The predicted mannosyltransferase gt69-2 antagonizes rfw-1 to regulate cell fusion in neurospora crassa

Filamentous fungi undergo somatic cell fusion to create a syncytial, interconnected hyphal network which confers a fitness benefit during colony estab-lishment. However, barriers to somatic cell fusion between genetically different cells have evolved that reduce invasion by parasites or exploitation by maladapted genetic entities (cheaters). Here, we identified a predicted mannosyltransferase, gly-cosyltransferase family 69 protein (GT69-2) that was required for somatic cell fusion in Neurospora crassa. Cells lacking GT69-2 prematurely ceased chemotropic signaling and failed to complete cell wall dissolution and membrane merger in pairings with wild-type cells or between Δgt69-2 cells (self fusion). However, loss-of-function muta-tions in the linked regulator of cell fusion and cell wall remodeling-1 (rfw-1) locus sup-pressed the self-cell-fusion defects of Δgt69-2 cells, although Δgt69-2 Δrfw-1 double mutants still failed to undergo fusion with wild-type cells. Both GT69-2 and RFW-1 localized to the Golgi apparatus. Genetic analyses indicated that RFW-1 negatively regulates cell wall remodeling-dependent processes, including cell wall dissolution during cell fusion, separation of conidia during asexual sporulation, and conidial ger-mination. GT69-2 acts as an antagonizer to relieve or prevent negative functions on cell fusion by RFW-1. In Neurospora species and N. crassa populations, alleles of gt69-2 were highly polymorphic and fell into two discrete haplogroups. In all isolates within haplogroup I, rfw-1 was conserved and linked to gt69-2. All isolates within haplogroup II lacked rfw-1. These data indicated that gt69-2/rfw-1 are under balancing selection and provide new mechanisms regulating cell wall remodeling during cell fusion and conidial separation.