MINING EXPRESSED SEQUENCE TAGS (ESTS) LEADS TO IDENTIFICATION OF PUTATIVE FUM CLUSTER TRANSCRIPTION FACTOR

Authors: Brown, Daren; Butchko, Robert; Busman, Mark; Proctor, Robert

Submitted to: Aflatoxin Workshop
Publication Type: Abstract Only
Publication Acceptance Date: 10/25/2005
Publication Date: 10/25/2005
Citation: Brown, D.W., Butchko, R.A., Busman, M., Proctor, R. 2005. Mining Expressed sequence tags (ESTs) leads to identification of putative fum cluster transcription factor [abstract]. Proceedings of the 4th Annaul Fungal Genomics, 5th Annual Multicrop Fumonisin, and 17th Annual Aflatoxin Elimination Workshops. p. 38.

Interpretive Summary:

Technical Abstract: Fumonisins are a family of mycotoxins produced by Fusarium verticillioides (teleomorph Gibberella moniliformis) and can be found contaminating maize throughout the world. F. verticillioides generally grows in maize tissue without causing disease symptoms, but under some conditions, can cause seedling blight and ear, root and stalk rots of maize as well as synthesize fumonisins. Fumonisins cause several different toxicoses in animals and, in humans, are epidemiologically associated with esophageal cancer and birth defects in some regions of the world. Fumonisins are synthesized via enzymes encoded by genes localized within a 42.5 kb portion of the F. verticillioides genome. Understanding fumonisin biosynthesis and its genetic regulation may lead to the development of novel methods to prevent contamination of maize with fumonisins and thereby eliminate the toxins from the animal and human food chain. Over four years ago, we initiated a project with The Institute for Genomic Research (TIGR) to sequence thousands of F. verticillioides cDNAs in order to generate an expressed sequence tag (ESTs) database. The primary goal of this project was to identify genes that regulate fumonisin biosynthesis. We generated eight cDNA libraries, each from a different culture condition, from which TIGR sequenced over 111,000 clones. After analysis, over 87,000 high quality sequences (e.g. ESTs) were generated that correspond to 11,119 unique sequences. Transcripts corresponding to genes within the fumonisin gene cluster were found primarily in four of the eight libraries. Three of these libraries were constructed from RNA harvested from F. verticillioides grown in GYAM, a liquid medium that supports fumonisin production, for 24, 48/72, or 96 hours. The fourth library was constructed from growth on maize meal, which also supports fumonisin biosynthesis. We reasoned that a comparison of the ESTs from different libraries would identify differentially expressed genes important to fumonisin biosynthesis. Preliminary analysis has found a number of candidate fumonisin regulatory genes and functional analysis is in progress. This report describes analysis of a set of ESTs that match genomic sequence adjacent to the fumonisin gene cluster. BLAST analysis of the genomic sequence corresponding to the ESTs indicated that it did not share similarity with any previously characterized sequence in GeneBank. In contrast, BLAST analysis of the ESTs indicated that they share significant similarity with transcriptional factors of the Zn(II)2Cys6 family. Analysis of the gene structure identified eight introns of which two are located within the sequence encoding the Zn(II)2Cys6 motif. A majority of the ESTs are alternative splice forms (ASFs) where an intron was either not excised or utilized an alternative 3’ splice sequence. All of the ASFs are unable to encode the predicted full length FUM21 protein. The distribution of ASFs in the GYAM libraries is consistent with the pattern of ASF ESTs of four fumonisin biosynthetic genes and further support our hypothesis that ASFs serve a biological function. Gene deletion studies indicate that FUM21 plays an important but not absolute role in fumonisin biosynthesis as deletion of FUM21 reduced fumonisin production to 30% of wild-type. Studies are in progress to examine if the FUM21 ASFs serve a biological function.