The transformation of protoplasts of Leptosphaeria maculans to hygromycin B resistance

Conditions are described for the efficient isolation and regeneration of protoplasts of a fungal pathogen of brassicas, Leptosphaeria maculans. Treatment of the protoplasts with DNA of the plasmid pAN7-1 (containing an E. coli hygromycin phosphotransferase gene with Aspergillus nidulans expression signals) and plating under selective conditions resulted in the formation of hygromycin 13-resistant colonies. Southern blot analysis of resistant colonies indicated that single copies of the plasmid had integrated into different sites in the genome. In twelve of the transformants analysed so far, the integration is stable through mitosis. The demonstration of efficient transformation is an essential first step in the molecular analysis of pathogenicity of this commercially important pathogen.