Characterization of the Tri10 gene from Fusarium sporotrichioides
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The trichothecene mycotoxins are secondary metabolites produced by a variety of fungal genera including Fusarium, Myrothecium, Trichothecium, and Stachybotyris, that are toxic to humans and animals that ingest them by consumption of contaminated grain. This work details the characterization of a novel regulatory gene from Fusarium sporotrichioides, Tri10, which is located in the trichothecene gene cluster. Northern analysis of Tri10 deletion strains, Tri10 overexpressing strains, and a Tri6 deletion strain indicated that Tri10 is required for wild-type trichothecene gene expression and for wild-type expression of a primary metabolic gene, Fpps. Analysis of these mutants also provided evidence for a regulatory feedback loop where Tri10 is required for the expression of Tri6 and Tri6 negatively regulates Tri10. Furthermore, under certain growth conditions the sensitivity of Tri10 and Tri6 strains to T-2 toxin was increased. Analysis of mutants altered in the expression and genomic position of Tri10 revealed that placing Tri10 under the control of an exogenous promoter resulted in the overexpression of Tri10 and the other Tri genes whether this construct was located inside or outside of the Tri gene cluster. Work outside of this study has shown that in addition to Fpps, three other primary metabolic genes from the isoprenoid pathway feeding into trichothecene biosynthesis (Acat, Mk, Hmgs) are also influenced by the expression of Tri10 and Tri6. In the present study, targeted cDNA microarrays were used in conjunction with multiple mutants to reveal a large group of genes, containing both trichothecene and primary metabolic genes, which were positively influenced by Tri10 expression. At the same time, a small group of genes negatively influenced by Tri10 expression was observed. These results were in agreement with observations made outside of this study and validated the use of targeted cDNA microarrays for further studies. Additional analysis of the regulatory network linking trichothecene secondary metabolism and isoprenoid primary metabolism revealed that in a mutant blocked in the first step of the pathway, and therefore in the absence of trichothecene production, this regulatory link is mediated by Tri10 and Tri6.
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