Fungus-specific sirtuin HstD coordinates secondary metabolism and development through control of LaeA

Abstract

The sirtuins are members of the NAD(+)-dependent histone deacetylase family that contribute to various cellular functions that affect aging, disease, and cancer development in metazoans. However, the physiological roles of the fungus-specific sirtuin family are still poorly understood. Here, we determined a novel function of the fungus-specific sirtuin HstD/Aspergillus oryzae Hst4 (AoHst4), which is a homolog of Hst4 in A. oryzae yeast. The deletion of all histone deacetylases in A. oryzae demonstrated that the fungus-specific sirtuin HstD/AoHst4 is required for the coordination of fungal development and secondary metabolite production. We also show that the expression of the laeA gene, which is the most studied fungus-specific coordinator for the regulation of secondary metabolism and fungal development, was induced in a ΔhstD strain. Genetic interaction analysis of hstD/Aohst4 and laeA clearly indicated that HstD/AoHst4 works upstream of LaeA to coordinate secondary metabolism and fungal development. The hstD/Aohst4 and laeA genes are fungus specific but conserved in the vast family of filamentous fungi. Thus, we conclude that the fungus-specific sirtuin HstD/AoHst4 coordinates fungal development and secondary metabolism via the regulation of LaeA in filamentous fungi.

Fig 1

Phylogenetic analysis of histone deacetylase in A. oryzae. Accession numbers and HDAC names are indicated for each branch. The HDAC names of S. cerevisiae or H. sapiens with the species name indicated are followed by a slash. The numbers at the nodes are bootstrap values obtained from 1,000 replicates and are indicated as percentages. Scale bar, a distance corresponding to 0.2 amino acid substitution per site. The class or subclass of HDACs is shown on the right. These classes of HDACs are referred to in previous phylogenetic studies (4, 6, 34). AoHDACs are indicated by underlines. Abbreviations of AoHDAC gene names are as follows: hda, histone deacetylase; hst, homolog of sirtuin. The class to which hstD belongs is surrounded by a gray border. The gene names and their accession numbers are identified in Table S2 in the supplemental material. Sc, Saccharomyces cerevisiae; An, Aspergillus nidulans; Nc, Neurospora crassa; Hs, Homo sapiens; Ao, Aspergillus oryzae.

Fig 2

hstD/Aohst4 and hdaD/Aohos2 regulate SM production and development. (A) The morphological phenotype on N agar medium (MM) and results of the kojic acid production plate assay (KA) are provided for the indicated strains. (B, C) Radial growth and conidiation of the indicated strains on N agar medium, respectively. (D) Time course characteristics of kojic acid production of the indicated disruptants. (E) Expression profiles of kojic acid cluster genes represented by Northern hybridization. The culture times of the indicated strains are shown at the top of the panel. The analyzed gene is indicated on the left side of each blot. The results for rRNA, used as the loading control, are shown. (F) Bioassay of penicillin production of the ΔhstD strain. (G) Northern hybridization of the penicillin biosynthetic gene ipnA in the ΔhstD strain. The results for rRNA, used as the loading control, are shown. The adeA⁺ strain was used as a control in the experiment whose results are presented in this figure. All data are represented as means ± SDs (n = 3); *, P < 0.01, t test.

Fig 3

Complementation analysis of hstD. (A) Analysis of the morphology and SM production of the ΔhstD and hstD⁺ strains. MM, morphological phenotype of the indicated strain on N agar medium; ×100, closeup stereomicroscopic images of the strains on N agar medium (magnification, ×100; bar, 500 μm; red arrows, examples of conidia); KA and PEN, plate assay or bioassay of kojic acid and penicillin, respectively. (B, C) Quantification of colony diameter and rate of conidiation of ΔhstD and hstD⁺ strains, respectively. (D) The expression profiles of the kojic acid cluster genes were determined by Northern hybridization. The culture time of the indicated strain is shown at the top. The analyzed gene is indicated on the left side of each blot. The results for rRNA, used as the loading control, are shown. (E) Quantification of kojic acid production. The adeA⁺ sC⁺ strain was used as the control, and the ΔhstD sC⁺ strain represents the ΔhstD strain. All data are represented as means ± SDs (n = 3); *, P < 0.01, t test.

Fig 4

Enrichment analysis of the FunCat categorization of the microarray analysis. Significantly enriched FunCat level 1 and level 2 categories of genes upregulated (A, B) or downregulated (C, D) by hstD/Aohst4 deletion are shown. The FunCat is the organism-independent functional description of proteins (33). FunCat consists of 28 main functional categories (level 1). Level 1 is the most general one, whereas level 2 shows much more detail. The percentage indicated for each category contributes to the total mapping. Insignificant FunCat categories are indicated as insignificant in the pie charts. Significantly enriched categories were extracted by FungiFun software (cutoff P value, <0.05; Fisher's exact test) (32). Details of the enrichment analysis are available at the FungiFun website (https://sbi.hki-jena.de/FungiFun/FungiFunHelp.html).

Fig 5

Genetic interaction between hstD and laeA. (A) Expression profile of laeA under the KA-producing condition. The adeA⁺ strain was used as a control. The culture time of the indicated strain is shown at the top of the panel. The results for rRNA, used as the loading control, are shown. (B) Analysis of morphology and SM production of the ΔhstD, ΔlaeA, and ΔhstD ΔlaeA strains. MM, morphological phenotype of the indicated strain on N agar medium; ×100, closeup stereomicroscopic images of the strains on N agar medium (magnification, ×100; bar, 500 μm; red arrows, examples of conidia); KA and PEN, plate assay or bioassay of kojic acid and penicillin, respectively. (C, D) Quantification of the conidiation rate and kojic acid production of the ΔhstD, ΔlaeA, and ΔhstD ΔlaeA strains, respectively. Except for panel A, the adeA⁺ sC⁺ strain was used as the control, and the ΔhstD sC⁺ and ΔlaeA sC⁺ strains represent the ΔhstD and ΔlaeA strains, respectively, in this figure. All data are represented as means ± SDs (n = 3); *, P < 0.01, t test.

Fig 6

Epistatic relationship between hstD and laeA. (A) Analysis of the morphology and SM production of the ΔhstD OE::laeA and ΔlaeA OE::hstD strains. MM, morphological phenotype of the indicated strain on N agar medium; ×100, closeup stereomicroscopic images of the strains on N agar medium (magnification, ×100; bar, 500 μm; red arrows, examples of conidia); KA and PEN, plate assay or bioassay of kojic acid and penicillin, respectively. (B, C) Quantification of the conidiation rate and kojic acid production of the ΔhstD OE::laeA and ΔlaeA OE::hstD strains, respectively. The adeA⁺ pUSA⁺ strain was used as the control, and the ΔhstD pUSA⁺, OE::laeA adeA⁺, ΔlaeA pUSA⁺, and OE::hstD adeA⁺ strains represent the ΔhstD, OE::laeA, ΔlaeA, and OE::hstD strains, respectively, in this figure. The amyB promoter was used to drive overexpression of laeA and hstD. All data are represented as means ± SDs (n = 3); *, P < 0.01, t test.

Fig 7

Schematic model of the regulation of SM production and development by HstD/AoHst4 through LaeA. An unknown signal induces or suppresses the function of HstD/AoHst4. Suppression of HstD/AoHst4 leads to expression of LaeA. This activation stimulates fungal development and secondary metabolite production. However, there is the possibility of an HstD/AoHst4 competitive mechanism by an unknown factor (described as factor X in this figure).