2008 Annual Report
NP 108, Component: 2, Problem Statement: 2.1, 2.2, 2.4, 2.5.
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:.
NP 108, Component: 2, Problem Statement: 1, 2, 4, 5.2. 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). The only exceptions were 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.
NP 108, Component: 2, Problem Statement: 2.1, 2.2, 2.4, 2.5.3. 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.
NP 108, Component: 2, Problem Statement: 1, 2, 4, 5.4. 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 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.
NP 108, Component: 2, Problem Statement: 1, 2, 4, 5.5. 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, as well as the importance of the protein encoded by the genes NorA, NorB and NadA in the final steps in formation of the toxic metabolites aflatoxins. Development of rational intervention strategies to prevent preharvest aflatoxin contamination depends on a full understanding of aflatoxin biosynthesis. Knockout 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. We are using 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 would be subjected.
NP 108, Component: 2, Problem Statement: 2.1, 2.2, 2.4, 2.5.
Chang, P., Wilkinson, J.R., Horn, B.W., Yu, J., Bhatnagar, D., Cleveland, T.E. 2007. Genes differentially expressed by Aspergillus flavus strains after loss of aflatoxin production by serial transfers. Applied Microbiology and Biotechnology. 77(4):917-925. Crawford, J.M., Vagstad, A.L., Ehrlich, K., Townsend, C.A. 2008. Starter unit specificity directs genome mining of polyketide synthase pathways in fungi. Bioorganic Chemistry. 36:16-22. Crawford, J.M., Vagstad, A.L., Ehrlich, K., Udwary, D.W., Townsend, C.A. 2008. Acyl-carrier protein - Phosphopantetheinyltransferase partnerships in fungal fatty acid synthases. ChemBioChem. 9(10):1559-1563. Crawford, J.M., Vagstad, A.L., Whitworth, K.P., Ehrlich, K., Townsend, C.A. 2008. Synthetic strategy of nonreducing iterative polyketide synthases and the origin of the classical “starter-unit effect." ChemBioChem. 9(7):1019-1023. Bhatnagar, D., Rajasekaran, K., Cary, J.W., Brown, R.L., Yu, J., Cleveland, T.E. 2008. Molecular Approaches to Development of Resistance to Preharvest Aflatoxin Contamination. In: Leslie, J.F., Bandyopadhyay, R., and Visconti, A. (eds.) Mycotoxins: Detection Methods, Management, Public Health, and Agricultural Trade. Wallingford, Oxfordshire, UK:CABI Publishing. p. 257-276. Bhatnagar, D., Rajasekaran, K., Brown, R.L., Cary, J.W., Yu, J., Cleveland, T.E. 2008. Genetic and Biochemical Control of Aflatoxigenic Fungi. In: Wilson, C.L. (editor). Microbial Food Contamination. Boca Raton, Fl: CRC Press. 17:395-425. Bhatnagar, D., Rajasekaran, K., Payne, G.A., Brown, R.L., Yu, J., Cleveland, T.E. 2008. The "omics" tools: genomics, proteomics, metabolomics and their potential for solving the aflatoxin contamination problem. World Mycotoxin Journal. 1(1):3-12. Cary, J.W., OBrian, G.R., Nielsen, D.M., Nierman, W., Harris-Coward, P.Y., Yu, J., Bhatnagar, D., Cleveland, T.E., Payne, G.A., Calvo, A.M. 2007. Elucidation of veA-dependent genes associated with aflatoxin and sclerotial production in Aspergillus flavus by functional genomics. Applied Microbiology and Biotechnology. 76:1107-1118. Klich, M.A., Frisvad, J.C., Peterson, S.W., Varga, J., Geiser, D.M., Samson, R.A. 2007. The Current Status of Species Recognition and Identification in Aspergillus. Studies in Mycology. 59:1-10. Rokas, A., Payne, G., Fedorova, N.D., Baker, S.E., Machida, M., Yu, J., Georgianna, D.R., Dean, R.A., Bhatnagar, D., Cleveland, T.E., Wortman, J.R., Maiti, R., Joardar, V., Amedeo, P., Denning, D.W., Nierman, W.C. 2007. What Can Comparative Genomics Tell Us About Species Concepts in the Genus Aspergillus?. Studies in Mycology. 59:11-17. Kale, S.P., Cary, J.W., Hollis, N., Wilkinson, J.R., Bhatnagar, D., Yu, J., Cleveland, T.E., Bennett, J.W. 2007. Analysis of aflatoxin regulatory factors in serial transfer-induced non-aflatoxigenic Aspergillus parasiticus. Journal of Food Additives & Contaminants. 24(10):1061-1069. Chang, P. 2008. A highly efficient gene-targeting system for aspergillus parasiticus. Letters in Applied Microbiology. 46:587-592.