罗伯茨绿僵菌定植昆虫血腔的机制研究

Mycoinsecticides are environmentally friendly alternatives to chemical insecticides in biocontrol programs for agricultural pests and vectors of disease. In addition, it is hard for insect pests to develop resistance to mycoinsecticides. However, mycoinsecticides currently have a small market share, mainly due to slow killing speed. Entomopathogenic fungi initiate infection via conidia that adhere to the insect cuticle and produce germ tubes that meander across the cuticle until they find a suitable site for penetration into the insect hemocoel. During hemocoel colonization, the fungi change from filament to blastospores, and the insect is killed by a combination of the proliferation of blastospores and the production of toxins. For many entomopathogenic fungi such as Metarhizium robertsii, penetration of the insect cuticle is quick, and most of time for the whole pathogenesis is spent on colonization of the hemocoel. The ability to colonize hemocoel largely determines the killing speed of the entomopathogenic fungi. Therefore, we propose to investigate the mechanisms for hemocoel colonization by entomopathogenic fungi using M. robertsii as a model. Firstly, a series of genes involved in hemocoel colonization will be identified by screening relevant mutants from a random T-DNA insertion library and a mutant library constructed by genome-wide target gene disruption, and by genome-wide profiling of gene expression during pathogenesis with RNA-seq. These genes encode components in signaling pathways, transcription factors or their downstream effectors. Secondly, molecular and biochemical analyses will be conducted to characterize the functions of key downstream effectors and their interactions. Finally, we will investigate how the signaling pathways and transcription factors regulate the downstream effectors. Completion of this project will provide advanced insights into mechanisms for hemocoel colonization by entomopathogenic fungi, which will facilitate improving killing speed of mycoinsecticides by reducing the time for hemocoel colonization using genetic engineering.

真菌杀虫剂具有对环境友好和寄主难以产生抗性等优点，但它们所占市场份额较小，一个主要原因是昆虫病原真菌的杀虫速度慢。昆虫病原真菌致病过程主要分为穿透体壁和定植血腔两个阶段，杀死昆虫所需时间主要消耗在血腔定植阶段；因此，定植昆虫血腔时间的长短，是决定杀虫速度快慢的主要因素。为此，本项目以罗伯茨绿僵菌为材料，研究昆虫病原真菌定植昆虫血腔的机制。首先，利用正向遗传学（筛选突变体库）和反向遗传学（转录组分析）等手段，鉴定系列血腔定植基因：包括信号转导途径，转录因子和它们所调控的下游血腔定植基因。然后，研究下游血腔定植基因的功能和基因间的互作理。最后，研究信号转导途径和转录因子对下游血腔定植基因的调控机理。项目的完成，将阐明罗伯茨绿僵菌定植昆虫血腔的关键机理，为缩短昆虫病原真菌定植昆虫血腔的时间、提高真菌杀虫剂的杀虫速度提供理论支撑。

昆虫病原真菌致病过程分为穿透体壁和血腔定殖两个阶段，它们杀虫所需时间大部分用于血腔定殖阶段，因此，研究血腔定殖机制可为加快真菌杀虫剂的杀虫速度提供理论依据。本研究通过突变体库筛选和转录组分析等技术，在昆虫病原真菌的模式材料罗伯茨绿僵菌中鉴定了16个血腔定殖相关基因，并解析了其中6个基因的功能，取得了如下主要结果。其一，发现控制罗伯茨绿僵菌适应血腔环境的新调控通路，该通路含4个组分：组蛋白乙酰基转移酶HAT1，组蛋白脱乙酰酶HDAC1，转录因子COH2和调控蛋白COH1。在昆虫体壁上，转录因子COH2激活体壁穿透基因的表达；真菌进入血腔后，由于组蛋白脱乙酰酶HDAC1表达下调，提高了调控蛋白COH1基因启动子部分组蛋白乙酰化水平上升，使得后者表达。HDAC1表达的下调是由于控制其启动子部分组蛋白乙酰化水平的组蛋白乙酰基转移酶HAT1表达水平下降所致。在血腔中表达的COH1与转录因子COH2互作，降低了COH2蛋白的稳定性，从而使得体壁穿透基因表达水平下降，同时解除了COH2对血腔定殖相关基因的抑制，使得破坏素Destruxin合成基因等血腔定殖基因表达。其二，发现了一个血腔定殖时起重要作用的效应蛋白EF1。EF1含一个信号肽，实验证明可以分泌到细胞外。EF1含有细胞穿透的motif (RXLR)，实验证明它能够穿过昆虫细胞膜，进入寄主细胞内。Pull-down和酵母双杂交实验发现EF1能与昆虫的Lim蛋白互作。其三，发现了抑制血腔定殖的次生代谢产物BA，它能削弱罗伯茨绿僵菌定殖昆虫血腔的能力，从而减少杀虫能力。与野生型菌株相比，BA合成突变体杀虫能力更强。生化分析表明，BA是一个法尼基转移酶抑制剂，由聚酮合酶PKS3合成。本项目的三个发现显著深化了对罗伯茨绿僵菌血腔定殖机制及其调控机制的认识。