核盘菌分泌蛋白SSITL抑制寄主抗病反应的分子机理研究

Sclerotinia sclerotiorum is an important plant pathogenic fungus and has a wide range of host, including oilseed rape and soybean. Sclerotinia diseases are stubborn fungal diseases. As a typical necrotrophic fungus, S. sclerotiorum has long been regarded as a destructive plant pathogen by secretion of oxalic acid and cell wall-degrading enzymes facilitating infection. But emerging evidence showed that secretory proteins also play crucial roles in the pathogenesis of S. sclerotiorum. Previously, we reported that a secretory protein SSITL (Ss-Integrin-like protein) can suppress host resistance at the early stage to facilitate the infection of S. sclerotiorum. Over expression of SSITL in host can lead them to be more susceptible to S. sclerotiorum. So SSITL is considered to be a potential effector. This result indicates that S. sclerotiorum is more complex and subtle in pathogenic tactics. In a follow-up study, the target proteins of SSITL in host were screened through an IP-Mass method. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) experiments showed that Calcium Sensing Receptor (CAS) interact with SSITL. In this project, we plan to further clarify the key domain between the interaction of SSITL and CAS. Meanwhile, we also will work out the effects of the interaction on the pathogenesis, plant resistance, CAS protein location, and the chloroplast structure. Finally, we manage to illuminate the functions and molecular mechanisms of SSITL in the host resistance suppression. The results will help us to more deeply understand the mechanism of pathogenesis in S. sclerotiorum, which gives us an important theoretical significance and application value for developing new technology to control Sclerotinia diseases. In addition, the results also will provide important information on clarifying the mechanism of pathogenesis in necrotrophic fungus.

核盘菌是重要的死体营养型病原真菌，寄主范围广泛，由其引起的作物菌核病难以控制。研究表明草酸及细胞壁降解酶是核盘菌的主要致病因子，但越来越多的证据表明分泌蛋白在核盘菌致病过程中也发挥重要作用。我们报道了核盘菌分泌蛋白SSITL在侵染早期抑制寄主的抗病反应，超表达SSITL的寄主对核盘菌更加感病，SSITL发挥类似效应子（effector）的功能；后续研究中我们筛选到并证实了钙受体蛋白CAS与SSITL互作。在此基础上，本项目拟进一步定位SSITL与CAS互作的功能域或位点，并研究SSITL与CAS互作对核盘菌致病、寄主抗病反应、CAS蛋白定位及叶绿体形态等方面的影响，明确SSITL与CAS互作的生物学意义，阐明SSITL抑制寄主抗病反应的分子机理。项目的完成对深入了解核盘菌的致病机制有重要促进作用，并可为菌核病的安全防控提供新的靶标，同时将为解析死体营养型病原真菌的致病机制提供重要参考。

核盘菌是一种死体营养型植物病原真菌，其致病机理非常复杂。前期研究发现分泌蛋白SSITL在核盘菌侵染初期可以显著抑制寄主的免疫反应，然而其作用机理尚未阐明。本项目深入研究了SSITL抑制寄主抗性的分子机制，主要取得以下结果：. 1. 证明了SSITL能够在大麦下表皮细胞间进行跨膜转运，发现SSITL与拟南芥钙受体蛋白（CAS）存在相互作用，两者共定位于叶绿体；截短试验表明只有全长的SSITL与CAS互作。. 2. 35S:AtCAS植株对核盘菌抗性明显增强，植株中水杨酸信号通路相关基因（ICS1、PAD4、 PBS3、EDS5和PR1）的表达量以及水杨酸浓度均显著升高，而突变体cas-1对核盘菌更加感病，同时水杨酸信号出现明显缺陷，表明CAS通过调控水杨酸的合成参与植物对核盘菌的抗性反应。. 3. 35S:SSITL植株对核盘菌更加感病，接种核盘菌后35S:SSITL植株水杨酸信号明显受损，与突变体cas-1表型一致。而35S:SSITL-NT1和35S:SSITL-CT1植株对核盘菌的抗性与野生型相似，表明SSITL-CAS互作是导致寄主植物免疫缺陷的直接因素。. 4. 证实水杨酸信号在植物对核盘菌的免疫反应中发挥重要作用；发现几丁质处理可诱导水杨酸信号途径基因的表达，但在35S:SSITL和cas-1植株中上述基因的诱导表达量均显著下降，表明CAS在几丁质激活的水杨酸信号相关植物免疫中发挥重要作用，而SSITL与CAS互作可抑制该信号的有效传导。. 5. 发现细胞膜钙离子通道阻断剂LaCl3处理可使35S:ATCAS植株对核盘菌的抗性减退，表明该抗性依赖于植物钙离子信号；在35S:SSITL和cas-1植株上外源施加LaCl3不能增强植株对核盘菌的感病性，表明35S:SSITL和cas-1植株中的钙离子信号存在缺陷，而SSITL很可能抑制了CAS调控的钙离子信号传导。. 总之，该研究在前期基础上解析了核盘菌分泌蛋白SSITL抑制寄主抗病反应的分子机理。发现SSITL与CAS在寄主植物的叶绿体上发生互作，CAS作用于水杨酸信号的上游并调控其生物合成并正向调控植物对核盘菌的抗性，其介导的抗病信号能够被几丁质所激活。而SSITL可通过与CAS互作以抑制其介导的水杨酸信号，从而促进核盘菌的侵染，是一个重要的效应蛋白。