Abstract
The recently identified phylogenetic subgroup B5 of fungal glycoside hydrolase family 18 genes encodes enzymes with mannosyl glycoprotein endo-N-acetyl-β-D-glucosaminidase (ENGase)-type activity. Intracellular ENGase activity is associated with the endoplasmic reticulum associated protein degradation pathway (ERAD) of misfolded glycoproteins, although the biological relevance in filamentous fungi is not known. Trichoderma atroviride is a mycoparasitic fungus that is used for biological control of plant pathogenic fungi. The present work is a functional study of the T. atroviride B5-group gene Eng18B, with emphasis on its role in fungal growth and antagonism. A homology model of T. atroviride Eng18B structure predicts a typical glycoside hydrolase family 18 (αβ)8 barrel architecture. Gene expression analysis shows that Eng18B is induced in dual cultures with the fungal plant pathogens Botrytis cinerea and Rhizoctonia solani, although a basal expression is observed in all growth conditions tested. Eng18B disruption strains had significantly reduced growth rates but higher conidiation rates compared to the wild-type strain. However, growth rates on abiotic stress media were significantly higher in Eng18B disruption strains compared to the wild-type strain. No difference in spore germination, germ-tube morphology or in hyphal branching was detected. Disruption strains produced less biomass in liquid cultures than the wild-type strain when grown with chitin as the sole carbon source. In addition, we determined that Eng18B is required for the antagonistic ability of T. atroviride against the grey mould fungus B. cinerea in dual cultures and that this reduction in antagonistic ability is partly connected to a secreted factor. The phenotypes were recovered by re-introduction of an intact Eng18B gene fragment in mutant strains. A putative role of Eng18B ENGase activity in the endoplasmic reticulum associated protein degradation pathway of endogenous glycoproteins in T. atroviride is discussed in relation to the observed phenotypes.
Highlights
Enzymes with mannosyl glycoprotein endo-N-acetyl-b-D-glucosaminidase (ENGase)-type activity (EC.3.2.1.96) are found in glycoside hydrolase (GH) families 18, 73 and 85 [1]
The translated Eng18B 337 amino acid sequence was analysed for conserved domains using SMART which identified a single GH family 18 module between aa positions 12–269 (Pfam00704), including a putative catalytic motif DGLDLDVE and a putative substrate binding site SLGG
The lack of signal peptide or GPI-anchor signal suggests a cytosolic localization of T. atroviride Eng18B, in contrast to the secreted ENGase Eng18A (homologous to T. reesei Eng18A (Endo T) [4])
Summary
Enzymes with mannosyl glycoprotein endo-N-acetyl-b-D-glucosaminidase (ENGase)-type activity (EC.3.2.1.96) are found in glycoside hydrolase (GH) families 18, 73 and 85 [1]. Characterized fungal ENGase sequences are reported from the GH family 85 Endo M from Mucor hiemalis [3], and recently from the GH family 18 members Trichoderma reesei Eng18A, ( referred to as Endo T, [4]), and Flammulina velutipes Endo FV [5]. Both T. reesei Eng18A and F. velutipes Endo FV belongs phylogenetically to the fungal GH family 18 subgroup B5 [6]. T. reesei Eng18A may be responsible for postsecretorial modifications of glycan structures on endogenous T. reesei glycoproteins such as cellulases, or participate in hydrolysis of the oligosaccharide-protective coat of foreign glycoproteins to generate nutrients
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