Genetic transformation of the vascular wilt fungusVerticillium dahliae

Abstract

To facilitate genetic analysis of pathogenicity of Verticillium dahliae, a vascular wilt pathogen, a DNA-mediated transformation system has been developed. Resistance to hygromycin B was obtained by transforming spheroplasts with the cosmid vector pAN7-2. Transformation efficiencies ranged between 3 and 5 transformants/μg vector DNA. The transforming DNA was integrated into the V. dahliae genome, in single and multiple copies and in tandem array. In several multicopy transformants, minor alterations in the integrated DNA sequences were evident following extensive vegetative growth in the absence of hygromycin B. Electrophoretic karyotype analysis also provided direct evidence of chromosome rearrangements in two transformants. The availability of a transformation system for V. dahliae will facilitate the cloning and characterization of genes that are important for pathogenicity and development. Key words: Verticillium wilt, fungal transformation, electrophoretic karyotype, hygromycin B resistance, chromosome rearrangement.