核盘菌效应蛋白S1和S2诱导植物细胞死亡的分子机制研究

Sclerotinia stem rot caused by the broad host range necrotrophic fungus Sclerotinia sclerotiorum is one of the most damaging disease of rapeseed, however, the mechanisms of its pathogenesis is unclear. Effector proteins play a key role in the pathogenesis of plant pathogens. The genome of Sclerotinia sclerotiorum encodes hundreds of potential effector proteins, but their biological functions and action mechanisms are largely unknown. In our previous work, we systematically cloned nearly one hundred possible effector-encoding genes of Sclerotinia sclerotiorum, and identified two new effector proteins S1 and S2 that can induce plant cell death. Furthermore, we found that S1 and S2 positively regulate Sclerotinia sclerotiorum pathogenesis, indicating that S1 and S2 may promote Sclerotinia sclerotiorum infection by initiatively inducing plant cell death. In this project, we intend to use protein interaction techniques including yeast two-hybrid (Y2H) and immunoprecipitation-mass spectrometry (IP-MS), combined with protein subcellular localization and transcriptomic data, to determine the target proteins of S1 and S2 in plant cells. After genetic verification of the targets, we will further explore the mechanisms underlying the interactions between the effectors and their target proteins, thereby revealing how S1 and S2 induce plant cell death to promote Sclerotinia sclerotiorum infection. This study will expand our understanding of the pathogenesis of broad host necrotrophic pathogens represented by Sclerotinia sclerotiorum, and provide theoretical basis and target genes for molecular breeding of anti-sclerotinia rapeseed.

广寄主性腐生型真菌核盘菌造成的菌核病是油菜重要病害，但其致病机制尚不清楚。效应蛋白在病原微生物致病过程中发挥关键作用，核盘菌基因组编码上百个潜在的效应蛋白，但它们的生物学功能及作用机制大多未知。在前期的研究工作中，我们系统克隆了近百个可能的核盘菌效应蛋白编码基因，从中筛选到2个能够诱导植物细胞死亡的新的核盘菌效应蛋白S1和S2，并发现S1和S2对核盘菌侵染具有明显的促进作用，提示S1和S2可能通过主动诱导植物细胞死亡促进侵染。本项目拟利用酵母双杂交、免疫共沉淀-质谱联用等蛋白互作技术，结合蛋白亚细胞定位和转录组学数据，确定S1和S2在植物中的靶标；对靶标进行遗传学验证后，进一步探究S1、S2与其靶标互作的具体机制，从而揭示S1、S2如何诱导植物细胞死亡以促进核盘菌侵染。本研究将拓展人们对核盘菌等广寄主性腐生型植物病原菌致病机制的认识，并为油菜抗菌核病分子育种提供理论基础和基因靶点。

由腐生真菌核盘菌导致的菌核病是油菜、向日葵等农作物的重要病害，严重影响其产量和品质，但核盘菌的致病机制尚不十分清楚。病原菌效应蛋白研究对揭示病原菌致病机制具有重要意义。在前期研究中，我们对核盘菌效应蛋白组进行了系统分析，并克隆了近百个功能未知的候选效应蛋白基因。本项目中，我们着重对核盘菌效应蛋白S1和S2的功能与作用机制进行了研究。利用信号肽预测和酵母分泌实验，证实了S1和S2具有分泌特性。通过构建核盘菌S1和S2基因的敲除菌株，我们发现敲除S1和S2对核盘菌离体生长没有明显影响，但会显著降低核盘菌的致病力，说明S1和S2对核盘菌侵染具有促进作用。有趣的是，对S1和S2敲除突变体以及野生型核盘菌菌株侵染后的油菜叶片进行转录组分析表明，S1和S2均对植物乙烯途径具有抑制作用。我们进一步筛选到了S2在植物中的一个靶标蛋白MF2。利用VIGS敲低植物MF2基因的表达会抑制植物乙烯途径并促进核盘菌侵染，说明S2可能通过与MF2互作，干扰MF2的功能，从而抑制植物乙烯途径，进而促进核盘菌侵染。我们的研究揭示两个新的核盘菌效应蛋白S1和S2的功能和作用机制，拓展了人们对核盘菌等腐生真菌致病机制的认识。