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Research Project: Genetic and Environmental Factors Controlling Aflatoxin Biosynthesis

Location: Food and Feed Safety Research

2019 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 was made in all three objectives, which fall under National Program 108 Food Safety, Component 1, Foodborne Contaminants. For Objective 1, ARS researchers in New Orleans, Louisiana, continue to pursue their mission to control aflatoxin contamination of crops through multiple intervention strategies. ARS researchers are analyzing data from ribonucleic acid (RNA)-sequencing experiments (RNA-Seq; a means of determining levels of activity of individual genes in organisms) to study the activity of all genes during the corn-Aspergillus (A.) flavus interaction. A. flavus is a fungus that produces aflatoxins (potent cancer-causing compounds that are also toxic to humans and animals) during growth on crops such as peanut and corn. In collaboration with researchers at Louisiana State University, Baton Rouge, Louisiana, RNA-seq data of infected corn plants showed that an A. flavus gene known as medA was highly active during the infection process. ARS researchers found that a medA mutant was less tolerant to fungicides than the wild-type A. flavus (a natural, non-mutated form of the fungus) under hypoxia (low oxygen levels that exist inside corn seed). A significant reduction in the ability of the medA mutant to infect corn seed suggests that this gene plays a role in A. flavus’ ability to successfully colonize corn seeds and therefore may serve as a target for intervention strategies. In collaboration with researchers at Northern Illinois University (NIU), Dekalb, Illinois, a number of rmtA-dependent genes were identified in A. flavus. RmtA produces a protein that can modify the structure of proteins associated with DNA, resulting in modulation of gene activity. Of particular interest was the discovery of genes in rmtA knock out mutants that were down-regulated in the uncharacterized A. flavus secondary metabolite gene cluster 21. Comparison of metabolite profiles from crude extracts of control A. flavus strains with those of a Cluster 21 gliP gene knock out mutant allowed presumptive identification of the cluster metabolite. Further purification of the metabolite is underway to better elucidate its chemical structure and biological function in the fungus. Researchers in New Orleans, Louisiana, and NIU are also characterizing the biological function of numerous homeobox 1 (hbx1)-dependent genes. One hbx1-dependent gene of interest was forkhead A (fkhA). FkhA is a putative transcription factor (TF) gene (TFs are a class of genes that control the activity of other genes in an organism) shown to be involved in development in some fungi but not yet characterized in A. flavus. FkhA knock out mutants were generated in A. flavus and they were analyzed for developmental defects and aflatoxin production. It was shown that fkhA mutants no longer produced sclerotia (fungal survival structures) but did still produce normal levels of spores and aflatoxin, indicating that the fkhA gene would not be a good target for our aflatoxin control strategies. Researchers in New Orleans, Louisiana, in collaboration with researchers at the University of South Carolina have performed a chromatin immunoprecipitation (ChIP) experiment (a method to determine the identity of genes that a specific TF can bind) to study the interaction of a green fluorescent protein (GFP)-tagged homeobox1 (Hbx1) DNA-binding protein with A. flavus genes. The ChIP samples have been sent to the University of Illinois-Chicago for sequencing and bioinformatic analysis. Plasmid vectors (circular, double stranded DNA molecules used to introduce genes into an organism) for studying protein-protein interactions of RtfA with other A. flavus proteins were constructed and introduced into A. flavus strains. These fungal strains are currently being analyzed for expression of the tagged proteins. Under Objective 2, ARS researchers in New Orleans, Louisiana, continued investigating the identity of metabolites produced by uncharacterized Aspergillus (A.) flavus secondary metabolite gene clusters (a closely grouped set of genes that together are required for production of compounds termed secondary metabolites that are often toxic and can also be involved in fungal development, survival and virulence). Comparison of metabolites produced by an A. flavus rmtA mutant to that of wild-type A. flavus indicated the production of a toxic compound similar in structure to epicorazine A (an antibiotic produced by a different fungus). We are purifying the compound to allow for a more accurate determination of its structure and biological activities. ARS researchers in New Orleans, Louisiana, in collaboration with researchers at University of California, Los Angeles, California, have induced the expression of a gene from the uncharacterized A. flavus Cluster U in a strain of Aspergillus nidulans (a well-characterized model Aspergillus strain often used to study genes in other Aspergillus species). Preliminary chemical analysis of the A. nidulans strain demonstrated that a unique metabolite was being produced. The putative Cluster U metabolite is being purified in enough quantity to allow for identification of its chemical structure and determination of its biological activity. In other experiments aimed at identifying novel secondary metabolites in A. flavus, the fungus was grown in the presence of insects, bacteria, or chemicals that modify proteins associated with DNA. The metabolites produced under these various growth conditions were extracted from the fungal cultures. The extracts are currently being analyzed for production of novel secondary metabolites that, if found, can then be analyzed for their contribution to the fungus’ ability to survive, colonize host crops and produce aflatoxin. In regard to Objective 3, ARS researchers in New Orleans, Louisiana, in collaboration with researchers at Cranfield University in the United Kingdom, continue to analyze the impact of altered environmental conditions (i.e., elevated temperature and carbon dioxide [CO2] levels and decreased moisture) on Aspergillus (A.) flavus growth, development, and virulence on corn seed. Using ribonucleic acid (RNA)-seq, the expression patterns of individual genes in several A. flavus secondary metabolite gene clusters including the aflatoxin cluster, were determined under variable levels of temperature, water and CO2. In addition, several gene networks (a collection of genes that demonstrate comparable expression patterns under each environmental condition tested) controlling DNA replication, amino acid synthesis, and conidia production were identified. Finally, ARS researchers in New Orleans, Louisiana, have completed the set up and environmental control modifications for a large, walk-in growth chamber where corn plants can be infected with A. flavus and environmental conditions such as carbon dioxide, light, and temperature can be accurately controlled and monitored. Once corn plants have reached the desired developmental stage, they will be infected with a green fluorescent protein (GFP)-expressing A. flavus strain and levels of fungal growth and aflatoxins in the infected kernels will be determined. The development of this highly complex system will allow for continued progression of host plant-pathogen studies conducted under variable environmental conditions. The information gained from these studies will aid in the development of computer models that can predict how future global environmental conditions may impact the geographical distribution and severity of aflatoxin contamination in food and feed crops.

4. Accomplishments
1. Genes identified in the fungus, Aspergillus (A.) flavus control its ability to infect crops and produce toxic compounds. Aflatoxin contamination in crops such as corn, cottonseed, peanut, and tree nuts caused by A. flavus is a worldwide food safety problem. Aflatoxins are potent carcinogens that adversely impact human and animal health. Additionally, contamination of crops with aflatoxins costs stakeholders tens of millions of dollars annually due to economic losses from the devaluation or destruction of adulterated crops. In order to develop strategies to mitigate aflatoxin contamination of food and feed crops, it is important to decipher the complex molecular mechanisms that govern the fungus’ ability to infect plants and produce aflatoxins. Using a sophisticated molecular technique known as ribonucleic acid (RNA) sequencing (a means of determining levels of activity of individual genes in organisms), ARS researchers in New Orleans, Louisiana, have identified thousands of genes in A. flavus that are under control of the homeobox1 (hbx1) gene, a global regulator of A. flavus growth and aflatoxin production. Numerous genes involved in production of fungal secondary metabolites (compounds that are often toxic and can be involved in fungal development, survival and infectivity) whose activities are dependent on hbx1 were identified. Another study showed that inactivation of the A. flavus medA gene resulted in reduced ability of the fungus to colonize corn seed suggesting medA plays a role in fungal virulence. Due to their key roles in A. flavus’ capacity to colonize seed tissues and produce toxic compounds, both the hbx1 and medA genes are good candidates as targets of control strategies to interrupt the ability of the fungus to infect and contaminate corn and other crops with aflatoxins.

Review Publications
Xie, H., Wang, X., Zhang, L., Wang, T., Zhang, W., Jiang, J., Chang, P.-K., Chen, Z.-Y., Bhatnagar, D., Zhang, Q., Li, P. 2018. Monitoring metabolite production of aflatoxin biosynthesis by orbitrap fusion mass spectrometry and a D-optimal mixture design method. Analytical Chemistry. 90:14331-14338.
Cary, J.W., Entwistle, S., Satterlee, T., Mack, B.M., Gilbert, M.K., Chang, P.-K., Scharfenstein, L.L., Yin, Y., Calvo, A. 2019. The transcriptional regulator Hbx1 affects the expression of thousands of genes in the aflatoxin-producing fungus Aspergillus flavus. G3, Genes/Genomes/Genetics. 9(1):167-178.
Ojiambo, P.S., Battilani, P., Cary, J.W., Blum, B.H., Carbone, I. 2018. Cultural and genetic approaches to manage aflatoxin contamination: recent insights provide opportunities for improved control. Phytopathology. 108:1024-1037.
Chang, P.-K., Scharfenstein, L.L., Mack, B.M., Wei, Q., Gilbert, M.K., Lebar, M.D., Cary, J.W. 2019. Identification of a copper-transporting ATPase involved in biosynthesis of A. flavus conidial pigment. Applied Microbiology and Biotechnology. 103:4889-4897.
Majumdar, R., Minocha, R., Lebar, M.D., Rajasekaran, K., Long, S., Carter-Wientjes, C.H., Minocha, S., Cary, J.W. 2019. Contribution of maize polyamine and amino acid metabolism toward resistance against Aspergillus flavus infection and aflatoxin production. Frontiers in Plant Science. 10:692.
Leslie, J.F., Lattanzio, V., Audenaert, K., Battilani, P., Cary, J.W., Chulze, S.N., De Saeger, S., Gerardino, A., Karlovsky, P., Liao, Y.-C., Maragos, C.M., Meca, G., Medina, A., Moretti, A., Munkvold, G., Mule, G., Njobeh, P., Pecorelli, I., Perrone, G., Pietri, A., Palazzini, J.M., Proctor, R.H., Rahayu, E.S., Ramirez, M.L., Samson, R., Stroka, J., Sulyok, M., Sumarah, M., Waalwijk, C., Zhang, Q., Zhang, H., Logrieco, A.F. 2018. MycoKey round table discussions of future directions in research on chemical detection methods, genetics and biodiversity of mycotoxins. Toxins. 10(3):109.
Lohmar, J.M., Puel, O., Cary, J.W., Calvo, A.M. 2019. The Aspergillus flavus rtfA gene regulates plant and animal pathogenesis and secondary metabolism. Applied and Environmental Microbiology. 85(6):e02446-18.
Ibarra, B.A., Lohmar, J.M., Satterlee, T., McDonald, T., Cary, J.W., Calvo, A.M. 2018. The 14-3-3 protein homolog ArtA regulates development and secondary metabolism in the opportunistic plant pathogen Aspergillus flavus. Applied and Environmental Microbiology. 84(5):e02241-17.