The bZIP Transcription Factor AflRsmA Regulates Aflatoxin B1 Biosynthesis, Oxidative Stress Response and Sclerotium Formation in Aspergillus flavus

Abstract

Fungal secondary metabolites play important roles not only in fungal ecology but also in humans living as beneficial medicine or harmful toxins. In filamentous fungi, bZIP-type transcription factors (TFs) are associated with the proteins involved in oxidative stress response and secondary metabolism. In this study, a connection between a bZIP TF and oxidative stress induction of secondary metabolism is uncovered in an opportunistic pathogen Aspergillus flavus, which produces carcinogenic and mutagenic aflatoxins. The bZIP transcription factor AflRsmA was identified by a homology research of A. flavus genome with the bZIP protein RsmA, involved in secondary metabolites production in Aspergillusnidulans. The AflrsmA deletion strain (ΔAflrsmA) displayed less sensitivity to the oxidative reagents tert-Butyl hydroperoxide (tBOOH) in comparison with wild type (WT) and AflrsmA overexpression strain (AflrsmA^OE), while AflrsmA^OE strain increased sensitivity to the oxidative reagents menadione sodium bisulfite (MSB) compared to WT and ΔAflrsmA strains. Without oxidative treatment, aflatoxin B₁ (AFB₁) production of ΔAflrsmA strains was consistent with that of WT, but AflrsmA^OE strain produced more AFB₁ than WT; tBOOH and MSB treatment decreased AFB₁ production of ΔAflrsmA compared to WT. Besides, relative to WT, ΔAflrsmA strain decreased sclerotia, while AflrsmA^OE strain increased sclerotia. The decrease of AFB₁ by ΔAflrsmA but increase of AFB₁ by AflrsmA^OE was on corn. Our results suggest that AFB₁ biosynthesis is regulated by AflRsmA by oxidative stress pathways and provide insights into a possible function of AflRsmA in mediating AFB₁ biosynthesis response host defense in pathogen A. flavus.

Keywords: Aspergillus flavus; aflatoxin B1; bZIP transcription factor; oxidative stress response; sclerotium formation.

Figure 1

Gene structure, functional domain, and phylogenetic analysis of A. flavus AflRsmA. (A) Gene structure of A. flavus AflrsmA. The upper lane indicates gene structure of AflrsmA (AFLA_133560) and the adjacent gene (AFLA_133570) from NCBI database. The lower lane indicates gene structure of AflrsmA identified in this study. The blue box and the line represent the exons and the introns of AflrsmA gene, respectively. (B) Functional domains of A. flavus bZIP-type TF AflRsmA. bZIP, basic leucine zipper domain; Redox domain, Yap1 redox domain. The number under the protein indicates the position of domain and the protein length. The protein domains were predicted manually using InterProScan 5 on EBI web server [36]. Candida albicans FCR3 (Q8X229), S. cerevisiae Yap3 (NP_011854.1); A. nidulans RsmA (AN4562.2, XP_662166), A. fumigatus RsmA (AFUA_2G02540, XP_749389), M. oryzae MoRsmA (MGG_02632, XP_003721157.1), and F. graminearum GzbZIP020 (FGSG_13313, ESU14603). (C). Phylogenetic analysis of bZIP-type TF RsmA from and RsmA orthologs that have been functionally verified in different fungi. The protein sequences were aligned with Clustal X and the maximum likelihood tree was generated using MEGA7.0 software. RsmA from A. flavus is in bold.

Figure 2

Generation of AflrsmA deletion and overexpression strains. (A). Transcription level analysis of AflrsmA by RT-PCR. (B). Schematic illustration of the deletion and overexpression of AflrsmA gene. The selection marker is the pyrG gene from A. fumigatus. (C). AflrsmA deletion strain verified by southern blot analysis. PCR fragment of 3′ flanking region was used as the probe. Genomic DNA from WT and ΔAflrsmA strains were digested with XhoI. The expected size is 8.2 kb and 3 kp for WT and for ΔAflrsmA respectively. (D). Transcription level of AflrsmA gene in overexpression strain verified by RT-PCR. The internal control gene is actin gene.

Figure 3

Comparison of the oxidative stress tolerance of A. flavus WT strains and mutant. (A) Mycelia growth of the A. flavus WT and AflrsmA mutants under oxidative stress. Two hundred conidia of A. flavus WT and AflrsmA mutants were cultured on YGT media supplemented with or without MSB (0.2 mM), H₂O₂ (6 mM), or tBOOH (1.2 mM) at 29 °C for five days. (B) Statistical analysis of colony diameter of the testing strains measured on the 5th day. Each treatment included three replicates. Error bars represent the standard deviations.

Figure 4

Assessment of Aflatoxin B₁ production of A. flavus WT and AflrsmA mutants under various conditions by thin-layer chromatography. All testing strains were cultured on YES medium, or YGT medium with or without MSB (0.2 mM), H₂O₂ (6 mM) and tBOOH (1.2 mM) for five days at 29 °C. AFB₁ = aflatoxin B₁ standard.

Figure 5

Sclerotial formation by A. flavus WT and AflrsmA mutants. (A) Visual phenotype of sclerotia production. 10³ conidia/plate was incubation on Wickerham medium and cultured for seven days at 37 °C. (B) Variation in sclerotial production by the WT and AflsrmA mutants on the Wickerham medium for seven days. The letter represents a significant difference at the level p < 0.05. Errors bars represent standard errors from three replicates.

Figure 6

Effect of AflrsmA on A. flavus Pathogenicity. (A) Growth of fungal colonies on living corn after five days of inoculation. (B) Spore production on corn after five days of inoculation. Data were analyzed using the GraphPad Instat software package version 5.01 (Graph Pad software Inc., San Diego, CA, USA) according to the Tukey–Kramer multiple comparison test at p < 0.05. The letter indicates statistical significance at p < 0.05. (C) Thin layer chromatography analysis of aflatoxin B₁ extracted from host plant corn. AFB₁ = aflatoxin B₁ standard.