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

Allorecognition is the ability of organisms/cells to differentiate self from non-self. In the fungus 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 analysis of wild 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 will fuse with previously HG incompatible cells, indicating cwr negatively regulates cell fusion. CWR-1 encodes a polysaccharide monooxygenase (PMO) domain that oxidatively degrades chitin; the PMO domain is sufficient to cause fusion arrest and confers allelic specificity by interacting in trans with CWR-2, a predicted transmembrane protein. However, the catalytic activity of CWR-1 is not required for triggering a block in cell fusion. The L2 and LC regions of the CWR-1 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, but not quite at wild type levels, 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 haplogroups also altered allelic specificity. SummaryAllorecognition or nonself recognition enables fungi to distinguish genetically different individuals, thereby regulating cooperation to form mycelial networks. This study focused on the cell wall remodeling checkpoint (cwr), where genetic differences in two genes, cwr-1 and cwr-2, triggers allorecognition. Upon cell contact, CWR-1 in one cell functions in trans with CWR-2 in a second cell to confer a block in cell fusion. Chimeric proteins were created and tested to pinpoint domains involved in allelic specificity. For CWR-1, the L2 and LC domains are critical, while for CWR-2, the ED2 and ED4 domains have an important role in regulating cell fusion. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/633681v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@7e150org.highwire.dtl.DTLVardef@1596b00org.highwire.dtl.DTLVardef@1800a37org.highwire.dtl.DTLVardef@11ea8d4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG