Histone 2-Hydroxyisobutyryltransferase Encoded by Afngg1 Is Involved in Pathogenicity and Aflatoxin Biosynthesis in Aspergillus flavus

Abstract

Aflatoxin, a carcinogenic secondary metabolite produced by Aspergillus flavus, is a significant threat to human health and agricultural production. Histone 2-hydroxyisobutyrylation is a novel post-translational modification that regulates various biological processes, including secondary metabolism. In this study, we identified the novel histone 2-hydroxyisobutyryltransferase Afngg1 in A. flavus, and explored its role in cell growth, development and aflatoxin biosynthesis. Afngg1 gene deletion markedly decreased lysine 2-hydroxyisobutyrylation modification of histones H4K5 and H4K8 compared with the control strain. Additionally, Afngg1 deletion inhibited mycelial growth of A. flavus, and the number of conidia and hydrophobicity were significantly decreased. Notably, aflatoxin B₁ biosynthesis and sclerotia production were completely inhibited in the ΔAfngg1 strain. Furthermore, the pathogenicity of the ΔAfngg1 strain infecting peanut and corn grains was also diminished, including reduced spore production and aflatoxin biosynthesis compared with A. flavus control and Afngg1 complementation strains. Transcriptome analysis showed that, compared with control strains, differentially expressed genes in ΔAfngg1 were mainly involved in chromatin remodelling, cell development, secondary metabolism and oxidative stress. These results suggest that Afngg1 is involved in histone 2-hydroxyisobutyrylation and chromatin modification, and thus affects cell development and aflatoxin biosynthesis in A. flavus. Our results lay a foundation for in-depth research on the 2-hydroxyisobutyrylation modification in A. flavus, and may provide a novel target for aflatoxin contamination prevention.

Keywords: 2-hydroxyisobutyryltransferase; Afngg1; Aspergillus flavus; aflatoxin; pathogenicity.

Figure 1

Bioinformatics analysis, subcellular localisation, and modification sites identification of Afngg1. (A) Phylogenetic tree based on sequence alignment. The numbers on the branches indicate bootstrap support value. (B) The domains of Afngg1 from the above nine species were further visualised by DOG2.0. The green area represented the Ada3 domain. (C) Subcellular localization of Afngg1. (D, E) Level of 2-hydroxyisobutyrylation modification of histones H4K5 and H4K8 in A. flavus control and ΔAfngg1 strains. *** represents p < 0.001.

Figure 2

Effects of Afngg1 on the growth, conidia and aflatoxin biosynthesis of A. flavus. (A) The colony (a), stereoscopic microscope (b) and conidiophores (c) analysis. A. flavus control strain, ΔAfngg1 and Afngg1-Com strains were cultivated on PDA medium at 37 °C for 5 days. (B) SEM analysis of conidia (a) and conidial heads (b). (C) Determination of colony diameter cultured on PDA medium. (D) Determination of the number of conidia from different strains. (E) TLC analysis of AFB₁ production. *** represents p < 0.001.

Figure 3

Analysis of hydrophobicity and sclerotium yield. (A) Determination of hydrophobicity of A. flavus control strain, ΔAfngg1 and Afngg1-Com strains. Colony morphology (a), microscope (b) and stereoscopic microscopic (c) analysis after Bromophenol blue treatment. (B) Colony morphology of different strains after 7 days of culture on PDA medium. The plate was sprayed with 70% alcohol to expose the sclerotium. (C) Determination of the number of sclerotium in (B). *** represent p < 0.001.

Figure 4

Effect of Afngg1 on the ability of A. flavus to infect crops. (A) Colonisation of peanut and corn grains by A. flavus control, ΔAfngg1 and ΔAfngg1-Com strains. (B) The number of conidia number was determined from the infected peanut and corn grains; (C) TLC analysis of AFB₁ production from infected peanut and corn grains. *** represents p < 0.001.

Figure 5

Sample correlation and gene expression analysis. (A) Person correlation coefficient. (B) Violin plot. (C) Volcano map of DEGs. (D) Total number of DEGs.

Figure 6

Biological process (A), cellular component (B), molecular function (C) and KEGG pathway (D) analysis of DEGs.