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United States Department of Agriculture

Agricultural Research Service

Research Project: Control of Aflatoxin Production by Targeting Aflatoxin Biosynthesis

Location: Food and Feed Safety Research

2012 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 project is to identify, by A. flavus 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. We have also performed studies to determine the molecular role of key specific regulatory factors in the initiation of aflatoxin biosynthesis. Some of these factors are potential targets for intervention to prevent aflatoxin production on plants. In collaboration with J. Craig Venter Institute (JCVI), the new generation sequencing technologies were used to investigate the mechanisms of gene expression regulation in aflatoxin production. A new methodology, called ribonucleic acid (RNA) sequencing (Illumina RNA-Seq) which can provide access to a cell’s entire transcriptome (gene expression profile) with almost infinite resolution, was employed to characterize the A. flavus transcriptome under conditions conducive and non-conducive to aflatoxin production. Sequencing of two 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 will help us to understand the mechanism of infection and the mechanism of genetic regulation on aflatoxin production. The other strain is A. parasiticus which infects peanut pod in the soil. Comparative studies and analysis of A. flavus/parasiticus genomes reveals several gene clusters that are predicted to potentially produce a variety of secondary metabolites, some of which could be toxic. Experiments designed and initiated to evaluate the potential for A. flavus/parasiticus to produce mycotoxins include: (1) In collaboration with the National Institute of Advanced Industrial Science and Technology, Japan, we have comprised the gene expression profiles between the field fungus (A. flavus) and its domesticated cousin (A. oryzae) to provide insights into any competitive advantage that A. flavus may possess as it survives in field conditions. (2) In collaboration with National Peanut Research Laboratory (NPRL), we identified the gene cluster involved in cyclopiazonic acid (CPA) formation. (3) In collaboration with Biological Control of Pests Research Unit at Jamie Whitten Delta States Research Center, we confirmed the genetic defects associated with the inability to produce aflatoxins and CPA by A. flavus K49.

1. Sequencing of all the Deoxyribonucleic Acid (DNA) of the fungus Aspergillus (A.) flavus and its applications. Knowing the genetic make up of this fungus is important to know how and why this fungus makes the potent carcinogen aflatoxin when it invades crops. Two different format whole genome microarrays (slides containing spots of DNA corresponding to fragments of all unique genes) have been designed recently, and used to identify critical genes involved in fungal response to various environmental factors favoring toxin production. These microarray resources have been used in large scale functional genomics studies by Agricultural Research Service (ARS) scientists at New Orleans, LA, and our collaborators to analyze which genes are affected under varying conditions:.
1)nutritional (high or low carbon source);.
2)environmental (temperature);.
3)developmental (veA mutant); and.
4)during the fungus-corn interaction (cooperation with ARS scientists and faculty, Mississippi State University) that affect toxin production. The long-term survival of aflatoxin producing A. flavus strains in comparison with non-producing strains has indicated that under temperature stress (47°C), spores of toxigenic strains survived longer than non-aflatoxin producers; and there is no difference in survival under ultraviolet light for aflatoxin-producing strains vs. non-aflatoxin-producing strains. From these microarray studies, over a hundred genes were identified that may have some impact on aflatoxin production and fungal survival. The database will be accessible through a website which is expected to be housed at the ARS, Mid South Area genomics facility, Stoneville, MS, and provide genomic information to all researchers worldwide working with this fungus. These studies carried out by ARS scientists are providing significant insights into what genes are involved in the interaction between the fungus and the crop.

2. Proof of involvement of genes in aflatoxin synthesis and fungal development obtained. Knowing the genetic make up of this fungus is important to know how and why this fungus makes the potent carcinogen aflatoxin when it invades crops. Using sophisticated molecular techniques, ARS scientists at New Orleans, LA, have tested specific interactions of key aflatoxin developmental regulatory factors. Further, the genetic basis for loss of aflatoxin production in toxin-deficient mutants of Aspergillus parasiticus (generated by physical manipulation of toxin-producing strains) has been investigated using microarrays and metabolic profiling, and specific regulatory genes causing this loss have been identified. ARS scientists at New Orleans, LA, have found that many factors are needed for complete regulation of the turning on and off of aflatoxin production. Through the use of these technologies we will rapidly assess the critical role of several genes of interest in aflatoxin formation in crops.

3. Cyclopiazonic acid is another toxic compound that the fungus Aspergillus (A.) flavus produces along with aflatoxins. In collaboration with National Peanut Research Laboratory (NPRL), ARS scientists at New Orleans, LA, have identified the gene cluster involved in cyclopiazonic acid (CPA) formation and characterized associated biosynthetic genes. The CPA gene cluster is located next to the aflatoxin gene cluster on the A. flavus genome. Using this information, scientists confirmed that the biopesticide Afla-Guard® (active ingredient is A. flavus NRRL21886), developed at NPRL and commercialized by Syngenta Crop Protection, does not contain the CPA and aflatoxin A. flavus gene clusters, which ensures the safe application of this genuinely nontoxic A. flavus strain in the field as a biocontrol agent. The colocation of the aflatoxin and CPA biosynthetic gene clusters on the fungal chromosome suggests how the fungus could make these two harmful compounds together based on one signal that allows the “turning on” of the genetic machinery within the fungus.

4. Deoxyribonucleic Acid (DNA) probes (primer sets) identified for universal screening for genetic variability of Aspergillus (A.) group fungi. Each year millions of dollars are lost to crops contaminated with toxic and carcinogenic aflatoxins produced by A. flavus. ARS scientists at the Southern Regional Research Center in New Orleans, LA, have conducted studies on the molecular characterization of the aflatoxin biosynthetic pathway from the aflatoxigenic cousin of A. flavus, namely toxin-producing A. ochraceoroseus, A. rambelli, as well as, nontoxigenic A. oryzae, were done to determine if aflatoxin production provides a competitive advantage to A. flavus for its ability to survive in field conditions. Strains of an A. parasiticus isolate with specific deletions of aflatoxin pathway genes have been compared for morphological and physiological differences due to the knocking-out of critical pathway genes. This will help us understand if the deletion of these pathway genes has an effect on the fungus in its ability to invade crops.

Review Publications
Yu, J., Nierman, W.C., Fedorova, N.D., Bhatnagar, D., Cleveland, T.E., Bennett, J.W. 2011. What can Aspergillus flavus genome offer for mycotoxin research? Mycological Society of China. 2(3):218-236.

Chang, P., Abbas, H.K., Weaver, M.A., Ehrlich, K., Scharfenstein, L.L., Cotty, P.J. 2012. Identification of genetic defects in the atoxigenic biocontrol strain Aspergillus flavus K49 reveals the presence of a competitive recombinant group in field populations. International Journal of Food Microbiology. 154:192-196.

Last Modified: 4/23/2014
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