ISOLATION OF A BENOMYL-RESISTANT ALLELE OF THE BETA-TUBULIN GENE FROM SEPTORIA-NODORUM AND ITS USE AS A DOMINANT SELECTABLE MARKER

We have developed a homologous transformation system for the wheat-pathogenic fungus Septoria nodorum based on a benomyl-(MBC-) resistant allele of the beta-tubulin gene. The beta-tubulin gene was isolated by heterologous hybridization from a cosmid library prepared from an MBC-resistant mutant. Cosmids carrying the gene conferred MBC resistance when introduced into a sensitive strain, demonstrating that resistance to MBC fungicides in S. nodorum may be determined by the beta-tubulin gene. This MBC resistant allele of the beta-tubulin gene (tubA(R)) was subcloned into pUC18 and used as a dominant selectable marker for transformation of wild-type sensitive strains. Transformants arose at frequencies of approximately 5 per mu-g of DNA, were integrative in nature and were mitotically stable. Some transformants showed a marked reduction in vigour, both in the presence and absence of MBC; this is thought to arise from overproduction of beta-tubulin. The S. nodorum tubA(R) gene also conferred MBC resistance on the related species Leptosphaeria maculans, a pathogen of Brassica, following its introduction by cotransformation. Probing digested S. nodorum DNA with tubA(R) at low stringency revealed only a single beta-tubulin gene. We anticipate that tubA(R) will prove a useful tool for the investigation of the pathogenicity of S. nodorum and other fungi.