Some plant proteases contain a latent sequence known as the plant-specific insert (PSI) that, upon release from the full protease sequence, initiates membrane fusion to defend from pathogens. However, the mechanism by which it exerts its effects has been unclear. Zhao et al. report an elegant integration of biophysical experiments and molecular dynamics simulations to reveal events leading up to PSI-mediated membrane fusion. Their results demonstrate a pH-dependent monomer-to-dimer transition, clear evidence of membrane association, and probable structures of prefusion intermediates. These data expand our understanding of the elusive PSIs and may provide new directions for antimicrobial development.