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Identification of AflR binding sites in the genome of Aspergillus flavus by ChIP-seq

ABSTRACT: We report here the AflR binding motif of Aspergillus flavus for the first time with the aid of ChIP-seq analysis. Of the 540 peak sequences associated with AflR binding events, 66.8% were located within 2 kb upstream (promoter region) of translational start sites. The identified 18-bp binding motif was a perfect palindromic sequence, 5′-CSSGGGWTCGAWCCCSSG’3′ with S representing G or C and W representing A or T. On closer examination, we hypothesized that the 18-bp motif sequence identified contained two identical parts (here called motif A and motif B). Motif A was in positions 8–18 on the upper strand, while motif B was in positions 11-1 on the bottom strand. The inferred length and sequence of the putative motif identified in A. flavus were similar to previous findings in A. parasiticus and A. nidulans. Gene ontology analysis indicated that AflR bound to other genes outside the aflatoxin biosynthetic gene cluster.

ORGANISM(S): Aspergillus flavus

PROVIDER: GSE149696 | GEO | 2020/05/02

REPOSITORIES: GEO

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Project description:Purpose: Aflatoxin B1 is the most toxic and carcinogenic compound in nature produced by Aspergillus fungi. In our study, we applied RNA-seq to compare the transcriptomic profiles of Aspergillus flavus strains in the presence and absence of medicinal plant Zanthoxylum bungeanum. Methods: mRNA profiles of Aspergillus flavus supplemented with 250 µg/ml of methanolic extract fraction (treated samples) or DMSO (control samples) were generated in triplicate, by an Illumina platform using paired-end 150 bp sequencing strategy. Clean paired-end reads were mapped to the reference genome of A. flavus NRRL3357. Gene expression quantification was calculated by FPKM (Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced). The differentially expressed genes (DGEs) between the control and test samples were analyzed using the DESeq2 R package. Results: RNA-seq produced 24.5–32.1 million clean paired-end reads (150 bp read length) per sample and most of them (83–91%) were uniquely mapped to the reference genome of A. flavus NRRL3357. Eighty-two percent of genes displayed FPKM value ≥1 and were thus classified as expressed genes. Ninety-six percent of the expressed genes were expressed both in the control and test groups whereas 2.3% and 1.8% of the genes were expressed only in the control or test group, respectively. With the combination of FPKM fold change ≥2 and adjusted p-value <0.05, we found in total 950 DEG’s. Among them, 515 genes were downregulated and 435 genes were upregulated. We used FungiFun software to analyze the functions of the DEGs based on FunCat pathways and categories. About half of the DEG’s had a relevant annotation in the FunCat database, and 60–70% of the annotated DEG’s were found to be enriched in specific functional pathways. Conclusions: we showed that simple organic extracts from Z. bungeanum inhibit both the growth and aflatoxin production by Aspergillus flavus. The repression of AF pathway is mediated by global regulators instead of pathway specific regulators AflR or AflS. Consistently, the expression of Velvet complex, a prominent regulator of fungal secondary metabolism and development, was substantially reduced. Natural compound extracts from Z. bungeanum have potential to facilitate the development of safe and economical control strategies that shutdown aflatoxin production in aflatoxigenic Aspergillus species.