Structural and Functional Characterization of EXPO‐Derived Extracellular Vesicles in Plants

In this study, 3D electron tomography (ET), cryo‐ET, and immunogold transmission electron microscopy (TEM) are employed to characterize plant extracellular vesicles (EVs) under physiological conditions. EVs are classified into three distinct um EVs are shown to originate from the plant‐specific exocyst‐positive organelle (EXPO), potentially contributing to plant stress responses. ABSTRACT Extracellular vesicles (EVs) are increasingly being recognized as important mediators of intercellular communication in plants, but their biogenesis, heterogeneity, and membrane origins remain poorly understood. Here, the structural diversity, formation mechanisms and potential functions of EVs from various plant cell types under normal physiological conditions are demonstrated using 3D electron tomography (ET), cryo‐ET, and immunogold transmission electron microscopy (TEM). The EVs are classified into three distinct –200 nm in diameter, lacking organelle content), Exo70E2‐positive medium EVs (∼200–500 nm in diameter, containing ribosomes), and PEN1‐positive large EVs/Extracellular tubules (∼500–2000 nm in diameter, containing ribosomes and small vesicles). The Exo70E2‐positive medium EVs originate from the plant‐specific exocyst‐positive organelle (EXPO), and isolated EXPOs carry cargoes associated with stress responses. Subsequent transcriptomic profiling and pathogen‐resistance assays in exo70e2 mutants indicate that EXPO‐derived EVs contribute to plant defense, potentially by delivering defense‐related proteins during pathogen infection. Collectively, these findings provide a framework for understanding EV heterogeneity in plants and highlight EXPO‐derived EVs as a potential key component of plant defense. In this study, 3D electron tomography (ET), cryo-ET, and immunogold transmission electron microscopy (TEM) are employed to characterize plant extracellular vesicles (EVs) under physiological conditions. EVs are classified into three distinct um EVs are shown to originate from the plant-specific exocyst-positive organelle (EXPO), potentially contributing to plant stress responses. ABSTRACT Extracellular vesicles (EVs) are increasingly being recognized as important mediators of intercellular communication in plants, but their biogenesis, heterogeneity, and membrane origins remain poorly understood. Here, the structural diversity, formation mechanisms and potential functions of EVs from various plant cell types under normal physiological conditions are demonstrated using 3D electron tomography (ET), cryo-ET, and immunogold transmission electron microscopy (TEM). The EVs are classified into three distinct –200 nm in diameter, lacking organelle content), Exo70E2-positive medium EVs (∼200–500 nm in diameter, containing ribosomes), and PEN1-positive large EVs/Extracellular tubules (∼500–2000 nm in diameter, containing ribosomes and small vesicles). The Exo70E2-positive medium EVs originate from the plant-specific exocyst-positive organelle (EXPO), and isolated EXPOs carry cargoes associated with stress responses. Subsequent transcriptomic profiling and pathogen-resistance assays in exo70e2 mutants indicate that EXPO-derived EVs contribute to plant defense, potentially by delivering defense-related proteins during pathogen infection. Collectively, these findings provide a framework for understanding EV heterogeneity in plants and highlight EXPO-derived EVs as a potential key component of plant defense. Advanced Science, EarlyView.