Impact of the sterol O-acyltransferase gene (Cmare2) on morphology and stability of the mushroom Cordyceps militaris.

Strain degeneration in mushroom cultivation can lead to significant commercial losses, yet the genetic factors remain elusive. This study identified the sterol O-acyltransferase gene, Cmare2, which is associated with colony sectorization in Cordyceps militaris, using a T-DNA insertion-mutant library. CmARE2 is conserved across Ascomycota and Basidiomycota. Deletion of Cmare2 via CRISPR/Cas9 resulted in degenerative phenotypes, including colony sectorization, hyphal adhesion, reduced conidia production, and abnormal fruiting body development. Scanning electron microscopy revealed surface perforations, cobweb-like filaments connecting the adhering hyphae, and eventual hyphal impairment. These phenotypes may be attributed to disturbance of sterol homeostasis, which impairs cell membrane fluidity and permeability. Overexpression of Cmare2 via CRISPR/Cas9 led to a more stable strain with consistent morphology, reduced reactive oxygen species level, and robust mycelia during successive subculturing. This research integrates forward and reverse genetics to unravel the molecular mechanisms behind strain degeneration, providing valuable insights for developing stable, non-degenerative mushroom strains.