几丁质合成对绿僵菌侵染致病的作用机制

Entomopathogenic fungi are capable of naturally regulating insect populations and have been considered as a promising alternative or supplement to chemical pesticides because of its environmental safety, high insect speci?city and low likelihood of the development of insect resistance. However, the disadvantages have retarded widespread application, including poor efficacy and sensitivity to environmental conditions. Thus, an understanding of the fungal pathogenesis is necessary for commercial development and 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 as a result of the modification of culture conditions and environmental stresses. Chitin, a β(1,4)-linked polymer of N-acetylglucosamine (GlcNAc), is an important structural component of fungal cell wall and can account for up to 15% of the total fungal cell wall polysaccharide. Thus, chitin synthesis is crucial for fungal development and pathogenicity. Chitin is synthesised by large families of chitin synthase (CHS) enzymes that fall into seven discernable classes, with classes III, V, VI, and VII being typical for filamentous fungi. Studies in numerous fungi have shown that the deletion of CHSs results in a weakening of the cell wall and causes abnormal growth of the fungal hyphae, as well as appressoria. Consequently, CHSs perform crucial roles in fungal pathogenicity. In entomopathogenic fungi, the roles of CHSs 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 seven putative genes encoding seven classes of CHSs, respectively. In this project, the expression pattern of CHSs would be investigated during infection processes in M. acridum. The functions of CHSs would be characterized during infection using disruption and complementation strategies. The effect of chitin synthesis on fungal pathogenesis and insect immune response would be elucidated. The structure and components of fungal cell wall would be clarified using transmission electron microscope (TEM), atomic force microscope (AFM), fluorescent staining and two-dimensional electrophoresis. The relationship between the components and structure of cell wall and fungal pathogenicity would be analyzed. The mechanisms of chitin synthesis on pathogenesis in M. acridum would be elucidated. And the knowledge will provide theoretical foundation and candidate gene for developing stable, high-efficiency mycoinsecticide.

几丁质是真菌细胞壁的主要结构成分，几丁质合成影响真菌细胞壁组成和结构，与人和植物病原真菌的侵染致病关系密切，对昆虫病原真菌侵染致病的影响尚不清楚。目前，从细胞壁组成和结构与侵染致病关系的层面，研究几丁质合成对真菌侵染致病的作用机制还不够深入和系统。绿僵菌是重要的昆虫病原真菌，其几丁质合成酶家族含有7个基因。本项目拟采用融合GFP的方法，结合激光共聚焦显微观察技术，分析7个几丁质合成酶基因在绿僵菌侵染致病过程中的表达特征；采用基因敲除和回复策略，明确影响绿僵菌毒力的几丁质合成酶基因；结合电镜观察、荧光抗体染色及数字表达谱等技术，研究几丁质合成对绿僵菌侵染行为、寄主免疫反应及重要侵染阶段菌体细胞壁组成和结构的影响，分析细胞壁组成和结构与绿僵菌侵染致病的关系，阐明几丁质合成对绿僵菌侵染致病的作用机制。本研究的完成，将有助于理解昆虫病原真菌的侵染致病机制，可望为菌株改良提供理论依据和目的基因。

阐明昆虫病原真菌侵染致病的分子机制对于充分挖掘其生防潜力十分必要。几丁质合成在维持病原真菌正常侵染和抵御寄主免疫反应等方面起重要作用。由于侵染过程存在差异，不同病原真菌中影响侵染致病的几丁质合成酶基因的数量和种类不尽相同。目前，几丁质合成酶家族影响昆虫病原真菌侵染致病的机制尚未报道。本项目以蝗绿僵菌为实验材料，采用基因敲除和回复策略分析了几丁质合成酶家族各成员的功能。几丁质合成酶家族MaChsI-VII各基因的缺失对蝗绿僵菌的萌发、紫外和湿热的耐受能力的影响存在差异。在产孢量方面，MaChs家族各基因敲除转化子的产孢量与WT和CP相比均显著降低。通过点滴和注射两种方式接种蝗虫进行各菌株的毒力测定，结果发现只有MaChsIII、MaChsV、MaChsVII三个基因被敲除后影响蝗绿僵菌的毒力，而其他成员敲除后对蝗绿僵菌毒力无影响。真菌细胞壁组成成分分析发现，ΔMaChsV、ΔMaChsVII几丁质的含量增高，ΔMaChsVI各菌株几丁质的含量降低；ΔMaChsIII、ΔMaChsV、ΔMaChsVII各菌株β-1,3-葡聚糖的含量增高；ΔMaChsIII甘露糖蛋白的含量增高，ΔMaChsVI甘露糖蛋白的含量降低。透射电镜观察菌细胞壁结构发现，MaChsIII、MaChsV、MaChsVII 的缺失导致蝗绿僵菌细胞壁的厚度变薄。荧光标记凝集素或抗体染色结果发现，MaChsIII、MaChsV、MaChsVII 的缺失致使蝗绿僵菌孢子细胞壁表面结构发生不同程度改变，从而影响了蝗绿僵菌躲避寄主免疫识别的能力。该研究结果对于明确几丁质合成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转录因子MaSte12的功能研究，敲除MaSte12后蝗绿僵菌的毒力显著降低，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所致，并通过RNA-seq鉴定出了可能受到MaSte12调控的下游基因。同时，本项目还进行了蝗绿僵菌中转录调控因子MaSom1的功能研究，敲除MaSom1影响蝗绿僵菌的生长发育、产孢、抗逆以及侵染致病能力均显著降低。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新的候选基因。

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