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

Lea Atanasova,Clemens Grünwald-Gruber,Dubraska Moreno-Ruiz,Susanne Zeilinger,Viktoria Hell

doi:10.3389/fmicb.2021.601113

Lea Atanasova, Clemens Grünwald-Gruber + Show 3 more

Open Access

https://doi.org/10.3389/fmicb.2021.601113

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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.

Highlights

The use of antagonistic microbes for plant disease management is a sustainable alternative to chemical fungicides (Benítez et al, 2004)
We show that the T. atroviride homolog of N. crassa Dfg5 is a member of the glycoside hydrolase family 76 (GH76) Dfg5 subfamily required for hyphal morphogenesis and osmoregulation in this prominent mycoparasite
Dfg5 orthologs have already been functionally characterized in several fungal organisms such as S. cerevisiae (Kitagaki et al, 2002), C. albicans (Spreghini et al, 2003), N. crassa (Maddi et al, 2012b), A. nidulans, and A. fumigatus (Muszkieta et al, 2019)

Summary

Introduction

The use of antagonistic microbes for plant disease management is a sustainable alternative to chemical fungicides (Benítez et al, 2004). Trichoderma (teleomorph Hypocrea, Ascomycota) species are efficient necrotrophic mycoparasites. These fungi are able to parasitize and kill other fungi, a behavior that makes Trichoderma species attractive biocontrol agents for plant disease control. The different stages during the mycoparasitic attack include sensing of the prey fungus, attachment to the prey’s hyphae, degradation of the prey’s cell wall, and killing of the prey (Chet et al, 1981; Inbar and Chet, 1992; Rocha-Ramirez et al, 2002; Lu et al, 2004). Several genes coding for chitinases, β-(1,4)-, β-(1,3)-, and β-(1,6)-glucanases, and proteases are induced under mycoparasitism-related growth conditions, and the respective enzymes degrade the cell wall of the prey fungus during mycoparasitism (Benítez et al, 2004; Kubicek et al, 2011; Gruber and Seidl-Seiboth, 2012; Atanasova et al, 2013)

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