Progress 06/14/16 to 04/18/21
Outputs
PROGRESS REPORT 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. 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. This is the final report for the Project 6054-41420-008-00D terminated in April 2021, which has been replaced by new Project 6054-41420-009-00D. For additional information, see the new project report. Significant progress was made by ARS scientists at New Orleans, Louisiana in all three objectives, which fall under National Program 108 Food Safety, Component 1, Foodborne Contaminants. To understand the preharvest aflatoxin (a toxic and carcinogenic compound) contamination process and develop effective aflatoxin mitigation strategies, it is important to understand the genetic make-up and the gene expression profile of the aflatoxin-producing fungus, Aspergillus (A.) flavus, under various environmental conditions (including changing climate), especially during interaction of the fungus with the host plant (for example, corn). Objective 1, ARS scientists in New Orleans, Louisiana, have made significant progress in identifying key genes from A. flavus that are involved in controlling the ability of the fungus to grow, infect crops and produce aflatoxins. ARS scientists used improved methods such as ribonucleic acid-sequencing (RNA-seq; a technique that is used to measure the level of activation of genes) and specifically looked at the level of expression of genes in the fungus during colonization of corn seed. These RNA-seq studies resulted in identification of several genes that regulate of A. flavus growth, virulence and aflatoxin production. ARS scientist will try to inactivate the A. flavus spermidine synthase gene (spds; needed to make a chemical involved in fungal virulence and aflatoxin production) using a strategy known as host-induced gene silencing. Evaluation of RNA-seq data led to the identification of a number of fungal genes whose expression correlated with active infection of corn seed. These included genes responsible for the production of proteins termed transcription factors that serve as key regulators of fungal growth and toxin production. Additionally, a number of genes involved in the production of peptide (a small protein) and terpene (a class of compounds often associated with plant essential oils) secondary metabolites (compounds not required for fungal growth but are needed for virulence and survival and can often be toxic to other organisms) were also shown to be highly expressed during infection of corn seed by the fungus and are believed to play a role in the ability of the fungus to successfully invade seed tissues. Genes responsible for production of two of the peptide compounds produced during infection of corn have been knocked out to better understand their biological role in the fungus. Using comparative RNA-seq of infected kernels from corn lines susceptible or resistant to A. flavus infection and aflatoxin contamination, ARS scientists have identified and are studying four fungal genes that are expressed at a higher level during infection of resistant lines compared to a susceptible line and therefore may be responsible in part for the ability of the fungus to infect corn. Objective 2, ARS scientists have performed a number of experiments to identify other chemicals (not aflatoxin) produced by A. flavus during infection of corn. These studies led to the identification of aspergillic acid and ferriaspergillin (both toxic compounds) which play a role in the ability of A. flavus to infect corn. Additional studies led to the development of a more sensitive assay for detection of aspergillic acid and similar compounds in plant tissues such as corn seed. Further, ARS scientists in New Orleans, Louisiana, have identified a gene cluster that so far has only been found in a single A. flavus strain whereas most gene clusters are commonly found in all A. flavus strains. In a collaboration with scientists at the University of California Los Angeles, ARS scientists want to determine if the presence of this unique gene cluster provides the fungus with the ability to compete more successfully for infection of corn. A number of small molecules that inhibit histone modifying enzymes (proteins that can modify amino acid residues associated with chromosomal DNA termed histones which in turn can impact gene expression) were analyzed to assess their effects on fungal secondary metabolism. Aflatoxin-producing A. flavus strains were grown in the presence of the inhibitors and metabolic extracts have been prepared. Data acquisition and analysis is being performed by ARS scientists to determine if addition of the inhibitors leads to production of novel secondary metabolites that normally are not produced by the fungus during growth on standard nutrient-based growth media. Objective 3, ARS scientists related to predicted climate change conditions such as low moisture, 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 dry conditions and high CO2 levels resulted in earlier than normal production of aflatoxins in A. flavus during infection of individual corn kernels germinating in an incubator. 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 higher CO2 levels. To better understand the impact of altered CO2 levels on the ability of A. flavus to infect and produce aflatoxins during growth on corn cobs, ARS scientists modified a large walk-in plant growth chamber that now allows us to grow corn to maturity under altered temperature, moisture and CO2 levels. This enables scientists 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. Using this chamber scientists have inoculated kernels present on ears of corn with A. flavus under conditions of 350 ppm CO2 (roughly the current global level of atmospheric CO2) or at 1000 ppm (levels predicted to be present at the end of this century). After 3 days of infection, individual seeds were harvested, flash frozen in liquid N2 and stored at - 80 for later analysis of gene expression in the fungus as well as determine the level of aflatoxin being produced. The overall impact of the research arising from these three objectives is to support our biological control research and to transfer basic information to our Host Resistance project for application in the development of improved host plant aflatoxin resistance strategies. In collaboration with ARS scientists in Peoria, Illinois, Maricopa, Arizona, and scientists at Wageningen University, Food Safety Research (WFSR) in The Netherlands, ARS scientists at New Orleans, Louisiana will attempt to develop a mobile application with a predictive model and decision support system to aid farmers in determining if environmental conditions will be conducive to aflatoxin/fumonisin contamination of their corn in the field and decide on best practices that can be applied to mitigate contamination. Record of Any Impact of Maximized Teleworking Requirement: Due to the maximized telework posture that has been in effect during FY2021, ARS researchers at New Orleans, Louisiana, were limited in the time that they could spend in the laboratory conducting research on the project⿿s three objectives that require microbiological, molecular and chemical techniques to be performed in a laboratory setting on an ongoing, daily basis to achieve optimal productivity. Professional networking and attendance in scientific meeting were also limited resulting in a significant reduction in productivity. ACCOMPLISHMENTS 01 Common bacteria found in corn kernels may reduce levels of toxic compounds. ARS scientists at New Orleans, Louisiana, believe a toxin, called aflatoxin, produced by the fungus Aspergillus (A.) flavus during growth on corn is a worldwide food safety problem. Aflatoxins are potent carcinogens that adversely impact human and animal health and contamination of crops with aflatoxins costs stakeholders tens of millions of dollars annually. Management of aflatoxin contamination in corn is difficult so understanding what contributes to kernel resistance is of great importance in developing control strategies. Using sophisticated DNA sequencing technologies, ARS scientists in New Orleans, Louisiana, were able to identify groups of bacteria that were naturally present in greater numbers in kernels of resistant corn lines compared to non-resistant lines. Continued evaluation of bacteria predicted to possess antifungal and anti-aflatoxigenic properties will aid in their development as effective agents for enhanced resistance to A. flavus infection and aflatoxin contamination thus ensuring a safer and more secure food supply. 02 Genetic switches turn off production of a deadly fungal toxin. ARS scientists at New Orleans, Louisiana, suggests the fungus, Aspergillus (A.) flavus, produces a toxic and carcinogenic family of compounds known as aflatoxins during growth on crops such as corn. Consumption of aflatoxin contaminated corn can lead to adverse health effects in humans and animals such as immunosuppression, stunting of growth in children and liver cancer. It is important to understand the complex genetic mechanisms that govern the fungus⿿ ability to infect corn and produce aflatoxin to develop effective aflatoxin control strategies. Using sophisticated molecular techniques, ARS scientists have found several novel genes that function as key genetic ⿿switches capable of controlling A. flavus growth and aflatoxin production. Information gained from these studies is being used to develop corn with enhanced resistance to A. flavus infection and aflatoxin contamination which will positively impact our stakeholders and the general public in the form of increased food safety and security.
Impacts
(N/A)
Publications
- Chang, P.-K., Chang, T.D., Katoh, K. 2020. Deciphering the origin of Aspergillus flavus NRRL21882, the active biocontrol agent of Afla-Guard®. Letters in Applied Microbiology. https://doi.org/10.1111/lam.13433.
- Chang, P.-K. 2021. Authentication of Aspergillus parasiticus strains in the genome database of the National Center for Biotechnology Information. BMC Research Notes. 14:111. https://doi.org/10.1186/s13104-021-05527-6.
- Gebru, S.T., Mammel, M.K., Gangiredla, J., Tartera, C., Cary, J.W., Moore, G.G., Sweany, R.R. 2020. Draft genome sequences of 20 Aspergillus flavus isolates from corn kernels and cornfield soils in Louisiana. Microbiology Resource Announcements. 9(38):e00826-20. https://doi.org/10.1128/MRA.00826- 20.
- Majumdar, R., Kandel, S.L., Cary, J.W., Rajasekaran, K. 2021. Changes in bacterial endophyte community following aspergillus flavus infection in resistant and susceptible maize kernels. International Journal of Molecular Sciences. 22(7). Article 3747. https://doi.org/10.3390/ ijms22073747.
- Fountain, J.C., Clevenger, J.P., Nadon, B.D., Youngblood, R.C., Chang, P., Starr, D., Wang, H., Wiggins, R., Kemerait, R.C., Bhatnagar, D., Ozias- Akins, P., Varshney, R.K., Scheffler, B.E., Vaughn, J.N., Guo, B. 2020. Two new Aspergillus flavus reference genomes reveal a large insertion potentially contributing to isolate stress tolerance and aflatoxin production. Genes, Genomes, and Genomics. 10(9). https://doi.org/10.1534/ g3.120.401405.
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Progress 10/01/19 to 09/30/20
Outputs
Progress Report 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. 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. 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 01 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.
Impacts
(N/A)
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.
- 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.
- 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.
- Chang, P.-K., Cary, J.W., Lebar, M.D. 2020. Biosynthesis of conidial and sclerotial pigments in Aspergillus species. Applied Microbiology and Biotechnology.
- 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.
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Progress 10/01/18 to 09/30/19
Outputs
Progress Report 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. 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. 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. Accomplishments 01 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.
Impacts
(N/A)
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.
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Progress 10/01/17 to 09/30/18
Outputs
Progress Report 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. 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. 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 01 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. 02 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.
Impacts
(N/A)
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.
- 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.
- 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.
- 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.
- 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.
- 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.
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Progress 10/01/16 to 09/30/17
Outputs
Progress Report 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. 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. 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. Accomplishments 01 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. 02 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.
Impacts
(N/A)
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. doi:10.3920/WMJ2016.2049.
- 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.
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