膜蛋白OPY2调控罗伯茨绿僵菌侵染昆虫的分子机制研究

Fungi are the most common insect pathogens, and some of them have been developed as insecticides for controlling insect pests and vector-borne diseases. Better understainding of fungal pathogenesis in insects is alleviating efficicay limitations by allowing construction of transgenic strains with improved ability to kill. In our previous study, we obtained from a random T-DNA insertion library of Metarhizium robertsii (formerly named as Metarhizium anisopliae) a mutant that was incapable of producing appressorium and thus unable to infect insects. The gene disrupted in this mutant is a homologue of yeast membrane protein gene Opy2 that is responsible for tolerance to high osmolarity by recruiting HOG-MAPK pathway. Unlike yeast OPY2 that is constitutively and stably expressed at all tested conditions, expression of Metarhizium's OPY2 (MrOPY2) is highly up-regulated in appressoria and in blastospores in the insect hemolymph, indicating that MrOpy2 is related not only to tolerance to high osmolarity but also to almost whole infection process of insects by M. robertsii. Based on the results obtained, we propose to further elucidate molecular mechanisms by which the fungus regulates the expression pattern of MrOpy2, characterize the relationship between MrOPY2 and its interacting downstream signal pathways, and profile MrOPY2-regulated pathogenicity genes. Completion of this proposal will not only provide a significant insight into pathogenesis of M. robertsii in insects (specifically), but also benefit the investigation of pathogenesis of human and plants fungi (generally) that also have OPY2-like proteins.

真菌是最常见的昆虫病原微生物，其中一些已被开发为真菌杀虫剂用于防治害虫和虫媒疾病,但杀虫速度慢等因素限制了它们的应用。研究昆虫病原真菌的致病机理有助于利用遗传重组技术创建高效安全的真菌杀虫剂。前期工作中，我们从罗伯茨绿僵菌(原金龟子绿僵菌)的突变库中筛选到一株致病突变体，并克隆了其内被破坏的膜蛋白基因Opy2，这是首次发现该基因与病原真菌致病力相关。OPY2是信号转导途径上感知胞外信号的因子,它在附着胞和芽生孢子内高效表达，表明它几乎调控整个致病过程。在此基础上,本项目将进一步研究OPY2调控致病过程的分子机制, 并利用深度测序技术挖掘由OPY2调控的下游毒力基因。完成本项目将显著加深对罗伯茨绿僵菌致病过程的认识。罗伯茨绿僵菌的致病机理与植物和人类病原真菌的均类似，而后两类真菌也有OPY2类蛋白，因此, 研究罗伯茨绿僵菌OPY2的功能具有普遍意义，能推动其它病原真菌致病机理的研究。

本项目以罗伯茨绿僵菌为材料研究昆虫病原真菌的致病机理，旨在为创建高效安全的真菌杀虫剂提供理论依据。前期工作发现膜蛋白OPY2是一个新致病因子，但机制不清。因此本项目提出三个研究内容：一是研究调控OPY2蛋白水平的机制；二是研究OPY2与下游信号转导途径的互作机制；三是挖掘受OPY2调控的下游致病基因。重要结果如下。其一，OPY2是控制真菌由腐生向寄生生活方式转换的关键基因，决定罗伯茨绿僵菌附着胞形成的起始。较低OPY2蛋白水平保证正常腐生生长， OPY2蛋白水平上升是启动附着胞形成所必须的。在寄生阶段OPY2基因通过alternative transcription start sites机制产生两条大小不同的转录本，而在腐生阶段只有其中的长转录本。短转录本中的主ORF翻译效率高，长转录本5UTR中的uORFs抑制下游主ORF的翻译，最终导致OPY2蛋白在寄生阶段水平高，腐生阶段水平低。其二、OPY2调控不同的MAPK途径以适应不同的环境。腐生阶段，OPY2调控Hog1-MAPK；附着胞形成阶段，OPY2负调控FUS3-MAPK；血腔定植阶段，OPY2调控HOG1-MAPK。其三、基于RNA-Seq的转录组分析发现， OPY2基因在不同的阶段调控特有的基因，以适应相应的环境。分析受OPY2调控的基因，我们发现了一个调控附着胞形成的新转录因子AFTF1。AFTF1表达水平提高是附着胞正常形成所需要的，但OPY2限制它在附着胞形成时水平过高；过高的AFTF1抑制附着胞的形成。其三、揭示了MAPK信号转导途径对罗伯茨绿僵菌致病过程的调控机制和它们在昆虫病原真菌毒力进化中的多样性。Fus3-，Hog1-，和Slt2 -MAPK途径均与致病相关。Fus3-MAPK途径是附着胞形成所必需的。Hog1-和Slt2-MAPK途径既参与控制附着胞形成，又调控定植昆虫血腔。RNA-seq分析发现，保守的MAPK信号转导途径在不同的昆虫病原真菌中调控不同的下游致病因子；而在不同昆虫病原真菌之间保守的下游致病因子受多样的信号转导途径调控。最后，绿僵菌通过基因水平转移机制从寄主昆虫中获得甾醇转运蛋白基因Mr-NPC2a，它帮助绿僵菌更好地适应血腔环境，从而成功侵染；该研究不但发现了重要的致病因子，而且首次从功能研究水平报道基因水平转移是推动真核病原物的致病能力的一个机制。