Location: Food and Feed Safety Research2013 Annual Report
1a. Objectives (from AD-416):
1. Use data from genome-wide systematic analysis to determine the molecular and biological changes that occur in A. flavus upon infection of corn and other crops. 2. Identify mechanistic and molecular requirements for transcriptional regulation of aflatoxin biosynthesis and fungal survival to develop targets for intervention. 3. Establish effects of abiotic (environmental, nutritional) factors on fungal development and toxin production by aflatoxin-producing fungi.
1b. Approach (from AD-416):
Aflatoxins (AFs) are polyketide-derived, toxic, and carcinogenic secondary metabolites produced by Aspergillus flavus on corn, peanuts, cottonseed, and tree nuts. While biosynthesis of these toxins has been extensively studied, much less is known about what causes the fungi to produce AFs under certain environmental conditions and only on certain plants. Our goal is to determine the dynamics of interaction among the key nutritionally and environmentally induced transcription factors necessary for production of AF in order to develop novel inhibitors to one or more of these factors to prevent AF formation in crops. We will use gene microarray, yeast two-hybrid, and chromatin immunoprecipitation assays to determine which critical AF transcription-associated proteins are affected by physiological stress, environmental and soil conditions, and interactions of the fungus with plants. Interactions among key known or to be discovered AF biosynthesis regulatory factors, such as LaeA, VeA, AflJ, and AflR, will be examined by these methods. We will examine the effects of known natural (plant-derived, such as volatile aldehydes) inhibitors of AF production on key components of the AF transcription machinery to ultimately design safe, inexpensive chemicals that inhibit proteins unique to fungal secondary metabolite biosynthesis. We expect to identify safe and effective inhibitors for applications on crops intended for consumption by humans or animals.
3. Progress Report:
The reason why Aspergillus (A.) flavus and A. parasiticus produce aflatoxins on certain plants is finally being understood by scientists in this unit. The goal of the project is to identify, by genomic technologies, the complex array of genes involved in fungal virulence, aflatoxin formation, response to the environment, and development/reproduction/survival, as well as molecular and biological changes that occur in A. flavus upon infection of corn and other crops. ARS scientists at the Southern Regional Research Center in New Orleans, LA, are continuing to perform studies in collaboration with Xavier University and the University of Northern Illinois to examine the mode of action of the role of key specific global regulatory factors in the initiation of aflatoxin biosynthesis in response to environmental signals. Some of these factors are potential targets for intervention to prevent aflatoxin production on plants. In collaboration with J. Craig Venter Institute (JCVI), using the new generation sequencing technologies, called ribonucleic acid (RNA) sequencing (Illumina RNA-Seq), ARS scientists at the Southern Regional Research Center in New Orleans, LA, are gaining access to the entire transcriptome (gene expression profile) of A. flavus cells, with almost infinite resolution, under conditions conducive and non-conducive to aflatoxin production. Sequencing of two additional aflatoxin-producing Aspergillus strains were carried out and completed in cooperation with JCVI; one of the two strains is a S strain of A. flavus (named because it produces small over-winter bodies called sclerotia), which is more virulent and also produces more aflatoxins than the L (large sclerotia producing) strain which we have already sequenced. Comparison of the two S and L types of strains is being carried out and will help us to understand the mechanism of infection and the mechanism of genetic regulation on aflatoxin production. Another strain that we have recently sequenced is A. parasiticus which is found associated with peanut pods in the soil. Comparative studies and analysis of A. flavus/parasiticus genomes reveals several (55) gene clusters that are predicted to potentially produce a variety of secondary metabolites, some of which could be toxic. The metabolites produced by the active clusters are being evaluated with the assistance of a collaborator from the University of Ghent, Belgium. A. flavus cells can contain more than one nuclei, but the role of these nuclei are not known. In collaboration with North Carolina State University, ARS scientists at the Southern Regional Research Center in New Orleans, LA, have developed a method for staining each nuclei with different dyes so that ARS scientists at the Southern Regional Research Center in New Orleans, LA, can try and understand what role, if any, is played by different nuclei in each cell. In collaboration with the University of Pittsburg, ARS scientists at the Southern Regional Research Center in New Orleans, LA, have made a great deal of progress in developing economic models of world food trade, and showing the crucial role of U.S. maize and pistachio industries in global trade of these foodstuffs.
Yu, J., Bhatnagar, D., Cleveland, T.E., Payne, G., Nierman, W.C., Bennett, J.W. 2012. Aspergillus flavus genetics and genomics in solving mycotoxin contamination of food and feed. In: Benkeblia, N. (ed). Omics Technologies: Tools for Food Science. CRC Press, Taylor & Francis Group, Boca Raton, FL. p. 367-402.