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United States Department of Agriculture

Agricultural Research Service

2011 Annual Report

1a.Objectives (from AD-416)
(1) Understand the molecular basis for fungal responses to conditions encountered during invasion of crops in order to identify and modify the factors in corn and other crops that could induce or impede aflatoxin formation or fungal development. Accomplishment of objective 1 could lead to generic approaches to enhance resistance in all crops vulnerable to aflatoxin contamination. (2) Determine, by genetic and physiological studies of diverse aflatoxin producing species, whether aflatoxin production provides an adaptive advantage for fungal survival and invasion of crops, particularly because many natural isolates of A. flavus do not produce aflatoxins. (3) Determine the molecular responses of aflatoxin producing fungi to stress factors, particularly with regard to developing an understanding of the ability of the fungi to adapt and produce toxins. (4) Undertake and utilize newly available sequences of genomic DNA from Aspergillus species to develop rapid and highly sensitive PCR based tests to identify aflatoxigenic fungi and their toxins in contaminated food products.

1b.Approach (from AD-416)
(1) Microarray experiments will be used to identify candidate genes in A. flavus that are turned on or off during a variety of environmental and nutritional conditions that are known to alter gene expression affecting aflatoxin biosynthesis. The DNA microarrays will exploit the available genomic resources of A. flavus ESTs and the whole genome sequences, combined with statistical analysis of up and down regulated signals on the chips. The candidate genes identified from microarrays (both A. flavus EST and whole genome) will be verified or confirmed through RT-Q- PCR or other well established methods. For further analysis, to identify specific genes, targeted gene mutagenesis will be necessary to determine their biological function. Comparison of the gene expression data under these aflatoxin-producing and non-producing conditions will allow us to identify the specific genes expressed under aflatoxin-producing conditions. (2) The adaptive advantage of aflatoxin production in certain environments will be determined by genetic and physiological studies. It will be ascertained if gene cluster imparts some fitness advantage in some environmental niches, particularly when aflatoxin production does not appear to be a virulence factor for crop infestation. Isogenic isolates will be developed in A. flavus for this experiment. Fungal viability will be measured for up to 12 months with these isolates. The measure of longevity proposed in the present study as a measure of fitness will be an important determinant for understanding aflatoxin production in Aspergillus populations in crop soils. Comparisons of genomic sequences will be made between toxigenic A. flavus found in agricultural fields and other Aspergillus species (non-toxigenic and domesticated) to determine what genes are involved in fungal virulence and toxin production of A. flavus, as well as its ability to survive in the field. (3) Stress cues that change the activity of proteins in the biosynthetic pathway and gene transcription will be evaluated. The chromatin structure of the gene cluster and adjacent regions will be studied after exposure to aflatoxin inducing conditions. Parameters such as DNA methylation patterns, that alter chromatin structure, will be explored in these adjacent regions to determine how aflatoxin biosynthesis is turned on in the fungus. Understanding environmental stress cues affecting aflatoxin gene expression may help to develop strategies to reduce aflatoxin contamination of corn under field conditions. It will be determined if the drought tolerant varieties have lower levels of aflatoxin contamination because the host-fungus interaction somehow alters the ability of the fungus to initiate toxin production at the DNA level. (4) The use of PCR to quantify the level of toxigenic fungi in foods through the use of Taqman primer-probe assay will be assessed to provide information on fungal bioburden, but not the level of aflatoxin contamination. The test will be designed to measure the potential of the commodity to become severely contaminated with the fungus (and consequently toxin) if stored under conditions suitable for subsequent growth of the fungus.

3.Progress Report
This is the final report for the project 6435-41420-005-00D, which has been replaced by new project 6435-41420-006-00D. By molecular analyses of the fungus Aspergillus (A.) flavus, the complex array of genes involved in fungal virulence, aflatoxin formation, response to the environment, and development/reproduction/survival were identified. Our genomics program on sequencing of the entire A. flavus genome was done at J. Craig Venter Institute (JCVI) in collaboration with North Carolina State University (NCSU). Further, we have constructed several microarrays (glass slides containing many individual genes) and used them in large scale functional genomics studies, to identify a set of fungal genes that are affected under varying nutritional, environmental or developmental conditions, and during the fungus-corn interaction that affect toxin production. The molecular role of key transcription regulatory factors (responsive to environmental and developmental signals) in the initiation of aflatoxin biosynthesis was determined; these factors are potential targets for intervention to prevent aflatoxin production on plants. Additionally, with JCVI, we used new generation sequencing technologies (called Illumina RNA-Seq) which can provide access to a cell’s entire transcriptome (all genes that are expressed) to investigate the mechanisms of gene expression regulation under conditions conducive and non-conducive to aflatoxin production. For understanding the ability of Aspergillus species to produce G1 type of aflatoxins, chemical studies revealed that aflatoxins G1 and B1 are independently produced from a common precursor, through a branching step, and at least three enzymes are required for production of G1. A putative oxidative stress-induced signaling pathway was discovered in cooperation with the Western Regional Research Center scientists by mutation studies of an A. flavus gene homologous to a known yeast stress response gene; results implied that the signaling pathway is suppressed by plant antioxidants affecting programmed fungal growth and development. In collaboration with the National Institute of Advanced Industrial Science and Technology, Japan, we used A. oryzae (a closely related atoxigenic strain in the A. flavus family) whole genome databases for comparison of the gene expression profiles between the field fungus (A. flavus) and A. oryzae; for insights into any competitive advantage that A. flavus may possess as it survives in field conditions. In collaboration with National Peanut Research Laboratory, we identified the gene cluster involved in cyclopiazonic acid (CPA) formation and characterized associated biosynthetic genes. The role of multiple nuclei in the A. flavus vegetative cells as well as in their spores (i.e. like seeds) in multiplication of these fungi or survival in their environment is unknown. In collaboration with NCSU, we have been successful in color-coding these nuclei so that we can track them at different stages of fungal growth and development to determine the role of these multiple nuclei on fungal growth and development and in response to various stresses that the fungus encounters in the field.

1. Cyclopiazonic acid is another toxic compound that the fungus Aspergillus (A.) flavus produces along with aflatoxins. In collaboration with National Peanut Research Laboratory (NPRL), ARS scientists at New Orleans, LA, have identified the gene cluster involved in cyclopiazonic acid (CPA) formation and characterized associated biosynthetic genes. The CPA gene cluster is located next to the aflatoxin gene cluster on the A. flavus genome. Using this information, scientists confirmed that the biopesticide Afla-Guard® (active ingredient is A. flavus NRRL21886), developed at NPRL and commercialized by Syngenta Crop Protection, does not contain the CPA and aflatoxin A. flavus gene clusters, which ensures the safe application of this genuinely nontoxic A. flavus strain in the field as a biocontrol agent. The co-location of the aflatoxin and CPA biosynthetic gene clusters on the fungal chromosome suggests how the fungus could make these two harmful compounds together based on one signal that allows the “turning on” of the genetic machinery within the fungus.

2. Sequencing of all the Deoxyribonucleic Acid (DNA) of the fungus Aspergillus (A.) flavus and its applications. Knowing the genetic make up of this fungus is important to know how and why this fungus makes the potent carcinogen aflatoxin when it invades crops. Two different format whole genome microarrays (slides containing spots of DNA corresponding to fragments of all unique genes) have been designed recently, and used to identify critical genes involved in fungal response to various environmental factors favoring toxin production. These microarray resources have been used in large scale functional genomics studies by Agricultural Research Service (ARS) scientists at New Orleans, LA, and our collaborators to analyze which genes are affected under varying conditions:.
1)nutritional (high or low carbon source);.
2)environmental (temperature);.
3)developmental (veA mutant); and.
4)during the fungus-corn interaction (cooperation with ARS scientists and faculty, Mississippi State University) that affect toxin production. The long-term survival of aflatoxin-producing A. flavus strains in comparison with non-producing strains has indicated that under temperature stress (47°C), spores of toxigenic strains survived longer than non-aflatoxin producers; and there is no difference in survival under ultraviolet light for aflatoxin-producing strains vs. non-aflatoxin-producing strains. From these microarray studies, over a hundred genes were identified that may have some impact on aflatoxin production and fungal survival. The database will be accessible through a website which is expected to be housed at the ARS, Mid South Area genomics facility and provide genomic information to all researchers worldwide working with this fungus. These studies carried out by ARS scientists are providing significant insights into what genes are involved in the interaction between the fungus and the crop.

3. Proof of involvement of genes in aflatoxin synthesis and fungal development obtained. Knowing the genetic make up of this fungus is important to know how and why this fungus makes the potent carcinogen aflatoxin when it invades crops. Using sophisticated molecular techniques, ARS scientists at New Orleans, LA, have tested specific interactions of key aflatoxin developmental regulatory factors. Further, the genetic basis for loss of aflatoxin production in toxin-deficient mutants of Aspergillus parasiticus (generated by physical manipulation of toxin-producing strains) has been investigated using microarrays and metabolic profiling, and specific regulatory genes causing this loss have been identified. ARS scientists at New Orleans, LA, have found that many factors are needed for complete regulation of the turning on and off of aflatoxin production. Through the use of these technologies we will rapidly assess the critical role of several genes of interest in aflatoxin formation in crops.

4. Details of the aflatoxin biosynthetic pathway further elucidated. Knowing the genetic make up of this fungus is important to know how and why this fungus makes the potent carcinogen aflatoxin when it invades crops. Studies have been conducted to understand the roles of hypothetical genes (genes whose function is not yet known) in aflatoxin biosynthesis. Development of rational intervention strategies to prevent preharvest aflatoxin contamination depends on a full understanding of aflatoxin biosynthesis.

5. Deoxyribonucleic Acid (DNA) probes (primer sets) identified for universal screening for genetic variability of aspergillus (A.) group fungi. Studies on the molecular characterization of the aflatoxin biosynthetic pathway from the aflatoxigenic cousin of A. flavus, namely toxin-producing A. ochraceoroseus, A. rambelli, as well as, non-toxigenic A. oryzae, were done to determine if aflatoxin production provides a competitive advantage to A. flavus for its ability to survive in field conditions. Strains of an A. parasiticus isolate with specific deletions of aflatoxin pathway genes have been compared for morphological and physiological differences due to the knocking-out of critical pathway genes. This will help us understand if the deletion of these pathway genes has an effect on the fungus in its ability to invade crops.

6. Antioxidants inhibit aflatoxin production. Aflatoxins are cancer causing compounds produced by the fungus Aspergillus flavus when it invades crops. Strategies are being developed to prevent the fungal growth or its ability to make the toxin. Caffeic acid, an antioxidant, was found to reduce >95% of aflatoxin production by Aspergillus flavus without affecting fungal growth. Genetic studies suggest that antioxidants may trigger induction of the enzymes, alkyl hydroperoxide reductases, that protect the fungus from oxidizing agents that are produced when the fungus invades the crop. Consequently, aflatoxin synthesis is prevented through molecular regulation of toxin synthesis. ARS scientists at New Orleans, LA, and Western Regional Research Center, Albany, CA, have, therefore, discovered how to prevent aflatoxin production with safe, common natural chemicals. In addition, we have shown how these compounds work in the fungus so as to turn-off the aflatoxin biosynthetic machinery of the fungus. In short, the compounds trick the fungus into "thinking" that it does not need to produce aflatoxin, which are produced by the fungi to protect them from chemical attacks from plants. This information should help in devising methods of breeding crop plants to prevent aflatoxin contamination. It also provides us with significant insights as to how to control the genes that trigger biosynthesis of aflatoxins.

Review Publications
Chang, P.-K., Scharfenstein, L.L., Luo, M., Mahoney, N.E., Molyneux, R.J., Yu, J., Brown, R.L., Campbell, B.C. 2010. Loss of msnA, a putative stress regulatory gene, in Aspergillus parasiticus and Aspergillus flavus increased production of conidia, aflatoxins and kojic acid. Toxins. 3:82-104.

Wu, F., Bhatnagar, D., Bui-Klimke, T., Carbone, I., Hellmich II, R.L., Munkvold, G., Paul, P., Payne, G., Takle, E. 2011. Climate change impacts on mycotoxin risks in US maize. World Mycotoxin Journal. 4(1):79-93.

Last Modified: 8/3/2015
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