Labeled trichothecenes for detoxification studies

Plant pathogenic fungi of the genus Fusarium produce toxins of the diverse class of trichothecenes. These metabolites (mycotoxins) can accumulate in agricultural products used as feed and food and create a health risk to animals and humans. The production of trichothecenes, which inhibit eukaryotic protein biosynthesis, is contributing to virulence of the plant pathogenic fungi. On the other hand, the ability to inactivate toxin (by glycosylation and other unknown mechanisms) seems to be an important resistance mechanism in plants. The goal of part A of this interdisciplinary project is to utilize metabolic engineering to convert a Fusarium sulphureum strain, which produces large amounts of the trichothecene diacetoxyscirpenol (DAS) in liquid culture, into a producer of deoxynivalenol (DON) and nivalenol (NIV), which are typically F. graminearum toxins. The strain will be further modified to produce DON-3-O-glucoside by introduction of a plant glucosyltransferase. Since the wild-type F. sulphureum can produce a glucosidic derivative of DAS, the expectation is that the engineered strain can produce NIV-glucosides (of unknown structure). The toxin-glucosides, which are potentially "masked mycotoxins" that can be reactivated in the digestive tract of animals, will be purified for toxicological studies. The engineered F. sulphureum should also be utilized to produce 13C labeled DON, which is useful as an internal standard for exact toxin determination by HPLC-MS/MS. Also radiolabeled 14C-DON will be produced for DON degradation studies. In project part B the produced DON will be utilized to identify and characterize bacteria with the ability to detoxify DON, and yeast mutants with increased detoxification capability. The aim of this project part is to identify a gene encoding an enzyme irreversibly inactivating DON. In part C the isolated toxins DON, NIV and the radiolabeled DON obtained by feeding of radioactive precursors, and chemically synthesized radiolabeled 3-acetyl-DON will be used to search for new detoxification mechanisms in plants, and to test whether genetic differences in this metabolic capability exists in germplasm used for resistance breeding and in experimental segregating populations of wheat. The mechanism of a wheat cDNA that confers DON resistance in yeast will be investigated. The information gained in this project should lead to new insights regarding risk assessment of Fusarium toxins regarding the problem of "masked toxins". The projects results should provide plant breeders with new tools to measure detoxification based resistance components and provide new ways to produces reference substances for analytical purposes. Potential uses for cloned microbial detoxification genes range from expression in yeast strains used for bioethanol formation (where toxins accumulate in the protein rich residue used as feed), or the production of transgenic plants that prevent trichothecene formation already in the field.