2013 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.
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.
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, Agricultural Research Service (ARS) scientists in 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 in 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 are rapidly assessing the critical role of several genes of interest in aflatoxin formation in crops.
Function of other gene clusters. A comparison of the Aspergillus (A.)flavus/parasiticus genomes has indicated that these organisms contain several gene clusters that are predicted to potentially produce a variety of secondary metabolites, some of which could be toxic. Experiments have been designed and initiated to evaluate the potential for A. flavus/parasiticus to produce mycotoxins other than aflatoxins, or other useful compounds that could have industrial applications (work being carried out in collaboration with Ghent University, Belgium). These newly defined gene clusters are involved in biosynthesis of compounds such as ditryptophenaline, a diketopiperazine dimer with potential anti-inflammatory and anticancer properties. Diketopiperazines are also involved in signaling pathway in aspergilli that are potentially important for fungal plant recognition. Another metabolite is another compound called an ergot alkaloid; this is the first identification of such a metabolite produced by A. flavus. A third metabolite is an anthraquinone (a class of colored compounds) that is the precursor for sclerotial pigmentation and an important element in sclerotial stability under ultra violet light, heat and insect predation.
Role of multiple nuclei in Aspergillus (A.) flavus cells. The fungus A. flavus has multiple nuclei in their vegetative cells as well as in their spores (equivalent of seeds in higher plants). The role of all these nuclei in multiplication of these fungi or survival in their environment is unknown and is being investigated in collaboration with scientists at North Carolina State University. To this end, we have been successful in generating yellow and cyan fluorescent reporter (genetically modified) strains in this fungus. Our ability to track nuclei at different stages of fungal growth and development will allow us to test the hypotheses on the role of these multiple nuclei and variation in type of nuclei on fungal growth and development and in response to various stresses that the fungus encounters in the field. In addition, some uninucleate mycelia are diploids and may have a higher ability to infect plants.
Sequencing of all the Deoxyribonucleic Acid (DNA) of different strains of the fungus Aspergillus (A.) flavus and one strain of A. parasiticus. Knowing the genetic make-up of these fungi is important to know how and why this fungus makes the potent carcinogen aflatoxin when it invades crops. Earlier, two different format whole genome microarrays (slides containing spots of DNA corresponding to fragments of all unique genes) were designed, 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 in New Orleans, LA, and our collaborators to analyze which genes are affected under varying conditions:.
1)nutritional (high or low carbon source);.
3)developmental (veA mutant); and.
4)during the fungus-corn interaction (cooperation with ARS scientists and faculty, Mississippi State University) that affect toxin production. From these microarray studies, over a hundred genes were identified that may have some impact on aflatoxin production and fungal survival. These results have been published. The genome sequencing data has been deposited at the ARS, Mid South Area Genomics facility, Stoneville, MS, and is currently being analyzed by ARS scientists at the Southern Regional Research Center in New Orleans, LA, to provide significant insights into what genes are involved in the interaction between the fungus and the crop.
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.
Yu, J. 2012. Current understanding on aflatoxin biosynthesis and future perspective in reducing aflatoxin contamination. Toxins. 4(11):1024-1057.
Kong, Q., Zhai, C., Guan, B., Li, C., Shan, S., Yu, J. 2012. Mathematic modeling for optimum conditions on aflatoxin B1 degradation by the aerobic bacterium Rhodococcus erythropolis. Toxins. 4:1181-1195.
Moore, G.G., Mack, B.M., Beltz, S.B. 2013. Testing the efficacy of eGFP-transformed Aspergillus flavus as biocontrol strains. Food and Nutrition Sciences. 4:469-479.