Basidiomycete-specific chitin synthase genes have clade-specific roles in cell wall formation and hyphal development in Pleurotus ostreatus

Chitin, an essential structural component of most fungal cell walls, is produced by transmembrane proteins called chitin synthases (CHSs). Our previous study identified novel basidiomycete-specific <i>chs</i>s (<i>chsb</i>) clades (BI-BIII) and suggested functional differences between the <i>chsb</i> genes of clade BII and BIII. This study, together with our previous work, presents the first comprehensive functional analysis and identification of clade-specific roles of <i>chsb</i> genes in vegetative hyphae of the white-rot fungus <i>Pleurotus ostreatus</i>. Using homologous recombination, we disrupted <i>chsb1</i> (clade BI) and simultaneously disrupted <i>chsb2</i> and <i>chsb3</i> (clade BII) to investigate their roles in vegetative growth, cell wall biosynthesis and stress response. Deletion of <i>chsb1</i> led to reduced colony growth, impaired aerial hyphae formation, thinner cell walls and significant cell wall remodelling. In contrast, single disruptions of <i>chsb2</i> or <i>chsb3</i> caused mild phenotypes, while double disruption of these genes resulted in severe growth defects, complete loss of aerial hyphae, and abnormal septation. Relative amounts of chitin were increased in the Δ<i>chsb1</i> and Δ<i>chsb2</i>Δ<i>chsb3</i> strains, whereas β-glucan was decreased, which is likely related to cell wall thinning. Together with our previous study, these results reveal clear functional differentiation between <i>chsb</i> clades: clade BI influences the relative percentage of cell wall components, as well as radial and aerial hyphal growth; clade BII affects overall growth, cell wall component and septum formation; and clade BIII appears to have a more specific role in aerial hyphae development. These findings advance our understanding of cell wall evolution in filamentous basidiomycetes.