Comparative proteomics reveals protein signatures shared between and unique to bona fide plant EVs and other plant-derived vesicles.

Extracellular vesicles are membrane-bound particles that mediate intercellular communication by transporting bioactive molecules. In plants, extracellular vesicles are essential for defence responses, cell wall remodeling, and interkingdom interactions, holding significant promise in agriculture, biotechnology, and nanomedicine. However, the characterization of plant extracellular vesicles, including their nomenclature and biological function, remains challenging. Nondestructive isolation methods typically yield more bona fide extracellular vesicles (genuine EVs or gEVs), while destructive approaches often result in a heterogeneous mixture of intracellular, extracellular, and artificial vesicles, collectively termed plant-derived vesicles, along with co-purifying contaminants released from disrupted cells. In this context, the identification and characterization of conserved proteins associated with extracellular vesicles, and the distinction of potential nonvesicular contaminants, are needed to provide critical insights into their biological roles and the functional significance of extracellular vesicles. Herein, we conducted an in silico comparative analysis using previously published proteomic datasets from gEVs and plant-derived vesicles. Using phylogenetic orthology inference and protein domain analysis, we identified conserved and distinct molecular features differentiating gEVs from plant-derived vesicles, alongside proteins likely co-purified as contaminants. Signal peptide prediction, transmembrane domain, and motif analyses were performed to uncover specific traits of genuine EV and plant-derived vesicle protein cargo. Our findings reveal substantial variability among plant-derived vesicles and identify evolutionarily conserved functions and molecular features specifically associated with genuine extracellular vesicles. Furthermore, our analyses shed light on potential secretion pathways of different classes of extracellular vesicle-associated proteins, as well as on their transport modes, likely distinguishing between luminal and membrane-associated cargo. Collectively, these results pave the way for a more comprehensive understanding of plant extracellular vesicle biology.