控制罗伯茨绿僵菌穿透昆虫体壁的新调控通路研究

Application of mycoinsecticides can reduce the amount of chemical insecticides used for pest control, protecting environments. However, the slow killing speed of mycoinsecticides is limiting their widespread usage. Understanding of how entomopathogenic fungi infect insects provides theoretical bases for improving efficacy of mycoinsecticides. Penetration of the insect cuticle is the initial step in pathogenesis and determines whether or not the infection is successful. Metarhizium robertsii has been used as a model for studying fungal pathogenesis in insects. In our previous studies, we identified two new transcription factors that are necessary for the fungus to penetrate the insect cuticle, and they were regulated by Slt2-MAPK, Fus3-MAPK or the membrane protein Mr-OPY2, forming novel circuits for regulating cuticle penetration. In this proposal, we will identify more components in the regulatory circuits, reveal the molecular interactions between the components and profile the downstream pathogenicity genes regulated by the circuits. We have three research aims in this proposal. First, we will identify the upstream components that regulate AFTF1 and AFTF2 and elucidate the regulatory mechanisms. Secondly, we will identify regulatory components, such as transcription factors and signaling pathways, which are controlled by AFTF1 or AFTF2, and reveal mechanisms for AFTF1 and AFTF2 to regulate these components. Finally, we will functionally characterize novel pathogenicity genes controlled by the above regulatory circuits. Completion of this project will provide significant insights into mechanisms for M. robertsii to penetrate the insect cuticle and advances our understanding of how the fungus infects insects.

研究昆虫病原真菌的致病机理可为提高真菌杀虫剂的效率提供理论支撑。穿透昆虫体壁是启动致病过程的关键步骤，成功与否决定感染能否进行下去。罗伯茨绿僵菌是研究昆虫病原真菌致病机理的模式材料；在前期工作中，项目组发现了调控该菌穿透昆虫体壁的新转录因子AFTF1和AFTF2和调控它们的部分上游基因，这些组分构成了控制穿透昆虫体壁的两个新调控通路的雏形。本项目将进一步完善这两个调控通路，研究通路内的调控机制及通路间的关系，并研究受通路调控的新致病基因的作用机制。包括三个研究内容。其一，挖掘调控AFTF1和AFTF2的更多上游组分，并研究它们对AFTF1和AFTF2的调控机制。其二，研究受AFTF1和AFTF2控制的下游调控组分和控制机制；并研究两个通路间的关系。其三，研究上述调控通路所控制的新致病基因的作用机制。完成本项目将揭示控制罗伯茨绿僵菌穿透昆虫体壁的新调控通路，加深对其致病机理的认识。

环境友好的真菌杀虫剂是化学杀虫剂的补充甚至是替代品，但这类杀虫剂存在杀虫速度慢和抗逆性不够强等问题。阐明昆虫病原真菌致病机制可为提高真菌杀虫剂的效率提供新理论支撑。体壁是昆虫抵御病原真菌的第一道屏障，因此能否穿透昆虫体壁就决定了昆虫病原真菌感染的成败。罗伯茨绿僵菌是研究昆虫病原真菌致病机理的模式材料。本项目发现了控制该菌穿透昆虫体壁和孢子形成的新调控通路。转录因子RNS1是MAPK级联体下游的一个新中心调控蛋白。在穿透昆虫体壁时，RNS1的215位苏氨酸和226位丝氨酸被Fus3-MAPK磷酸化，它因此能进入细胞核与自身启动子上的片段BM2结合、诱导自身的表达水平。RNS1进一步启动近50个体壁水解酶基因（如蛋白酶和及几丁质酶等）的表达。除了在穿透体壁过程中起重要作用之外，Fus3-MAPK/RNS1通路还可启动相关基因来利用蛋白质和几丁质等非最适营养物质。在含最适碳源葡萄糖的PDA培养基上，RNS1的306位丝氨酸被Slt2-MAPK磷酸化，进而诱导自身的表达，RNS1然后诱导BrlA转录本的表达，进而激活孢子形成调控中心通路启动分生孢子产生途径。本项目还发现了另外一个控制罗伯茨绿僵菌穿透昆虫体壁的调控通路Fus3-MAPK/STE12/AFTF1。Fus3-MAPK磷酸化STE12，后者结合到转录因子基因Aftf1的启动子区域，进而诱导Aftf1的表达。综上，项目组发现了MAPK级联体下游两个分别控制罗伯茨绿僵菌穿透昆虫体壁和产孢的新调控通路，并发现已知通路Fus3-MAPK/STE12上的下游新组分转录因子AFTF1，显著深化了对罗伯茨绿僵菌致病机制和孢子机制的认识。