O-甘露糖基化修饰影响绿僵菌细胞壁组成、结构及侵染致病的作用机制

Entomopathogenic fungi have been considered as a promising alternative or supplement to chemical pesticides. However, the disadvantages have retarded widespread application, including poor efficacy and sensitivity to adversities. Thus, an understanding of the pathogenesis in M. acridum is necessary for improvement of the biocontrol fungus. The fungal cell wall, which comprises a polysaccharide-protein complex, can protect the cell from harsh environments and plays a prominent role in resisting host response. The cell wall is now seen as a dynamic structure that is continuously changing to respond to the various extracellular stimuli. Glycosylation is an extremely common and highly conserved type of protein modi?cation involving the addition of carbohydrate residues to target proteins. It plays a critical role in determining the structure and function of numerous secreted and membrane-bound proteins. O-mannosylation is an important type of glycosylation. Many roles have been described for fungal O-mannosylation, including in cell wall integrity, cell morphology, and localization of proteins. The initial O-mannosylation reaction is performed by protein O-mannosyltransferases (PMTs), which are integral endoplasmic reticulum membrane proteins that add the ?rst mannose to Ser or Thr residues of target proteins. Studies in numerous fungi have shown that PMTs are involved in the cell wall synthesis and fungal pathogenicity. In entomopathogenic fungi, the characterization of PMTs has not been investigated, and their roles in infection have been still a mystery. Metarhizium, one of the most important genera of entomopathogenic fungi, has emerged as an excellent model organism for exploring many questions at the molecular and biochemical levels. In our previous work, the whole genomic DNA of M. acridum has been sequenced. From the whole genomic sequence of M. acridum, we found three putative genes encoding PMTs. In this project, the expression pattern of PMTs genes would be investigated during infection processes in M. acridum. The functions of PMTs in fungal pathogenicity and sensitivity to adversities would be confirmed using disruption/interference and overexpression strategies. The structure and components of cell wall of various strains, including MNTs transformants and PMTs transformants, would be characterized by transmission electron microscope (TEM), atomic force microscope (AFM), fluorescence staining, two-dimensional electrophoresis (2-DE) and LC-MS. The relationship between the components of cell wall and fungal pathogenicity would be analyzed. Some component related to fungal pathogenicity would be identified. The mechanism would be clarified that O-mannosylation influences the structure and components of cell wall and fungal pathogenicity. The results will help to furhter elucidate the mechanisms of fungal pathogenesis. The knowledge will provide theoretical foundation for developing stable, high-efficiency mycoinsecticide.

昆虫病原真菌是重要生防微生物，其细胞壁主要由多糖和蛋白组成，糖基化修饰对细胞壁合成至关重要。在模式真菌中，O-甘露糖基转移酶（PMTs和MNTs）是蛋白O-糖基化修饰的重要酶类，与真菌的生长发育、细胞壁合成及侵染致病密切相关。在昆虫病原真菌中，PMTs的功能研究未见报道，O-甘露糖基化修饰影响其细胞壁组成、结构及侵染致病的机制还缺乏研究。经分析发现，绿僵菌中存在3个PMTs基因。本项目将采用real-time RT-PCR、基因敲除或干扰及超表达的方法，分析PMTs基因的表达特征及其对绿僵菌抗逆和毒力的影响；以绿僵菌MNTs和PMTs系列转化子为研究材料，利用透射电镜观察、原子力显微观察、荧光染色、双向电泳和液质联用等技术，测定不同菌株细胞壁的组成结构。阐明O-甘露糖基化修饰对绿僵菌细胞壁组成、结构及侵染致病的作用机制。本研究将有助于理解昆虫病原真菌的侵染致病机制，为菌株改良提供理论依据。

昆虫病原真菌作为重要的生防资源，潜力巨大，阐明其侵染致病的分子机制十分必要。O-甘露糖基转移酶PMT家族是真菌进行蛋白糖基化修饰的重要酶类，在植物和人类病原真菌中，PMT影响真菌的生长发育，抗逆和毒力，但在昆虫病原真菌中研究较少。本研究以蝗绿僵菌为实验材料，采用基因敲除、干扰及回复策略分析了该家族的功能。研究发现，敲除MaPmt1对蝗绿僵菌的萌发和产孢量无影响；敲除MaPmt4后，蝗绿僵菌萌发速度减慢，产孢量无变化；干扰MaPmt2后，蝗绿僵菌萌发速度减慢，产孢量降低。MaPmt1/2/4的缺失或干扰后蝗绿僵菌对紫外和湿热的耐受能力下降。敲除MaPmt1主要通过影响附着胞的膨压降低蝗绿僵菌毒力；干扰MaPmt2主要通过影响附着胞的形成和膨压降低蝗绿僵菌毒力；敲除MaPmt4主要通过影响附着胞的形成和膨压以及昆虫血淋巴中的生长降低蝗绿僵菌毒力。MaPmt1/2/4敲除或干扰菌株细胞壁变薄，细胞壁表面结构发生改变，胞壁蛋白、几丁质含量显著降低，葡聚糖含量无变化。该研究结果对于明确O-甘露糖基化修饰的改变对蝗绿僵菌细胞壁重要成分、结构及侵染致病能力的影响，以及细胞壁组成、结构与真菌侵染致病的关系具有重要意义。另外，本项目还进行了蝗绿僵菌中丝氨酸-苏氨酸蛋白激酶Snf1的功能研究，敲除MaSnf1蝗绿僵菌的产孢量下降、萌发延迟和逆境敏感性增加，影响真菌对蔗糖、乙醇、甘油、海藻糖和蝗蜕等碳源的利用。敲除MaSnf1导致蝗绿僵菌毒力降低，是由于真菌在寄主体表的孢子萌发率和附着胞形成率的降低以及体内定殖阶段真菌利用昆虫血淋巴中的碳源（海藻糖等）能力下降所致，说明蝗绿僵菌中的海藻糖代谢相关基因可能是重要的毒力因子。因此，本项目进行了蝗绿僵菌中酸性海藻糖酶MaATM1的功能研究，敲除MaATM1影响蝗绿僵菌对海藻糖的利用，致使真菌在蝗虫体内生长较慢，毒力显著降低。本项目的研究结果有助于进一步明确昆虫病原真菌侵染致病的分子机制，为杀虫真菌的改良提供了候选基因。