Cell wall-resident proteins with internal repeats (PIRs) show an inverted architecture in Neurospora crassa, but maintain their role as wall stabilizers.

Proteins with internal repeats (PIRs) are the second most abundant class of fungal cell wall resident proteins. In yeasts, PIRs preserve the stability of the cell wall under stressful conditions. They are characterized by conserved N-terminal amino acid sequences repeated in tandem (PIR motifs), and a cysteine (Cys)-rich C-terminal domain. PIRs have been identified in several filamentous fungi genomes; however, they have not been studied beyond yeasts. In this work, the diversity, evolution, and biological role of PIRs, with a particular focus on a new PIRs class, was addressed. Bioinformatic inference of PIRs in fungi indicated they were an innovation in Ascomycota. Predicted PIRs clustered in two main groups: classical yeasts PIRs (N-terminal PIR motifs; C-terminal Cys-rich domain), and PIRs from filamentous fungi with an inverted architecture (N-terminal Cys-rich domain; C-terminal PIR motifs), which could harbor additional glycosylphosphatidylinositol (GPI) addition-signals. As representatives of the second group, Neurospora crassa (Nc) PIR-1 (NCU04033) and PIR-2 (NCU07569) were studied. Confocal microscopy of eGFP-labeled Nc PIR-1 and Nc PIR-2 revealed they accumulate in apical plugs; additionally, PIR-1 requires the Kex2 processing site for correct maturation and harbors a predicted GPI modification signal. Moreover, Nc Δpir-1 and Δpir-2 single mutants showed a growth rate similar to that of Nc wild-type (WT), but the double mutant Nc Δpir-1/Δpir-2 grew significantly slower. Similarly, Nc Δpir-1 and Nc Δpir-2 were mildly sensitive to calcofluor white, although Nc Δpir-1/Δpir-2 double mutant was severely impaired. Despite the inverted architecture of Nc PIR-1 and Nc PIR-2, they maintain a role as cell wall stabilizers like classical yeast PIRs.