvrE靶向植物的TOPP蛋白和上调叶片脱落酸通路促进侵染的致病机理。