In filamentous fungi, communication is essential for the formation of an interconnected, multinucleate, syncytial network, which is constructed via hyphal fusion or fusion of germinated asexual spores (germlings). Anastomosis in filamentous fungi is comparable to other somatic cell fusion events resulting in syncytia, including myoblast fusion during muscle differentiation, macrophage fusion, and fusion of trophoblasts during placental development. In Neurospora crassa, fusion of genetically identical germlings is a highly dynamic and regulated process that requires components of a MAP kinase signal transduction pathway. The kinase pathway components (NRC-1, MEK-2 and MAK-2) and the scaffold protein HAM-5 are recruited to hyphae and germling tips undergoing chemotropic interactions. The MAK-2/HAM-5 protein complex shows dynamic oscillation to hyphae/germling tips during chemotropic interactions, and which is out-of-phase to the dynamic localization of SOFT, which is a scaffold protein for components of the cell wall integrity MAP kinase pathway. In this study, we functionally characterize HAM-5 by generating ham-5 truncation constructs and show that the N-terminal half of HAM-5 was essential for function. This region is required for MAK-2 and MEK-2 interaction and for correct cellular localization of HAM-5 to "fusion puncta." The localization of HAM-5 to puncta was not perturbed in 21 different fusion mutants, nor did these puncta colocalize with components of the secretory pathway. We also identified HAM-14 as a novel member of the HAM-5/MAK-2 pathway by mining MAK-2 phosphoproteomics data. HAM-14 was essential for germling fusion, but not for hyphal fusion. Colocalization and coimmunoprecipitation data indicate that HAM-14 interacts with MAK-2 and MEK-2 and may be involved in recruiting MAK-2 (and MEK-2) to complexes containing HAM-5.

Somatic cell fusion and conspecific cooperation are crucial social traits for microbial unicellular-to-multicellular transitions, colony expansion, and substrate foraging but are also associated with risks of parasitism. We identified a cell wall remodeling (cwr) checkpoint that acts upon cell contact to assess genetic compatibility and regulate cell wall dissolution during somatic cell fusion in a wild population of the filamentous fungus Neurospora crassa. Non-allelic interactions between two linked loci, cwr-1 and cwr-2, were necessary and sufficient to block cell fusion: cwr-1 encodes a polysaccharide monooxygenase (PMO), a class of enzymes associated with extracellular degradative capacities, and cwr-2 encodes a predicted transmembrane protein. Mutations of sites in CWR-1 essential for PMO catalytic activity abolished the block in cell fusion between formerly incompatible strains. In Neurospora, alleles cwr-1 and cwr-2 were highly polymorphic, fell into distinct haplogroups, and showed trans-species polymorphisms. Distinct haplogroups and trans-species polymorphisms at cwr-1 and cwr-2 were also identified in the distantly related genus Fusarium, suggesting convergent evolution. Proteins involved in chemotropic processes showed extended localization at contact sites, suggesting that cwr regulates the transition between chemotropic growth and cell wall dissolution. Our work revealed an allorecognition surveillance system based on kind discrimination that inhibits cooperative behavior in fungi by blocking cell fusion upon contact, contributing to fungal immunity by preventing formation of chimeras between genetically non-identical colonies.