Classical and Modern Genetic Approaches Reveal New Gene Associations with Aflatoxin Biosynthesis in Aspergillus parasiticus and A. flavus.

Abstract

Production of aflatoxin (AF) in Aspergillus species is a highly regulated process involving transcriptional and post-transcriptional controls. Most of the regulation of AF production is focused through the pathway-specific transcriptional regulator aflR. While much is understood about the steps involved in biosynthesis, less is known about the regulatory circuits controlling AF production. A targeted cDNA microarray consisting of 768 genes was developed to investigate the effect of nitrogen source, carbon source, culture temperature and culture pH on AF production in A. parasiticus. Seventeen genes were identified as consistently differentially expressed with respect to AF, including three of the AF pathway structural genes. One of these genes, CA747470 was consistently downregulated with AF and was shown to repress AF production when overexpressed in A. flavus.

Using an expanded cDNA microarray consisting of 5002 genes from an EST sequencing project (USDA-ARS, SRRC), we investigated the impact of aflR deletion on the transcriptome of A. parasiticus. In addition to the AF pathway genes, five additional genes were found to be regulated by aflR: niiA, hlyC, hypA, nadA, and hypB. These additional genes all possess putative consensus binding sites for AflR. The expression data from this study was also compared to the previous targeted array study by looking at expression of 324 genes shared by both microarrays. Expression profiles for the AF genes present on both arrays were consistent between experiments. CA747470 was shown to be highly expressed in all conditions. Overexpression of CA747470 resulted in increased radial growth and decreased AF production.

Finally, a putative Rho-GDP dissociation inhibitor (Afrdi1) was deleted in A. flavus that was found to share a transcription profile with aflR with respect to AF. The Afrdi1 deletion strain exhibited repressed AF production, as well as a severe growth defect on minimal medium. The deletion mutant was phenotypically similar to the bem4 deletion strain of S. cerevisiae. The implication of this gene in AF regulation provides a direct link between vegetative growth and secondary metabolism in A. flavus.

This work provides insight into the regulatory networks responsible for regulation of AF production in Aspergillus species, and indicates where future investigations are needed to understand the biology of this important mycotoxin.