小G蛋白Ras1的氧化还原修饰对灰霉菌致病力的调控机制研究

Botrytis cinerea is a significant pathogen causing devastating diseases on various fruits such as grapes, strawberries and tomatoes. The monomeric G protein Ras was reported to be involved in the regulation of the pathogenic signaling pathways of B. cinerea. Our previous studies showed that the regulatory subunit of NADPH oxidases, which played a crucial role in the virulence of B. cinerea, affected the redox status of cysteine on Ras1. Therefore, it is speculated that the redox modification of Ras1 played an important role in the regulation of the virulence of B. cinerea. This project will construct site specific mutants by transforming the seven cysteines on Ras1 to serines, respectively. The key redox modification sites of Ras1 and their roles in the virulence of B. cinerea will be determined by exogenous reactive oxygen species treatment and phenotype detection of the single amino acid mutants. Then, the regulation mechanism of different cysteines on Ras1 will be investigated through examining the activity and localization of Ras1 in the single amino acid mutants. We will also explore the genes/proteins regulated by the redox modification of Ras1 using transcriptomics and proteomics and determine their functions. This project will reveal the regulatory mechanism of redox modification on Ras1 and its pathogenic signaling pathways in B. cinerea, providing theoretical foundations for developing effective technologies for postharvest disease control.

灰霉菌是能引起葡萄、草莓和番茄等多种果实灰霉病的重要致病真菌。小G蛋白Ras参与了灰霉菌致病信号通路的调控。前期发现NADPH氧化酶复合物（Nox）的调节亚基在影响灰霉菌致病力的同时，可显著改变小G蛋白Ras1上半胱氨酸（Cys）的氧化还原状态，推测Ras1的氧化还原修饰在灰霉菌的致病力调控中有重要作用。本项目将构建Ras1中7个Cys的单氨基酸突变株，通过外源活性氧处理和表型检测，找到Ras1的关键氧化还原修饰位点，探究关键氧化还原修饰位点在灰霉菌致病力中的作用；通过研究单氨基酸突变株中Ras1的活力和定位情况，明确单氨基酸位点的作用；利用转录组学和蛋白质组学方法，比较野生型和Ras1单氨基酸突变株中的差异基因和蛋白，敲除可能与致病力相关的基因并确定其功能。本项目有望揭示Ras1的氧化还原修饰在灰霉菌致病力中的调控机制，为研发有效防治果实采后病害的绿色新型保鲜技术提供理论依据。

灰霉菌是一种造成果蔬采后巨大损失的重要致病真菌，且防治困难。深入解析灰霉菌的致病分子机制可为高效防治灰霉菌提供理论依据。小G蛋白Ras是真菌细胞中普遍存在的单体G蛋白，参与了多种真菌致病信号通路的调控。揭示灰霉菌中小G蛋白的功能及其下游信号通路，将为深入解析灰霉菌的致病机制提供突破口。本项目通过同源重组的方法构建了小G蛋白Ras1的缺失突变株，通过表型检测，发现Ras1与灰霉菌的生长、菌落形态和致病力密切相关。通过转录组学的方法，比较了野生型和Ras1突变株中的差异表达基因，发现Ras1缺失突变株与野生型差异表达的基因有1428个。这些基因主要参与了活性氧、MAPK等信号通路。其中，活性氧通路相关的差异表达基因有456个，包括负责活性氧产生的NADPH氧化酶复合物（Nox）的多个亚基的编码基因。选取关键差异表达基因Ras2进行深入分析。敲除Ras2基因后，发现Ras2缺失突变株生长速率显著降低、菌核产量明显增加，对氧化胁迫的抵抗力减弱，而对细胞壁胁迫的抵抗力增强。此外，Ras2缺失促进了菌核中黑色素相关基因的表达，并降低了分生孢子中黑色素基因的表达。研究首次发现灰霉菌的Ras2基因与环境胁迫和黑色素调控途径有关，为进一步研究环境条件对灰霉菌黑色素和致病力的调控机制奠定了基础。研究部分揭示了灰霉菌中小G蛋白调控病原菌致病力的分子网络，为研制果实采后病害的有效技术提供了理论依据。