Location: Food and Feed Safety Research
2018 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, Agricultural Research Service (ARS) researchers in New Orleans, Louisiana, continue to pursue their mission to control of aflatoxin contamination of crops through multiple intervention strategies. ARS researchers have begun to analyze data from a ribonucleic acid (RNA)-sequencing experiment (RNA-Seq; a means of determining levels of activity of individual genes in organisms) to study the activity of all genes that are expressed 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. We are comparing expression of genes in the A. flavus fungus during its growth on two lines of corn, one that is resistant and one that is susceptible to infection by the fungus. Examination of the RNA-seq data has identified a number of candidate genes that may function as global regulators of A. flavus growth and aflatoxin biosynthesis during its interaction with corn kernels as well as developmental and virulence (ability to cause infection) factors that can serve as targets for intervention strategies. A number of these candidate genes have functions that are known while others are unknown but in some cases putative functions have been ascribed. Studies were completed on some of these previously uncharacterized genes including the identification and characterization of the A. flavus homeobox 1 (hbx1) gene (homeobox genes are a class of genes known to be involved in development in fungi, insects and mammals). This gene was shown to be required for the fungus to produce conidia (asexual reproductive structures also known as spores), sclerotia (fungal survival structures) and aflatoxins. In addition, inactivation of the hbx1 gene reduced the ability of the fungus to infect corn kernels. An RNA-seq study was performed to identify other genes in A. flavus that are under the control of hbx1. Analysis of data from the RNA-seq experiment has identified a number of putative regulatory genes that may be involved in controlling the ability of the fungus to infect corn and produce aflatoxin. A gene, ecm33, was shown to be responsible for proper cell wall composition in the fungus as well as regulating fungal growth, development and aflatoxin production. This gene was also shown to be required for normal levels of corn seed colonization. Biosynthesis of the polyamine (PAs; small positively charged molecules derived from amino acids) spermidine (SPD) has been shown to play a significant role in fungal cell growth and pathogenesis. It was shown that inactivation of the gene, spds (spermidine synthase), required by A. flavus to produce SPD, resulted in a severe reduction in fungal growth and development as well as aflatoxin biosynthesis both on synthetic growth media as well as during growth on corn kernels.
Under Objective 2, Agricultural Research Service (ARS) researchers in New Orleans, Louisiana, continued investigating the Aspergillus (A.) flavus secondary metabolite gene cluster (a closely grouped set of genes that together are required for production of compounds that are often toxic and can also be involved in fungal development, survival and infectivity) #11 metabolite, aspergillic acid (a toxic compound). The role of aspergillic acid in corn infection was difficult to study because no aspergillic acid was detected following infection of corn kernels with A. flavus. ARS researchers have now infected corn kernels with an A. flavus mutant that produces very high levels of aspergillic acid and were able to detect a large amount of aspergillic acid in the infected corn kernels. By comparing this data with earlier kernel infection data from wild-type (non-mutant) A. flavus –infected corn kernels using a highly sensitive mass spectrometer (an instrument used to identify extremely small quantities of chemical compounds), ARS researchers were able to identify low levels of aspergillic acid in the wild-type strains. Because of this, ARS researchers can now detect low levels of aspergillic acid in infected corn samples allowing us to further probe how aspergillic acid affects the ability of the fungus to infect corn. Progress has been made to identify the compound produced by the A. flavus strain 70 secondary metabolite gene cluster designated “Cluster U." Previous attempts to isolate and identify the cluster product using gene knockout mutants (a technique to inactivate a gene in the fungus) of the fungus were unsuccessful, however introduction of the gene (pks 181) encoding the core polyketide synthase (PKS, a key protein needed to produce the final secondary metabolite compound) into another A. flavus strain (a strain that does not produce aflatoxin, thus simplifying detection) identified a novel peak and potential product of the PKS protein that is being analyzed. ARS researchers have since developed a collaboration with researchers at University of California, Los Angeles, who are introducing the pks 181 gene into a specialized fungal strain to aid in identification of the unknown Cluster U secondary metabolite compound.
In regard to Objective 3, progress has been made on the analysis of Aspergillus (A.) flavus growth, development, and virulence on corn seed under altered environmental conditions (i.e., elevated temperature and carbon dioxide ([CO2] levels and decreased water availability). Chemical analyses demonstrated that A. flavus aflatoxin production increased with elevated CO2 levels under differing temperature and water availability conditions. Production of aflatoxin correlated with an increase in the expression of genes involved in aflatoxin biosynthesis. This information can be used to predict how future alterations in global environmental conditions may impact the geographical distribution and ability of A. flavus to grow and produce aflatoxins in crops such as corn.
Accomplishments
1. Involvement of a regulatory gene, ecm33, 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, ARS researchers 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 the identification of a gene, ecm33, which is required for production of normal levels of conidia (asexual reproductive structures also known as spores) and sclerotia (fungal survival structures). The ecm33 gene was also found to be required for production of normal levels of aflatoxins and for the ability of the fungus to colonize corn seed. This research provides additional information on genes involved in the regulation of A. flavus development, aflatoxin production and pathogenicity and the ecm33 gene can serve as a target of control strategies to interrupt the ability of the fungus to colonize and contaminate corn with aflatoxins.
2. The impact of atmospheric carbon dioxide (CO2) levels on the response of the fungus, Aspergillus (A.) flavus, to temperature and water availability. The influence of predicted increases in global CO2 levels on the environment as it relates to the geographical distribution of A. flavus and outbreaks of aflatoxin contamination of crops is unknown. The impact of temperature and water on the ability of A. flavus to grow, infect crops, and produce toxins has been well characterized. However, the impact these two environmental factors under increased CO2 conditions have only recently been characterized. Agricultural Research Service (ARS) researchers in New Orleans, Louisiana, demonstrated that increased CO2 can lead to an increase in aflatoxin (a potent cancer-causing compound) production. The expression patterns of genes present in several secondary metabolite (compounds that are often toxic and can also be involved in fungal development, survival and infectivity) gene clusters including the aflatoxin cluster were modified due to increased CO2 levels. Finally, several gene networks controlling fungal biological processes such as deoxyribonucleic acid (DNA) replication, amino acid synthesis, and conidia (asexual reproductive structures also known as spores) production were also affected. These results demonstrate the impact that elevated CO2 levels can have on important fungal biological processes. These data are being used by modelers for predicting the levels of toxin contamination under various environmental conditions. These models are providing insight on how remediation efforts will be influenced by future global environmental conditions.
Review Publications
Majumdar, R., Lebar, M.D., Mack, B.M., Minocha, R., Minocha, S., Carter-Wientjes, C.H., Sickler, C.M., Rajasekaran, K., Cary, J.W. 2018. The Aspergillus flavus spermidine synthase (spds) gene, is required for normal development, aflatoxin production, and pathogenesis during infection of maize kernels. Frontiers in Plant Science. 9:317. https://doi.org/10.3389/fpls.2018.00317.
Cary, J.W., Gilbert, M.K., Lebar, M.D., Majumdar, R., Calvo, A.M. 2018. Aspergillus flavus secondary metabolites: more than just aflatoxins. Food Safety. 6(1):7-32. https://doi.org/10.14252/foodsafetyfscj.2017024.
Lebar, M.D., Cary, J.W., Majumdar, R., Carter-Wientjes, C.H., Mack, B.M., Wei, Q., Uka, V., De Saeger, S., Diana Di Mavungu, J. 2018. Identification and functional analysis of the aspergillic acid gene cluster in Aspergillus flavus. Fungal Genetics and Biology. 116:14-23.
Chang, P.-K., Zhang, Q., Scharfenstein, L.L., Mack, B.M., Yoshimi, A., Miyazawa, K., Abe, K. 2018. Aspergillus flavus GPI-anchored protein-encoding ecm33 has a role in growth, development, aflatoxin biosynthesis, and maize infection. Applied Microbiology and Biotechnology. 102:5209-5220. https://doi.org/10.1007/s00253-018-9012-7.
Cary, J.W., Harris-Coward, P.Y., Scharfenstein, L.L., Mack, B.M., Chang, P.-K., Wei, Q., Lebar, M.D., Carter-Wientjes, C.H., Majumdar, R., Mitra, C., Banerjee, S., Chanda, A. 2017. The Aspergillus flavus homeobox gene, hbx1, is required for development and aflatoxin production. Toxins. 9(10):315. https://doi.org/10.3390/toxins9100315.
Bhatnagar, D., Rajasekaran, K., Gilbert, M.K., Cary, J.W., Magan, N. 2018. Advances in molecular and genomic research to safeguard food and feed supply from aflatoxin contamination. World Mycotoxin Journal. 11(1):47-72. https://doi.org/10.3920/WMJ2017.2283.
Gilbert, M.K., Medina, A., Mack, B.M., Lebar, M.D., Rodriguez, A., Bhatnagar, D., Magan, N., Obrian, G., Payne, G. 2018. Carbon dioxide mediates the response to temperature and water activity levels in Aspergillus flavus during infection of maize kernels. Toxins. 10(1):5. https://doi.org/10.3390/toxins10010005.
