Submitted to: Applied and Environmental Microbiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 1/28/2000
Publication Date: N/A
Citation: Interpretive Summary: The mold Fusarium graminearum is an important pathogen of crop plants causing wheat head scab, maize root, stalk, and ear rot, and barley head blight. Production of the trichothecene mycotoxin deoxynivalenol (DON) by F. graminearum reduces the quality of infected grain and represents the major source of trichothecene contamination in these crops. Limited information is available concerning the nutritional and environmental factors that influence trichothecene biosynthesis. The lack of a liquid shake culture method for trichothecene production in F. graminearum has limited the molecular studies of trichothecene biosynthesis in F. graminearum and has increased the difficulties associated with the large-scale preparation of trichothecenes such as DON. Strains of F. graminearum engineered with a gene promoter from F. sporotrichioides produce trichothecenes in liquid culture and are ideal for molecular studies. Strains engineered to contain both the gene promoter and a regulatory gene from F. sporotrichioides produce high concentrations of 15-ADON in liquid culture. These new strains will be useful to molecular biologists who are characterizing the genes that control DON production and to chemists developing immunochemical detection methods for mycotoxins.
Technical Abstract: The TRI5 and TRI6 genes reside within the trichothecene pathway gene clusters of both Fusarium sporotrichioides (FS) and F. graminearum (FG). These genes encode trichodiene synthase (TRI5) and a transcription factor (TRI6) required for pathway gene expression. Transformation of F. graminearum with plasmids containing either a 749 bp FGTRI5 promoter fragment (FGTRI5P) or a FGTRI5P/GUS reporter gene resulted in the isolation of several transformants capable of producing significantly higher levels of 15-acetyldeoxynivalenol (15-ADON) in liquid culture. Increased 15-ADON production was only observed in transformants where integration occurred through FGTRI5P sequence. Changes in 15-ADON production were not correlated with increased beta-D-glucuronidase (GUS) expression in liquid culture. Further increases in 15-ADON production of up to 20-fold were observed following introduction of the FSTRI6 gene into transformants. Effects of FSTRI6 and 15-ADON production in liquid culture were influenced by whether or not plasmid integration occurred via FGTRI5P and involved in a 100-fold increase in GUS expression. Increased trichothecene production by FSTRI6 transformants was also found in cultures grown on several solid substrates including corn, wheat, and and rice. High level production of 15-ADON in liquid shake cultures by transformants provides a convenient method for large-scale trichothecene production and will facilitate studies of trichothecene biosynthesis in this fungus. The results suggest that targeting transformation vector integration to FGTRI5P alters pathway gene expression and are consistent with the proposed conservation of TRI6 function between Fusarium species.