Disruption of Cerevisin via Agrobacterium tumefaciens-mediated transformation affects microsclerotia formation and virulence of Verticillium dahliae

Agrobacterium tumefaciens-mediated transformation (ATMT) was used to obtain a large number of Verticillium dahliae (Vd991) T-DNA insertion mutants that were randomly integrated. Insertion mutants that produced significantly fewer microsclerotia were chosen for further analysis. Mutant T0065 was identified as having the Cerevisin gene interrupted by T-DNA, and it was named cerevisin. The cerevisin protein showed a high amino acid sequence similarity with the vacuolar protease B. The mutant strain cerevisin displayed decreased production of microsclerotia and conidia, significantly reduced growth rate and reduction in virulence compared to the wild type. Moreover, the composition of secreted proteins differed between the cerevisin mutant and the wild type. Loss of function of Cerevisin decreased secretion of proteins of low molecular weight (14-25 kDa). Upon treatment with the secreted proteins of the mutant, the degree of leaf wilting decreased, indicating that Cerevisin is implicated in production of these proteins, which are putative pathogenicity factors of V. dahliae. The results suggest that Cerevisin is involved in controlling multiple processes of development and metabolism, plays an important role in vegetative growth and microsclerotia formation and affects virulence of V. dahliae.