The GPI-Anchored GH76 Protein Dfg5 Affects Hyphal Morphology and Osmoregulation in the Mycoparasite Trichoderma atroviride and Is Interconnected With MAPK Signaling

Abstract

The fungal cell wall is composed of a cross-linked matrix of chitin, glucans, mannans, galactomannans, and cell wall proteins with mannan chains. Cell wall mannans are directly attached to the cell wall core, while the majority of mannoproteins is produced with a glycosylphosphatidylinositol (GPI) anchor and then transferred to β-1,6-glucan in the cell wall. In this study, we functionally characterized the transmembrane protein Dfg5 of the glycoside hydrolase family 76 (GH76) in the fungal mycoparasite Trichoderma atroviride, whose ortholog has recently been proposed to cross-link glycoproteins into the cell wall of yeast and fungi. We show that the T. atroviride Dfg5 candidate is a GPI-anchored, transmembrane, 6-hairpin member of the GH76 Dfg5 subfamily that plays an important role in hyphal morphology in this mycoparasite. Alterations in the release of proteins associated with cell wall remodeling as well as a higher amount of non-covalently bonded cell surface proteins were detected in the mutants compared to the wild-type. Gene expression analysis suggests that transcript levels of genes involved in glucan synthesis, of proteases involved in mycoparasitism, and of the Tmk1 mitogen-activated protein kinase (MAPK)-encoding gene are influenced by Dfg5, whereas Tmk3 governs Dfg5 transcription. We show that Dfg5 controls important physiological properties of T. atroviride, such as osmotic stress resistance, hyphal morphology, and cell wall stability.

Keywords: Trichoderma atroviride; cell wall; glycoside hydrolase family 76; mitogen-activated protein kinase; mycoparasitism.

FIGURE 1

Phylogenetic inference of Dfg5 orthologs from N. crassa OR74A (Neucr2), A. nidulans FGSC A4, S. cerevisiae S288C, C. albicans SC5314, and T. atroviride IMI206040 using recombination network analysis in SplitsTree4 (Huson and Bryant, 2006) program. The tool was used as a framework for interfering with the phylogenetic relation between the multiple T. atroviride Dfg5 orthologs and already characterized Dfg5 and Dcw1 proteins.

FIGURE 2

Transcript levels of Dfg5 subfamily member genes in T. atroviride wild-type (WT) and a Δdfg5 deletion mutant upon cultivation in potato dextrose broth (PDB) with and without the addition of the cell wall stressor Congo red (CR). The asterisks (*) indicate statistical significance in regulation of the respective genes (–1.5 < log2(FC) > 1.5, where FC indicates fold change) toward the untreated control and the sar1 normalizer. The lines above the bars indicate standard error (SE) as calculated by the Relative Expression Software Tool REST using SE estimation via a Taylor algorithm (Pfaffl et al., 2002).

FIGURE 3

Growth rate (A) and macromorphology (B) of T. atroviride Δdfg5 deletion mutants and the wild-type (WT) upon cultivation on potato dextrose agar (PDA). (C) Biomass production of Δdfg5, the overexpression strain dfg5OE, and the WT upon cultivation in liquid culture [potato dextrose broth (PDB) or PDB supplemented with the glucan synthase inhibitor caspofungin (CAS)]. Due to the highly similar phenotype of the two Δdfg5 mutants (Δdfg5-2-2 and Δdfg5-8-1), results of Δdfg5-8-1 are presented in the following unless otherwise stated.

FIGURE 4

(A) Hyphal morphology of T. atroviride Δdfg5, Δtmk1, and Δtmk3 deletion mutants as well as of the wild-type (WT) after growth on potato dextrose agar (PDA) for 48 h. Δdfg5 mutants showed thin hyphae with a “curled” hyphal phenotype. Images were taken using a laser scanning confocal microscope. The bars annotate 50 μm. The bar chart illustrates hyphal diameters of the tested strains, which were measured using Fiji software. p-value (^∗∗∗) ≤ 0.001. (B) Hyphal branching in WT as well as Δtmk1, Δtmk3, and Δdfg5 mutants. Bright field microscopy was used to assess branching in colonies that developed from one single conidium. One conidium of each strain was inoculated on PDA and observed after 16 h (upper image row) and 36 h (lower image row) of incubation at 25°C. Scale bar: 10 μm. The bar chart illustrates the branching frequency of the tested strains after 16 and 24 h of incubation on PDA (the quantification at 36-h time point was unsuitable due to Δdfg5 mutant’s dense growth). p-value (^∗∗∗) ≤ 0.001.

FIGURE 5

Confocal laser scanning fluorescence microscopy of T. atroviride wild-type (WT), Δtmk3, and Δdfg5 mutants after staining their cell wall with 0.5% CFW solution. The Δdfg5 mutant hyphae showed a “curly” shape, which was associated with patchy cell wall accumulations. The arrows indicate sites with a thicker deposition of chitin in the cell wall. Scale bar: 50 μm.

FIGURE 6

Involvement of Dfg5 in the response to osmotic stress and in antagonism of T. atroviride against R. solani. (A) Cultures of Δdfg5, dfg5OE, Δtmk1, and Δtmk3, as well as the wild-type (WT) on potato dextrose agar (PDA) without any stressor and on PDA supplemented with 50 mM sorbitol or 1 M NaCl. Plates were incubated at cycling daylight for 10 days at 25°C. Graphs represent the colony diameters of the tested strains under the different growth conditions (black bar, growth on PDA; light gray bar, growth in the presence of 50 mM sorbitol; dark gray bar, growth in the presence of 1 M NaCl) as an indicator of their stress sensitivity. Error bars represent standard deviations derived from at least three biological replicates. (B) Plate confrontation assays against R. solani (back and front sides of the plates are shown). The arrows mark the clearing zone devoid of sporulation of the Δdfg5 mutant upon contact with R. solani. Due to the highly similar behavior of the two Δdfg5 mutants, only results of Δdfg5-8-1 are presented.

FIGURE 7

Transcript levels of genes putatively involved in signaling and cell wall remodeling in T. atroviride, as well as of two proteases known to be involved in mycoparasitism, in Δdfg5 and the Δtmk1 and Δtmk3 mitogen-activated protein kinase (MAPK)-deficient mutants, normalized to the transcriptional levels of the wild-type (WT). (A) Transcriptional regulation of tmk1, tmk2, and tmk3 MAPK-encoding genes, dfg5, two chitin synthase genes (chs1 and 2), genes involved in glucan synthesis (fks1, gel1, and smi1), and prb1 and s8 protease genes in the indicated mutants normalized to the WT. The filled bars indicate statistically significant regulation of the respective genes [–1.5 < log2(FC) > 1.5, where FC indicates fold change] toward the untreated control and the normalizer; unfilled bars represent non-significant changes. The lines above the bars indicate standard error (SE) as calculated by the Relative Expression Software Tool REST using SE estimation via a Taylor algorithm (Pfaffl et al., 2002). (B) Schematic representation of the proposed interplay between MAPKs and the genes involved in cell wall remodeling upon deletion of dfg5, tmk1, and tmk3, respectively. The connecting lines between the objects annotate statistically significant activating (arrows) or suppressing (dots) relations between particular proteins in a hyphal cell. The single connecting dashed line symbolizes non-significant regulation of dfg5 in the Δtmk1 mutant. The colored (green, orange, red, and dark gray) and light gray forms annotate transcriptional upregulation and downregulation of a particular gene in a certain strain, respectively. Crossed dashed-line objects indicate deletion of this particular gene.