Identification of regions required for allelic specificity at the cell wall remodeling allorecognition checkpoint in Neurospora crassa

Abstract Allorecognition is the ability of organisms/cells to differentiate self from non-self. In Neurospora crassa, allorecognition systems serve as checkpoints to restrict germling/hyphal fusion between genetically incompatible strains. The cell wall remodeling (cwr) checkpoint functions after chemotrophic interactions and is triggered upon cell/hyphal contact, regulating cell wall dissolution and subsequent cell fusion. The cwr region consists of two linked loci, cwr-1 and cwr-2, that are under severe linkage disequilibrium. Phylogenetic analyses of N. crassa populations showed that cwr-1/cwr-2 alleles fall into six different haplogroups (HGs). Strains containing deletions of cwr-1 and cwr-2 fuse with previously HG incompatible cells, indicating cwr negatively regulates cell fusion. CWR-1 encodes a chitin polysaccharide monooxygenase (PMO); the PMO domain confers allelic specificity by interacting in trans with the predicted transmembrane protein, CWR-2, from a different HG. However, catalytic activity of CWR-1 is not required for triggering a block in cell fusion. The CWR-1 L2 and LC regions of the PMO domain show high levels of structural variability between different HGs. CWR-1 chimeras containing a LC region from a different HG were sufficient to trigger a cell fusion block, suggesting that the complete PMO structure is necessary for allorecognition. Modeling of the transmembrane protein CWR-2 revealed allelic variability in the two major extracellular domains (ED2/ED4). Chimeras of CWR-2 with swapped ED2 or ED4 or ED2/ED4 domains from different cwr-2 HGs also altered allelic specificity. This work identified key regions of CWR-1 and CWR-2 that contribute to allorecognition specificity, providing insight into the molecular basis of this process.