|Woloshuk, Charles - PURDUE UNIVERSITY|
|Bluhm, Burton - PURDUE UNIVERSITY|
Submitted to: Aflatoxin Workshop
Publication Type: Abstract Only
Publication Acceptance Date: October 25, 2005
Publication Date: January 25, 2005
Citation: Butchko, R.A.E, Proctor, R.H., Brown, D.W., Woloshuk, C.P., Bluhm, B.H., Busman, M. 2005. Using genomics approaches to characterize potential fumonisin regulatory genes [abstract]. Proceedings of the 4th Annual Fungal Genomics, 5th Annual Multi-Crop Fumonisin, and 17th Annual Aflatoxin Elimination Workshops. p. 60. Technical Abstract: Fusarium verticillioides can cause seedling disease, stalk rot and ear rot of maize and also produces the mycotoxins fumonisins. Fumonisins are polyketide derived secondary metabolites synthesized through a multi-step biosynthetic pathway by enzymes encoded by a co-regulated cluster of genes (FUM gene cluster). Fumonisins are toxic to both humans and animals and have most recently been described as teratogenic, causing neural tube defects in mice. In an effort to reduce or eliminate fumonisin contamination of maize we are employing genomic resources to elucidate the genetic regulation of fumonisin production. Three types of F. verticillioides genomic resources are available: expressed sequence tag (ESTs) libraries, microarrays and whole genome sequence. We have developed an EST library containing over 87,000 sequences, in collaboration with The Institute for Genomic Research (TIGR), which represents 11,119 different sequences. The cDNA libraries were constructed from mRNAs isolated from eight different growth conditions. The publicly accessible TIGR F. verticillioides Gene Index (FvGI) incorporates all available sequence data into one database and at present, includes 11,126 total unique sequences. We have utilized the EST libraries to identify possible regulatory genes. 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 genes with similarity to regulatory type genes which may have a role in the transcriptional regulation of the FUM gene cluster. We have disrupted a number of these candidates, six of which have an effect on the transcription of the FUM genes. We have also generated a NimbleGen oligonucleotide microarray, in collaboration with TIGR, based on the FvGI. The microarrays consist of approximately 180,000 24-base pair probes or features, with each sequence in the FvGI represented by a set of 12 probes. We have validated the microarrays using mRNA generated from wild-type F. verticillioides cultured on fumonisin-inducing media. mRNA was isolated at 6 time points over 5 days and was used to probe the microarrays. FUM genes exhibited patterns of expression expected based on previous Northern analysis. Analysis of this time course experiment will allow us to set baseline levels of expression across the set of genes represented on the chip for comparison in other experiments. We are currently investigating differential gene expression between wild-type F. verticillioides and a FCK1 mutant cultured on whole maize kernels, as well as on dissected endosperm and germ tissue. Recently, 4X coverage of the Fusarium verticillioides genome generated at Syngenta and assembled at the Broad Institute was made available to the public. The intersection of whole genome sequence, EST libraries and microarrays is allowing us to more comprehensively define genes and describe their expression at the transcription level.