核盘菌小分泌蛋白SsCVNH影响其致病的分子机制的研究

Sclerotinia sclerotiorum (Lib.) de Bary is a typical necrotrophic phytopathogenic fungus with a remarkably broad host range. White mould or Sclerotinia stem rot caused by this pathogen often leads to huge economic damage to a wide range of crops. The molecular mechanisms of the interaction between S. sclerotiorum and its hosts are unclear, as is posing big challenges on disease resistance breeding and prevention and treatment of the disease. S. sclerotiorum secretes oxalic acid (OA) and cell wall-degrading enzymes (CWDEs) during infection, which are two important pathogenic factors, however, more and more evidence indicated secreted proteins are also necessary for its pathogenicity. We have reported that a small secreted protein SsSSVP1 played crucial roles in the necrotrophic interaction between S. sclerotiorum and its host plants, just like the action of “effectors” in biotrophic and hemi-biotrophic phytopathogenic pathogens. But the two kinds of interactive patterns, modes and results in necrotrophic and biotrophic pathogens are completely different. In a recent report, we showed another small secreted protein SsCVNH could be secreted from the hyphal tips and significantly facilitated the infection of S. sclerotiorum. In this project, we plan to further study the acting site and the molecular target of SsCVNH in host plant cells, and the biological significance of the interaction between SsCVNH and its host target. These anticipated results will deepen our understanding of the molecular mechanisms underlying the pathogenesis and the host range determinant factors of the necrotrophic fungi, and provide valuable clues for the control of these kinds of diseases.

核盘菌是寄主范围广泛的典型的死体营养型植物病原真菌，由其引起的菌核病每年给我国粮油和蔬菜生产造成巨大损失。目前对核盘菌与寄主植物互作的分子机理知之甚少，造成针对菌核病的抗病育种和防治的极大困难。研究表明草酸和细胞壁降解酶是其重要的致病因子，但近期研究表明分泌蛋白在核盘菌致病过程中的作用也是必不可少的。申请者前期报道了核盘菌中小分泌蛋白SsSSVP1也可以像活体和半活体营养型植物病原菌中的“效应因子”那样与寄主靶标互作，但互作的方式、类型和结果与后者迥然不同。申请者前期又报道了另外一个小分泌蛋白——SsCVNH也可以显著影响核盘菌的致病力。本项目拟在此基础上深入研究SsCVNH在寄主植物细胞中的作用位点和作用靶标，揭示SsCVNH与寄主靶标互作的生物学意义。项目的完成将有助于加深理解核盘菌致病的分子机理和死体营养型植物病原菌寄主范围的决定因素，为该类病害的防控提供新的靶标和思路。

核盘菌是一种能够引起多种作物严重菌核病的死体营养型病原真菌。核盘菌的多核特性使对其进行基因敲除等遗传操作耗时费力。在前期研究中，项目负责人利用基因沉默（RNAi）技术发现一些小分泌蛋白如SsSSVP1和SsCVNH在核盘菌致病过程中起着重要作用。对SsSSVP1致病分子机理的解析已经发表，但对SsCVNH的致病分子机制还知之甚少。在本研究中，项目成员成功获得了SsCVNH基因敲除纯合转化子和互补转化子，SsCVNH的敲除使核盘菌致病力显著下降，而互补转化子则可以恢复其致病力，再次说明SsCVNH在核盘菌致病过程中发挥重要作用，与前期研究结果一致。利用酵母双杂交（Y2H）筛库技术筛选到了与SsCVNH互作的寄主靶标——AtPRX71，该蛋白是III型过氧化物酶家族中的一员。该试验结果又进一步被Pull down、荧光双分子互补（BiFC）和荧光蛋白标记共定位实验所证实。Y2H和BiFC试验又将SsCVNH的寄主靶标扩展至多个其它III型过氧化物酶家族成员。III型过氧化物酶是分泌性的、分布于质外体或细胞壁上的多功能蛋白家族，BiFC和荧光蛋白标记共定位实验证实它们与SsCVNH的互作发生在质外体或细胞壁，这与前期研究中发现的核盘菌在与寄主互作时SsCVNH主要分布于菌丝尖端与植物细胞相接触的界面这一结果是一致的。在成功获得表达SsCVNH的拟南芥转基因植株的基础上，项目成员发现SsCVNH与III型过氧化物酶家族的互作可以抑制病原体相关分子模式(PAMP)触发的植物细胞中活性氧的积累，影响寄主植物的免疫应答，使其更利于病原菌的侵染，从而揭示了它们之间互作的生物学意义。此外，本研究还证实植物细胞中表达的SsCVNH存在两个糖基化修饰位点，但是SsCVNH的糖基化修饰在核盘菌与植物互作过程中的生物学意义还有待进一步研究。