On the evolution, function and cellular fate of Neurospora crassa ACW-1 and NCW-3, proteins with different cell wall interaction mechanism

The fungal cell wall is populated by proteins (CWPs), mostly uncharacterized, that show an atypical evolutionary behavior. Most CWPs are glycosylphosphatidylinositol(GPI)-proteins, followed by proteins with internal repeats (PIR), and non-covalently attached proteins that harbor carbohydrate binding domains (CBM). Several structural CWPs are initially bound to the same wall carbohydrates, but either covalently or non-covalently. However, it is not clear whether they work in the same way and if they are subjected to the same evolutionary constraints. In Neurospora crassa, CWPs ACW-1 (NCU08936) and NCW-3 (NCU07817) bind to {beta}-1,3-glucans through a GPI anchor or a predicted CBM-52 domain, respectively. Here, the evolutionary trajectories and functional roles of both CWPs were analyzed. Both proteins localized primarily to distal septa and hyphal wall surfaces. Morphological characterization and stress cell wall assays suggested that both proteins contribute to cell wall integrity, but NCW-3 likely plays a more prominent role. ACW-1 and NCW-3 homologues were predominantly identified in Ascomycota. ACW-1 displayed a broader distribution than NCW-3, whose homologues were largely restricted to Sordariales. Despite these differences, both protein families exhibited similar moderate global conservation and signatures of purifying selection within shared taxa. Nevertheless, a divergence gradient was identified within ACW-1, related to its tandem leucine-rich repeat (LRR) regions. A similar local accumulation of evolutionary change was not observed within NCW-3. These findings suggested that distinct CWP architectures can accommodate different patterns of sequence diversification despite sharing similar global evolutionary change.