组蛋白H3K4甲基化修饰在罗伯茨绿僵菌与宿主昆虫互作中的调控机制研究

Histone lysine methylation is an important epigenetic modification, which plays vital role in growth and development, response to environmental stresses and biological interactions. Despite a growing body of evidence points towards epigenetic mechanisms being responsible for a wide range of biological phenomena in model organisms, the role of histone lysine methylation in shaping host-fungal pathogen interactions has received comparatively little attention. In our previous research, disrupting the Set1 gene responsible for H3K4 methylation (MrSet1) in the entomopathogenic fungus Metarhizium robertsii reduced its virulence to Anopheles mosquitoes and silkworms, inferring that H3K4 methylation might regulate the interactions between fungi and insect. Therefore, firstly in this study, the function of H3K4 methylation in M. robertsii is confirmed by phenotype analyses including growth and development test, appressorium induction and hypha body formation in insect hemolymph of mutant ΔMrSet1 and its complementary strain compared with the wild type strain (WT). Secondly, the expression patterns of WT and mutant under different culture conditions are investigated by RNA-seq. Finally, ChIP-qPCR is applied to confirm the expression pattern of the candidate genes identified in RNA-seq and their marks with H3K4 methylation. This study will provide new insights into the regulation mechanism in host-fungal pathogen interaction at epigenetics level and illustrate the theoretical basis for biocontrol application of entomopathogenic fungi.

组蛋白甲基化是一种重要的表观遗传修饰，在生物生长发育、响应环境胁迫和生物互作中发挥重要作用。尽管组蛋白甲基化修饰在模式生物中已有大量研究，但在丝状真菌与宿主互作中的表观遗传调控机制知之甚少。我们前期研究发现，罗伯茨绿僵菌H3K4甲基转移酶基因（MrSet1）敲除后，杀虫毒力显著降低，表明MrSet1参与绿僵菌与昆虫互作的调控。因此，本项目以罗伯茨绿僵菌为研究对象，通过对野生型、MrSet1突变株和互补菌株的侵染结构附着胞的形成、虫体内发育分化和杀虫毒力等表型分析，详细研究H3K4甲基化修饰在绿僵菌发育和致病中的功能；通过RNA-seq明确野生型和突变株在有表型差异条件下的基因表达模式和调控网络；通过ChIP-qPCR验证差异表达基因的调控受到H3K4甲基化的影响，从而揭示H3K4甲基化修饰在真菌发育和致病中的作用和调控机制，也为深入了解昆虫真菌发育及其与宿主互作的分子机理提供新的视角。

蚊虫可传播疟疾、登革热、黄热病和寨卡等多种疾病，严重威胁人类的生命健康。蚊虫控制是阻断蚊媒传染病的重要措施。罗伯茨绿僵菌是一种重要的昆虫病原真菌，在蚊虫生物防治和阻断疾病传播上具有巨大优势，被认为是最有潜力的下一代生物杀蚊剂之一。然而，罗伯茨绿僵菌与蚊虫互作过程中的表观遗传调控机制尚不清楚。本项目在罗伯茨绿僵菌中鉴定到了一个组蛋白H3K4甲基转移酶MrKMT2基因，研究表明它在真菌侵染蚊虫体壁阶段发挥重要的调控作用。敲除Mrkmt2基因后，罗伯茨绿僵菌侵染结构附着胞的形成显著减少，杀蚊毒力显著降低。转录组测序（RNA-seq）分析表明，分别有770和728个昆虫体壁诱导上调和下调的差异基因依赖于MrKMT2的表达。这些基因主要参与脂类、碳水化合物的代谢和转运。染色质免疫共沉淀测序（ChIP-seq）一共鉴定了2,987个基因受到MrKMT2介导的H3K4三甲基化（H3K4me3）的修饰。RNA-seq和ChIP-seq关联分析表明，MrKMT2通过修饰转录因子编码基因Mrznf的H3K4me3直接激活Mrznf的转录。突变株ΔMrznf与ΔMrkmt2有同样的表型缺陷；在ΔMrkmt2中过表达MrZnF可以恢复敲除Mrkmt2的表型缺陷，从而在遗传水平上证明了MrZnF是MrKMT2调控的关键靶标基因。因此，罗伯茨绿僵菌组蛋白甲基转移酶MrKMT2通过H3K4me3修饰激活转录因子MrZnF来诱导蚊虫体壁侵染相关基因的表达，调控真菌在体壁侵染过程中附着胞的分化，最终影响杀蚊真菌致病力的形成。本研究首次揭示了组蛋白甲基化修饰在病原真菌侵染宿主蚊虫体壁过程中的调控作用，不仅为病原真菌与宿主昆虫互作提供新机制，而且为高效杀蚊真菌遗传改良提供新的靶点基因和改造策略。