We had previously identified several yeast deletion mutants that conferred resistance to trichothecin (Tcin), a type B trichothecene and DON congener, which revealed a critical role for mitochondria in trichothecene-toxicity (1). Mitochondrial translation was directly inhibited prior to damage to mitochondrial membrane integrity and independent of cytosolic translation inhibition (2). To further explore the molecular mechanism of trichothecene toxicity we screened the yeast diploid knockout (YKO) library to identify deletion mutants that exhibited increased sensitivity to Tcin at concentrations that are sublethal to nonlethal (0.5-2µM) to the wild-type parental strain BY4743. We identified 121 deletion mutants which were disrupted in functions involved in cellular damage control from the toxic effects of trichothecenes, including DNA repair and response (15.7%), RNA degradation and stability (29.8%), ribosome biogenesis and protein degradation (10.7%). Saccharomyces Genome Database (SGD) phenotypic analysis revealed that a large fraction (42%) of the Tcin-sensitive mutants exhibited high sensitivity to oxidative stress. Oxidant-sensitive 2’, 7’-dichlorofluorescin diacetate (DCFH-DA) staining of these mutants revealed that Tcin-induced ROS generation was up to 3-fold higher relative to BY4743. We observed a significant and dose-dependent increase in ROS levels (2-4 folds) in BY4743 treated with DON, T-2 and DAS and found a strong correlation between ROS generation and cell death. Moreover, treatment with antioxidants, such as ascorbic acid and vitamin E increased cell survival 3-4-fold in T-2 treated cells and 6-9-fold in Tcin treated cells, suggesting a direct role for ROS in trichothecene-mediated cell death. Trichothecenes failed to generate ROS in the petite strain lacking mitochondrial DNA (ρo) or when mitochondrial membrane potential (ψ mito ) was depolarized with the ionophore FCCP (carbonilcyanide p-triflouromethoxyphenylhydrazone), suggesting the mitochondrial origin of trichothecene-induced ROS. REFERENCES

Trichothecenes constitute a large family of low-molecular-weight sesquiterpenoid mycotoxins produced by various species of Fusarium, Trichoderma, Cephalsporium, and other fungi. Identified by their characteristic trichothecene ring, these toxins include deoxynivalenol (DON), diacetoxyscierpenol (DAS), T-2, and trichothecin (T-cin). Each toxin varies with regard to toxicity and as a group is known to have a wide array of effects in plants, animals, and humans. The plant pathogen Fusarium gramineraum causes Fusarium head blight (FHB) in both wheat and barley resulting in reduced plant yield and contamination of cereal grains with trichothecenes, in particular DON. DON is an inhibitor of translation. However, the inhibitory effects of trichothecenes are often not limited to translation and information on other downstream targets of trichothecenes is lacking. To obtain a comprehensive picture of the pathways involved in trichothecene metabolism and resistance, we have used the yeast, Saccharomyces cerevesiae, as a model organism to study the impact of trichothecences on eukaryotic cells. We screened the yeast knockout (YKO) collection, composed of 4700 strains, to identify mutants that exhibit hypersensitivity to T-cin. We selected T-cin over DON to screen the library since yeast is sensitive to micromolar levels of T-cin compared to mM levels of DON. Bioinformatic analyses of the select sensitive mutants have revealed components of pathways that play a role in trichothecene resistance, such as MAP kinases, components of protein synthesis, vacuolar protein sorting, and ribotoxic stress pathways, suggesting a cellular-wide response. These genes represent new candidates for engineering resistance to DON and FHB in cereals. Further characterization of these genes will provide important new insights into the trichothecene metabolism. ACKNOWLEDGEMENT AND DISCLAIMER This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 590790-6-069. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture.