Submitted to: Aflatoxin Elimination Workshop Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 10/1/2004
Publication Date: 10/25/2004
Citation: Brown, D.W., Cheung, R.H., Proctor, R., Butchko, R.A., Zheng, L., Lee, Y., Utterback, T., Smith, S., Feldblyum, T., Glenn, A.E., Plattner, R.D., Kendra, D.F., Town, C.D., Whitelaw, C.A. 2004. Comparative analysis of 87,000 expressed sequence tags from the fumonisin-producing fungus fusarium verticillioides. Aflatoxin Elimination Workshop. Interpretive Summary:
Technical Abstract: Fusarium verticillioides is a pathogen of maize worldwide and produces fumonisins, a family of mycotoxins that cause several animal diseases and is epidemiologically associated with human esophageal cancer and birth defects in some regions of the world. This fungus is generally an endophyte of corn, but under some conditions, it can cause seedling blight and ear, root and stalk rots. Fumonisins are often found in diseased tissue and sometimes in corn kernels with little-to-no disease symptoms. One of the primary goals of our labs is to eliminate fumonisins contamination of corn and corn products, and thus prevent them from entering animal and human food supplies. We believe that understanding how and why these toxins are produced as well as the biology of F. verticillioides-maize (should be consistent corn vs. maize) interactions will allow us to develop novel strategies to limit fumonisin contamination and corn diseases caused by the fungus. We, in collaboration with The Institute for Genomic Research (TIGR), have sequenced the 5'ends (and in some cases the 3'ends) of F. verticillioides cDNAs derived from eight different fungal growth conditions. Three cDNA libraries were constructed from RNA after growth for different lengths of time (e.g. 24, 48/72, and 96 hours) on a synthetic medium that supports fumonisin synthesis between 72 and 96 hours. We reasoned that a comparison of these ESTs would identify differentially expressed genes important in fumonisin biosynthesis. Four libraries were constructed from RNA after growth on maize-derived media. We hoped to identify genes that were differentially expressed and involved in fungal response to maize as well as required for growth on maize as a sole substrate. The final library was a suppressive subtracted library that was prepared from fungal culture with or without 2-benzoxazolinone (BOA), an antimicrobial compound produced by maize. In this case, we sought to identify specific and general fungal genes that provide protection from plant defense molecules. A total of over 87,000 expressed sequence tags (ESTs) were generated which corresponded to 11,119 unique sequences. A comparative analysis between the eight different library sequences found thousands of differentially expressed genes including all 15 genes in the fumonisin gene cluster. We identified numerous candidate fumonisin regulatory genes and a number of genes that may play a role in F. verticillioides-plant disease process. Analysis of over 700 FUM gene ESTs led to the discovery of a new FUM gene designated FUM20 and to the presence of alternative splice form transcripts (ASFs). ASFs are transcripts with unspliced introns or spliced introns with different 3' splice sites. Most ASFs would yield truncated proteins because the retained sequence introduces stop codons and/or frameshifts. ASFs appear to be differentially expressed as more were present in libraries derived from older cultures. This trend was not observed for ASFs of genes located outside the cluster. This, coupled with the high frequency of occurrence of some ASFs suggests they serve a biological role.