Functional and proteomic characterization of Phytophthora nicotianae extracellular vesicles and putative cargo-sorting signals in filamentous pathogens.

Extracellular vesicles (EVs) are lipid-bound structures that transport bioactive molecules and are increasingly recognized as key mediators of plant-pathogen interactions. Although EV secretion has been reported in filamentous pathogens, their roles in plant immunity, microbial interactions and cargo-sorting mechanisms remain poorly understood. This study aimed to characterize the functional roles and composition of EVs produced by P. nicotianae, and to investigate shared EV-associated protein categories and putative cargo-sorting signals, including KFERQ-like (Lys-Phe-Glu-Arg-Gln) motifs, across filamentous pathogens. EVs were isolated and characterized using transmission electron microscopy (TEM) and Nanoparticle tracking analysis (NTA). TEM confirmed the presence of membrane-bound vesicles with both single- and double-membrane morphologies, while NTA demonstrated a reproducible and heterogeneous particle population. Functional assays demonstrated that P. nicotianae EVs elicit immune-associated responses in Nicotiana benthamiana, including cell death, ROS accumulation, and callose deposition. In addition, the EVs exhibited antimicrobial activity against five soil-associated bacterial isolates. Proteomic analysis identified 2,324 proteins, revealing a diverse EV cargo enriched in metabolic enzymes, protein-modifying enzymes, and transporters, along with virulence-associated proteins. Comparative analysis across selected filamentous pathogens identified shared EV-associated protein categories, including ATP synthase subunits, elongation factors, and heat shock protein 70 (HSP70), present across all examined species. Notably, approximately 78% of EV-associated proteins from nine filamentous pathogens contained putative KFERQ-like motifs. Collectively, these findings show that P. nicotianae secretes structurally heterogeneous EV-like particles with biological activity in plant and bacterial assays, and suggest potential roles in host interaction, microbial competition, and putative conserved features that may be involved in cargo loading.