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ARS Home » Southeast Area » New Orleans, Louisiana » Southern Regional Research Center » Food and Feed Safety Research » Research » Research Project #430859

Research Project: Genetic and Environmental Factors Controlling Aflatoxin Biosynthesis

Location: Food and Feed Safety Research

2017 Annual Report

1a. Objectives (from AD-416):
Objective 1. Identify key genes, using transcriptome analysis of Aspergillus flavus and Aspergillus flavus-crop interaction that are involved in fungal growth, morphogenesis, toxin production and virulence which can be used as targets for intervention strategies. Objective 2. Identify metabolites produced by predicted secondary metabolic gene clusters in Aspergillus flavus, characterize the molecular regulation of their biosynthesis, and determine if they contribute to the fungus’ ability to survive, colonizes host crops and produce aflatoxin. Objective 3. Examine the role of climatic and environmental pressures on the growth, virulence, toxigenic potential, geographical distribution and aflatoxin production by Aspergillus flavus.

1b. Approach (from AD-416):
Aflatoxin contamination in crops such as corn, cottonseed, peanut, and tree nuts caused by Aspergillus (A.) flavus is a worldwide food safety problem. Aflatoxins are potent carcinogens and cause enormous economic losses from the destruction of contaminated crops. While biosynthesis of these toxins has been extensively studied, much remains to be determined regarding regulatory factors, their interactions and gene networks that respond to environmental cues governing fungal development and aflatoxin production. Using an –omics approach (transcriptomics, interactomics, proteomics, metabolomics), fungal genes/proteins will be identified and functionally characterized that are critical for successful host plant colonization and aflatoxin production during interaction of A. flavus with the plant. Interactions of regulatory proteins involved in fungal growth and toxin production, such as AflR and other velvet (VeA)-dependent proteins with global regulators, will be examined to elucidate novel mechanisms governing aflatoxin production and fungal morphogenesis. We will also identify and characterize the biological roles of other secondary metabolites produced by A. flavus, their impact on aflatoxin production and food safety in general. Further, we will better define the molecular mechanisms affected by physiological stress (i.e. changing environmental conditions) to the fungus and plant. We expect to utilize the fundamental knowledge gained from the proposed studies for development of targeted strategies (biological control or host-resistance) to significantly reduce pre-harvest aflatoxin contamination of crops intended for consumption by humans or animals.

3. Progress Report:
Progress on this project focuses on preventing pre-harvest aflatoxin contamination of food a feed crops. Under Objective 1, Agricultural Research Service (ARS) scientists in New Orleans, Louisiana, made significant progress in identifying genes from the fungus Aspergillus (A.) flavus that are critical for the fungus to grow, survive and produce aflatoxins (compounds that are toxic and carcinogenic to humans and animals). To study the effect of environmental stress on the infection of corn, we used ribonucleic acid sequencing (RNA-Seq; a technique that can be used to measure the activation of genes) and identified a number of A. flavus genes that showed changes in expression during growth on stressed (no watering) and unstressed (watered) corn plants. Many of these genes were of unknown function while others were identified as having functions involved in metabolite biosynthesis (production of toxins like aflatoxins), stress response (important for survival), and gene regulation (important to all aspects of the biology of the fungus). A similar expression study was conducted to identify A. flavus genes that are differentially regulated by the fungal gene rmtA. We found that several genes present in secondary metabolite gene clusters (a closely grouped set of genes that together are required for production of compounds that are often toxic and can also be involved in development, survival and infectivity) were down regulated in the absence of rmtA, including those involved in the production of aflatoxin and other fungal toxins. In addition, gene expression data revealed that rmtA also regulates numerous genes involved in the response to several environmental stresses, including oxidative and osmotic stress, thermal stress and starvation, as well as genes involved in DNA (deoxyribonucleic acid) repair mechanisms. Progress was made on additional A. flavus genes that are required for normal development and toxin production that we can target for intervention strategies. We identified an A. flavus homeobox gene (a class of genes known to be involved in development in insects and mammals) that is required for the fungus to produce conidia (asexual reproductive structures also known as spores), sclerotia (fungal survival structures) and aflatoxins. This work represented the first report of a single fungal gene that is required for production of conidia, sclerotia and aflatoxins. Lastly, we also identified and characterized the A. flavus gene, designated aswA, which is critical for normal sclerotial formation. Inactivation of aswA led to poorly formed sclerotia that lacked key, sclerotium-specific toxic compounds, though aflatoxin was still present. Under Objective 2, Agricultural Research Service (ARS) scientists in New Orleans, Louisiana, have made significant progress on the identification of secondary metabolite gene clusters and their associated metabolites. We showed that the A. flavus 55 gene clusters (#11) is responsible for the production of aspergillic acid (one of a toxic compound). Chemical analyses of extracts from the fungus showed that this cluster also produced the metabolite, ferriaspergillin, which is capable of binding atoms of iron. Using a kernel screening assay (KSA) we inoculated corn kernels with a control (normal) A. flavus strain or a mutant strain that was unable to make aspergillic acid/ferriaspergillin. Seven days post-infection the kernels were collected and the degree of fungal infection and aflatoxin production determined. Significantly lower levels of fungal growth and aflatoxin were observed from kernels inoculated with the mutant compared to the control strain which suggests that aspergillic acid is serving as a virulence (infection promoting) factor. Progress was also made in identifying the compound produced by the gene cluster designated “U” (for Unique) that we have only been able to detect in one isolate of A. flavus, strain AF70. Because this gene cluster is unique to AF70 and not found in other A. flavus strains we are interested in determining what metabolite(s) is being produced by the gene cluster and what role it plays in the biology of the fungus. Several attempts to identify the cluster U metabolite(s) in control and mutant AF70 strains using liquid chromatography-mass spectrometry (LC-MS; sophisticated instruments for detection of secondary metabolites) failed. We are now placing the gene required for the first step in the biosynthesis of the unknown AF70 metabolite into a yeast (yeast do not normally produce many secondary metabolites) with hopes that we will be able to detect production of a novel metabolite. In regard to Objective 3, progress was made to examine the impact of environmental conditions on A. flavus growth, development, and aflatoxin production during its interaction with corn kernels. By altering temperature (30° and 37°C), water availability (0.91 and 0.99 Aw), and carbon dioxide levels (350, 650, and 1000 ppm), we identified a number of A. flavus genes that demonstrated altered expression levels during infection of corn. An initial RNA-Seq experiment analyzed the effects of water and temperature on gene expression. Genes involved in a number of A. flavus biological processes were identified, especially those involved in carbohydrate metabolism and these are being targeted for future studies. A second study aimed at integrating the effect of increasing carbon dioxide levels has also been conducted. Interestingly, biological pathways involving genes related to spore production such as those encoding a histone deacetylase gene (hosB), transcription factor gene (abaA) and a hydrophobin gene (rodA), showed altered expression profiles as a result of increased carbon dioxide. The RNA-Seq gene expression data for these three candidate genes and others has been validated by quantitative PCR (a technique to accurately monitor expression levels of a selected gene). These genes are being knocked-out (inactivated) in the host A. flavus strain so that we can confirm their roles in spore production. Lastly, the creation of acclimatized strains has been accomplished through 20 generations of subculturing (repeated transfer of the fungus to growth media) in a “stressful” environment (37°C, 0.91Aw, and 1000 ppm CO2). We are currently characterizing these strains phenotypically (visually) and genetically (at the level of the DNA). To date we have observed changes in aflatoxin production and spore production, with corresponding changes in the expression of spore production-related genes.

4. Accomplishments
1. Involvement of a regulatory gene in Aspergillus (A.) flavus development and aflatoxin production. It is important to decipher the complex molecular mechanisms that govern the fungus’ ability to infect plants and produce aflatoxin (a potent cancer-causing compound). Using sophisticated molecular techniques, Agricultural Research Service (ARS) scientists in New Orleans, Louisiana, have identified a number of novel genes that are key regulators of A. flavus growth and aflatoxin production. Of particular interest was our finding of a gene, hbx1, which is required for production of conidia (asexual reproductive structures also known as spores), sclerotia (fungal survival structures) and aflatoxins. The equivalent of this gene has been found in a number of other fungi and will provide researchers the opportunity to study its role in disease development and production of toxic and carcinogenic compounds in A. flavus and other fungal pathogens.

2. Identification of a secondary metabolite gene cluster in Aspergillus (A.) flavus responsible for the production of a toxic compound. Analysis of the A. flavus genome has indicated that it contains several gene clusters (groups of genes) that are predicted to potentially produce a variety of metabolites, some of which could be toxic. Many of the metabolites produced by these gene clusters are unknown with respect to their structure and impact on the biology of the fungus. It has been determined by Agricultural Research Service researchers in New Orleans, Louisiana, that a gene cluster in A. flavus produces toxic compounds other than aflatoxins. One of these compounds is capable of binding iron and may be involved in toxicity to humans and animals and also in helping the fungus to infect crops. This work was carried out in collaboration with Ghent University, Belgium.

Review Publications
Chang, P.-K., Scharfenstein, L.L., Ehrlich, K., Diana Di Mavungu, J. 2016. The Aspergillus flavus fluP-associated metabolite promotes sclerotial production. Fungal Biology. 120:1258-1268.

Satterlee, T., Cary, J.W., Calvo, A.M. 2016. RmtA, a putative arginine methyltransferase, regulates secondary metabolism and development in Aspergillus flavus. PLoS One. 11(5):e0155575. doi:10.1371/journal.pone.0155575.

Lohmar, J.M., Harris-Coward, P.Y., Cary, J.W., Dhingra, S., Calvo, A.M. 2016. rtfA, a putative RNA-Pol II transcription elongation factor gene, is necessary for normal morphological and chemical development in Aspergillus flavus. Applied Microbiology and Biotechnology. 100(11):5029-5041. doi:10.1007/s00253-016-7418-7.

Zhuang, Z., Lohmar, J.M., Satterlee, T., Cary, J.W., Calvo, A.M. 2016. The master transcription factor mtfA governs aflatoxin production, morphological development, and pathogenicity in the fungus Aspergillus flavus. Toxins. 8(1):29. doi:10.3390/toxins8010029.

Arroyo-Manzanares, N., Diana Di Mavungu, J., Uka, V., Malysheva, S.V., Cary, J.W., Ehrlich, K.C., Vanhaecke, L., Bhatnagar, D., De Saeger, S. 2015. Use of UHPLC high-resolution Orbitrap mass spectrometry to investigate the genes involved in the production of secondary metabolites in Aspergillus flavus. Food Additives & Contaminants. Part A, 32(10):1656-1673. doi:10.1080/19440049.2015.1071499.

Chang, P.-K., Scharfenstein, L.L., Li, R.W., Arroyo-Manzanares, N., De Saeger, S., Diana Di Mavungu, J. 2017. Aspergillus flavus aswA, a gene homolog of Aspergillus nidulans oefC, regulates sclerotial development and biosynthesis of sclerotium-associated secondary metabolites. Fungal Genetics and Biology. 104:29-37.

Chang, P.-K., Hua, S.T., Sarreal, S.L., Li, R.W. 2015. Suppression of aflatoxin biosynthesis in Aspergillus flavus by 2-phenylethanol is associated with stimulated growth and decreased degradation of branched-chain amino acids. Toxins. 7:3887-3902. doi:10.3390/toxins7103887.

Gilbert, M.K., Mack, B.M., Payne, G.A., Bhatnagar, D. 2016. Use of functional genomics to assess the climate change impact on Aspergillus flavus and aflatoxin production. World Mycotoxin Journal. 9(5):665-672.

Gilbert, M.K., Mack, B.M., Wei, Q., Bland, J.M., Bhatnagar, D., Cary, J.W. 2016. RNA sequencing of an nsdC mutant reveals global regulation of secondary metabolic gene clusters in Aspergillus flavus. Microbiological Research. 182:150-161.

Boue, S.M., Fortgang, I., Levy, R.J., Bhatnagar, D., Burow, M., Fahey, G., Heiman, M.L. 2016. A novel gastrointestinal microbiome modulator from soy pods reduces absorption of dietary fat in mice. Obesity. 24(1):87-95.

Chalivendra, S.C., DeRobertis, C., Chang, P.-K., Damann, K.E. 2017. Cyclopiazonic acid is a pathogenicity factor for Aspergillus flavus and a promising target for screening germplasm for ear rot resistance. Molecular Plant-Microbe Interactions. 30(5):361-373.

Medina, A., Gilbert, M.K., Mack, B.M., OBrian, G.R., Rodriguez, A., Bhatnagar, D., Payne, G., Magan, N. 2017. Interactions between water activity and temperature on the Aspergillus flavus transcriptome and aflatoxin B1 production. International Journal of Food Microbiology. 256:36-44.