The putative forkhead transcription factor FhpA is necessary for development, aflatoxin production, and stress response in Aspergillus flavus

Abstract

Forkhead transcription factors regulate several important biological processes in many eukaryotic species including fungi. Bioinformatic analysis of the Aspergillus flavus genome revealed four putative forkhead transcription factor genes. Genetic disruption of (AFLA_005634), a homolog of the Aspergillus nidulans fhpA/fkhA gene (AN4521), revealed that the fhpA gene is a negative regulator of both asexual spore production and aflatoxin B1 production in A. flavus. Furthermore, disruption of the fhpA gene caused a complete loss of sclerotial formation. Overexpression of the fhpA gene caused A. flavus to become more sensitive to sodium chloride whereas disruption of the fhpA gene did not change the ability of A. flavus to respond to any osmotic stress agent tested. Interestingly, both disruption and overexpression of the fhpA gene led to increases in sensitivity to the oxidative stress agent menadione. Overall, these results suggest that fhpA is an important regulator of morphological and chemical development in addition to stress response in A. flavus.

Fig 1. Phylogenetic analysis of fungal Fox TFs.

A phylogenetic tree based on the Clustal Omega MSA was constructed using the maximum-likelihood (ML) method with a bootstrap analysis (1,000 replicates) with the support values shown at the nodes. Gene accessions listed in the tree correspond to those listed in Table 2. Boxes around the “AFLA” accession numbers correspond to the Fox TF proteins in the A. flavus genome.

Fig 2. Genetic disruption of the fhpA gene leads to increases in conidiospore production in A. flavus.

(A) The WT, ΔfhpA, and OEfhpA A. flavus strains were center point inoculated in triplicate onto PDA medium. Photographs of the cultures were taken after 7 days of incubation at 30 °C under light conditions. (B) Conidia was quantified in the strains by taking agar pieces from the cultures and quantifying the number of conidia present utilizing an automated cell counter. Error bars represent standard error and letters over the bars indicate statistical significance. Different letters indicate data that was statistically significant (p value ≤  0.05).

Fig 3. Sclerotial formation is abolished in the absence of the fhpA gene.

(A) The A. flavus wild-type, ΔfhpA strains, and OEfhpA strains were center-point inoculated onto Wickerham agar medium in triplicate then incubated for 7 days at 30 °C in the dark. Photographs were taken before and after 70% ethanol washes that were used to visualize sclerotial production. Micrographs were taken of each culture after ethanol washing. (B) Quantification of sclerotia from the same cultures displayed in Fig 3, Panel A was carried out by collecting a 7 mm agar pieces approximately 1.0 cm away from the center of the colony and physically counting the number of sclerotia on each agar piece. Error bars represent standard error and letters over the bars indicate statistical significance. Different letters indicate data that was statistically significant (p value ≤  0.05).

Fig 4. fhpA negatively regulates the production of AFB₁ biosynthesis in A. flavus.

The A. flavus WT, ΔfhpA 1, and OEfhpA 4 strains were center point inoculated on YES medium and incubated under dark conditions for 7 days. AFB₁ levels were analyzed by UPLC analysis and represented as ppb (ng/g of sample). Different letters represent statistical significance (p ≤  0.05), and error bars represent standard error.

Fig 5. Overexpression of the fhpA gene affects the expression of specific developmental and AFB₁ regulatory genes.

Relative gene expression levels of (A) flbB (B) brlA (C) nsdD (D) sclR (E) aflR (F) aflC and (G) aflM were analyzed from mycelium that was harvested from liquid static PDB cultures that were inoculated at a concentration of 1.0 x 10⁶ spores/mL and incubated at 30°C under dark condition for 3 days. Statistical significance (p ≤  0.05) is represented by different letters placed over the top of standard error bars.

Fig 6. Overexpression of the fhpA gene alters the ability to A. flavus to tolerate high concentrations of sodium chloride.

(A) The A. flavus strains were center point inoculated onto PDA medium with and without 2.7 M NaCl in triplicate. The cultures were grown for 4 days under dark conditions at 30°C prior to being photographed. (B) Radial colony growth of the cultures was measured (in cm). Data are represented as the percentage of reduction in the growth of strains on PDA medium versus PDA medium containing 2.7 M NaCl. Error bars represent the standard error. Columns with different letters represent values that are statistically different (P <  0.050).

Fig 7. Disruption and overexpression of the fhpA gene increases A. flavus’s sensitivity to oxidative stress.

(A) The A. flavus WT, ΔfhpA 1, and OEfhpA 4 strains were center point inoculated in triplicate onto PDA medium with and without 1.2 M menadione supplementation. The cultures were incubated in the dark for 4 days at 30°C prior to being photographed. (B) After incubation, the radial colony growth of each culture was measured (in cm). Data are represented as the percentage of reduction in the growth of strains on PDA medium versus PDA medium containing 2.7 M NaCl. Error bars represent the standard error. Columns with different letters represent values that are statistically different (P <  0.050).