2012 Annual Report
1a.Objectives (from AD-416):
To determine the molecular basis for the phenomenon of non-production of aflatoxin in certain members of the Aspergillus (A.)flavus group of the fungi with a view to understanding the global regulation of toxin synthesis and the convergent evolution of aflatoxin biosynthesis.
1b.Approach (from AD-416):
The present proposal is centered upon previously isolated Apergillus (A.) parasiticus sec- (for secondary metabolism minus) strains, that display altered morphology and sporulation. Recent work from our laboratory has shown that a mutation in aflR coding or promoter region is not responsible for the sec-phenotype. Yet, the aflR expression is lowered in the sec-strains. There is no expression of the aflatoxin pathway genes, and aflR overexpression does not reverse the sec- phenotype. Preliminary results have also revealed clear differences between the sec- and their parental sec+ (for secondary metabolism plus) total protein-profiles. Proteomics and microarray technology will be used to determine the differences between sec+ and sec- strains at the molecular level. These differences will provide insights into the global regulatory mechanisms governing aflatoxin synthesis.
Aflatoxins are carcinogenic secondary metabolites produced by the saprophytic fungi (ones that live on decaying or organic debris), Aspergillus (A.) flavus and A. parasiticus. Due to the health impacts of aflatoxins, strict food regulations are enforced to minimize exposure. However, the regulations also reduce the profitability of affected crops. In our earlier studies, when normal development of A. parasiticus (sec+) was thwarted, by forced repeated mycelial (filamentous threads of the fungus) transfer, the resulting isolate (sec-) permanently lost some of its normal developmental functions, including the ability to produce aflatoxins. The defects, if any, created by mycelial transfer in these atoxigenic isolates (sec-) have been examined using microarray technique to examine expression of many genes at once. These (sec-) variants still produced gene expression transcripts (indication of gene expression) of aflatoxin biosynthesis pathway regulatory genes that may trigger aflatoxin production, aflR and aflJ. From previous studies at ARS we know that to produce aflatoxins, a set of 29 genes are turned on almost simultaneously to produce the necessary proteins (enzymes) required to make the toxin. Although we have found that there are two genes (aflJ and aflR) that regulate the genes needed for toxin synthesis, another protein (LaeA) has recently been identified as a key regulator of many secondary metabolites (compounds not required for growth, such as fungal toxins, mycotoxins). In this project, we have found that the protein LAEA binds to the two earlier identified regulatory proteins, AFLR and AFLJ, to affect the “turning on” of the toxin production. In other studies with ARS we showed that two other regulatory genes NsdC and NsdD are able to bind the global regulator of secondary metabolism and development, LaeA and affect fungal development. We now have a better understanding of the complex machinery that is needed by the fungus to allow it to start making the toxin. Through these studies, we hope to identify factors in the fungus that could be targeted for controlling aflatoxin formation, and consequently contamination of crops.