Abstract Description

Bacterial extracellular vesicles (EVs) are increasingly recognised as carriers of functional biomolecules, yet their roles in plant-pathogenic bacteria remain poorly characterised. Dickeya dadantii and Pectobacterium brasiliense are soft rot pathogens that cause tissue maceration in a wide range of plants. This study aimed to characterise EV production in these species and examine how vesicle traits respond to chemical stress. Scanning and transmission electron microscopy were used to visualise vesicle release and morphology, while nanoparticle tracking analysis determined vesicle size and abundance. Cultures were exposed to chemical stress conditions affecting osmotic balance, membrane stability, and fluidity (NaCl, EDTA, and ethanol) to assess their impact on vesicle production. EVs were isolated under standard conditions, and their nucleic acid content was examined. DNA and RNA were extracted from EVs and whole cells to compare size distributions and prepare for downstream sequencing. Both D. dadantii and P. brasiliense released EVs from the cell surface, exhibiting single or double membrane layers consistent with outer membrane vesicles (OMVs) and outer–inner membrane vesicles (OIMVs). D. dadantii produces larger but fewer EVs than P. brasiliense. Under chemical stress, the yield and protein profiles of EVs differed, indicating that stress modulates EV production and cargo composition. DNA and RNA fragments detected in EVs were shorter than those in corresponding whole-cell extracts. This suggests that EVs may transport DNA elements involved in gene transfer and small RNAs that could modulate gene expression in recipient cells.  These findings advance the understanding of EV-mediated processes in plant-pathogenic bacteria and highlight their potential role in stress adaptation.