Deletion of VdKu80 enhances targeted gene replacement in Verticillium dahliae

Abstract

Verticillium dahliae is a notorious phytopathogenic fungus and causes severe Verticillium wilt diseases worldwide. The available genomic sequence of V. dahliae has facilitated genome-wide investigation of its life cycle and disease process. However, inefficient targeted gene replacement hampers gene functional analysis of V. dahliae. Ku heterodimer genes Ku70 and Ku80 have been proved to be involved in the recognition of DNA double-strand breaks, which will reduce the rate of homologous recombination. Here, in order to improve the frequency of the homologous recombination, we identified and deleted the V. dahliae VdKu80 with bioinformatics methods and protoplast-mediated transformation, respectively. The phenotypes of VdKu80 deletion mutants, such as fungal growth, microsclerotia formation and virulence, were similar to those of the wild type strain. Remarkably, the gene replacement frequencies of two genes (VDAG_00736 and VDAG_07169 which encode the cell division control protein) were increased by about 20% with VdKu80 deletion mutant as a recipient strain compared with that of the wild type strain. The results suggest that deletion of VdKu80 triggered the enhancement of the gene replacement frequency, indicating that the ∆VdKu80 strain can be used as an efficient recipient for the targeted gene manipulation in V. dahliae.