The chitin synthase regulator CSR-3 promotes cellular integrity during cell-cell fusion in the filamentous ascomycete fungus Neurospora crassa.

Cell-cell fusion in plants and fungi requires localized cell wall dissolution at the contact site to allow direct plasma membrane contact and subsequent membrane merger. Since cell wall removal carries the risk of cell rupture, the process must be tightly regulated to permit localized fusion pore formation while preserving cellular integrity. While the molecular events guiding cell-cell signaling leading to contact between fusing fungal cells have begun to unfold, the post-contact mechanisms stabilizing the forming fusion pore remain largely unknown. Here, we identify the chitin synthase regulator CSR-3 as a molecular factor promoting stable pore formation during somatic fusion in the fungal cell fusion model Neurospora crassa. CSR-3 specifically accumulates at the contact zones of fusing cells and contributes to fusion fidelity by preventing membrane rupture and lysis, particularly under calcium-limited conditions. Loss of CSR-3 leads to elevated fusion-induced lysis, a phenotype rescued by osmotic stabilization, suggesting a cell wall defect. Beyond fusion, CSR-3 is involved in septum formation, septal pore plugging, conidiation, and the response to biotic and abiotic cell wall stress. These observations support a broader role for CSR-3 in chitin-mediated cell wall remodeling. Our data indicate that CSR-3 dynamics at fusion sites depend on the MAP kinase MAK-1, implicating cell wall integrity signaling in post-contact fusion events. Consistent with this finding, phospho-mimetic analysis suggests a regulatory role for CSR-3 phosphorylation. Co-localization and genetic analyses identify the chitin synthase CHS-2 as a likely downstream target of CSR-3, with both proteins functioning in the same pathway. Together, our findings reveal that CSR-3 coordinates cell wall remodeling during cell fusion and stress responses, uncovering a crucial regulatory layer that safeguards fungal cellular integrity during dynamic developmental processes. Our observations support a model in which cell wall biosynthesis plays a critical role in cell wall remodeling during fusion pore formation.