Characterization of GFP-AtPEN1 as a marker protein for extracellular vesicles isolated from Nicotiana benthamiana leaves

Plant extracellular vesicles (EVs) are cell-secreted membrane structures enclosing cytosolic components, including pathogenesis-related proteins, tiny RNAs, and microRNAs et al. Their roles are shown to be involved in plant-microbe interactions. Albeit several marker proteins were developed for EVs labeling for Arabidopsis thaliana and other plant species, we lack similar knowledge on EVs isolated from model plant Nicotiana benthamiana, which serves as an excellent host for plant pathogen studies. Here, we transiently expressed two arabidopsis EV markers AtPEN1 and AtTET8 and one ESCRT protein VPS4 in Nicotiana benthamiana leaves and tested for their ability in EV labeling. We found that GFP tagged AtPEN1 expression in Nicotiana benthamiana leaves is more stable than other proteins tested, and GFP-AtPEN1 accumulated in Nicotiana benthamiana EVs. Furthermore, we showed that EVs isolated from Nicotiana benthamiana leaf apoplast have typical EV density and vesicle-like morphology. Our finding demonstrates that GFP-AtPEN1 can be used as an excellent marker protein to label Nicotiana benthamiana EVs.