稻曲病菌致病关键效应蛋白的鉴定及其毒性功能的分子机理

Rice false smut, caused by the fungal pathogen Ustilaginoidea virens (Cooke) Takah, has become one of the most important diseases in rice. Effector proteins secreted by phytopathogenic fungi play essential roles in the host-pathogen interaction. Some effectors have been demonstrated to suppress plant immunity and promote fungal infection using different molecular strategies. However, little is known about pathogenicity mechanisms of U. virens and virulence functions of its effector proteins given that it is a recently emerging disease. In this project, experiments will be first designed to screen important effector candidates, which inhibit non-host resistance and host immunity, through multiple high throughput screening systems. The secretion of these effector candidates will be subsequently confirmed through various secretion systems. Second, virulence functions of these effectors will be investigated using the transgenic rice plants conditionally expressing these effectors. Meanwhile, the effector genes essential for full virulence and pathogenicity will be determined through investigating virulence of gene-deletion mutants of these effector genes. Third, the host targets of effector proteins required for virulence will be identified and characterized. Putative biochemical functions of the effectors and host targets will be also explored. Finally, the function(s) of host targets in plant immunity will be studied using T-DNA insertion mutants or gene knockout lines to determine biological significance of the protein-protein interaction. The expected results will facilitate understanding molecular mechanisms underlying virulence and pathogenicity of U. virens and provide important information for development of effective disease management strategies.

由病原真菌Ustilaginoidea virens引起的稻曲病已成为我国水稻生产上最重要的病害之一。植物病原真菌分泌到寄主体内的效应蛋白参与其与寄主的互作，其中一些以不同的机制抑制寄主的免疫反应，在病原菌的致病性中起到关键作用。由于研究基础薄弱，对稻曲病菌致病机理及其效应蛋白毒性功能的了解十分缺乏。本项目首先将通过多条途径大规模筛选抑制非寄主抗性和寄主免疫的稻曲病菌候选效应蛋白，并对其分泌进行确认；其次，在水稻中异源表达效应蛋白而确定其毒性功能，并结合稻曲病菌基因敲除技术，确定对致病起关键作用的效应蛋白；第三，筛选并鉴定关键效应蛋白的寄主靶标，解析关键效应蛋白及其寄主靶标可能具有的生化功能；最后，用突变体或基因敲除体研究靶标蛋白在寄主抗病性中的作用，确定效应蛋白与靶标蛋白间互作的生物学意义。研究结果将为深入揭示稻曲病菌致病的分子机理提供理论依据，进而为制定稻曲病的综合防控策略奠定基础。

稻曲病是由活体营养型真菌稻曲病菌侵染水稻小花而导致的穗部病害，是水稻上最重要的病害之一。稻曲病菌编码大量效应蛋白可能在其侵染的过程中扮演着重要作用。本研究首先通过突变体致病性分析等多种方法鉴定出在稻曲病菌致病性中发挥关键作用的效应蛋白SCRE1/2/4/6，这些蛋白在侵染过程中能分泌到水稻体内，定位于质外体、细胞质或细胞核中；这些蛋白能抑制非寄主植物本式烟细胞过敏性坏死反应和病原相关分子模式（PAMP）诱导的水稻免疫反应，从而抑制水稻的抗病性。.此外，发现SCRE1与水稻基础免疫正调控因子ATPase XB24互作且阻止XB24与ATP的结合，从而抑制XB24酶活；发现水稻中调节能量信号蛋白激酶SnRK1A与XB4互作；该激酶通过磷酸化XB24 的Thr83残基提高XB24的酶活；还发现SCRE1通过阻止SnRK1A与XB24互作而抑制其对XB24的磷酸化，从而抑制XB24的酶活。研究结果揭示了SCRE1作用于SnRK1A-XB24磷酸化免疫途径抑制水稻免疫的新机制。.通过生物化学和生物信息学分析，阐明了SCRE6是一个以水稻OsMPK6为靶标的新型酪氨酸磷酸酶。发现在水稻中过量表达OsMPK6显著促进病原真菌和细菌的侵染，说明OsMPK6是免疫负调控因子；OsMPK6通过26S蛋白酶体途径降解。当稻曲病菌菌侵染时，PAMP诱导OsMPK6的磷酸化促进其降解，提高水稻抗病性；然而，病原菌分泌的SRCE6能去磷酸化OsMPK6，抑制OsMPK6的降解，促进病菌侵染。在稻曲菌中还发现3个与SCRE6行使同样的功能的同源蛋白。综上，SCRE6属于新型酪氨酸磷酸酶家族，通过去磷酸化抑制寄主免疫负调节因子的降解，从而促进致病性。.研究结果揭示了SCRE1与SCRE6在水稻中的毒性靶标，阐明稻曲病菌促进侵染的新机制，为该病害的绿色防控以及抗病新种质的创制提供了重要理论支撑。