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.