Interplay between GPI-anchored α-1,6-mannanase Dfg5 and MAPK signaling in cell wall remodeling of the mycoparasite Trichoderma atroviride

Lea Atanasova Susanne Zeilinger
Institute of Microbiology, University of Innsbruck, Innsbruck, Austria

The cell wall plays a critical role in fungal cells. It not only protects the cell from hazardous environmental factors but also allows the fungus to assess its environment and activate signaling pathways in response to it. The wall consists of a cross-linked matrix of glucans, chitins, and cell wall proteins. Many of the integral cell wall proteins are produced as glycosylphosphatidylinositol (GPI)-anchored proteins, which are membrane-bound proteins with enzymatic, antigenic and adhesive function.

Using a membrane-based yeast two-hybrid screening in Trichoderma atroviride – a prominent mycoparasite that kills and feeds on other fungi - we identified a GPI-anchored glycosidase protein of the GH76 family as a putative interactor of the Gpr1 membrane receptor. GH76 proteins are α-1,6-mannanases suggested to be involved in cross-linking of glycoproteins into the cell wall, where they are proposed to act as transglycosylases.

Our structural and functional analyses show that T. atroviride α-1,6-mannanase is a GPI-anchored Dfg5 protein that is required for hyphal morphogenesis and cell wall remodeling. Δdfg5 mutants exhibited reduced radial growth, likely due to a hyphal elongation defect, however were not impaired in biomass production. Further, Δdfg5 mutants better assimilated glucose and some of its related substances compared to the WT, but were impaired in utilization of sucrose and some polyols. Increased release of mannose was detected after addition of an Aspergillus niger mannosidase to the mutants’ fermentation broth, what supports the hypothesis that mannose-containing oligosaccharides (e.g. galactomannans) are incorporated to the cell wall by the Dfg5. In addition, elevated levels of non-covalently entrapped cell wall proteins but a decrease in total proteins secreted into the growth medium were detected in Δdfg5 strains. We show that expression of dfg5 is Tmk1 dependent, whereas the chitin and glucan synthases are under its suppression. Further, the expression of glucan synthase genes gel1 and smi1 is governed by Dfg5.