Knock-out technique as reverse genetics tool for genome-wide functional analysis of phytopathogen Verticillium albo-atrum

Knock-out strains of V. albo-atrum

Verticillium albo-atrum is an important soil-borne fungal pathogen that causes vascular wilt diseases in a large variety of important crop plants.

Verticillium wilt diseases are difficult to control, therefore molecular analysis of V. albo-atrum pathogenicity contributes to the development of effective and environmentally sound means for mitigating the impact of V. albo-atrum on agriculture.

Our protocol for generating knock-outs of the fungal pathogen V. albo-atrum comprises two methods: the creation of knock-out plasmids by the USER Friendly cloning technique and transformation of the fungal pathogen by Agrobacterium tumefaciens-mediated transformation (ATMT). Knock-out strains of V. albo-atrum are made by site directed modifications of the pathogen genome by means of homologous recombination and achieved by introducing a DNA fragment containing two homologous recombination sequences flanking a selection marker.

The pathogenicity of knock-outs is then tested on tomato plants. Reduced virulence against tomato host plants is indicator, that knock-out of investigated gene resulted in loss of pathogenicity.