Location: Food and Feed Safety Research
2020 Annual Report
Objectives
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.
Approach
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.
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 (RNA-Seq; a means of determining levels of activity of individual genes in organisms) and other specialized experiments to identify and study the biological activity of all fungal (mold) genes during the corn-Aspergillus (A.) flavus interaction under various environmental conditions. Genes shown to be critical in the regulation of fungal growth and aflatoxin production will be used as targets for our approaches to control A. flavus infection of crops and subsequent contamination with aflatoxin.
Aspergillus 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. ARS reseachers at New Orleans, Louisiana, found that the A. flavus cpcA gene is involved in amino acid utilization and it’s expression increases during early stages of infection of corn by the fungus. Inactivation of the cpcA gene resulted in A. flavus strains (termed cpcA mutants) that demonstrated reduced growth on synthetic media, however no differences in growth or aflatoxin production were observed between the cpcA mutant and wild-type A. flavus (a natural, non-mutated form of the fungus) during growth on live corn kernels. Another gene of interest in A. flavus was also identified in experiments conducted by ARS researchers in New Orleans, Louisiana. The DNA present in the genomes of two A. flavus isolates, one producing high amounts of aflatoxin and another producing low amounts, were sequenced. Comparison of the two sequences revealed a large insertion of DNA in the genome of the high-producer strain. This inserted region of DNA contained numerous genes including one predicted to control the expression of other genes, designated atfC. Studies suggested the atfC gene might be associated with the high aflatoxin producer’s ability to infect plants and tolerate environmental stress. Additional experiments are underway to confirm these observations. ARS reseachers at New Orleans, Louisiana, in collaboration with researchers at Northern Illinois University (NIU), Dekalb, Illinois, the rmtA gene, previously shown to be involved in fungal growth and aflatoxin production, was specifically shown to control expression of a gene designated gliP. Preliminary chemical analysis suggests that this gene plays a role in the synthesis of two novel compounds in A. flavus. Further chemical analyses are being conducted to identify the structure of these compounds and their role in the biology of the fungus. ARS reseachers at New Orleans, Louisiana, working with researchers at NIU have also examined another A. flavus gene, designated lreC. The LreC protein is similar to the known fungal light-sensing proteins LreA and LreB and its expression is known to be dependent on the RmtA regulator protein. Preliminary studies of an A. flavus strain with an inactivated lreC gene suggests a role for this gene in A. flavus development and possibly production of aflatoxins. The function of the lreC gene is being further characterized. Researchers in New Orleans, Louisiana, and NIU continued studies on characterizing the biological function of numerous genes whose level of expression is dependent on the homeobox1 (hbx1) gene. It was revealed that hbx1 is needed for normal expression of the hdt1 gene which is necessary for normal aflatoxin production and development in A. flavus and is currently being analyzed in more detail. ARS researchers in New Orleans, Louisiana, in collaboration with researchers at the University of South Carolina have performed experiments to study the interaction of the A. flavus Hbx1 protein with its own genes. Analysis of the gene DNA sequences that bound with the Hbx1 protein allowed identification of a number of genes that play roles in metabolism and development in A. flavus including those that are required for the formation of small storage compartments within individual fungal cells known as endosomes that house the proteins required for aflatoxin biosynthesis.
Objective 2, ARS researchers in New Orleans, Louisiana, continued investigating the identity and biological function of metabolites produced by Aspergillus (A.) flavus. Comparison of metabolites produced by an A. flavus strain with an inactivated rmtA gene to that of wild-type (natural, non-mutated version) A. flavus indicated the production of two yet to be identified compounds from extracts of the wild-type culture. Cultures for production of the unidentified metabolites were scaled up and fractions containing the two metabolites were purified and sent to collaborators for more sensitive chemical analyses. Additionally, ARS researchers in New Orleans, Louisiana, have grown A. flavus on insect larvae commonly associated with corn. Growth of the fungus on maize weevil carcasses resulted in production of a compound, similar in structure to previously identified antiinsectan compounds. Scale-up of cultures were performed and extracts were sent to collaborators at the University of Ghent for more in depth chemical analysis.
In regard to Objective 3, ARS scientists in New Orleans, Louisiana, working with scientists at Cranfield University, U.K., have analyzed the impact of altering environmental factors such as low water, high temperature, and elevated CO2 levels on the ability of A. flavus to produce aflatoxins and other toxic metabolites during infection of corn kernels. The data indicate that a combination of low water stress and high CO2 levels resulted in earlier than normal production of aflatoxins in A. flavus during infection of corn kernels. Further, analysis of gene expression data of A. flavus growing under differing CO2 levels identified several genes that are potentially responsible for controlling the expression of fungal genes associated with response to elevated CO2 levels. These fungal genes are being inactivated in order to determine their role in the fungus’ ability to respond to altered CO2 levels. Finally, in the past year ARS reseachers at New Orleans, Louisiana, established a plant growth chamber that allows us to grow corn to maturity under altered temperature, moisture and CO2 levels. This allows us to conduct A. flavus infection of developing corn plants under environmental conditions simulating predicted climate change that cannot be simulated during infection of individual corn kernels.
Accomplishments
1. A gene identified in the mold, Aspergillus (A.) flavus controls its ability to infect crops and produce toxic compounds. Aflatoxin contamination in crops such as corn, cottonseed and peanut caused by A. flavus is a worldwide food safety problem as they are potent carcinogens that adversely impact human and animal health. Additionally, contamination of crops with aflatoxins costs tens of millions of dollars every year due to economic losses from the damaged crops that cannot be sold or are sold at a lower price. In order to develop plans to mitigate aflatoxin contamination of food and feed crops, it is important to understand 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 a number of genes in A. flavus that are under control of the homeobox1 (hbx1) gene, known to regulate A. flavus growth and aflatoxin production. Collaborating with scientists at Northern Illinois University, ARS reseachers at New Orleans, Louisiana, have continued studies on characterizing the biological function of a number of genes regulated by hbx1. It was revealed that hbx1 affects the expression of a gene designated hdt1. Analyses showed that hdt1 is necessary for normal aflatoxin production and formation of reproductive structures termed conidia in A. flavus. Due to the role of hdt1 in A. flavus’ capacity to reproduce and generate aflatoxin, it is a good candidate as a target of control strategies to interrupt the ability of the fungus to infect and contaminate corn and other crops with aflatoxins. Information gained from our studies will positively impact our stakeholders and the general public in the form of increased food safety and security.
Review Publications
Satterlee, T., Entwistle, S., Yin, Y., Cary, J.W., Lebar, M.D., Losada, L., Calvo, A.M. 2019. rmtA-dependent transcriptome and its role in secondary metabolism, environmental stress, and virulence in Aspergillus flavus. G3, Genes/Genomes/Genetics. 9(12):4087-4096. https://doi.org/10.1534/g3.119.400777.
Chang, P.-K., Cary, J.W., Lebar, M.D. 2020. Biosynthesis of conidial and sclerotial pigments in Aspergillus species. Applied Microbiology and Biotechnology. 104:2277-2286. https://doi.org/10.1007/s00253-020-10347-y.
Kong, Q., Chang, P.-K., Li, C., Hu, Z., Zheng, M., Sun, Q., Shan, S. 2020. Identification of AflR binding sites in the genome of Aspergillus flavus by ChIP-Seq. The Journal of Fungi. 6:52. https://doi.org/10.3390/jof6020052.
Chang, P.-K., Scharfenstein, L.L., Abbas, H.K., Bellaloui, N., Accinelli, C., Ebelhar, M.W. 2020. Prevalence of NRRL21882-like (Afla-Guard®) Aspergillus flavus on sesame seeds grown in research fields in the Mississippi Delta. Biocontrol Science and Technology. 30:1090-1099. https://doi.org/10.1080/09583157.2020.1791798.
Musungu, B.M., Bhatnagar, D., Payne, G.A., O'Brian, G., Quiniou, S., Geisler, M., Fakhoury, A. 2020. Use of dual RNA-seq for systems biology analysis of zea mays and Aspergillus flavus interaction. Frontiers in Microbiology. 11:853.
