Abstract: Plants are constantly challenged by diverse pathogens and respond through multilayered immune mechanisms described by the classical “zig-zag” model. Apoplast nanoparticles (ANs), extracellular vesicle (EV)-like entities, are dynamically remodeled during plant–pathogen interactions. Although EVs are known to carry immune-regulatory cargos, the specificity and coordination of ANs protein responses to distinct pathogens remain largely unresolved. Here, we show that the glycosylation profiles of ANs and their N-glycoproteins undergo distinct remodeling upon infection with Pseudomonas syringae and Botrytis cinerea, as revealed by lectin microarray and mass spectrometry analyses. We identify SPILR, an ANs protein that modulates bacterial morphology and motility by reducing polygalacturonase activity and inhibiting the bacterial flagellar P-ring protein, FlgI. Additionally, the ESM1 protein enhances Arabidopsis resistance to B. cinerea through modulation of lipase activity and lipid metabolism. Disruption of N-glycosylation sites on ANs proteins compromises their antimicrobial function and alters host resistance to both bacterial and fungal pathogens. Together, our findings uncover N-glycosylation as a critical determinant of ANs-mediated extracellular immunity, highlighting glycosylation as an integrative mechanism linking vesicle biology, pathogen specificity, and immune signaling. This work establishes a framework for glycoengineering-based strategies to enhance crop resistance and advance nano-agricultural applications.

Key words: apoplast, extracellular vesicles, nanoparticles, N‐glycosylation, plant immunity