High resistance of Isaria fumosorosea to carbendazim arises from the overexpression of an ATP-binding cassette transporter (ifT1) rather than tubulin mutation

Aims: Probing possible mechanisms involved in the resistance of entomopathogenic fungus Isaria fumosorosea to carbendazim fungicide. Methods and Results: A carbendazim-sensitive strain (If116) selected from 15 wild-type strains was subjected to NaNO2-induced mutagenesis, yielding nine mutants with carbendazim resistance increased by 82- to 830-fold and thermo-tolerance decreased by 15-51%. Comparing the protein sequences deduced from the alpha-and beta-tubulin genes of If116 and its mutants revealed no traceable site mutation relating to the enhanced resistance although the transcripts levels of b-tubulin gene in all mutants were 0 87- to 7 16-fold of that in If116. Three examined mutants showed multidrug resistance because they were significantly more resistant to glufosinate, imidacloprid and other six fungicides than If116 during growth. Further examination of rhodamine-stained blastospores revealed existence of drug efflux pump protein(s) in all carbendazim-resistant mutants. Thus, the sequences of an ATP-binding cassette (ABC) transporter gene (ifT1) and its promoter region cloned from the wild-type and mutant strains were analysed. Three common point mutations were located, respectively, at the binding sites of Gal4, Abf1 and Raf, which are crucial transcription factors in the regulative network of numerous protein loci. Such point mutations elevated the ifT1 expression by 17 to 137-fold in all the mutants. Conclusions: The overexpression of the ABC transporter caused by the point mutations at the binding sites was responsible for the fungal resistance to various pesticides including carbendazim. Significance and Impact of Study: The transporter-mediated multidrug resistance found for the first time in entomopathogenic fungi is potential for use in improving mycoinsecticide compatibility with chemical pesticides.