Goals: Mycotoxins are metabolites of fungi that can adversely affect animal and human health. Mycotoxins can be produced in grain during storage or processing, but are most frequently associated with fungal infection that occurs before harvest. Environmental factors that determine fungal infection and mycotoxin production are complex. Generally, a basal level of mycotoxins is present in US grain; however, in some years, environmental conditions lead to localized or widespread outbreaks of mycotoxin contamination. However, there is no organized monitoring system for tracking the incidence and severity of mycotoxin contamination at either the national or the regional levels. Although breeding and transgenic technologies have shown promise for reducing the risk of mycotoxin contamination of grain, to date no commercial variety of any major US crop is available with either genetic or transgenic resistance to mycotoxin contamination. These studies will provide new information on mycotoxin detection, detection of fungi that produce mycotoxins, and the non-destructive detection of contaminated grain. <P>The goal of the team working on Objective 4 is to provide basic knowledge about the biochemical and molecular factors that regulate the biosynthesis of aflatoxins and fumonisins. This will reveal critical points in the regulation where targeted inhibitors could be designed. The importance of the work, and consequences if it is not done: Hazard assessment includes exposure assessment and evaluations of toxicity, both are essential. The proposed research is wide-ranging and could lead to negative consequences if not completed. First of all, the presence of mycotoxins is an important health hazard. We propose basic research to define the toxicity of several important mycotoxins. Without this information, it is impossible to assess risks associated with mycotoxins. Without a proactive research program to find innovative ways to monitor and treat mycotoxins, US agriculture faces the consequence of being unprepared for a mycotoxin outbreak, regardless of its origin. Finally, the production of mycotoxins represents a basic aspect of agricultural science. Improving our understanding of how mycotoxin biosynthesis is regulated will not only lead to novel treatment strategies, but may also advance our understanding of fungal pathogenesis in general. <P>Objective 4: Define the Regulation of Mycotoxin Biosynthesis and the Molecular Relationships Between Mycotoxigenci Fungi. <P>Outputs: 1. Refereed journal publications; many will be co-authored by the members from multiple states. <BR> 2. Validation of new management tools for diagnostics and prevention of mycotoxin contamination. <BR>3. Transfer of information that is generated to grain producers and food producers during extension programs.
NON-TECHNICAL SUMMARY: There have been at least 7 known host-parasite interactions in which host-specific toxins produced by Alternaira alternara pathotypes are responsible for plant disease. The information makes them relatively unique and interesting system for addressing the evolution of virulence and host specialization. Toxins produced by A. alternate pathotypes are mainly low molecular weight secondary metabolites. Some toxins have been shown to be a sphingolipid-like molecule structurally similar to fumonisins. We have optimized a relatively simple and economical PEG-mediated protoplast transformation method to disrupt individual targeted genes with typically 100% efficiency 1. This method depends on an unconventional linear construct, named linear minimal element (LME), comprised of an antibiotic resistance selectable marker gene at one side and a 250-600bp long partial targeted gene sequence on the other. This method is advantageous for a high throughput approach for the initial screening of virulence factors because disruption mutants are highly stable during virulence assays 1-3. The LME mediated gene disruption may be viewed overall as more desirable than other methods including KU mutants that have similar gene knockout efficiency 4, 5 in its simplicity to create the constructs, to isolate pure mutants, and in the tendency of a single copy insertion of the disruption constructs into the genome. We have knockout out all polyketide synthase genes identified in the Alternaria brassicicola draft genome and finished preliminary pathogenicity assays. However, none of them turned out host specific toxins. Proposed work to generate knockout mutants for a cycline and cycline kinase will allow us to investigate the toxin synthesis in a new perspective. The results from this work will also provide valuable comparison opportunities to compare the toxin synthesis and cell cycle regulation in two different systems Alternaria and Fusarium. In addition, for grain and livestock producers, the most important issues are preventing mycotoxin contamination and reducing the effects of mycotoxins on livestock. For grain buyers and food processors, the primary issue is being able to rapidly assess the quality of grain as pertaining to mycotoxins and mycotoxigenic fungi. The worst-case scenario for these stakeholders is to own millions of bushels of corn contaminated with unacceptable levels of aflatoxins and fumonisins, or wheat with excessive concentrations of deoxynivalenol (DON). Rapid methods to detect mycotoxins at the first points of sale (elevators) as well as methods to detect mycotoxigenic fungi in the commodity (e.g. DON-producing Fusarium in barley) would address these concerns. Additionally, these stakeholders need cost-effective methods to detoxify mycotoxins and prevent further deterioration of contaminated grain. Our proposed work will significantly contribute to the general goal of food security by providing more information regarding toxin synthesis pathways.
<P>APPROACH: Methods for objective 4: Stations participating in objective 4 (IN, TX, ARS, WI) will collaborate to 1) generate disruption mutants for genes expressed during fumonisin biosynthesis and 2) evaluate gene expression profiles in wild type and fck1 mutant of F. verticillioides cultured on various corn kernel tissues. Through microarray techniques and statistical analysis, IN identified 17 genes that displayed expression patterns similar to the FUM genes. The goal of this project is to mutate these genes by homologous recombination in F. verticillioides and to determine any function in fumonisin biosynthesis. IN, TX will construct disruption vectors consisting of a hygromycin-resistance cassette flanked by about 500 bp of each EST sequence. Transformants of F. verticillioides will be obtained by a PEG-mediated method, and disrupted genes will be identified by PCR and Southern analyses. We will determine the effects of mutations on conidiation and fumonisin production when grown on kenels as well as defined medium at pH 3 and pH 9. Of those affected in fumonisin biosynthesis, we will monitor expression of fumonisin biosynthetic genes by northern analysis and/or qPCR. We will also complement the mutations with the corresponding wild-type genes. Analysis of two genes (a cyclin (fcc1) and a cyclin-dependent kinase (fck1) indicates that both genes are needed for fumonisin production during colonization of corn kernels. In addition, gene expression in the wild-type fungus is greatly affected by the tissue type, genotype, and developmental stage of the colonized kernel. To illuminate other genes whose expression might be dependent on or influenced by the Fcc1/Fck1 complex as well as tissue specific gene expression, IN, TX, ARS and WI will utilize the F. verticillioides microarrays to analyze and identify genes differentially expressed when the fck1 mutant and wild type are grown under differing corn kernel environments. Fusarium verticillioides microarrays have been developed at the USDA-NCAUR utilizing an EST library of F. verticillioides genes. In our lab, we plan to utilize Alternaria brassicicola as a model organism in understanding its toxin synthesis to compare with Fusarium. We plan to identify the homolgs for homologs of cyclin (Fcc1) and cyclin kinase (Fck1) genes in the publicly available draft genome sequence and knock out them to examine their ability to produce secondary metabolites (toxins) as well as to test their contribution to the pathogenicity. We are especially interested in the comparisons to the molecular modification of the secondary metabolites in the mutants compared to the wild type whether it is comparable to the Fumonisin production. At the same time, we will examine the expression patterns of the target genes during the host development as well disease cycles, using quantitative real time PCR. We will monitor the complementation of the mutants with the corresponding wild type as well as the Fusarium homolog.