自噬相关基因ATG8通过非自噬途径参与球孢白僵菌应对氧化胁迫的分子机制

Beauveria bassiana, an important filamentous entomopathogenic fungus, performs a saprophytic and parasitic growth in its lifecycle, and has a great potential for the biological control of insect pests. Fungal resistance to oxidative stress plays an important role in the environmental fitness and pathogenicity of entomopathogenic fungi. The preliminary research indicated that BbATG8 (an ortholog of autophagy-related gene 8 in B. bassiana) mediates fungal response to oxidative stress via a mechanism independent of the autophagic process. This study is sought to elucidate the BbATG8-mediated mechanisms involved in B. bassiana resistance to oxidative stress. First, it is to identify the actual down-stream target proteins/pathways affected by BbATG8 via quantitative comparative proteomic analysis between wild type and knockout strain. Second, the affinity purification and related techniques are adopted to characterize the interacting proteins of BbATG8p. Their roles in fungal antioxidant capability will be elucidated by strategy of gene disruption. Third, the mechanisms of interaction between BbATG8p and its interaction protein were elucidated by the biochemical and genetic techniques. Fourth, all these results are used to develop a BbATG8-dependant network in B. bassiana under oxidative stress. These results will highlight for the first time that the non-autophagic roles of ATG proteins significantly contribute to stress response and pathogenesis of filamentous mycopathogen, and add the new knowledge to the mechanistic diversities of environmental fitness of mycopathogens. Such theory will provide a new genetic strategy to improve the environmental fitness of entomopathogenic fungi.

球孢白僵菌是生活史中含有腐生和寄生阶段的丝状真菌，在害虫微生物防治中具有广阔的应用前景。本课题以球孢白僵菌为模型，围绕丝状真菌的氧化胁迫耐受力决定其环境适应性这一主题，探求自噬相关基因ATG8参与真菌应对氧化胁迫的新机制。第一，通过分析野生及敲除菌株在氧化胁迫条件下的差异蛋白质组，筛选ATG8所介导的潜在下游蛋白，并通过基因功能分析阐明关键的下游蛋白或途径；第二，利用亲和层析技术并结合相应的生化技术，阐明在氧化胁迫条件下与ATG8蛋白互作的蛋白及其功能；第三，通过生化分析，阐明ATG8蛋白与互作蛋白的结合模式及其对下游蛋白的调控方式；最后，构建ATG8基因介导菌体应对氧化胁迫的遗传网络。本课题的实施和完成将首次揭示自噬相关蛋白通过非自噬途径调控丝状真菌抗氧化系统的分子机制，为进一步认识动物病原真菌环境适应性的多样化机制提供新理论。同时，也为通过遗传改造提高昆虫病原真菌环境适应性提供新策略。

球孢白僵菌是生活史中含有腐生和寄生阶段的丝状真菌，在害虫微生物防治中具有广阔的应用前景。本课题以球孢白僵菌为模型，围绕丝状真菌的氧化胁迫耐受力决定其环境适应性这一主题，探求自噬相关基因ATG8参与真菌应对氧化胁迫的新机制。主要研究内容包括：第一，通过分析菌株在寄主体内增殖过程中的转录组，发现可变剪接机制参与调控ATG8基因非自噬功能；第二利用酵母双杂技术并结合相应的生化技术，分离鉴定与ATG8蛋白互作的蛋白及其功能；第三，通过生化分析，阐明ATG8蛋白与互作蛋白的结合模式；最后，通过ATG8互作蛋白介导的转录组，构建ATG8基因介导菌体应对氧化胁迫的遗传网络。本课题的实施和完成将首次揭示自噬相关蛋白通过非自噬途径调控丝状真菌抗氧化系统的分子机制，为进一步认识动物病原真菌环境适应性的多样化机制提供新理论。同时，也为通过遗传改造提高昆虫病原真菌环境适应性提供新策略。