Characterization of a transcriptional activator controlling trichothecene toxin biosynthesis

Trichothecene biosynthetic pathway genes are localized within a gene cluste r in Fusarium sporotrichioides and require the zinc-finger containing prote in, TRI6, for expression. We show here that TRI6 is able to bind within the promoter regions of nine different pathway genes and that TRI6 binding is involved in pathway gene activation. TRI6 binding occurs at three distinct sites in the TRI5 promoter, all of which contain the sequence TNAGGCCT. DNA fragments from the promoter regions of six other pathway genes containing this sequence are also substrates for TRI6 binding. Specific nucleotide cha nges in the TNAGGCCT sequence dramatically reduced TR16 binding. Analysis o f TR16 binding within the TRI3 and TRI11 promoters and the TRI4-TRI6 interg enic region which do not contain the TNAGGCCT motif suggests that the minim um sequence required for TRI6 binding is YNAGGCC. Two potential TRI6 bindin g sites, T4A and T4B, were identified within the intergenic region for the divergently transcribed TRI4 and TRI6 genes. Alteration or deletion of the T4A site resulted in the loss of nearly all in vitro TR16 binding and was c orrelated with the loss of promoter activity in vivo as measured by the exp ression of mutant TRI4(p)/GUS fusions, This establishes a physiological rol e for TRI6 binding and demonstrates that TRI6 is directly involved in the r egulation of pathway gene expression. To determine if a predicted CYs(2)His (2) zinc-finger motif at the C-terminus of TRI6 is involved in DNA binding, a C187A mutant was constructed in TRI6 using site-directed mutagenesis. Th e C187A mutant did not bind promoter DNA fragments, supporting the role of C187 in DNA binding. In addition, a TR16 homologue in the distantly related macrocyclic trichothecene pathway of Myrothecium roridum (MRTRI6) was also shown to bind to the same TRI5 and TRI4 promoter fragments bound by TRI6, Together, these data confirm our previous proposal that TRI6 is an activato r of trichothecene pathway gene expression and that DNA binding employs the C-terminal region of TRI6 containing three predicted Cys(2)His(2) zinc fin gers. (C) 1999 Academic Press.