STRIPAK Dependent and Independent Phosphorylation of the SIN Kinase DBF2 Controls Fruiting Body Development and Cytokinesis during Septation and Ascospore Formation in Sordaria macrospora

Abstract

The supramolecular striatin-interacting phosphatases and kinases (STRIPAK) complex is highly conserved in eukaryotes and controls diverse developmental processes in fungi. STRIPAK is genetically and physically linked to the Hippo-related septation initiation network (SIN), which signals through a chain of three kinases, including the terminal nuclear Dbf2-related (NDR) family kinase DBF2. Here, we provide evidence for the function of DBF2 during sexual development and vegetative growth of the homothallic ascomycetous model fungus Sordaria macrospora. Using mutants with a deleted dbf2 gene and complemented strains carrying different variants of dbf2, we demonstrate that dbf2 is essential for fruiting body formation, as well as septum formation of vegetative hyphae. Furthermore, we constructed dbf2 mutants carrying phospho-mimetic and phospho-deficient codons for two conserved phosphorylation sites. Growth tests of the phosphorylation mutants showed that coordinated phosphorylation is crucial for controlling vegetative growth rates and maintaining proper septum distances. Finally, we investigated the function of DBF2 by overexpressing the dbf2 gene. The corresponding transformants showed disturbed cytokinesis during ascospore formation. Thus, regulated phosphorylation of DBF2 and precise expression of the dbf2 gene are essential for accurate septation in vegetative hyphae and coordinated cell division during septation and sexual spore formation.

Keywords: SIN kinase DBF2; STRIPAK; Sordaria macrospora; fungal development.

Figure 1

Septation of mycelial hyphae. Images from vegetative cells from wild type and Δdbf2 strains were taken after 48 h of incubation on complete medium (BMM) and stained with calcofluor-white (CFW). The bar indicates 20 µm.

Figure 2

Sexual development of wild type, Δdbf2 and complemented strains. Sexual development of all strains was observed after three (ascogonia), five (unpigmented and pigmented protoperithecia) and seven days (perithecia) on BMM media. Scale bars indicate 20 μm (white) or 100 μm (black). Septation of ascogonia was monitored after staining with CFW.

Figure 3

Comparison of DBF2 homologs from ascomycetous fungi. (A) Primary structure of DBF2 proteins from S. macrospora (S.m) DBF2, N. crassa (N.c) DBF-2, and S. cerevisiae (S.c) DBF-2p. All homologs contain a Mob-binding domain found in fungal Sid2p-like serine/threonine protein kinases (MobB_sid2p) and a serine/threonine-protein kinase (STK_sid2p). Phosphorylation sites of S. macrospora DBF2 are indicated that were investigated in this study. (B) Multiple sequence alignment of DBF2 homologs from proto- and euascomycetes. Framed are the conserved phosphorylation residues S104 and S502. Abbreviations: S.m, Sordaria macrospora (XP_003347031.1); N.c, Neurospora crassa (XP_964888.1); P.a, Podospora anserina (CDP24309.1); P.g, Pyricularia grisea (XP_030980211.1); A.n, Aspergillus nidulans (XP_050467798.1); S.p, Schizosaccharomyces pombe (NP_592848.1); S.c, Saccharomyces cerevisiae (CAA97095.1).

Figure 3

Comparison of DBF2 homologs from ascomycetous fungi. (A) Primary structure of DBF2 proteins from S. macrospora (S.m) DBF2, N. crassa (N.c) DBF-2, and S. cerevisiae (S.c) DBF-2p. All homologs contain a Mob-binding domain found in fungal Sid2p-like serine/threonine protein kinases (MobB_sid2p) and a serine/threonine-protein kinase (STK_sid2p). Phosphorylation sites of S. macrospora DBF2 are indicated that were investigated in this study. (B) Multiple sequence alignment of DBF2 homologs from proto- and euascomycetes. Framed are the conserved phosphorylation residues S104 and S502. Abbreviations: S.m, Sordaria macrospora (XP_003347031.1); N.c, Neurospora crassa (XP_964888.1); P.a, Podospora anserina (CDP24309.1); P.g, Pyricularia grisea (XP_030980211.1); A.n, Aspergillus nidulans (XP_050467798.1); S.p, Schizosaccharomyces pombe (NP_592848.1); S.c, Saccharomyces cerevisiae (CAA97095.1).

Figure 4

Expression of phospho-mutated variants of DBF2-GFP fusion proteins in strains as indicated. Strains were cultured on malt-cornmeal medium (BMM) as a surface culture for three days. Crude protein extracts, consisting of 10 μg of protein from each strain, were prepared and separated using SDS-PAGE. Western blot analysis was conducted using an anti-GFP antibody, with an anti-α-tubulin (55 kDa) antibody serving as a control. The DBF2-GFP fusion protein, with a molecular weight of 107 kDa, was detected in all phospho-mutant strains (S104A, S104E, S502A, S502E), as well as in the complemented strain. The wild type strain (wt) was used as a control.

Figure 5

Sexual development and hyphal fusion in wild type, Δdbf2, complemented transformants, phospho-mimetic (S104E, S502E) and phospho-deficient dbf2 mutants (S104A, S502A). (A) Images of colony morphology and protoperithecia distribution were taken after incubation on BMM medium at 27 °C for seven days. Bar indicates 500 µm. (B) For microscopic analysis of perithecia and ascospores, strains were grown on BMM medium at 27 °C for 10 d. Bar indicates 100 µm. (C) For investigation of hyphal fusion, strains from (A) were grown on a layer of cellophane on MMS for two days. Investigation of hyphal fusion events (arrowheads) took place in a region 5–10 mm off the colony edges. Arrowheads mark hyphal anastomosis. Strains were grown on cellophane-coated MMS medium at 27 °C for 2–4 days. Scale bar is 20 µm.

Figure 5

Sexual development and hyphal fusion in wild type, Δdbf2, complemented transformants, phospho-mimetic (S104E, S502E) and phospho-deficient dbf2 mutants (S104A, S502A). (A) Images of colony morphology and protoperithecia distribution were taken after incubation on BMM medium at 27 °C for seven days. Bar indicates 500 µm. (B) For microscopic analysis of perithecia and ascospores, strains were grown on BMM medium at 27 °C for 10 d. Bar indicates 100 µm. (C) For investigation of hyphal fusion, strains from (A) were grown on a layer of cellophane on MMS for two days. Investigation of hyphal fusion events (arrowheads) took place in a region 5–10 mm off the colony edges. Arrowheads mark hyphal anastomosis. Strains were grown on cellophane-coated MMS medium at 27 °C for 2–4 days. Scale bar is 20 µm.

Figure 6

Vegetative growth and stress response of wild type, Δdbf2, complemented transformants and phospho-mimetic Δdbf2::OEdbf2-S104E, Δdbf2::OEdbf2-S502E (S104E, S502E), and phospho-deficient dbf2 mutants Δdbf2::OEdbf2-S104A, Δdbf2::OEdbf2-S502A (S104A, S502A). Vegetative growth and cell wall stress response of DBF2 kinase mutants. Sensitivity against Congo Red (0.01% (v/v) SDS plus 2 mg/mL CR) was monitored on petri dishes for 7 consecutive days. Shown are average growth rates on SWG (yellow bars) and SWG + CR (red bars) and standard deviations from three independent experiments are shown. Significant differences of growth length from that of the wild type are indicated by asterisks and were evaluated by a two-sided student’s t-test (***, p ≤ 0.001, **, p ≤ 0.01, *, p ≤ 0.05).

Figure 7

Septation phenotype of wild type, Δdbf2, complemented transformants and phospho-mimetic (S104E, S502E) and phospho-deficient dbf2 mutants (S104A, S502A). (A) Images of septation phenotypes from strains as indicated. Fluorescence microscopy was done with CFW stained mycelia. The scale bar corresponds to 20 μm (B) Quantitative data of septum distances. Significant differences of septum distances from that of the wild type and complemented strains are indicated by asterisks and were evaluated by a two-sided student’s t-test (*, p ≤ 0.01).

Figure 8

Nuclear division in asci from wild type and dbf2 overexpression strains. (A) Microscopic images show asci from wild type and dbf2 overexpression strains. In the latter, only large ascospores having a sausage-like shape are seen. (B) Schematic drawing of nuclear division during ascospore formation (Kück, unpublished). (C) DAPI staining to visualize nuclei in developing asci from wild type and dbf2 overexpression strains. a, b, c, and d indicate nuclear division stages, as shown in (C); scale bar is 20 µm.

Figure 8

Nuclear division in asci from wild type and dbf2 overexpression strains. (A) Microscopic images show asci from wild type and dbf2 overexpression strains. In the latter, only large ascospores having a sausage-like shape are seen. (B) Schematic drawing of nuclear division during ascospore formation (Kück, unpublished). (C) DAPI staining to visualize nuclei in developing asci from wild type and dbf2 overexpression strains. a, b, c, and d indicate nuclear division stages, as shown in (C); scale bar is 20 µm.