ARS | Publication request: Functional Analysis of Trichothecene Biosynthetic Genes Via Heterologous Expression in a Trichothecene-Nonproducing Fusarium Species

Submitted to: International Fusarium Workshop
Publication Type: Abstract
Publication Acceptance Date: December 15, 2004
Publication Date: December 15, 2004
Citation: Alexander, N.J., Mc Cormick, S.P., Proctor, R., Harris, L.J. 2004. Functional analysis of trichothecene biosynthetic genes via heterologous expression in a trichothecene-nonproducing fusarium species [abstract]. Proceedings of the 2nd International Symposium of Fusarium Head Blight. p. 552.

Technical Abstract: The biosynthesis of trichothecene mycotoxins by Fusarium sporotrichioides and F. graminearum involves a complex biochemical pathway that begins with the cyclization of farnesyl pyrophosphate to the sesquiterpene hydrocarbon trichodiene and continues with multiple oxygenation, cyclization and esterification reactions. While almost all of the steps in the pathway have been identified, there are still some questions regarding gene function, particularly of several of the P450 enzymes involved in the oxygenation steps. In previous studies using F. sporotrichioides, disruption of the P450 monooxygenase-encoding gene Tri4 blocked trichothecene production and led to the accumulation of trichodiene. Therefore, trichodiene is the likely substrate of the TRI4 protein. To further elucidate the function of the TRI4 protein, we heterologously expressed the F. graminearum Tri4 (FgTri4) in F. verticillioides, which does not produce trichothecenes. Transgenic F. verticillioides carrying FgTri4 under the control of a fumonisin biosynthetic gene (FUM8) promoter converted exogenous trichodiene to isotrichodermin. Conversion of trichodiene to isotrichodermin requires seven steps. Previous studies indicate that two of these reactions are non-enzymatic, and feeding studies done here indicate that wild-type F. verticillioides can convert isotrichodermol to isotrichodermin. Thus, the remaining four oxygenation reactions required for the conversion of trichodiene to isotrichotriol must be catalyzed by the TRI4 protein, suggesting that it is a multifunctional monooxygenase. Using a similar strategy, we analyzed the expression of FgTRI1 and FsTRI1. We have shown the usefulness of using a transgenic expression system to determine function of unknown genes which should be helpful in analyzing the many genes that are being identified in genomic projects.