Functional Analysis of the Class III Chitin Synthase Genes in the Filamentous Basidiomycete Pleurotus Ostreatus

Class III chitin synthases (CHSs) are specific to filamentous fungi and are associated with biosynthesis of chitin, a key structural component of fungal cell walls. While extensively studied in ascomycetes, their roles in basidiomycetes remain poorly understood. In this study, we present the first functional analysis of class III chss in the white-rot basidiomycete Pleurotus ostreatus. Through targeted single gene disruption of the class III chss, chs7 and chs9, as well as double disruption of chs7chs9, we demonstrate their distinct and sometimes overlapping roles in regulating hyphal growth, cell wall thickness, and stress resilience. Despite reduced cell wall thickness and decreased growth rates in disruption strains, compensatory upregulation of other chitin and glucan synthase genes maintains polysaccharide composition, revealing a robust regulatory network. Notably chs9 disruption strains had more severe impacts than those of chs7, suggesting that chs9 is the main class III chs and chs7 provides more of a supporting role. Double disruption strains also revealed straighter hyphae suggesting redundancy and highly specialized functions of clade III chss in hyphal straightness in P. ostreatus. Our findings highlight the evolutionary divergence and seemingly unique functions of class III CHSs in comparison to the previously analyzed basidiomycete-specific classes, providing foundational insights into filamentous basidiomycete cell wall dynamics.