Functional analysis of glycoside hydrolase family 18 and 20 genes in Neurospora crassa

Abstract

Glycoside hydrolase family 18 contains hydrolytic enzymes with chitinase or endo-N-acetyl-β-d-glucosaminidase (ENGase) activity, while glycoside hydrolase family 20 contains enzymes with β-N-acetylhexosaminidase (NAGase) activity. Chitinases and NAGases are involved in chitin degradation. Chitinases are phylogenetically divided into three main groups (A, B and C), each further divided into subgroups. In this study, we investigated the functional role of 10 Neurospora crassa genes that encode chitinases, 2 genes that encode ENGases and 1 gene that encode a NAGase, using gene deletion and gene expression techniques. No phenotypic effects were detected for any of the studied group A chitinase gene deletions. Deletion of the B group member chit-1 resulted in reduced growth rate compared with the wild type (WT) strain. In combination with the presence of a predicted glycosylphosphatidylinositol anchor motif in the C-terminal of chit-1, indicating cell wall localization, these data suggest a role in cell wall remodeling during hyphal growth for chit-1. Deletion of the ENGase gene gh18-10 resulted in reduced growth rate compared with WT, increased conidiation, and increased abiotic stress tolerance. In addition, Δgh18-10 strains displayed lower secretion of extracellular proteins compared to WT and reduced levels of extracellular protease activity. The connection between gh18-10 ENGase activity and the endoplasmic reticulum associated protein degradation process, a stringent quality control of glycoprotein maturation, is discussed. N. crassa group C chitinase genes gh18-6 and gh18-8 were both induced during fungal–fungal interactions. However, gh18-6 was only induced during interspecific interactions, while gh18-8 displayed the highest induction levels during self–self interactions. These results provide new information on functional differentiation of fungal chitinases.