大丽轮枝菌LysM蛋白毒力功能分析

Lysin motif (LysM) proteins may act as virulence factor in plant pathogenic fungus, but there is no findings reported in Verticillium dahliae. In previous studies, we de novo sequenced the high-virulence isolate VDG1, and completed the gene prediction and functional annotation. Further analysis showed that partial genes encoding secreted proteins were arranged in clusters. Two LysM protein genes, VdLysM1 and VdLysM2 locate in one gene cluster, are strongly induced during V. dahliae infection in cotton plants, indicating their potential roles of pathgenicity in V. dahliae. In this project, we will adopt target gene replacement technique to examine the virulence alteration when VdLysM1 and VdLysM2 are knock-out in wide-type strain VDG1. Meantime, the complementary experiment of VdLysM1 and VdLysM2 in the mutants and the low-virulent strain will be performed to further validate the virulence functions of VdLysM1 and VdLysM2. Second, the subcellular location of VdLysM1 and VdLysM2 will be tested to ensure that both of them are secreted proteins. Third, the VdLysM1 and VdLysM2 protein will be expressed and purified in yeast expression system to test wilt-inducing activity, the chitin oligosaccharides binding speciality, and suppression activity in hyphal tip hydrolysis by host chitinase enzymes. Finally, the polymorphism of VdLysM1 and VdLysM2 will be analyzed in the V. dahliae population to further identify the correlation of gene variation and virulence divergence. Through the experiments mentioned above, the virulence functions of VdLysM1 and VdLysM2 and their roles in population virulence divergence will be addressed, further suggesting the pathogenicity mechanism mediated by VdLysM1 and VdLysM2 through suppression of host immunity induced by sequestering chitin oligosaccharides.

LysM蛋白是植物病原真菌一类重要的毒力因子，在大丽轮枝菌中尚未报道。我们前期已经完成了高毒力大丽轮枝菌VDG1全基因组测序、基因预测和功能注释，进一步分析发现编码的分泌蛋白具有成簇分布的特点。其中1个基因簇含有2个LysM蛋白基因（VdLysM1和VdLysM2），在大丽轮枝菌侵染棉花条件下被显著诱导表达，呈现与毒力相关的特性。在此基础上，本项目拟通过基因敲除和功能互补实验确定VdLysM1和VdLysM2的毒力功能，亚细胞定位明确它们为分泌蛋白，酵母表达并纯化VdLysM1和VdLysM2，测定蛋白的致萎活性、结合几丁质寡糖和抑制植物几丁质酶降解真菌菌丝的特性，通过群体分析VdLysM1和VdLysM2基因的多态性与毒力差异的关系。从而初步鉴定大丽轮枝菌2个LysM蛋白基因的毒力功能及其与毒力变异的关系，探讨其通过与寄主受体竞争结合几丁质寡糖来抑制免疫反应激活的致病分子机理。

LysM 蛋白是植物病原真菌一类重要的毒力因子，在大丽轮枝菌中亦有发现。本项目围绕大丽轮枝菌的LysM基因簇上的2 个LysM 蛋白基因（VdLysM1 和VdLysM2）开展研究。VdLysM1缺失后突变体对棉花的致病力显著下降，而功能回补后能恢复大丽轮枝菌强致病力的表型，VdLysM2基因缺失后未影响病原的致病力，证明VdLysM1是大丽轮枝菌LysM基因簇上的毒力基因。通过毒力分化的群体分析发现，VdLysM1是一类保守的毒力基因，其单核苷酸变异与种群毒力分化相关；基因表达分析检测同样证明VdLysM1基因参与了大丽轮枝菌对寄主棉花的侵染过程，其在侵染12小时后即被诱导表达且持续至第5天。酵母信号肽跟踪系统检测证明VdLysM1基因编码的信号具有介导蛋白分泌到胞外的功能，为典型的胞外分泌蛋白。体表表达实验证明VdLysM1可以诱导棉花叶片发生典型的萎蔫坏死症状，同时VdLysM1具有结合几丁质寡糖和抑制植物几丁质酶降解真菌菌丝的特性。上述研究表明LsyM蛋白是大丽轮枝菌一类重要的毒力因子，这为大丽轮枝菌利用胞外分泌蛋白参与寄主侵染的致病分子机理研究提供理论依据。