title: TRI12, a trichothecene efflux pump from Fusarium sporotrichioides: gene isolation and expression in yeast.
abstract: Many of the genes involved in trichothecene toxin biosynthesis in Fusarium sporotrichioides 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 TRI3 substrate, 15-decalonectrin, cultures of yeast strains carrying TRI12 and TRI3 accumulated much higher levels of the acetylated product, calonectrin, than was observed for strains carrying TRI3 alone. PDR5, a transporter of the ABC superfamily, which is known to mediate trichothecene resistance in yeast, increased calonectrin accumulation in TRI12/TRI3 yeast strains but not in TRI3 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.
authors.affiliation: Mycotoxin Research Unit, USDA/ARS, National Center for Agricultural Utilization Research, Peoria, IL 61604, USA.
authors.forename: N J
authors.initials: NJ
authors.lastname: Alexander
authors.forename: S P
authors.initials: SP
authors.lastname: McCormick
authors.forename: T M
authors.initials: TM
authors.lastname: Hohn
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D003001:Cloning, Molecular; D004271:DNA, Fungal; D005656:Fungal Proteins; D005670:Fusarium / Q000235:genetics*; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014170:Transformation, Genetic; D014255:Trichothecenes / Q000235:genetics* / Q000378:metabolism*
chemical_list: D004271:DNA, Fungal; D005656:Fungal Proteins; D008565:Membrane Proteins; C408432:TRI102 protein, Gibberella zeae; D014255:Trichothecenes