关键词: 大丽轮枝菌 , 棉花黄萎病 , 蛋白激酶A催化亚基 , 微菌核 , 致病力 , 生物学性状

Abstract: 【Objective】The objective of this study is to investigate the roles of VdPKAC1, coding a catalytic subunit of cAMP-dependent protein kinase A, in the microsclerotial development, the conidia production and the virulence of Verticillium dahliae hyphal type strain. 【Method】Gateway® cloning technology from Invitrogen was used to construct the binary vector for the targeted deletion of VdPKAC1. In the T-DNA region of the vector, the 5′- and 3′-end homologous DNA sequences of VdPKAC1 were placed on the flank of the hygromycin resistance cassette. Mediated by the Agrobacterium tumefaciens AGL-1, this T-DNA was integrated into the genomic DNA of V07DF2, which is a highly aggressive defoliating V. dahliae strain and was isolated from the infected cotton in Jiangsu. Based on the PCR and the Southern blot analysis, five of six transformants with hygromycin resistance cassette were the targeted deletion mutants of VdPKAC1, and three of them were selected for further analysis. Seven days after cultured in PDB, the secretions of melanin was observed. Twenty-eight days after cultured on PDA plates, the growth of hypha and the formation of resting structure were investigated. Moreover, the ability of conidia production of mutants and wild type strain were assessed by the induction of cotton root extracts. And the virulence of the deletion mutants and wild type strain were also investigated. 【Result】Southern blot analysis indicated that the targeted deletion mutants 2B5 and C5 harbored a single insert of T-DNA, while in other mutants, ectopic integration were occurred. Compared with the wild type strain V07DF2, the mutants defective in VdPKAC1 produced more pigment in PDB, and formed chain-like resting structure which is different from normal microsclerotia. Moreover, the conidia production and pathogenicity of the mutants was reduced significantly against wild type strain V07DF2, but the surface of the mutant colonies was covered with more vigorous aerial hyphae. 【Conclusion】 The cAMP signaling pathway mediated by protein kinase A plays multiple roles in determining the traits of V. dahliae. It has been reported that VdPKAC1 (homolog VDAG_06474.1) is negatively regulated the formation of black microsclerotia in V. dahliae, its targeted deletion mutant produced more microsclerotia than wild type strain. To wild type strain V07DF2 which does not form any black microsclerotia or similar structure in the PDA plate, when the homologs gene of VdPKAC1 was knocked out, the targeted deletion mutant formed chain-like resting structure which is different from normal microsclerotia. The mutants with the novel phenotypes will be excellent materials for the identification of the functional genes involved in microsclerotia formation.

Key words: Verticillium dahliae , cotton Verticillium wilt , catalytic subunit of protein kinase A , microsclerotia , virulence , biological characteristic