HexA is required for growth, aflatoxin biosynthesis and virulence in Aspergillus flavus

Abstract

Background: Woronin bodies are fungal-specific organelles whose formation is derived from peroxisomes. The former are believed to be involved in the regulation of mycotoxins biosynthesis, but not in their damage repair function. The hexagonal peroxisome protein (HexA or Hex1) encoded by hexA gene in Aspergillus is the main and the essential component of the Woronin body. However, little is known about HexA in Aspergillus flavus.

Results: In this study, hexA knock-out mutant (ΔhexA) and complementation strain (ΔhexA^C) were produced using homologous recombination. The results showed that, ΔhexA and ΔhexA^C were successfully constructed. And the data analysis indicated that the colony diameter, stress sensitivity and the sclerotia formation of A. flavus were nearly not affected by the absence of HexA. Yet, the deletion of hexA gene reduced the production of asexual spores and lessened virulence on peanuts and maize seeds markedly. In addition, it was also found that there was a significant decrease of Aflatoxin B1 production in deletion mutant, when compared to wild type.

Conclusions: Therefore, it suggested that the hexA gene has an essential function in conidia production and secondary metabolism in A. flavus. The gene is also believed to be playing an important role in the invasion of A. flavus to the host.

Keywords: Aflatoxins; Aspergillus flavus; Virulence; hexA.

Fig. 1

Phylogenetic and structure analysis of HexA proteins. a Phylogenetic analysis of HexA proteins from Aspergillus members and other fungi. Bootstrap values were calculated using the neighbor-joining method with 1000 replicates. A. flavus was underlined in red. b Schematic diagrams of HexA proteins. Conserved domain was signed, respectively

Fig. 2

Construction, verification and growth of hexA mutants. a The scheme of deletion strategy for hexA. In the gene product of hexA, N terminal (N), C terminal (C) and conserved domain (grey) was signed. b q-PCR verification of hexA gene deletion and complementation strains. The actin gene was used as an inner reference. c The role of hexA gene in mycelium growth

Fig. 3

Conidia production is attenuated in null hexA mutants. a The electron micrograph of conidiophores of WT, ΔhexA and ΔhexA^C. Strains were pre-cultured on PDA and Yes were transferred on sterile slides under 37 °C overnight to induce conidiophores. (Magnification scale: ×200) b The number of conidia in WT, ΔhexA and ΔhexA^C strains was measured after grown on YES and PDA agar for 5 days at 37 °C. Values are a mean of four replicates. ***Significant difference between the WT and mutant strains at P < 0.001, as assessed by one-way ANOVA and Dunnett’s multiple-comparisons test

Fig. 4

Phenotype analysis of hexA deletion, WT and ΔhexA^C strains on sclerotia production. a Morphology of strains on sclerotia-inducing WKM medium after being washed by 75% ethanol and their detail views. b The number of sclerotia in WT, ΔhexA and ΔhexA^C strains was measured after grown on WKM medium for 7 days at 37 °C in dark

Fig. 5

Deletion of hexA results in reduced AF production. a AFB₁ production ofWT, ΔhexA and ΔhexA^C were detected by TLC after cultured in YES liquid media for 6 days. b Quantification of AFB₁ production as in a. c qPCR result of AF biosynthetic related genes (aflK, aflD, aflR and aflS) at 72 h. Each bar indicates the mean ± standard deviation (SD) of four replicate assay experiments. ***Significant difference between the WT and mutant strains at P < 0.001, as assessed by one-way ANOVA and Dunnett’s multiple-comparisons test

Fig. 6

The growth of WT, ΔhexA and ΔhexA^C response to stressors. a The strains grew on YES media with 1 M NaCl, 100 μg/mL CFW, 100 μg/mL CR and 100 μg/mL SDS, respectively. b Determination of growth inhibition rate of a

Fig. 7

The effect of hexA gene on the pathogenicity of WT, ΔhexA and ΔhexA^C of A. flavus. a The strains of the WT, ΔhexA and ΔhexA^C were grown on peanut seeds at 28 °C for 7 days. b Conidia production was assessed from the infected peanut seeds. c Quantification of AFB1 production as in d. d Aflatoxin was detected by TLC, which extracted from infected peanut seeds. e The strains of the WT, ΔhexA and ΔhexA^C were grown on maize seeds at 28 °C for 7 days. f Conidia production was assessed from the infected maize seeds. g Quantification of AFB1 production as in h. h Aflatoxin was detected by TLC, which extracted from infected maize seeds. ***The bars represent significantly different (P < 0.001)