Stage-Specific Lipidomes of Extracellular Vesicles Modulate Fungal Symbiosis.

The mycoheterotrophic orchid relies entirely on symbiosis with for nutrient acquisition during tuber development. The signaling mechanisms underlying this interaction have long been a research focus, and several pathways, such as phytohormone-mediated signaling, have been reported. However, the role of plant-derived extracellular vesicles (PDEVs) in - communication remains unexplored. In this study, we conducted a comprehensive lipidomic analysis of -derived extracellular vesicles (GDEVs) isolated from juvenile, immature (active symbiosis), and mature tubers. By employing high-resolution mass spectrometry and advanced statistical methods, we established a detailed EV lipidome profile for , identifying 996 lipid species spanning eight major classes. Distinct lipidomic remodeling was observed throughout tuber maturation. Notably, as the immature stage corresponds to the period of peak symbiotic activity, targeted lipidome comparisons enabled the identification of core lipid markers, particularly Glc-sitosterols and the polyketide 7,8-dehydroastaxanthin, which are highly enriched during active symbiosis and potentially associated with inter-kingdom communication. These findings suggest that developmentally regulated lipid transport via EVs plays a critical role in mediating - interaction. Our work not only illuminates the contribution of vesicle lipids to plant-fungal interaction but also provides a methodological foundation for investigating EV-mediated signaling in non-model plant-microbe systems.