Fumonisins are mycotoxins -produced mainly by Gibberella moniliformis (anamorph Fusarium verticillioides)- that contaminate maize and maize-based products and cause great concern for human and animal health. Several biosynthetic (FUM) genes are known; among them, FUM1 encodes a key polyketide synthase. To study the genetic and environmental factors linked to fumonisin production, we amplified the putative FUM1 promoter (pFUM1; about 1000 bp upstream of the start codogen) from a toxigenic F. verticillioides (Fv) strain. pFUM1 was used to generate transgenic Fv strains expressing the N-terminus of FUM1 translationally fused to GFP under the control of pFUM1. Currently, pFUM1-driven transcriptional activity is being tested in fumonisin-inducing and non-inducing conditions, to estimate the degree of correlation between the fluorescence observed and transgene transcription (quantified by RT-qPCR); and between the latter and transcription of the endogenous FUM1. If correlations turn out to be acceptable, these transgenic strains could be used as indicators of fumonisin production at the single-cell level during plant infection and tissue colonization. Moreover, bioinformatic analyses on the putative promoter sequences of all known FUM genes suggest the presence of a statistically over-represented, 6-bp sequence; this motif could cis-act on the regulation of FUM1 transcription, since it is repeated twice in pFUM1. At present, we are generating a synthetic pFUM1 version where this motif is mutated, and preparing promoter-reporter constructs as above. The analysis (by fluorescence microscopy and RT-qPCR) of the corresponding Fv transformants will allow an immediate comparison with the activity of the Wt promoter, and thus help us to validate the bioinformatic predictions.
Molecular factors affecting fumonisin biosynthesis in Fusarium verticillioides: a study on FUM1 putative promoter
VISENTIN, IVAN;MONTIS, Valeria;VALENTINO, Danila;TAMIETTI, Giacomo;CARDINALE, Francesca
2009-01-01
Abstract
Fumonisins are mycotoxins -produced mainly by Gibberella moniliformis (anamorph Fusarium verticillioides)- that contaminate maize and maize-based products and cause great concern for human and animal health. Several biosynthetic (FUM) genes are known; among them, FUM1 encodes a key polyketide synthase. To study the genetic and environmental factors linked to fumonisin production, we amplified the putative FUM1 promoter (pFUM1; about 1000 bp upstream of the start codogen) from a toxigenic F. verticillioides (Fv) strain. pFUM1 was used to generate transgenic Fv strains expressing the N-terminus of FUM1 translationally fused to GFP under the control of pFUM1. Currently, pFUM1-driven transcriptional activity is being tested in fumonisin-inducing and non-inducing conditions, to estimate the degree of correlation between the fluorescence observed and transgene transcription (quantified by RT-qPCR); and between the latter and transcription of the endogenous FUM1. If correlations turn out to be acceptable, these transgenic strains could be used as indicators of fumonisin production at the single-cell level during plant infection and tissue colonization. Moreover, bioinformatic analyses on the putative promoter sequences of all known FUM genes suggest the presence of a statistically over-represented, 6-bp sequence; this motif could cis-act on the regulation of FUM1 transcription, since it is repeated twice in pFUM1. At present, we are generating a synthetic pFUM1 version where this motif is mutated, and preparing promoter-reporter constructs as above. The analysis (by fluorescence microscopy and RT-qPCR) of the corresponding Fv transformants will allow an immediate comparison with the activity of the Wt promoter, and thus help us to validate the bioinformatic predictions.
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