ABSTRACT 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.

中文翻译：

---

表征 GFP-AtPEN1 作为从本氏烟草叶中分离的细胞外囊泡的标记蛋白

摘要 植物细胞外囊泡 (EVs) 是细胞分泌的膜结构，包裹着细胞质成分，包括发病机制相关的蛋白质、微小 RNA 和微小 RNA 等。它们的作用被证明与植物-微生物相互作用有关。尽管为拟南芥和其他植物物种的 EV 标记开发了几种标记蛋白，但我们缺乏对从模式植物本氏烟草中分离的 EV 的类似知识，该植物是植物病原体研究的极好宿主。在这里，我们在本氏烟草叶中瞬时表达了两种拟南芥 EV 标记 AtPEN1 和 AtTET8 以及一种 ESCRT 蛋白 VPS4，并测试了它们在 EV 标记中的能力。我们发现本氏烟草叶中 GFP 标记的 AtPEN1 表达比其他测试的蛋白质更稳定，和 GFP-AtPEN1 在本氏烟草 EV 中积累。此外，我们发现从本氏烟草叶质外体中分离的 EV 具有典型的 EV 密度和囊泡状形态。我们的发现表明 GFP-AtPEN1 可用作标记本氏烟草 EV 的极好标记蛋白。