Filamentous fungi are well-known producers of secondary metabolites (natural products) such as antibiotics, toxins, and pigments. These compounds often demonstrate no apparent advantage to the producing organisms and have complex biosynthetic pathways. Aflatoxins (AFs) are secondary metabolites produced by only certain Aspergillus species such as A. parasiticus and A. flavus and are highly toxic, mutagenic and carcinogenic to animals and humans. Many structural genes of the AF pathway have been cloned and characterized, but only one, the af/R gene, has been shown to positively regulate the transcription of the pathway structural genes. Mechanisms that govern af/R function however, remain largely unknown. Several recent studies includingours have suggested that aspects of fungal development and secondary metabolism are correlated. The main goals of this project are to identify new factors that act separately from, or in conjunction with af/R in controlling AF production and to determine the extent of correlation between AF regulation and fungal development. The present proposal is centered upon previously isolated A. parasiticus sec- (for secondary metabolism minus) strains, also displaying altered morphology and sporuiation. Recent work from our laboratory has shown that a mutation in af/R coding or promoter region is not responsible for the sec- phenotype. Yet, the af/R expression is lowered in the sec- strains, there is no expression of the AF pathway genes and af/R 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. In this study, the isogenic sec+/sec- pairs will be used in proteomics, specifically to identify and characterize the 'up' regulated proteins in the sec-strains since these are suggestive of negative regulators of af/R/AF biosynthesis. Through protein sequencing, identified genes will studied for their role in AF production and/or fungal development. Other experiments will include testing specific homologous and heterologous developmental genes [latter from a related sterigmatocystin (ST) pathway of A. nidulans] known to have a positive influence on secondary metabolism, for their possible role in the sec-reversal. Lastly, constructed A. flavus DNA microarrays (98 % homologous to A. parasiticus) will be used to identify the possible transcription-level differences between the isogenic sec+/sec- pairs and candidate genes will be isolated in future. Results obtained from this study will contribute toward its long-term goal of preventing preharvest AF contamination of susceptible crops by developing effective strategies such as 'designer' biocompetitive strains.
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