The STRIPAK component SipC is involved in morphology and cell-fate determination in the nematode-trapping fungus Duddingtonia flagrans

Abstract

The striatin-interacting phosphatase and kinase (STRIPAK) complex is a highly conserved eukaryotic signaling hub involved in the regulation of many cellular processes. In filamentous fungi, STRIPAK controls multicellular development, hyphal fusion, septation, and pathogenicity. In this study, we analyzed the role of the STRIPAK complex in the nematode-trapping fungus Duddingtonia flagrans which forms three-dimensional, adhesive trapping networks to capture Caenorhabditis elegans. Trap networks consist of several hyphal loops which are morphologically and functionally different from vegetative hyphae. We show that lack of the STRIPAK component SipC (STRIP1/2/HAM-2/PRO22) results in incomplete loop formation and column-like trap structures with elongated compartments. The misshapen or incomplete traps lost their trap identity and continued growth as vegetative hyphae. The same effect was observed in the presence of the actin cytoskeleton drug cytochalasin A. These results could suggest a link between actin and STRIPAK complex functions.

Keywords: Duddingtonia flagrans; fungal development; nematode-trapping fungi; septation; striatin-interacting phosphatase and kinase (STRIPAK) complex; trap formation.

Figure 1

Scheme of D. flagrans STRIPAK orthologs and expression of sipC in hyphae and traps. (A) Domain organization of D. flagrans STRIPAK components. The amino acids are numbered, and conserved domains are labeled. Abbreviations: WD40, WD or beta-transducin repeat sequence; FHA, forkhead-associated; HEAT, Huntingtin, elongation factor 3, protein phosphatase 2A, and the yeast kinase TOR1; Mob1, monopolar spindle one-binder protein; N1221, acidic domain with possible transmembrane domains; DUF3402, domain of unknown function 3402; PHAO03269, envelope glycoprotein C. (B) Visualization of the expression of sipC using a H2B-mCherry reporter construct. The expression of h2b-GFP under the h2b promoter was used as a control. Exposure time: mCherry 300 ms, GFP 200 ms. Fungal germlings were observed after 1–2 h of incubation on LNA. Old mycelium refers to at least 16 h postinoculation of spores.

Figure 2

Deletion of sipC impairs vegetative growth and septation. (A) Targeted deletion of the sipC gene in D. flagrans using homologous recombination. Left: scheme of the deletion strategy. Right: analytic PCR using primers indicated on the left (as arrows) and southern blot analysis (outer right panel) using the probe indicated in the scheme. Genomic DNA of wild type (WT) and transformant no. 6 (T6) was digested with XbaI before blotting and hybridizing. (B) Growth of D. flagrans WT, the ΔsipC mutant T6, and the re-complemented strain on PDA after 7 days. Additionally, top and bottom view of the ΔsipC mutant on PDA after 21 days are shown. (C) Septation of vegetative mycelium in WT, the ΔsipC mutant, and the re-complemented strain. Septa were stained with Calcofluor White. Asterisks indicate septa of the main hypha. Arrows indicate septa at branching hyphae. Quantification of compartment length in each strain. Each biological replicate is color-coded (orange, blue, gray). While each data point is depicted as dot, the averages are shown as triangles. Error bars show the standard deviation (SD). (D) Hyphal fusion events in WT, ΔsipC, and the re-complemented strain are labeled with white arrows.

Figure 3

SipC is required for asexual development and determines chlamydospore morphogenesis. (A) Quantification of the number of conidia and chlamydospores in WT, the ΔsipC mutant, and the re-complemented strain. (B) Microscopic pictures of conidia of the WT and the ΔsipC-mutant strain. (C) Microscopic pictures of chlamydospores of the WT and the ΔsipC-mutant strain. (D) Overview of chlamydospores of the WT and the ΔsipC-mutant strain. (E) Quantification of chlamydospore morphology in the ΔsipC strain and quantification of chlamydospore length of the WT, the ΔsipC mutant, and the re-complemented strain. Each biological replicate is color-coded (orange, blue, gray). While each data point is depicted as dot, the averages are shown as triangles. Error bars show the SD.

Figure 4

SipC is required for trap formation. (A) Comparison of trap formation in WT, the ΔsipC mutant, and the re-complemented strain. The asterisks indicate traps. Trapped C. elegans larvae are highlighted by arrows. (B) Overview of the trap formation in the ΔsipC strain. The asterisks indicate traps. A vegetative hypha is highlighted by an arrow. (C) Comparison of trapping networks in WT and the ΔsipC mutant. (D) Close-up comparison of traps in the WT, the ΔsipC mutant, and the re-complemented strain. (E) Visualization of nuclei inside of traps of the WT and the ΔsipC mutant. In the WT, the nuclei were labeled by H2B-mCherry (colored in yellow). In the ΔsipC mutant, the nuclei were labeled by H2B-GFP (colored in green). The cell wall was stained by CFW (colored in magenta). 3D reconstruction.

Figure 5

SipA determines trap morphology. (A) Traps of the ΔsipC mutant were divided into four groups according to their morphology. I, stick-like trap; II, ring-like trap; III, 90° bending; IV, ring with cell fusion. The cell wall was stained by CFW. (B) Quantification of the compartment length of traps in the WT, the ΔsipC mutant, and the re-complemented strain. See Table 4 for the quantification. The length of four compartments was measured. In the ΔsipC mutant, quantification of the most abundant groups I (stick-like traps) and II (ring-like traps) are displayed. Each biological replicate is color-coded (orange, blue, gray). While each data point is depicted as dot, the averages are shown as triangles. Error bars show the SD.

Figure 6

SipA controls the expression of trap-specific genes. (A) Trapping of C. elegans by traps of the ΔsipC mutant. The cell wall of the fungus is stained by CFW. A C. elegans strain expressing a C-terminal GFP fusion protein of the histone HIS-72 was used to distinguish between digested (surrounded by a dashed line) and trapped but alive (arrow) nematodes. (B) C-terminal mCherry tagging of the serine-protease P12 (colored in yellow) in the WT and the ΔsipC mutant. The arrow shows a septum and indicates the switch from a trap cell to vegetative growth. (C) The relative fluorescent intensity (RFI) along a ΔsipC trap was measured and plotted (x-axis displays the distance in µm; y-axis displays the measured RFI). Arrows indicate the septa of the trap. (D) Visualization of the expression of p12 during trapping of C. elegans using a H2B-mCherry reporter construct. The expression of H2B-GFP under the h2b promoter was used as control. Arrows indicate transition points of reduced p12-mCherry expression in vegetative hyphae.

Figure 7

Characterization of the microtubule cytoskeleton. (A) Visualization of an N-terminal mCherry fusion protein of the alpha-tubulin TubA in a vegetative hypha and trap of D. flagrans WT (colored in yellow). The cell wall was stained by CFW (colored in magenta). Asterisks indicate the localization of septal MTOCs. Three-dimensional reconstruction. (B) Microtubules in a forming trap, where still no septum was formed at the base (asterisk). (C) MTs in a bending trap. The asterisk indicates a septal MTOC and the presence of a septum. The arrow points to microtubule bending at the inner site of the trap. (D) Localization of mCherry-TubA in a trap of the ΔsipC mutant. (E) Hyperbranching of vegetative hyphae after 1 h treatment with the microtubule-depolymerizing drug benomyl (5 µg/ml). (F) The addition of benomyl (5 µg/ml) stopped trap morphogenesis in D. flagrans WT. Forming traps are labeled with an asterisk. (G) Disassembly of MTs after 5 min treatment with benomyl (5 µg/ml).

Figure 8

Characterization of the actin cytoskeleton. (A) Actin patches at a hyphal tip and an actin-ring during septum development visualized with Lifeact-GFP (colored in green). Three-dimensional reconstruction. (B) Actin during trap formation and in a mature trap. (C) Actin trap morphogenesis in the ΔsipC mutant strain. The white arrow indicates the septum at the base. The cell wall was stained by CFW. (D) Septum formation in a column-like trap in the ΔsipC mutant strain. The arrow indicates septum development by localization of Lifeact-GFP at a developing septum. The asterisk indicates a mature septum without GFP signal. (E) The addition of the actin-depolymerizing drug cytochalasin A (5 µg/ml) leads to swollen hyphal tips. As solvent control, hyphae were treated with 0.5% DMSO. (F) Loss of trap identity after the addition of cytochalasin A (5 µg/ml). After some time, traps continued to grow as vegetative hyphae (see also Supplementary Movie S1).

Figure 8

Characterization of the actin cytoskeleton. (A) Actin patches at a hyphal tip and an actin-ring during septum development visualized with Lifeact-GFP (colored in green). Three-dimensional reconstruction. (B) Actin during trap formation and in a mature trap. (C) Actin trap morphogenesis in the ΔsipC mutant strain. The white arrow indicates the septum at the base. The cell wall was stained by CFW. (D) Septum formation in a column-like trap in the ΔsipC mutant strain. The arrow indicates septum development by localization of Lifeact-GFP at a developing septum. The asterisk indicates a mature septum without GFP signal. (E) The addition of the actin-depolymerizing drug cytochalasin A (5 µg/ml) leads to swollen hyphal tips. As solvent control, hyphae were treated with 0.5% DMSO. (F) Loss of trap identity after the addition of cytochalasin A (5 µg/ml). After some time, traps continued to grow as vegetative hyphae (see also Supplementary Movie S1).