ENDOSOMAL SORTING COMPLEX REQUIRED FOR TRANSPORT machineries play crucial roles in vacuolar trafficking and cytokinesis in the moss Physcomitrium patens.

The ENDOSOMAL SORTING COMPLEX REQUIRED FOR TRANSPORT (ESCRT) proteins are essential for converting early endosomes (EEs) into late endosomes/multivesicular bodies (LEs/MVBs) during endocytosis. In animals, they also play noncanonical roles in cytokinetic abscission, membrane repair, and other membrane remodeling processes. However, their functions in plants, especially nonvascular plants, remain poorly understood. Here, we systematically investigate the localization and functions of ESCRT proteins in the moss Physcomitrium patens using fluorescent protein labeling, mutant analyses, interaction assays, and electron microscopy. We show that ESCRT-related proteins localize to endosomes and play a conserved role in endocytic trafficking. Notably, all tested ESCRT components also localize to the developing cell plate, whereas reporters involved in trans-Golgi network (TGN)-MVB transition do not. Disruption of VPS4, a key regulator of ESCRT-III, delays cytokinesis progression and alters the dynamics of cell plate assembly factors. We propose that the ESCRT machinery plays both conserved and previously unrecognized noncanonical roles in plants. By providing this evidence in a bryophyte, our work establishes a critical reference point for future functional studies in nonvascular plants and for evolutionary comparisons across plant lineages.