关键词: 棉花黄萎病, 大丽轮枝菌, VdHP1, 产孢, 致病力, 转录表达

Abstract:

【Objective】The objective of this study is to determine the function of a novel gene (VdHP1) in Verticillium dahliae causing cotton verticillium wilt, and to provide a basis for analyzing the pathogenic mechanism of V. dahliae and the prevention and treatment of cotton verticillium wilt.【Method】The full length of VdHP1 was cloned and sequenced from V. dahliae wild-type strain V592 genomic DNA and cDNA. The relative expression of VdHP1 in V592 strain induced by cotton roots for different times and from different tissues of V592 was measured by reverse transcription quantitative real-time PCR (RT-qPCR). VdHP1 gene knockout vector, complementary vector and overexpressed vector were constructed to produce VdHP1 gene knockout strains, complementary strains and overexpressed strains by Agrobacterium tumefaciens-mediated transformation, respectively. Taking wild-type strain V592 as the control, colony growth on PDA and hypha morphology were observed, microsclerotia production, conidial production and pathogenicity to cotton of VdHP1 gene knockout mutants and complementary strains were measured. The relative expression of other genes involved in pathogenicity in VdHP1 knockout mutants and overexpressed strains was measured by RT-qPCR.【Result】The full length of VdHP1 was determined to be 862 bp and deduced protein contained 268 amino acids, which shared no significant sequence similarity to any known annotated gene in GenBank. The transcriptional expression of VdHP1 was significantly up-regulated when V592 strain induced by cotton roots for 6-12 h, indicating that VdHP1 plays a role at the early stage of the infection. The transcriptional expression of VdHP1 in conidia was significantly higher than that in mycelia and microsclerotia, indicating that VdHP1 is differentially expressed in different tissues of V. dahliae. Compared with wild-type strain V592, VdHP1 gene knockout mutants showed significantly decreased conidia and conidiophores, branching hyphae were spirally shaped, and the pathogenicity to cotton was significantly decreased. The relative expression of genes involved in penetration peg formation (VdCrz1, VdNoxB, VdPls1), delivery of secretory protein (VdSep5) and conidial production (Vdpf, VdSge1, VGB, VdPLP, VdCYC8, VdNLP1, VdNLP2) was significantly down-regulated in VdHP1 knockout mutants, but was up-regulated in the overexpressed strains; whereas, the relative expression of genes involved in melanin synthesis (VdCmr1, VdSho1, VdLAC, VdPKS1) was significantly up-regulated in VdHP1 knockout mutants, and was down-regulated in the overexpressed strains. 【Conclusion】VdHP1 is participated in the production of conidia and conidiophores, and is involved in pathogenicity in V. dahliae. VdHP1 positively regulates the transcriptional expression of genes involved in penetration peg formation, delivery of secretory protein and conidial production, and negatively regulates the transcriptional expression of genes involved in melanin synthesis.

Key words: cotton verticillium wilt, Verticillium dahliae, VdHP1, conidiation, pathogenicity, transcriptional expression