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 categories based on their size, content, and molecular marker profiles. These are TET8‐positive small EVs (∼50–200 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.

Recent studies on membrane imaging in the plant endomembrane system by 2-D/3-D CLSM and TEM provide future perspectives of whole-cell ET and cryo-FIB-aided cryo-ET analysis.

Plant fertilization relies on controlled pollen tube growth that integrates membrane dynamics and cell wall expansion. We previously identified an unconventional exocytic pathway wherein Golgi-derived secretory vesicles (GDSVs) bypass the trans-Golgi network to deliver Nicotiana tabacum pectin methylesterase 1 (NtPPME1), thereby modulating cell wall rigidity. However, the mechanisms linking this pathway with membrane dynamics and signaling remain elusive. Here, we used cryo–focused ion beam–scanning electron microscopy and three-dimensional tomography to identified GDSVs as a distinct vesicle population at the pollen tube tip. We further demonstrated that tobacco LORELEI-like glycosylphosphatidylinositol-anchored protein 4 (NtLLG4), a key signaling molecule controlling membrane dynamics and integrity, functions as a receptor for NtPPME1, regulating its polar exocytosis via GDSVs to control cell wall stiffness. Furthermore, we identified trafficking signals that direct the unconventional exocytosis of NtPPME1 across intracellular organelles. Our findings reveal a crucial mechanism coupling cell wall rigidity with membrane signaling to control pollen tube growth and integrity during fertilization.

Sclerotinia sclerotiorum is a devastating fungal pathogen that can colonize numerous crops. Despite its economic importance, the regulation of its development and pathogenicity remains poorly understood. From a forward genetic screen in S. sclerotiorum, six UV mutants were identified with loss-of-function mutations in SsATG1, SsATG2, SsATG4, SsATG5, SsATG9, and SsATG26. Functional validation through gene knockouts revealed that each ATG is essential for sclerotia formation, although the morphology of appressoria was not significantly altered in the mutants. Different levels of virulence attenuation were observed among these mutants. Autophagy, monitored using GFP-ATG8, showed dynamic activities during sclerotia development. These findings suggest that macroautophagy and pexophagy contribute to sclerotia maturation and virulence processes. Future work will reveal how autophagy controls target organelle or protein turnover to regulate these processes.

Understanding cellular events in three dimensions (3D) is of great importance for the annotation and illustration of biological processes in a contextual way. Imaging techniques based on electron microscopy (EM), such as those derived from scanning electron microscopy (SEM) and transmission electron microscopy (TEM), provide various options to visualize biological samples at scales ranging from cells to macromolecules in situ . Recently, a series of cryogenic techniques has brought EM‐based imaging to a new level, enabling specimens to retain their hydrated state throughout the sample preparation and imaging steps, thereby offering a near‐native visualization of cellular events. The application of dual‐beam focused ion beam (FIB)‐SEM to biological samples has enabled high‐resolution reconstructions in 3D and streamlined sample preparation workflows for downstream cryo‐electron tomography (cryo‐ET) imaging. However, applications of these technologies to plant materials are limited due to intrinsic characteristics of plant cells (e.g., non‐adhesive growth, large size with a central vacuole, and the presence of cell walls). For the timely application of dual‐beam FIB‐SEM in three‐dimensional subcellular imaging of plant materials, we have recently tested and developed three major workflows with proof‐of‐concept evidence using developing anthers and in vitro ‐cultured pollen tubes based on Aquilos 2 Cryo‐FIB, including (1) room‐temperature FIB‐SEM volume imaging, (2) cryo‐lamellae preparation from cell suspension culture or high‐pressure‐frozen organs for cryo‐ET imaging, and (3) cryo‐FIB‐SEM volume imaging, which will facilitate structural studies of plant materials and provide technical guidance for the broader plant cell biology research community.