Submitted to: Aflatoxin Elimination Workshop Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 10/16/2006
Publication Date: 10/16/2006
Citation: Butchko, R.A., Brown, D.W., Busman, M., Proctor, R. 2006. Identification and characterization of regulators of FUM gene expression [abstract]. Aflatoxin Elimination Workshop. p. 36. Interpretive Summary:
Technical Abstract: Fumonisins are polyketide derived mycotoxins produced by the maize pathogen Fusarium verticillioides. The genes required for fumonisin production are clustered on chromosome I. Previous analysis showed that the expression of the FUM genes is coordinately regulated and expression of the FUM gene cluster first becomes detectable at approximately 48 hours after inoculation of a liquid medium, and peaks approximately at 96 hours. None of the FUM cluster genes was predicted to encode a pathway specific regulatory gene, however. In an effort to identify genes that have an effect on the expression of the FUM gene cluster, we hypothesized that, similar to other secondary metabolite pathways, a specific regulatory gene might have a similar expression pattern as the genes required for toxin biosynthesis. Two Expressed Sequence Tag (EST) libraries were compared, one from a 24 hour culture (FUM genes off) and the other from a 96 hour culture (FUM genes on). Twenty ESTs with homology to DNA binding proteins, transcription factors, regulatory genes, etc., were selected that are present in the 96 hour library, but absent in the 24 hour library. These candidate genes were disrupted in a strain containing a FUM1p:GUS fusion construct, and fungal transformants were screened initially for the loss of GUS expression. Disruption of six of the 20 ESTs that resulted in the loss of GUS expression also resulted in the loss of FUM gene expression and toxin production. With the release of the first draft of F. verticillioides genomic DNA sequence, sequences flanking the six ESTs were identified and used to create gene deletion mutants. Characterization of deletion mutants confirms the effect on the expression of the FUM genes and on toxin production.