Fumonisins are mycotoxins produced mainly by the pathogenic fungus Fusarium verticillioides (Fv; teleomorph Gibberella moniliformis) that contaminates maize-based food and feed, causing great concern for human and animal health. Genes in the fumonisin biosynthetic pathway are included in the so-called FUM gene cluster. Among them, FUM1 is key to fumonisin production, since strains of F. verticillioides with a disrupted FUM1 fail to produce detectable levels of fumonisins. With this work, we want to explore the possibility that both epigenetic mechanisms and pathway-specific transcription factors contribute to the observed transcriptional co-regulation of the genes in the FUM cluster. Namely, the tested hypotheses were: 1) the existence, and relevance for transcription, of cluster-specific cis-regulatory motifs on the FUM1 promoter region (pFUM1); and/or 2) the importance of epigenetic mechanisms (histone acetylation/deacetylation, DNA methylation) for FUM1 expression and therefore for fumonisin production. Results of an in silico analysis suggest the presence of a 6-bp sequence statistically over-represented in the FUM gene promoters, compared to the rest of the genome. This motif could cis-act on the regulation of FUM1, since it is repeated twice in pFUM1. To validate the in silico prediction, a targeted mutant version of it (pFUM1mut) was obtained. Both wt pFUM1 and pFUM1mut sequences were used to drive GFP expression in transgenic Fv strains. Molecular characterization of the Fv transformants is under way, including quantification of endogenous FUM1 and GFP transcripts by qRT-PCR; this will allow us to validate the role of the identified motif in FUM1 expression. In parallel, we followed FUM1 expression after treatment with a histone deacetylase inhibitor and investigated the pFUM1 methylation pattern both under fumonisin-inducing and non-inducing conditions. Preliminary results suggest that both histone acetylation and pFUM1 methylation may contribute to FUM1 expression under inducing conditions in vitro.
Epigenetic and cis-regulatory factors affecting FUM1 gene expression for fumonisins biosynthesis in Fusarium verticillioides
MONTIS, Valeria;VISENTIN, IVAN;CARDINALE, Francesca;TAMIETTI, Giacomo;
2010-01-01
Abstract
Fumonisins are mycotoxins produced mainly by the pathogenic fungus Fusarium verticillioides (Fv; teleomorph Gibberella moniliformis) that contaminates maize-based food and feed, causing great concern for human and animal health. Genes in the fumonisin biosynthetic pathway are included in the so-called FUM gene cluster. Among them, FUM1 is key to fumonisin production, since strains of F. verticillioides with a disrupted FUM1 fail to produce detectable levels of fumonisins. With this work, we want to explore the possibility that both epigenetic mechanisms and pathway-specific transcription factors contribute to the observed transcriptional co-regulation of the genes in the FUM cluster. Namely, the tested hypotheses were: 1) the existence, and relevance for transcription, of cluster-specific cis-regulatory motifs on the FUM1 promoter region (pFUM1); and/or 2) the importance of epigenetic mechanisms (histone acetylation/deacetylation, DNA methylation) for FUM1 expression and therefore for fumonisin production. Results of an in silico analysis suggest the presence of a 6-bp sequence statistically over-represented in the FUM gene promoters, compared to the rest of the genome. This motif could cis-act on the regulation of FUM1, since it is repeated twice in pFUM1. To validate the in silico prediction, a targeted mutant version of it (pFUM1mut) was obtained. Both wt pFUM1 and pFUM1mut sequences were used to drive GFP expression in transgenic Fv strains. Molecular characterization of the Fv transformants is under way, including quantification of endogenous FUM1 and GFP transcripts by qRT-PCR; this will allow us to validate the role of the identified motif in FUM1 expression. In parallel, we followed FUM1 expression after treatment with a histone deacetylase inhibitor and investigated the pFUM1 methylation pattern both under fumonisin-inducing and non-inducing conditions. Preliminary results suggest that both histone acetylation and pFUM1 methylation may contribute to FUM1 expression under inducing conditions in vitro.
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