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