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

Many of the genes involved in trichothecene toxin biosynthesis in Fusarium sporotrichioides are present within a gene cluster. Here we report the comp lete sequence for TRI12, a gene encoding a trichothecene efflux pump that i s located within the trichothecene gene cluster of F. sporotrichioides. TRI 12 encodes a putative polypeptide of 598 residues with sequence similaritie s 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 p roduction. Growth of tri12 mutants on trichothecene-containing media is inh ibited, suggesting that TRI12 may play a role in F. sporotrichioides self-p rotection against trichothecenes. Functional analysis of TRI12 was performe d by expressing it in yeast strains that were co-transformed with a gene (T RI3) encoding a trichothecene 15-O-acetyl-transferase. In the presence of t he TRI3 substrate, 15-decalonectrin, cultures of yeast strains carrying TRI 12 and TRI3 accumulated much higher levels of the acetylated product, calon ectrin, than was observed for strains carrying TRI3 alone. PDR5, a transpor ter of the ABC superfamily, which is known to mediate trichothecene resista nce in yeast, increased calonectrin accumulation in TRI12/TRI3 yeast strain s but not in TRI3 strains. These results confirm the involvement of TRI12 i n the trichothecene efflux associated with toxin biosynthesis, and demonstr ate the usefulness of yeast as a host system for studies of MFS-type transp orters.