2009 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, 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. 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, the complex array of genes that are 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. With the advances of the Southern Regional Research Center (SRRC) genomics program on deciphering genes involved in aflatoxin production in A. flavus, the entire A. flavus genome (i.e. all of the deoxyribonucleic acid (DNA) contained in the fungus) has been sequenced at John Craig Venter Institute (JCVI, previously named Institute for Genomic Research (TIGR)) in collaboration with North Carolina State University (NCSU). Two different format whole genome microarrays (slides containing spots of 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.
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) have 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 will potentially allow us to distinguish between aflatoxin-producing and non-producing Aspergillus species; the functional role of these specific genes is being determined. Specific gene sequences will allow us to distinguish toxigenic fungi from non-toxigenic fungi.
A putative oxidative stress-induced signaling pathway, discovered in cooperation with ARS/WRRC scientists through comparison of A. flavus and yeast genomic data, is suppressed by plant antioxidants. This finding through molecular examination of the stress signaling pathway governing aflatoxin production in the fungus 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. Microarray analysis of caffeic acid-treated A. flavus indicated expression of almost all genes in the aflatoxin biosynthetic cluster were down-regulated (decreased), with the exception of genes norB and the aflatoxin-pathway regulator-gene, aflJ, which showed low expression levels in both treated and control fungi. The secondary metabolism regulator-gene, laeA, also showed little change in expression levels. Alternatively, expression of genes in metabolic pathways of the fungus were up-regulated (increased). The most notable up-regulation of A. flavus expression occurred in four genes that encode specific enzymes, such as alkyl hydroperoxide reductases that detoxify organic peroxides (harmful chemical compounds produced in cells). These findings suggest 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. Microarray experiments have been completed and candidate gene expression profiles have been validated for the genes that are potentially involved in the control of fungal development and secondary metabolism (dependent on the global regulatory gene veA). The microarray data has been generated and we are currently analyzing this data to determine if there are any clusters of genes that are regulated by the gene veA and that demonstrate expression profiles expected for genes involved in sclerotial (over-wintering structures of the fungus) development or aflatoxin production. Using sophisticated molecular techniques, such as yeast two-hybrid system and immunoprecipitation, we have begun to test 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. Reproducible data using various molecular techniques suggested that although the regulatory genes aflR, aflJ, and laeA are necessary for aflatoxin production, they are not sufficient. Additional factors are needed for complete regulation of the turning on and off of aflatoxin production. Additionally, recently a highly-efficient gene targeting approach has been developed to investigate the functions of those candidate genes identified by the microarray DNA technology and potentially related to aflatoxin production. This technology will help us in rapidly assessing the critical role of several genes of interest.
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, are on-going. These studies are being conducted to determine if aflatoxin production provides a competitive advantage to A. flavus for its ability to survive in field conditions. In order to distinguish pathogenic Aspergillus species from non-pathogenic organisms, we have been looking for genes that contain SNPs (single nucleotide polymorphisms or variations in single bases in fungal DNA) unique to each type. Of the four common aflatoxin-producing Aspergilli: large sclerotia-producing A. flavus, small sclerotia-producing A. flavus (A. parvisclerotigenus), A. parasiticus, and (A. minisclerotigenes) (formerly the west African variant of A. parasiticus), we have identified usable polymorphisms (many different forms) in genes encoding enzymes, such as an amylase, a xylanase, and a methyl transferase. Some of these genes are easy to isolate from soil or food or feed samples by molecular techniques (such as, Polymerase Chain Reaction or PCR) and rapid sequencing with high throughput DNA sequencing methodology which allows rapid determination of type of fungus and source. Work continues on finding the aflatoxin pathway genes in A. ochraceoroseus, which will be compared to that of A. flavus to determine the evolutionary relationship of aflatoxin production in the two species. Strains of an A. parasiticus isolate with specific deletions of aflatoxin pathway genes are being compared for morphological and physiological differences due to the knock-outs. This will help us understand if these genes have effects on characteristics outside of the aflatoxin biosynthetic pathway.
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. Knock-out of these genes (that is, disrupting their functionality) in either A. parasiticus or A. flavus, has been successful and the metabolites made by these mutants have been characterized. In addition, aflatoxin non-producing mutants have been prepared in Aspergillus parasiticus by knocking out genes involved in early and late steps in the aflatoxin biosynthesis. These include mutants of the genes that code for key enzymes in the aflatoxin biosynthetic pathway namely, polyketide synthase, averantin oxidase, hydroxyversicolorin oxidase, versicolorin A oxidase, and O-methylsterigmatocystin oxidase. Similarly, atoxigenic mutant strains of A. flavus have been developed. We have used these in comparative studies to test the importance of these genes in fungal survival under a variety of growth conditions that mimic natural field conditions to which the fungi were subjected.
Sequencing of all the DNA of the fungus Aspergillus flavus and its applications: Two different format whole genome microarrays (slides containing spots of deoxyribonucleic acid (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 been studied. Preliminary results indicate that under temperature stress (47°C), spores of toxigenic strains survived longer than non-aflatoxin producers. Preliminary results showed 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. From this list, about 20 genes have been selected for further study. Assigning specific function to these genes in a complicated biological system is a limiting factor in further studies, because it is time consuming and requires careful interpretation of data. A database for storing and analyzing data relating to Aspergillus flavus is being 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.
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Chang, P.-K. 2008. Aspergillus parasiticus crzA, Which Encodes
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Chang, P.-K., Horn, B.W., Dorner, J.W. 2009. Clustered Genes Involved in Cyclopiazonic Acid Production are Next to the Aflatoxin Biosynthesis Gene Cluster in Aspergillus flavus. Fungal Genetics and Biology. 46:176-182.
Bhatnagar, D., Perrone, G., Visconti, A. 2008. The MYCOGLOBE Project: A European Union Funded Successful Experiment in Enhancing Cooperation and Coordination Amongst Mycotoxin Researchers Worldwide. World Mycotoxin Journal. 1(4):493-500.
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