球孢白僵菌特有疑似分泌性假定蛋白基因中高生防潜能因子的发掘

Beauveria bassiana is a classic entomopathogenic fungus widely applied in biological control programs of pest insects and mites. Its annotated genome contains up to 25% genes that encode hypothetical proteins with no recognizable domain or domains of unknown function and hence are obstacles to understanding molecular mechanisms involved in the fungal invasion and virulence. Our previous analysis of transcriptomes constructed in the infection course of B. bassiana against Plutella xylostella has unveiled 556 genes co-upregulated at 24, 36 and 48 h post inoculation. Of those, nearly half encode putative secretory proteins (PSP) likely involved in the fungal infection course. This project has two goals. The first goal is to discover new signalling factors or effectors crucial for biological control potential of B. bassiana by elucidating biological functions of about 50 PSP genes through multi-phenotypic analyses of their deletion/complement mutants in parallel with wild-type. The genes selected for study encode small hypothetical proteins without any recognizable domain, lack homologues in non-entomopathogenic fungi and hence are more likely involved in the fungal invasion. The second goal is to identify proteins and transcription factors that interact with the new signalling genes or mediate the new effector genes, to reveal cellular processes and events involved in their interactions and to explore possible mechanisms involved in their expression, regulation and function. The expected results include new virulence factors highly potential for use in genetic maintenance and improvement of fungal strains against arthropod pests and provide new insights into genetic mechanisms of B. bassiana pathogenicity and biocontrol potential.

球孢白僵菌是广泛用于农林害虫生物防治的昆虫病原真菌，其被注释的基因组中约有25%的基因编码结构域未知或根本缺乏可辨识结构的假定蛋白，成为探索其致病杀虫分子遗传机制的障碍。前期球孢白僵菌与小菜蛾侵染互作的转录组分析显示，在接种后24、36和48小时三个关键时刻显著上调即可能在侵染过程中扮演重要角色的基因多达556个，其中近半数编码小分子疑似分泌蛋白PSP。本项目选择在非昆虫病原真菌中无同源物即最可能在侵染昆虫过程中发挥特殊作用的50余个编码缺乏可辨识结构域假定蛋白的PSP基因进行研究。通过基因敲除株、回补株及野生株的多表型实验和多层次分析，从中发掘对生物防治潜能重要性状具有突出贡献和调控作用的新信号因子或效应因子，筛选与之互作的信号蛋白或上游转录因子，研究揭示新信号因子的作用机理、新效应因子的表达调控机理及其细胞生物学事件，由此深化球孢白僵菌致病杀虫机理及生物防治潜能遗传背景的科学认识。

本项目旨在对球孢白僵菌入侵昆虫过程中表达上调的50余个疑似分泌蛋白(PSP)基因进行研究，发掘对生物防治潜能具有突出贡献和调控作用的新信号或效应因子，实际完成100多个基因的研究工作。在50余PSP基因中，发现了一个小分子无半胱氨酸蛋白CFP是罕见的超级毒力因子，其同源物仅见于缺有性型的昆虫病原真菌中，其基因缺失突变导致毒力完全丧失，超表达则大幅增强毒力。分别编码BbRei1和Ssr4的PSP基因也被证明是白僵菌的重要毒力因子。无论半胱氨酸多寡，其余PSP均对宿主菌的毒力无实质性贡献。由此证明半胱氨酸多寡不是推断PSP为杀虫真菌毒力因子的依据。在扩展研究的50多个基因中，发现了一批调控球孢白僵菌生长发育、胁迫应答、寄主侵染及毒力的重要基因，深化了白僵菌害虫生物防治潜能遗传背景和调控机理的科学认识。共计75个基因的研究成果已在国际主流学术期刊发表有标注的论文34篇，每篇平均影响因子5.713，已被累计引用365篇次，培养博士生8人和硕士生6人。