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

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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 chs s ( chsb ) clades (BI-BIII) and suggested functional differences between the chsb 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 chsb genes in vegetative hyphae of the white-rot fungus Pleurotus ostreatus. Using homologous recombination, we disrupted chsb1 (clade BI) and simultaneously disrupted chsb2 and chsb3 (clade BII) to investigate their roles in vegetative growth, cell wall biosynthesis and stress response. Deletion of chsb1 led to reduced colony growth, impaired aerial hyphae formation, thinner cell walls and significant cell wall remodelling. In contrast, single disruptions of chsb2 or chsb3 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 chsb1 and Δ chsb2 Δ chsb3 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 chsb 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.