基于Ss-caf1基因的核盘菌侵染垫形成及其功能研究

The ascomycete Sclerotinia sclerotiorum (Lib.) de Bary, is an important plant-pathogenic fungus with worldwide distribution. Sclerotinia disease is induced by this pathogen and is responsible for significant annual yield loss of many economically important crops, including rapeseed, soybean. The previous research has been clearly confirmed that infection cushion plays important role during S. sclerotiorum infects health hosts without wound. The new control strategy will be applied via inhibition infection cushion formation. However, the molecular mechanism and biological function of infection cushion have not been clearly elucidated. We previously found that a mutant Sunf-MT6, that is incapable of forming infection cushion, cannot successfully infect health host, but can cause typical diseased lesion on wounded host. A gene Ss-caf1 associated infection cushion formation was disrupted by T-DNA in mutant Sunf-MT6, and we conjecture that infection cushion has important roles for secrete or transport proteins/ metabolites from fungi to plant. Therefore, in this research, a serial experiment will be conducted based on mutant Sunf-MT6 and its original strain Sunf-M. Firstly, to obtain new genes associated infection cushion formation, the proteins interacted with Ss-Caf1 will be identified. Secondly, the biological function of the candidate genes will be performed via gene silence in S. sclerotiorum. Firstly, to elucidate that infection cushion has biological function for transporting secretary proteins from S. sclerotiorum to its host, proteome and metabolome will be deeply analyzed during infection cushion formaion. The above predicted results will contribute to understand the biological function of Ss-caf1 during infection cushion formation. Our findings will offer new insights into development new strategies to control Sclerotinia disease.

核盘菌是重要的植物病原真菌，对油菜、大豆等作物安全生产造成严重威胁。侵染垫在核盘菌侵染健康寄主时至关重要，抑制侵染垫形成可成为新的菌核病防治策略，然而侵染垫形成机制及功能尚不清楚。在前期研究中发现T-DNA插入突变体Sunf-MT6不能形成侵染垫，且不能侵染健康植株，但可从伤口侵入形成典型病斑；以Sunf-MT6为材料鉴定了与侵染垫形成相关的新基因（Ss-caf1），并推测侵染垫可能参与合成或运输致病相关蛋白或代谢产物。本申请拟以Sunf-MT6及野生型菌株Sunf-M为材料，以基因Ss-caf1为线索：鉴定与Ss-Caf1互作的蛋白，并沉默互作蛋白编码基因，分析对侵染垫形成及致病力的影响，从而发现新的侵染垫形成相关基因；通过侵染垫形成阶段的蛋白质组及代谢组分析，评估侵染垫分泌致病相关物质的潜力。加深对侵染垫形成机制及功能的认识，为菌核病绿色防治提供理论依据。

核盘菌是一种重要植物病原真菌，由其引起的作物菌核病严重威作物的生产安全，研究核盘菌的致病机理有助于更好的防控菌核病。目前关于核盘菌致病机理的研究，主要集中于草酸和细胞壁降解酶类等的作用，而对侵染垫等致病相关因子的了解较少。前期研究中我们发现突变体Sunf-MT6由于T-DNA插入导致基因Ss-caf1功能缺失，造成突变体不能形成侵染垫且丧失致病力。以该突变体为研究材料，本项目对核盘菌与寄主互作早期侵染垫形成机制及功能进行了研究。.通过扫描电镜观察，结合前期研究结果（Xiao et al., 2014）我们明确了核盘菌与寄主植物互作的早期全过程及侵染垫的生物学功能。在侵染早期（病斑可见前），核盘菌在寄主上定殖主要依赖于侵染垫产生机械压力直接穿透寄主表皮细胞，且通过菌丝的蔓延以在新的位点形成更多侵染垫，从而建立更多侵入位点。侵染垫和菌丝表面覆盖的物质覆盖，可能有助于核盘菌更好附着植物表皮。当病斑形成后，核盘菌依靠以侵染点为中心形成大量菌丝在在寄主表皮下向四周辐射状扩展，导致病斑不断扩大。此外，我们发现核盘菌侵入寄主植物不依赖于从气孔侵入。.我们发现将核盘菌接种parafilm膜可以很好的模拟核盘菌侵染垫形成过程。利用该接种体系获取野生型菌株Sunf-M与突变体Sunf-MT6接种后不同阶段的转录组数据。从中筛选出了新的可能参与核盘菌与寄主互作早期的信号通路，及核盘菌侵染垫形成相关基因。结合转录组数据，通过CRISPR-Cas9技术进行基因敲除，鉴定出一个新的可影响侵染垫形成的致病相关分泌蛋白。此外，利用免疫沉淀、pull-down及酵母双杂交的方法，鉴定出了与Ss-Caf1存在直接相互作用的蛋白，并明确了互作蛋白所在信号通路在核盘菌侵染垫形成时期的表达动态。.本研究明确了核盘菌与寄主植物互作早期的过程，进一步明确了侵染垫具有重要生物学功能，获得了侵染垫形成过程中的基因表达动态谱，鉴定出新的核盘菌致病相关基因及与Ss-Caf1存在直接相互作用的蛋白，为后期深入解析核盘菌与寄主互作机制，安全防控作物菌核病奠定了基础。

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