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Aflatoxin biosynthesis is correlated with oxidative stress and is proposed to function as a secondary defense mechanism to redundant intracellular reactive oxygen species (ROS). We find that the antioxidant gallic acid inhibits aflatoxin formation and growth in Aspergillus flavus in a dose-dependent manner. Global expression analysis (RNA-Seq) of gallic acid-treated A. flavus showed that 0.8% (w/v) gallic acid revealed two possible routes of aflatoxin inhibition. Gallic acid significantly inhibited the expression of farB, encoding a transcription factor that participates in peroxisomal fatty acid β-oxidation, a fundamental contributor to aflatoxin production. Secondly, the carbon repression regulator encoding gene, creA, was significantly down regulated by gallic acid treatment. CreA is necessary for aflatoxin synthesis, and aflatoxin biosynthesis genes were significantly downregulated in ∆creA mutants. In addition, the results of antioxidant enzyme activities and the lipid oxidation levels coupled with RNA-Seq data of antioxidant genes indicated that gallic acid may reduce oxidative stress through the glutathione- and thioredoxin-dependent systems in A. flavus.

The study of aflatoxin in Aspergillus spp. has garnered the attention of many researchers due to aflatoxin's carcinogenic properties and frequency as a food and feed contaminant. Significant progress has been made by utilizing the model organism Aspergillus nidulans to characterize the regulation of sterigmatocystin (ST), the penultimate precursor of aflatoxin. A previous forward genetic screen identified 23 A. nidulans mutants involved in regulating ST production. Six mutants were characterized from this screen using classical mapping (five mutations in mcsA) and complementation with a cosmid library (one mutation in laeA). The remaining mutants were backcrossed and sequenced using Illumina and Ion Torrent sequencing platforms. All but one mutant contained one or more sequence variants in predicted open reading frames. Deletion of these genes resulted in identification of mutant alleles responsible for the loss of ST production in 12 of the 17 remaining mutants. Eight of these mutations were in genes already known to affect ST synthesis (laeA, mcsA, fluG, and stcA), while the remaining four mutations (in laeB, sntB, and hamI) were in previously uncharacterized genes not known to be involved in ST production. Deletion of laeB, sntB, and hamI in A. flavus results in loss of aflatoxin production, confirming that these regulators are conserved in the aflatoxigenic aspergilli. This report highlights the multifaceted regulatory mechanisms governing secondary metabolism in Aspergillus. Additionally, these data contribute to the increasing number of studies showing that forward genetic screens of fungi coupled with wholegenome resequencing is a robust and cost-effective technique.IMPORTANCE In a postgenomic world, reverse genetic approaches have displaced their forward genetic counterparts. The techniques used in forward genetics to identify loci of interest were typically very cumbersome and time-consuming, relying on Mendelian traits in model organisms. The current work was pursued not only to identify alleles involved in regulation of secondary metabolism but also to demonstrate a return to forward genetics to track phenotypes and to discover genetic pathways that could not be predicted through a reverse genetics approach. While identification of mutant alleles from whole-genome sequencing has been done before, here we illustrate the possibility of coupling this strategy with a genetic screen

Aspergillus flavus is a saprophytic soil fungus that poses a serious threat worldwide as it contaminates many food and feed crops with the carcinogenic mycotoxin called aflatoxin. This pathogen persists as sclerotia in the soil which enables fungal survival in harsh environmental conditions. Sclerotia formation by A. flavus depends on successful cell communication and hyphal fusion events. Loss of LaeA, a conserved developmental regulator in fungi, abolishes sclerotia formation in this species whereas overexpression (OE) of laeA results in enhanced sclerotia production. Here we demonstrate that sclerotia loss and inability to form heterokaryons in A. flavusΔlaeA is mediated by homologs of the Neurospora crassa ham (hyphal anastomosis) genes termed hamE-I in A. flavus. LaeA positively regulates ham gene expression and deletion of hamF, G, H, or I phenocopies ΔlaeA as demonstrated by heterokaryon and sclerotia loss and reduced aflatoxin synthesis and virulence of these mutants. Deletion of hamE showed a less severe phenotype. hamE-I homologs are positively regulated by the clock controlled transcription factor ADV-1 in N. crassa. Similarly, the ADV-1 homolog NosA regulates hamE-I expression in A. flavus, is required for sclerotial development and is itself positively regulated by LaeA. We speculate that a putative LaeA>NosA>fusion cascade underlies the previously described circadian clock regulation of sclerotia production in A. flavus.