一个编码富含丝氨酸蛋白的基因影响大丽轮枝菌的微菌核形成、产孢及致病力

A serine-rich protein identified in Verticillium dahliae affects microsclerotial formation,conidiation and pathogenicity

大丽轮枝菌是一种可在广泛的寄主范围内引发黄萎病的土传植物病原真菌,主要以微菌核的形式在土壤中存活多年,因此鉴定与微菌核形成及致病力相关的基因对防治该病害至关重要。本课题组从前期构建的棉花黄萎病菌T-DNA插入突变体库中筛选到一个微菌核明显减少的突变体,致病力测定结果表明,该突变体致病力明显下降。以棉花黄萎病菌野生型菌株V592的基因组DNA为模板,从棉花黄萎病菌中克隆到被T-DNA插入突变的基因(VdSRP1)编码区全长为415bp,包含一个外显子,编码一个富含丝氨酸蛋白,与任何已知的注释基因没有显著的序列相似性。为了明确VdSRP1基因在大丽轮枝菌中的功能,利用同源重组的原理及农杆菌介导的遗传转化方法获得了VdSRP1基因的2个敲除体菌株,与棉花黄萎病菌野生型菌株V592相比,VdSRP1基因敲除突变体的微菌核形成明显减少,产孢量及孢子萌发率下降,对棉花的毒力也显著下降。对野生型菌株V592及VdSRP1敲除突变体的转录分析表明,VdSRP1调控一系列与微菌核形成、孢子形成及致病力相关基因的表达。这些结果表明,VdSRP1基因影响大丽轮枝菌的致病力、微菌核形成、产孢及孢子萌发。

Verticillium dahliae is a soil-borne phytopathogenic fungus that causes vascular wilt disease on a broad range of hosts and survives for many years in soil in the form of microsclerotia.It is important for developing disease control strategies to identify genes invovled in microsclerotial formation as well as pathogenicity.Here,a T-DNA insertion mutant was found to be involved in microsclerotial formation based on screening of TDNA insertion library and the mutant displayed drastic reduction in virulence compared with the wild-type strain V592.Strain V592 genomic DNA was used as the template to clone and sequence the full-length of T-DNA insertion gene(VdSRP1).The full-length sequence of VdSRP1 was 415 bp with one exon encoding a serine-rich protein,which shared no significant similarity to any known annotated proteins.To explore the functions of VdSRP1 in V.dahliae,two VdSRP1 deletion mutants were generated by homologous recombination and Agrobacterium tumefaciens-mediated transformation.VdSRP1 deletion mutants produced reduced microsclerotia,conidia and conidial germination,and displayed drastic reduction in virulence compared with the wild-type strain V592.Transcription analysis of the wild-type strain and the VdSRP1 deletion mutants suggested that VdSRP1 regulates the expression of a series of genes involved in microsclerotial formation,conidial production and pathogenicity.These results indicated multiple roles of VdSRP1,including virulence,microsclerotial formation,conidiation and conidial germination in V.dahliae.