Abstract: 【Objective】 The objective of this study is to characterize Vdku80 of Verticillium dahliae, which encodes the protein involved in the nonhomologous end-joining double DNA break repair pathway, and to provide a recipient strain with enhanced gene knockout frequency. 【Method】 V. dahliae Vdku80 deletion mutant ΔVdku80 was constructed as previously reported. Briefly, Vdku80 gene knockout was accomplished through a combination of techniques, beginning in Vdku80 homologous DNA fragment construction. Individual V. dahliae cells were genetically transformed via Agrobacterium tumefaciens mediated transformation. Recombination then occurred in the region of that sequence within the gene of Vdku80, resulting in the insertion of hygromycin resistance gene to disrupt the gene of Vdku80. Vegetable growth, sporulation and pathogenicity of ΔVdku80 were studied compared with those of wild type strain. The effect of the absence of Vdku80 on gene knockout frequency was tested by disruption of ChsV and ChsVI, which encoded V. dahliae chitin synthases.【Result】Vdku80 deletion mutant strain (ΔVdku80) was successfully constructed. Deletion of Vdku80 had no effect on colony morphology and mycelial growth, as deduced from the monitoring of radial growth of wild type strain and ΔVdku80 on PDA media 8 days after inoculation. Both wild type strain and ΔVdku80 reached highest level of conidia production after growing for 5 days on Czapek media and showed no significant differences with respect to conidia number in this moment. Growth of wild-type strain and ΔVdku80 was inhibited by 0.02% MMS, and the inhibition rate was also similar. Finally, the Vdku80 deletion mutant ΔVdku80 was qualitatively and quantitatively as pathogenic as its corresponding wild-type strains. Altogether, these results showed that the deletion of Vdku80 did not affect mycelial growth, conidia production and pathogenicity of V. dahliae. Using wild-type strain as a gene knockout recipient, gene knockout frequency was 44% and 31% for ChsV and ChsVI, respectively, while using ΔVdku80 as gene knockout recipient, gene knockout frequency was 94% and 87% for ChsV and ChsVI, respectively.【Conclusion】Vdku80 deletion mutant ΔVdku80 displayed wild-type phenotypes regarding growth, sporulation and pathogenicity, but clearly improved gene knockout frequency of other genes. These results support that ΔVdku80 can be a recipient strain with enhanced gene knockout frequency in V. dahliae.

Key words: Verticillium dahliae , gene knockout frequency , homologous recombination , nonhomologous end-joining