Cloning and Functional Analysis of VdKeR in Verticillium dahliae
CHEN Rui, LI Biao, WANG Yuan, HUANG Jia-Feng*
College of Agriculture/Key Laboratory of Oasis Agricultural Pest Management and Plant Protection Resources Utilization, Xinjiang Uygur Autonomous Region, Shihezi University, Shihezi 832003, China
Abstract：Cotton verticillium wilt caused by Verticillium dahliae is an important soil-borne fungal vascular disease threatening cotton production in China. In previous studies, the transcriptional expression of a gene encoding hypothetical protein in the virulent defoliating V. dahliae V592 strain isolated from cotton (Gossypium hirsutum) was dramatically down-regulated when V592 strain was cultured with cotton roots. To determine the function of the gene, the full length of the gene was cloned from the genome of V592 strain. The target gene knockout plasmid was constructed based on homologous recombination, and its complementation plasmid containing the full length of the target gene encoding region was also constructed, then to generate the target knockout mutant and its complementary strain by Agrobacterium-mediated transformation. The growth phenotype characteristics and pathogenicity to cotton of the gene knockout mutant were assayed. The transcriptional expression of 4 genes involved in virulence in the target gene knockout mutants were measured by reverse transcription qPCR (RT-qPCR). The results showed that the target gene full length was determined to be 1 439 bp and the deduced protein contained 6 tandem Kelch motif and a galactose oxidase domains, and Kelch repeats formed a 6-bladed propeller construction. Thus, the gene was designated as VdKeR (GenBank No. MZ855770) due to the encoding protein containing Kelch repeats. On potato dextrose agar (PDA) media, VdKeR knockout mutant produced more aerial hyphae on the edge of the colony than the wild-type strain V592 and complementary strain, but the colony growth rate of VdKeR knockout mutant exhibited obviously lower than those of V592 strain and complementary strain, indicating that VdKeR gene knockout resulted in increased formation of aerial hyphae and slower growth rate in V. dahliae. VdKeR knockout mutant produced significantly less conidia than those of V592 strain and the complementary strain, suggesting that VdKeR gene knockout affected the conidiation in V. dahliae. In cotton seedling, VdKeR knockout mutant displayed significantly reduced disease severity compared with V592 strain and complementary strain, showing that VdKeR gene knockout led to reduced virulence to cotton. In VdKeR deletion mutants, the other 4 genes involved in virulence including the C subunit gene of cAMP-dependent protein kinase A (VdPKAC1), the G protein β subunit gene (VGB), the necrosis-and ethylene-inducing-proteins (Nep1)-like protein (NLP) family genes VdNLP1 and VdNLP2, the transcriptional expression of which were strongly down-regulated. These results indicated that VdKeR gene played an important role in the growth and pathogenicity and affected the transcriptional expression of other genes involved in virulence in V. dahliae. The results provide basic data for further study on the pathogenic mechanism of Verticillium dahliae.
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