On the evolution of Neurospora crassa ACW-1 and NCW-3, proteins with different cell wall interaction mechanisms

The fungal cell wall is populated with cell wall-resident glycoproteins (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, either covalently or non-covalently. However, it is not yet clear whether they are subjected to the same evolutionary constraints. In Neurospora crassa, CWPs ACW-1 (NCU08936) and NCW-3 (NCU07817) bind to β-1,3-glucans through a GPI anchor or a predicted CBM-52 domain, respectively. In this study, the evolutionary trajectories of both CWPs were analyzed. Both proteins were identified to localize primarily to distal septa and hyphal wall surfaces, and morphological characterization of ACW-1 and NCW-3 knockout mutants suggested that both proteins are involved in the cell wall organization, with NCW-3 likely having a more prominent role. Additionally, ACW-1 and NCW-3 homologs were shown to be predominantly encoded in the Ascomycota. ACW-1 displayed a broader distribution than NCW-3, whose homologs 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 for NCW-3. These findings suggested that distinct CWP architectures can accommodate different patterns of sequence diversification despite sharing similar global evolutionary change.