The filamentous fungus, Aspergillus parasiticus grows on a variety of susceptible food crops and produces aflatoxin, a carcinogenic secondary metabolite that is both a health and economic threat. Aflatoxin biosynthesis is one of the most well-characterized eukaryotic secondary metabolic pathways. Understanding how aflatoxin biosynthesis initiates and where aflatoxin is made are critical to control synthesis and export of fungal metabolites. A previous study conducted in the Linz laboratory using electrophoretic mobility shift analysis (EMSA) and chromatin immunoprecipitiation (ChIP) found that the bZIP transcription factor AtfB binds to promoters of specific aflatoxin and stress response genes. Once aflatoxin gene expression initiates, our lab showed that A. parasiticus performs synthesis, compartmentalization, and export of aflatoxin in subcellular organelles called toxisomes. This dissertation is dedicated to further characterizing the role of transcriptional regulation and subcellular localization of aflatoxin biosynthesis specifically through two cellular targets, AtfB and Vps34. In order to study function of these two targets, we exploited a novel and endogenous CRISPR/cas9-like system in A. parasiticus to down-regulate function of AtfB and Vps34. This system is an easy, rapid, and highly efficient co-transformation method; 1 out of every 3 niaD + (the gene encoding nitrate reductase [niaD] is the selectable marker) transformants exhibited an atypical phenotype that we later showed was associated with the presence and expression of the disruption construct. The disruption of AtfB resulted in a consistent phenotype of low aflatoxin production, lower conidiospore numbers, and lower levels of spore pigmentation. Down-regulation of AtfB targets involved in aflatoxin biosynthesis and stress response showed that the level of expression of target genes is consistent with the observed phenotype of the disruption strains. Of particular importance, global expression data and computer-based network analysis also suggested the AtfB network extends beyond mycotoxin biosynthesis and stress response. Interestingly, the disruption of Vps34 resulted in the opposite phenotype compared to disruption of AtfB. Vps34 transformants exhibited high aflatoxin production, high conidiospore numbers, and high levels of spore pigmentation. We conducted an independent experiment using 3-methyladenine, a biochemical inhibitor of Vps34. Treatment of A. parasiticus SU-1 with 3-methyladenine caused a 10-fold increase in aflatoxin levels detected in aflatoxin-inducing medium. This biochemical approach supports the phenotype of high aflatoxin levels observed in Vps34 disruption strains. We further propose that Vps34 negatively regulates the transport of toxisomes to the vacuoles. Vps34 also positively regulates the export pathway directing early endosomes carrying aflatoxin for export out of the cell. Fungal targets like AtfB and Vps34 that differentially regulate aflatoxin biosynthesis serve as useful tools to understand the molecular mechanisms that contribute to fungal virulence. From a public health perspective, our research goal is to use practical and sustainable natural inhibitors that target fungal specific cellular targets to block toxin production in the field and in storage.
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