Altered regulation of 15-acetyldeoxynivalenol production in Fusarium graminearum

Most Fusarium graminearum isolates produce low or undetectable levels of tr ichothecenes in liquid shake cultures, making it difficult to perform bioch emical studies of trichothecene biosynthesis, To develop strains with highe r levels of trichothecene production under liquid shake conditions we trans formed F. graminearum with both a reporter gene containing a homologous tri chothecene pathway gene promoter (TRI5) and a gene encoding a heterologous trichothecene pathway transcription factor (TRI6), The TRI5 and TRI6 genes are part of the trichothecene pathway gene clusters of both Fusarium sporot richioides and F. graminearum. These genes encode trichodiene synthase (enc oded by TRI5), the first enzyme in the trichothecene pathway, and a transcr iption factor (encoded by TRI6) required for pathway gene expression. Trans formation of F. graminearum with plasmids containing either an F, graminear um TR15 promoter fragment (FGTRI5(P)) or FGTRI5(P) coupled with the beta-D- glucuronidase (GUS) reporter gene resulted in the identification of several transformants capable of producing 45 to 200 mg of 15-acetyldeoxynivalenol (15-ADON)/liter in liquid shake culture after 7 days. Increased 15-ADON pr oduction was only observed in transformants where plasmid integration occur red through the FGTRI5(P) sequence and was not accompanied by increased GUS expression. 15-ADON production was further increased in liquid culture up to 1,200 mg/liter following introduction of the F. sporotrichioides TRI6 ge ne (FSTRI16) into F. graminearum. The effects of FSTRI6 on 15-ADON producti on also depended on plasmid integration via homologous recombination of the FGTRI5(P) fragment and resulted in a 100-fold increase in GUS expression. High-level production of 15-ADON in liquid shake cultures provides a conven ient method for large-scale trichothecene preparation, The results suggest that targeting transformation vector integration to FGTRI5(P) alters pathwa y gene expression and are consistent with the proposed conservation of TRI6 function between Fusarium species.