质外体效应蛋白SCR96参与恶疫霉致病的作用机制

The intercellular apoplastic space is the first line of plant defense against invasion and colonization by Phytophthora pathogens. To prevent initial pathogen invasion and control diseases, it is important for us to understand the pathogenesis of Phytophthora pathogens in the apoplastic space. In our previous study, we found that oomycetes including Phytophthora species secrete one class of small cysteine-rich proteins, namely PcF/SCR effectors, into the space. One of such effectors, SCR96, can trigger plant cell death in tomato and Nicotiana benthamiana, and is important for the pathogenicity of P. cactorum. However, it is unclear about how it is involved in the pathogenesis of the pathogen. In this project, we will elucidate its contribution to the pathogen’s pathogenicity by gene knockout and overexpression in P. cactorum; identify its functional domains and key residues based on its sequence polymorphism in the isolates from different host plants and geographical locations and in the isolates with different virulence, combined with replacement and disruption mutation analyses; specify its regulating effect on the plant immunity by analyzing its virulence function in promoting pathogens' infection and interactions with PTI and ETI signal pathways; identify its interacting targets in the plants using different methods, and dissect the functions of plant targets using virus induced gene silencing and transgenosis. The results will reveal the molecular mechanism that SCR96 is involved in the pathogenesis of P. cactorum. They will also provide guidance to design novel prevention and control strategies against plant Phytophthora diseases.

质外体空间是植物阻止疫霉菌侵入致病的第一道防线，揭示疫霉菌在该空间的致病机制是阻断其早期侵染、防治植物疫病的关键。我们前期研究发现，疫霉等卵菌均向植物质外体空间泌出一类分子量小且富含半胱氨酸的PcF/SCR型效应蛋白，其中恶疫霉的SCR96可引起茄科植物的细胞死亡，是一个致病关键的毒性因子，但不清楚它如何参与病原菌的致病过程。本项目拟在恶疫霉中敲除和过表达该基因，明确该效应蛋白在病原菌致病性中的作用；比较分析不同来源、不同致病力菌株中该效应蛋白的序列，结合替换和缺失突变分析，鉴定其功能域和关键位点；通过分析该效应蛋白与PTI、ETI信号途径的互作及促侵染表型，明确它对植物免疫反应的调控作用；筛选鉴定该效应蛋白在植物中的作用靶标，通过沉默和过表达靶标基因，分析它们在植物抗病性中的作用。研究结果不仅可明确质外体效应蛋白SCR96参与致病的作用机制，而且对于制定植物疫病防控策略具有指导意义。

质外体空间是植物阻止疫霉菌侵入致病的第一道防线，揭示疫霉菌在该空间的致病机制是阻断其早期侵染、防治植物疫病的关键。我们前期研究发现，疫霉等卵菌均向植物质外体空间泌出一类分子量小且富含半胱氨酸的PcF/SCR效应蛋白，其中恶疫霉的SCR96可引起茄科植物的细胞死亡（PCD），是一个致病关键的毒性因子，但不清楚它如何参与病原菌的致病过程。本项目的执行取得如下进展：（1）通过CRISPR/Cas9基因敲除实验，证实scr96基因的缺失显著削弱了恶疫霉对寄主植物的致病力，说明SCR96在恶疫霉侵染植物的过程中发挥重要作用；（2）利用农杆菌介导的瞬时表达、酵母以及哺乳动物细胞表达分析，发现异源表达的SCR96可以在茄科植物中诱导PCD，而且卵菌中SCR96的同源蛋白诱导PCD存在着功能分化；（3）通过酵母蔗糖酶分泌实验、删除/替换突变以及胞间液分析实验，明确了信号肽、第43至73位的氨基酸、半胱氨酸残基是SCR96引起PCD的关键功能区段和位点；（4）通过本氏烟上的VIGS分析发现，SCR96发挥PCD功能依赖HSP90和SGT1介导的信号途径；（5）利用农杆菌介导的瞬时表达以及酵母表达蛋白处理茄科植物，发现SCR96会诱导植物活性氧迸发、胼胝质沉积、防卫相关基因上调表达等免疫反应，导致植物对于辣椒疫霉和灰葡萄孢的侵染表现出更强的抗性；（6）通过胞间蛋白组Label-free profiling和酵母双杂交分析，发现SCR96靶向植物的过氧化物酶。本项目的研究结果明确了PcF/SCR类效应蛋白SCR96在恶疫霉侵染植物过程中起着重要的作用，初步探明了其参与致病的作用机制，以及它具备的诱导植物免疫抗性的功能。研究结果不仅为认识恶疫霉与植物胞间互作机制提供了重要的实验数据，而且为开发新型的植物免疫诱抗剂提供了重要的蛋白资源。