Characterization of EOP-1 reveals cell autonomous oscillations preceding somatic cell fusion in Neurospora crassa

Abstract

Cell fusion is a fundamental process essential for the development and proliferation of eukaryotic organisms. In the ascomycete fungus Neurospora crassa, germinating spores undergo chemotropic interactions and fusion to merge into a supracellular unit, which gives rise to the mycelial colony. Within mature colonies, hyphal branches fuse to form anastomoses between leading hyphae, enhancing the overall connectivity of the mycelium. Both germling and hyphal fusion rely on the same molecular machinery. The MAP kinase MAK-2 and the fungal-specific protein SO have been identified as key regulators of these processes, and their alternating recruitment to the plasma membrane at interacting cell tips suggests a dialog-like cell communication mechanism involving dynamic switches between signal sending and receiving. However, the mechanisms that trigger the onset of this intercellular communication are still not understood. This study identifies EOP-1 as an interaction partner of the SO protein and functionally characterizes its role in cell communication and fusion. Deletion of the eop-1 gene abolished germling fusion and chemotropic interactions, while live-cell imaging showed EOP-1 oscillating at interacting cell tips, coinciding with SO recruitment. Intriguingly, EOP-1 displayed a similar dynamic, oscillatory tip recruitment also in isolated, non-interacting germlings, setting it apart from previously characterized fusion factors in N. crassa. This observation suggests for the first time that spore germlings of N. crassa exhibit fusion related cell-autonomous oscillatory behavior and implicates EOP-1 in initiating intercellular communication. The oscillatory recruitment pattern of EOP-1 was dependent on the presence of SO, MAK-1, MAK-2, BEM1 and HAM-14 in the cell. Loss of EOP-1 strongly reduced MAK-1 phosphorylation, placing EOP-1 upstream of MAK-1 pathway activation. This work offers new insight into how genetically and developmentally identical cells initiate and coordinate their communication and mutual attraction.