2010 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.
The fungi, Aspergillus (A.) flavus and A. parasiticus, produce the potent cancer-causing compounds, aflatoxins, when these fungi invade crops. For the first time, we have deciphered how the toxins are made by these fungi, but it is still unknown why these fungi make the toxins. For this understanding, a set of non-aflatoxin mutants prepared through a number of different manipulations of the parent toxin-producing strains has been used to compare the ability of all these strains to withstand stress conditions and to adapt to a variety of environmental changes, such as soil conditions, ultra-violet (UV) light, and osmotic stress. Another project continues to identify, through Aspergillus flavus genomics (the study of the entire complement of genes in an organism) technologies carried out at John Craig Venter Institute (JCVI), previously named Institute for Genomic Research (TIGR)) in collaboration with North Carolina State University (NCSU), the complex array of genes that is involved in fungal virulence, aflatoxin formation, fungal reproduction/survival, as well as, in signals exchanged between the fungus and the host crop and the environment. Two different format whole genome microarrays (slides containing spots of deoxyribonucleic acid (DNA) corresponding to fragments of all unique genes) designed under this project have been used to identify critical genes involved in fungal response to various environmental factors favoring toxin production. The whole genome oligo microarrays (one type) containing over 12,000 A. flavus and A. oryzae unique genes (and containing a few relevant corn genes) has been constructed at JCVI. The other type whole genome microarray has been constructed at Affymetrix Company in collaboration with NCSU. The microarry studies are providing significant insights into global regulatory elements in the fungus that affect toxin production and fungal development.
In collaboration with the National Institute of Advanced Industrial Science and Technology, Japan, a comparison of the gene expression profiles between the field fungus (A. flavus) and its domesticated cousin (A. oryzae) has provided insights into any competitive advantage that A. flavus may possess as it survives in field conditions. Specific DNA sequences (genes) have been identified that are providing us with insights into our ability to distinguish between aflatoxin-producing and non-producing Aspergillus species; the functional role of these specific genes is being determined. Specific gene sequences will also allow us to distinguish toxigenic fungi from non-toxigenic fungi.
The finding that a putative oxidative stress-induced signaling pathway, discovered in cooperation with Agricultural Research Service/Western Regional Research Center scientists through comparison of A. flavus and yeast genomic data, is suppressed by plant antioxidants substantiates the correlation of highly expressed stress induced kernel proteins with a lowering of aflatoxin levels and points to solutions through breeding to enhance stress resistance traits that suppress toxin production.
Antioxidants Inhibit Aflatoxin Production. 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. We 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.
Proof of Involvement of Genes in Aflatoxin Synthesis and Fungal Development Obtained. Using sophisticated molecular techniques, we have tested specific interactions of key aflatoxin developmental regulatory factors. Further, the genetic basis for loss of aflatoxin production in toxin-deficient mutants of A. 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. We 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.
Deoxyribonucleic Acid (DNA) Probes (primer sets) Identified for Universal Screening for Genetic Variability of Aspergillus 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 these genes have effects on the fungus having any benefit in invasion of crops.
Details of the Aflatoxin Biosynthetic Pathway Further Elucidated. 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.
Cyclopiazonic Acid is Another Toxic Compound that the Fungus Aspergillus flavus Produces Along with Aflatoxins. In collaboration with National Peanut Research Laboratory (NPRL), we 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. Using this information, we 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 AF gene clusters, which ensures the safe application of this genuine 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.
Sequencing of All the Deoxyribonucleic Acid (DNA) of the Fungus Aspergillus flavus and Its Applications. 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 us and our collaborators to analyze which genes are affected under varying conditions:.
1)nutritional (high or low carbon source);.
3)developmental (veA mutant); and.
4)during the fungus-corn interaction (cooperation with Agricultural Research Service (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 have indicated that under temperature stress (47°C), spores of toxigenic strains survived longer than non-aflatoxin producers; and 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. A database for storing and analyzing data relating to Aspergillus flavus has been implemented at North Carolina State University under a specific cooperative agreement funded from this project’s funds. 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.
Yu, J., Nierman, W.C., Bennett, J.W., Cleveland, T.E., Bhatnagar, D., Campbell, B.C., Dean, R.A., Payne, G.A. 2010. Genetics and Genomics of Aspergillus flavus. In: Rai, M.K., Kovics, C., Editors. Progress in Mycology. Scientific Publishers (India). p.51-73.
Chang, P-K. 2010. Aflatoxin Biosynthesis and Sclerotial Development in Aspergillus flavus and Aspergillus parasiticus. In M. Rai and A. Varma (eds), Mycotoxins in Food, Feed and Bioweapons. Springer-Verlag Berlin Heidelberg. p 77-92.
Yu, J., Chang, P.-K., Cleveland, T.E., Bennett, J.W. 2010. Aflatoxins. In: Encyclopedia of Industrial Biotechnology: Bioprocess, Bioseparation, and Cell Technology, M.C. Flickinger, Editor. John Wiley & Sons, Inc.: Hoboken, New Jersey. pp. 1-12.
Klich, M.A. 2009. Health Effects of Aspergillus in Food and Air. Toxicology and Industrial Health. 25(9-10):657-667.
Chang, P-K., Ehrlich, K. 2010. What Does Genetic Diversity of Aspergillus flavus Tell Us About Aspergillus oryzae? International Journal of Food Microbiology. 138(3):189-199.
Chang, P-K., Ehrlich, K., Fujii, I. 2009. Cyclopiazonic Acid Biosynthesis of Aspergillus flavus and Aspergillus oryzae. Toxins. 1:74-99.
Georgianna, D., Fedorova, N.D., Burroughs, J.L., Dolezal, A.L., Bok, J., Horowitz-Brown, S., Woloshuk, C.P., Yu, J., Keller, N.P., Payne, G.A. 2010. Beyond aflatoxin: four distinct expression patterns and functional roles associated with Aspergillus flavus secondary metabolism gene clusters. Molecular Plant Pathology. 11(2):213-226.