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TRI12, a trichothecene efflux pump from Fusarium sporotrichioides: gene isolation and expression in yeast

Alexander, N.J., McCormick, S.P., Hohn, T.M.
MGG 1999 v.261 no.6 pp. 977
Fusarium sporotrichioides, genes, proteins, polypeptides, secretion, trichothecenes, nucleotide sequences, amino acid sequences, complementary DNA, mutants, introns, antifungal properties, genetic transformation, gene expression, Saccharomyces cerevisiae, plasma membrane, phenotype, biological resistance
Many of the genes involved in trichothecene toxin biosynthesis in Fusarium sprortrichioides are present within a gene cluster. Here we report the complete sequence for TRI12, a gene encoding a trichothecene efflux pump that is located within the trichothecene gene cluster of F. sporotrichioides. TRI12 encodes a putative polypeptide of 598 residues with sequence similarities to members of the major facilitator superfamily (MFS) and is predicted to contain 14 transmembrane-spanning segments. Disruption of TRI12 results in both reduced growth on complex media and reduced levels of trichothecene production. Growth of tri12 mutants on trichothecene-containing media is inhibited, suggesting that TRI12 may play a role in F. sporotrichioides self-protection against trichothecenes. Functional analysis of TRI12 was performed by expressing it in yeast strains that were co-transformed with a gene (TRI3) encoding a trichothecene 15-O-acetyltransferase. In the presence of the TR13 substrate, 15-decalonectrin, cultures of yeast strains carrying TRI12 and TR13 accumulated much higher levels of the acetylated product, calonectrin, than was observed for strains carrying TR13 alone. PDR5, a transporter of the ABC super-family, which is known to mediate trichothecene resistance in yeast, increased calonectrin accumulation in TRI12/TRI3 yeast strains but not in TR13 strains. These results confirm the involvement of TRI12 in the trichothecene efflux associated with toxin biosynthesis, and demonstrate the usefulness of yeast as a host system for studies of MFS-type transporters.