Capture of the SmSTRIPAK proxiome identifies the greenbeard proteins SmDOC1/2 as regulators of the MAK2 pathway to control sexual development in Sordaria macrospora

Hyphal fusion and sexual development in filamentous fungi rely on coordinated signaling of numerous conserved nodes such as the striatin interacting phosphatase and kinase (STRIPAK) complex or the pheromone response (PR) MAP kinase cascade (MIK2, MEK2, MAK2, HAM5). Here we used the homothallic ascomycete Sordaria macrospora (Sm) to screen for putative protein interactors of the SmSTRIPAK complex. Using the STRIPAK complex interactor 1 (SCI1) subunit of the complex as bait, we enriched and identified canonical SmSTRIPAK components and a determinant of communication (DOC) protein. The DOC proteins were previously described in the closely related and heterothallic species Neurospora crassa , functioning in allorecognition of germlings and hyphal fusions. We generated ΔSmdoc1, ΔSmdoc2 single deletion strains and the double deletion mutant ΔSmdoc1ΔSmdoc2 in S. macrospora . Deletion phenotypes were paradoxical: single knockouts (ΔSmdoc1 or ΔSmdoc2) were nearly sterile and sexual development was impaired, yet the double mutant (ΔSmdoc1ΔSmdoc2) exhibited wild-type fertility and development, demonstrating non-redundant and mutually antagonistic roles. Similarly, we demonstrated an impairment of the N. crassa Δ doc-2 mutant in sexual development. Using gene tagging at the native locus, we performed TurboID-based proximity mapping with SmDOC1 and SmDOC2 as bait proteins. This proximity mapping demonstrated close ties of SmDOC1/2 to components of the PR MAP kinase pathway and revealed mutual SmDOC1 - SmDOC2 proximity. Yeast Two-Hybrid experiments with SmDOC1 confirmed the direct interaction with the MAP kinases MEK2 and MAK2. Fluorescence microscopy revealed that SmDOC1-TagRFP-T localized to ring-like structures around septal pores. Our results demonstrate that the DOC system is not restricted to heterothallic N. crassa , but also plays an essential role in the development of fruiting bodies in the homothallic fungus S. macrospora . These findings suggest the DOC1/2 proteins as a novel system that integrates STRIPAK and PR pathways, providing a possible mechanistic explanation for their non-additive deletion strain phenotypes.