蛋白激酶A催化亚基VdPKAC1对菌丝型大丽轮枝菌V07DF2培养性状及致病力的调控

Regulation of Cultural Characteristics and Virulence in Verticillium dahliae Hyphal Type Strain V07DF2 by the Catalytic Subunit VdPKAC1 of cAMP-Dependent Protein Kinase A

【目的】通过同源重组敲除技术,解析菌丝型大丽轮枝菌中蛋白激酶A催化亚基基因VdPKAC1在调控微菌核发育、产孢及其致病力方面的作用。【方法】利用Invitrogen公司的Gateway技术构建VdPKAC1的同源重组敲除质粒。该质粒的潮霉素抗性基因盒两端分别为靶标基因5′和3′端各1 kb的DNA序列。通过农杆菌AGL-1的介导,将该质粒的T-DNA区域整合到来源于棉花的菌丝型大丽轮枝菌V07DF2菌株中。从6个随机选择的含有潮霉素抗性基因片段的转化子中,通过靶标基因的PCR检测,获得了5个VdPKAC1基因敲除突变体。经过Southern杂交检测,选择了3个敲除突变体材料(2B5、C5和HH2)进行突变表型的观察和分析。在液体PDB培养7 d后观察敲除突变体与野生型菌株的黑色素产生情况;在固体PDA培养28 d后,观察敲除突变体与野生菌株的菌丝生长和休眠结构形成情况。此外,利用棉花根提取物对液体Cazpek-Dox培养7 d的各菌株分别进行诱导处理,在处理24、72 h后分别统计分生孢子数量,以此对敲除突变体和野生菌株的分生孢子产生能力进行评估。最后,将孢子浓度为1×107个/mL的突变体和野生菌株的分生孢子液灌根接种2叶期的棉花,测定其致病力。【结果】Southern杂交结果表明,在上述5个敲除突变体中,只有2B5和C5两个菌株为T-DNA单拷贝插入,而其他菌株均存在T-DNA异位整合的情况。在PDA培养条件下,3个敲除突变体(2B5、C5和HH2)与野生型相比具有黑色素合成增加,气生菌丝更加发达的特点。其中,敲除突变体2B5和C5在平板培养条件下还形成了野生型菌株没有的、与典型微菌核形态不同的深褐色"链状休眠菌丝体结构"。在棉花根提取物的诱导处理下,3个敲除突变体产生的分生孢子数量少于野生型菌株。另外,致病力分析结果表明,敲除突变体材料仍然具有一定的致病力,但却显著弱于野生菌株V07DF2。【结论】在大丽轮枝菌中,通过蛋白激酶A介导的环腺苷酸信号途径控制多种重要性状,而VdPKAC1是这一途径中的重要成员。以前的报道证实,VdPKAC1负调控微菌核的数量,而本研究利用菌丝型菌株V07DF2在PDA培养基上不产生微菌核的这一特性,通过同源重组原理敲除该负调控基因,成功获得可以在PDA上产生休眠结构的菌丝型大丽轮枝菌菌株的突变体。这些突变体材料将为大丽轮枝菌微菌核发育信号途径及其调控基因的发掘提供平台和基础。

【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.