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Abstract
We have developed a homologous transformation system for the wheat-pathogenic fungus Septoria nodorum based on a benomyl-(MBC-) resistant allele of the β-tubulin gene. The β-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 β-tubulin gene. This MBC resistant allele of the β-tubulin gene (tubAR) was subcioned into pUC18 and used as a dominant selectable marker for transformation of wild-type sensitive strains. Transformants arose at frequencies of approximately 5 per μg 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 β-tubulin. The S. nodorum tubAR 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 tubAR at low stringency revealed only a single β-tubulin gene. We anticipate that tubAR will prove a useful tool for the investigation of the pathogenicity of S. nodorum and other fungi.
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