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United States Department of Agriculture

Agricultural Research Service

Title: Utilization of Fusarium Verticillioides Est's to Identify Potential Regulators of the Fum Gene Cluster

Authors
item Butchko, Robert
item Brown, Daren
item Proctor, Robert

Submitted to: Aflatoxin Elimination Workshop Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: October 28, 2004
Publication Date: October 25, 2004
Citation: Butchko, R.A.E., Brown, D.Q. and Proctor, R.H. 2004. Utilization of Fusarium verticillioides ESTs to identify potential regulators of the FUM gene cluster. Proceedings of the 4th Annual Fungal Genomics 5th Annual Multi-Crop Fumonisin 17th Annual Multi-Crop Aflatoxin Elimination Workshops. p. 5.

Technical Abstract: Fusarium verticillioides is the causal agent of stalk and ear rot of maize. F. verticillioides also produces the mycotoxins fumonisins which have been shown to cause diseases in horses and swine as well as cancer in laboratory animals. In the effort to reduce or eliminate fumonisin contamination of maize, we are interested in understanding the genetic regulation of fumonisin production. A fumonisin (FUM) biosynthetic gene cluster, consisting of 15 co-regulated genes, has been described in F. verticillioides and predicted amino acid sequences of these genes suggest that most are involved in fumonisin biosynthesis. Through gene deletion analysis, we have shown that many of the genes in the cluster are required for fumonisin production. Similarly, other genes have been shown to be required for certain steps in the biosynthetic pathway and accumulate fumonisin-like intermediates. However, absent from the FUM gene cluster is a transcription regulatory element. Expressed Sequence Tag (EST) libraries are useful tools for identifying genes expressed under different conditions. EST libraries have been constructed from Fusarium verticillioides mRNA's isolated from the fungus grown under various conditions (including synthetic fumonisin inducing liquid media and maize seedlings). Comparison of libraries from different growth conditions allows the identification of genes that are expressed under those conditions. Comparison of libraries from conditions where the FUM gene cluster is not transcribed and conditions where the FUM gene cluster is transcribed has allowed the identification of a number of regulatory type genes which may have a role in the transcriptional regulation of the FUM gene cluster. We have created disruption constructs using information from the EST libraries in order to study the role, if any, of candidate genes on the activity of the FUM gene cluster. Our disruption constructs contain a portion of the EST nucleotide sequence as well as an antibiotic resistance gene. Incorporation of these constructs into the genomic region of the genes of interest results in the disruption of the gene with the antibiotic resistance gene. The recipient strain for disruption analysis contains a FUM1::GUS fusion construct which allows us to assess FUM gene expression by the activity of the GUS gene product (beta-glucoronidase) under the control of the FUM1 promoter. Beta-glucoronidase catalyzes the conversion of the colorless substrate X-Gluc to a blue compound. This system allows us to use a blue/white screen for activity of the FUM1 promoter. We have disrupted a number of these candidates and are characterizing the affects of the mutation on toxin production and FUM gene cluster activity.

Last Modified: 7/31/2014
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