Abstract

Aspergillus flavus is a saprophytic fungus that can be found across the entire world. It can produce aflatoxin B1 (AFB1), which threatens human health. CreA, as the central factor in carbon catabolite repression (CCR), regulates carbon catabolism and AFB1 biosynthesis in A. flavus. Additionally, SsnF-RcoA are recognized as the corepressors of CreA in CCR. In this study, ssnF and rcoA not only regulated the expressions of CCR factors and hydrolase genes, but also positively affected mycelia growth, conidia production, sclerotia formation, and osmotic stress response in A. flavus. More importantly, SsnF and RcoA were identified as positive regulators for AFB1 biosynthesis, as they modulate the AF cluster genes and the relevant regulators at a transcriptional level. Additionally, the interactions of SsnF-CreA and RcoA-CreA were strong and moderate, respectively. However, the interaction of SsnF and RcoA was weak. The interaction models of CreA-SsnF, CreA-RcoA, and SsnF-RcoA were also simulated with a docking analysis. All things considered, SsnF and RcoA are not just the critical regulators of the CCR pathway, but the global regulators involving in morphological development and AFB1 biosynthesis in A. flavus.

Highlights

  • Aspergillus favus is an aerobic saprophytic fungus found in tropical and subtropical regions

  • Our results provide a comprehensive analysis of SsnF and RcoA in A. flavus and contribute to a better understanding of the relationship between the carbon catabolite repression (CCR) pathway and aflatoxin B1 (AFB1) biosynthesis

  • The recruiter proteins SsnF (AFLA_134730, 869 amino acid) in A. flavus is homologous with Cyc8/Ssn6 in S. cerevisiae or A. nidulans, and the transcriptional repressor RcoA (AFLA_054810, 586 amino acid) is derived from TupA in S. cerevisiae [16,18]

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Summary

Introduction

Aspergillus favus is an aerobic saprophytic fungus found in tropical and subtropical regions. The cluster-specific regulators, AflR and AflS, directly bind to the promoter regions and regulate the transcriptional expressions of AF’s biosynthetic genes [7]. Different environmental conditions, such as carbon sources, nitrogen sources, pH, light, temperature, water activity, phenolic compounds, lipids, and oxidative stress can affect AF’s production [8]. Carbon catabolite repression (CCR) is a critical regulatory pathway for preferential carbon utilization It is involved in fungal growth development and secondary metabolisms [9]. Our results provide a comprehensive analysis of SsnF and RcoA in A. flavus and contribute to a better understanding of the relationship between the CCR pathway and AFB1 biosynthesis

Results
SsnF and RcoA Affect the Expressions of Hydrolase Genes by Regulating CCR
Discussion
Sequence Analyses and Phylogenetic Tree Construction
Construction of the Deletion Cassette and Complementary Strains
The Extraction and Detection of AFB1 Production
RNA Extraction and RT-qPCR
Yeast Two-Hybrid Assay
Analysis of Osmotic Stress on Different Mutants
Protein Docking Analysis
Statistical Analysis
5.10. Experimental Design

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