Homeobox proteins are essential for fungal differentiation and secondary metabolism in Aspergillus nidulans

Abstract

The homeobox domain-containing transcription factors play an important role in the growth, development, and secondary metabolism in fungi and other eukaryotes. In this study, we characterized the roles of the genes coding for homeobox-type proteins in the model organism Aspergillus nidulans. To examine their roles in A. nidulans, the deletion mutant strains for each gene coding for homeobox-type protein were generated, and their phenotypes were examined. Phenotypic analyses revealed that two homeobox proteins, HbxA and HbxB, were required for conidia production. Deletion of hbxA caused abnormal conidiophore production, decreased the number of conidia in both light and dark conditions, and decreased the size of cleistothecia structures. Overexpressing hbxA enhanced the production of asexual spores and formation of conidiophore under the liquid submerged conditions. The hbxB deletion mutant strains exhibited decreased asexual spore production but increased cleistothecia production. The absence of hbxB decreased the trehalose content in asexual spores and increased their sensitivity against thermal and oxidative stresses. The ΔhbxA strains produced more sterigmatocystin, which was decreased in the ΔhbxB strain. Overall, our results show that HbxA and HbxB play crucial roles in the differentiation and secondary metabolism of the fungus A. nidulans.

Figure 1

The phylogenetic analyses and sequence features of the Homeobox proteins in A. nidulans. (A) The phylogenetic tree of the putative homeobox proteins in three Aspergillus species, including A. nidulans FGSC4, A. fumigatus AF293, and A. flavus NRRL 3357. (B) The domain architecture of the putative homeobox proteins in A. nidulans.

Figure 2

The roles of eight genes coding for homeobox-type proteins in A. nidulans. (A) The expression level of each gene coding for homeobox-type protein during the life cycle of A. nidulans was measured by qRT-PCR. C = Conidia, Veg = Vegetative growth, Asex = Asexual development. (B) The colony photographs of WT or deletion mutant strains point-inoculated on solid MMG and grown for 5 days at 37 °C in the light or dark. (C) Quantitative analysis of the conidia in the WT and mutant strains after 5 days of incubation at 37 °C in the light or dark. Differences between the WT and mutants, *p < 0.05, **p < 0.01, and ***p < 0.001).

Figure 3

Fungal development of the ΔhbxA mutant. (A) Colony morphology of WT (TNJ36), ΔhbxA (TYE14), and C’hbxA (TYE27) strains grown on MMG for 5 days at 37 °C in the light or dark. The middle panels show the magnified views of the middles of the plates (bar = 0.25 µm). The panels on the right show the morphologies of the WT and mutant conidiophores under a microscope (bar = 0.25 µm). Arrows indicate conidiophores. (B) Quantitative analysis of the conidia shown in (A). (Differences between the WT and mutants, ***p < 0.001). (C) qRT-PCR analysis for brlA mRNA levels in WT (TNJ36), ΔhbxA (TYE14), and C’hbxA (TYE27) strains after inducing asexual development. β-Actin was used as the endogenous control. (D) WT (TNJ36), ΔhbxA (TYE14), and C’hbxA (TYE27) strains were point-inoculated, and the plates were grown on SM for 7 days at 37 °C in the dark. The middle panels show the plates, which were washed with ethanol to observe the sexual structure. The panels on the right show the magnified views of the sexual structures in WT (TNJ36), ΔhbxA (TYE14), and C’hbxA (TYE27) strains (bar = 0.25 µm). (E) The sizes of the cleistothecia in WT (TNJ36), ΔhbxA (TYE14), and C’hbxA (TYE27) strains (differences between the WT and mutants, ***p < 0.001).

Figure 4

Effect of hbxA overexpression. (A) Control (TNJ36) and hbxA–overexpression (TYE19) strains were inoculated onto non-inducing (MMG) or inducing (MMT) condition media and photographed after 5 days of culture. The middle panels show the magnified views of the middles of the plates under the inducing conditions (bar = 0.25 µm). The panels on the right show the morphologies of the control (TNJ36) and OEhbxA (TYE19) conidiophores under the inducing conditions (bar = 0.25 µm). (B) Quantification of the number of conidia in the control (TNJ36) and OEhbxA (TYE19) strains shown in (A). Differences between the control and mutants, ***p < 0.001. (C) Photomicrographs of the mycelia in the control (TNJ36) and OEhbxA (TYE19) strains grown in liquid MMG (non-inducing) or MMT (inducing) media. Arrow indicates conidiospore.

Figure 5

Developmental phenotypes of the ΔhbxB mutant. (A) The colony photographs of WT(TNJ36), ΔhbxB (TSH1), and C’hbxB (TSH7) strains point-inoculated on solid MMG and grown for 5 days at 37 °C in the light or dark. The panels on the right show the magnified views of the plates grown in the light (bar = 0.25 µm). (B) Quantitative analysis of the number of conidia from WT(TNJ36), ΔhbxB (TSH1), and C’hbxB (TSH7) strains shown in (A). The number of conidia per plate was counted in triplicate. Differences between the WT and mutants, ***p < 0.001. (C) Quantitative analysis of the number of cleistothecia from WT (TNJ36), ΔhbxB (TSH1), and C’hbxB (TSH7) strains shown in (A). Differences between the WT and mutants, ***p < 0.001. (D) The colony morphologies of WT (TNJ36), ΔhbxB (TSH1), and C’hbxB (TSH7) strains after 7 days of culture at 37 °C in the dark. The colonies were washed to enable the visualization of the sexual structures (middle panels) and the magnified views of the edges of the plates (right panels, bar =200 μm). (E) The size of WT (TNJ36), ΔhbxB (TSH1), and C’hbxB (TSH7) strains. Differences between the WT and mutants, ***p < 0.001. (F,G) qRT-PCR analysis for brlA (F) and abaA (G) mRNA levels in WT (TNJ36), ΔhbxB (TSH1), and C’hbxB (TSH7) strains after inducing asexual development. β-Actin was used as the endogenous control.

Figure 6

Developmental phenotypes of the hbxB overexpression strain. (A) Control (TNJ36) and hbxB–overexpression (TSH13) strains were inoculated onto non-inducing (MMG) or inducing (YLC) condition media and photographed after 5 days of culture. The right panels show the magnified views of the middles of the plates under the inducing conditions (bar = 0.25 µm). (B–C) Quantification of the number of conidia (B) or cleistothecia (C) in the control (TNJ36) and OEhbxB (TSH13) strains shown in (A). Differences between the control and mutants, ***p < 0.001. All the experiments were carried out in triplicate.

Figure 7

The role of hbxB in trehalose biosynthesis and stress tolerance. (A) Trehalose amount of conidia in WT (TNJ36), ΔhbxB (TSH1), and C’hbxB (TSH7) strains (measured in triplicate) (***p < 0.001). (B) Tolerance of WT (TNJ36), ΔhbxB (TSH1), and C’hbxB (TSH7) conidia to thermal stress (50 °C, triplicate measurements). Differences between the WT and mutants, *p < 0.05, ***p < 0.001. (C) The oxidative stress response of WT (TNJ36), ΔhbxB (TSH1), and C’hbxB (TSH7) conidia (triplicate measurements). Differences between the WT and mutants, **p < 0.01. (D) The mRNA levels of tpsA, wetA, and vosA in WT (TNJ36), ΔhbxB (TSH1), and C’hbxB (TSH7) conidia.

Figure 8

Analysis of ST production in the ΔhbxA and ΔhbxB mutant. (A) Thin-layer chromatography (TLC) of ST from WT (TNJ36), ΔhbxA (TYE14), and C’hbxA (TYE27) strains was performed for 7 days in the dark. Arrow indicates ST. (B) Densitometry of the ST bands from the TLC plates shown in (A). Differences between the WT and mutants, **p < 0.01. (C) Thin-layer chromatography (TLC) of ST from WT (TNJ36), ΔhbxB (TSH1), and C’hbxB (TSH7) strains was performed for 7 days in the dark. Arrow indicates ST. (D) The densitometry of the ST bands from the TLC plates shown in (C). Differences between the WT and mutants, ***p < 0.001. (E) Relative mRNA levels of aflR in WT (TNJ36), ΔhbxB (TSH1), and C’hbxB (TSH7) strains.