大丽轮枝菌果胶裂解酶PL1家族基因VdPL1-4的致病功能研究

大丽轮枝菌Verticillium dahliae引起的黄萎病是一种传播迅速且为害广泛的土传维管束真菌病害。大丽轮枝菌分泌的水解酶在侵染中发挥重要作用，尤其是纤维素酶和果胶酶。大丽轮枝菌果胶裂解酶包括PL1、PL3、PL4、PL9和PL11 5个家族，其中PL1家族成员数目最多，但对该家族的致病功能还未见系统研究。本研究明确大丽轮枝菌PL1家族共包含17个成员，且多数成员在侵染早期上调表达，其中VdPL1-4和VdPL1-8在侵染早期强烈诱导表达。挑选VdPL1-4构建缺失突变体，发现VdPL1-4缺失后菌株对棉花的致病力显著下降，对复杂碳源(果胶、羧甲基纤维素、淀粉)的利用能力受限，该基因回补后菌株的毒力和碳源利用能力均得以恢复。信号肽介导的分泌活性验证表明VdPL1-4为分泌蛋白。以上研究表明，VdPL1-4是重要毒力因子，研究结果为后续系统解析果胶裂解酶在病原致病过程中发挥的作用提供了理论依据。

Functional analyses of pectate lyase family 1 gene VdPL1-4 in the pathogenicity of Verticillium dahliae

Verticillium wilt caused by Verticillium dahliae is a rapidly spreading soil borne vascular fungal disease. Hydrolytic enzymes, especially cellulases and pectases produced by V. dahliae, play an important role during infection. Pectate lyases in V. dahliae comprise PL1, PL3, PL4, PL9, and PL11 families. Among them, the PL1 family has the largest number of proteins, but there is no systematic understanding of the pathogenic function of this family. This study showed that the pectate lyase of PL1 family from V. dahliae contained 17 members, and most of them showed up regulation of expression in the early stage of infection. The expressions of VdPL1-4 and VdPL1 8 were strongly induced in the early stages of infection. VdPL1-4 was selected to construct deletion mutants, and it was found that the growth of the deletion mutants of VdPL1-4 was inhibited on different complex carbon sources (pectin, carboxymethyl cellulose, starch) and they were significantly less virulent. The virulence and carbon utilization capacity of the complementary strains were restored to the levels of the wild type strain. Verification of secretory activity mediated by signal peptide showed that VdPL1-4 was secretory protein. The gene VdPL1-4 is therefore an important virulence factor in V. dahliae, and this study provides a strong theoretical basis for the subsequent systematic analysis of the roles of the pectate lyase genes of PL1 family in V. dahliae pathogenesis.