大丽轮枝菌纤维素结合域蛋白VdCB1.1调控植物先天免疫的分子机制

Cellulose binding domain proteins (CBM1) are widely distributed in fungal cell wall degradation enzymes and have the ability to facilitate cell wall degradation, but the pathogenic function of CBM1 was unclear. In previous study, we found one glycoside hydrolases 12 (GH12) protein coupling CBM1 domain which functions as pathogen-associated molecular pattern (PAMP) to trigger the plant innate immunity during plant infection, but the coupling CBM1 domain have the ability to suppress GH12-triggered immunity. Further analysis confirmed that one CBM1 protein VdCB1.1 from Verticillium dahliae also has the ability to suppress the plant innate immunity. However, the mechanism of how CBM1 protein to suppress GH12-triggered immunity in pathogen is unknown. To uncover the regulation mechanism of plant innate immunity by VdCB1.1, the secretory characteristics, subcellular localization, and sequence variation of VdCB1.1 will be identified to demonstrate the gene characterization that function as one effector; the functional domain and recognition site of VdCB1.1 will be determined by the activity of suppressing GH12-induced cell death and plant innate immunity with the progressive truncated peptide and site-directed mutagenesis of VdCB1.1; to describe the molecular mechanism of VdCB1.1 suppressing plant innate immunity, the expression of disease-resistance related genes will be analyzed after infiltration of VdCB1.1, and the ability of CBM1 domain suppressing GH12-induced cell death and plant innate immunity will be identified by GH12 proteins coupled with multiple CBM1 domains; the virulence function of VdCB1.1 will be illustrated by gene knockout and heterologous overexpression in plant during infection. In conclusion, this program will help us get insight into the molecular mechanism of CBM1 protein regulating plant innate immunity and facilitating the infection progress during plant-pathogen interaction, and also provide primary theoretical foundations for developing the novel control strategy to Verticillium wilt.

纤维素结合域CBM1广泛存在于真菌细胞壁降解酶中，促进了细胞壁组份降解，但其参与侵染寄主的致病功能不清楚。本项目前期研究发现大丽轮枝菌1个糖苷水解酶GH12蛋白激活植物先天免疫的活性因藕联CBM1而被抑制，进而发现病原编码的CBM1蛋白VdCB1.1同样具有抑制活性，这种利用CBM1蛋白抑制先天免疫而促进侵染的致病机制未见报道。本项目拟通过分泌特性、亚细胞定位、基因变异等研究明确VdCB1.1作为效应子的特性；鉴定逐步缺失和点突变对抑制细胞死亡活性的影响，解析其抑制功能域和识别位点；分析VdCB1.1对抗病基因表达的影响，明确藕联CBM1抑制先天免疫的功能，阐明其抑制GH12蛋白激发先天免疫的分子机理；通过基因缺失、植物中过表达等研究揭示VdCB1.1参与侵染寄主的毒力功能。完成上述研究有助于揭示CBM1蛋白调控先天免疫参与病原侵染寄主的致病分子机理，为发展新的黄萎病防治策略提供理论依据。

项目按照研究计划完成了既定研究内容。通过VdCBM1信号肽分泌特性、植物中亚细胞定位等研究，明确了VdCBM1为典型的分泌蛋白；通过短肽缺失、定点突变等抑制GH12蛋白（VdEG1/VdEG3）诱导的细胞死亡活性分析，明确VdCBM1抑制活性的依赖于CBM1结构域的完整性及半胱氨酸位点；VdCBM1能够抑制VdXYN4、VdCB21和VdNEP2等效应蛋白激发的免疫反应，包括抗性相关基因表达、胼胝质沉积等。发现了GH12蛋白藕联CBM1结构域抑制了其激发PTI抗性的特性，揭示大丽轮枝菌利用CBM1蛋白或者藕联CBM1结构域抑制先天免疫反应的机理；通过基因敲除与功能互补、植物中过表达等研究明确VdCBM1具有毒性功能。通过上述研究，明确大丽轮枝菌纤维素结合域蛋白VdCB1.1的毒性功能及其调控GH12蛋白激发先天免疫反应的致病分子机理，为发展新的黄萎病防治策略提供理论依据，同时也为真菌利用CBM1和GH12蛋白协同互作干扰植物抗性反应的分子机理研究提供借鉴。