ENGINEERED EXTRACELLULAR VESICLES FOR RNA DELIVERY AGAINST DEVASTATING PLANT FUNGAL PATHOGENS

Abstract

Global crop losses to phytopathogenic fungi such as Botrytis cinerea, Verticillium dahliae, and Fusarium species threaten food security, yet conventional chemical fungicides face mounting challenges from resistance development and environmental toxicity. Spray-Induced Gene Silencing (SIGS) using double-stranded RNA offers a species-selective, environmentally sustainable alternative, but naked RNA undergoes rapid degradation under field conditions. This review examines the transformative potential of engineered extracellular vesicles (EVs) including plant-derived nanovesicles, bacterial EVs, and synthetic biomimetic artificial nanovesicles as protective carriers for RNA-based crop protection. We synthesize current understanding of cross-kingdom RNA interference (ckRNAi), wherein host plants package regulatory small RNAs and messenger RNAs into exosomes that traverse fungal cell walls via clathrin-mediated endocytosis to silence virulence factors. Nanoscale biophysical analyses reveal that fungal cell walls function as dynamic viscoelastic hydrogels permitting intact vesicle transit despite static pore size limitations. We critically evaluate nucleic acid loading technologies, demonstrating that hydrophobic co-incubation achieves superior encapsulation efficiency (30–60%) and preserves vesicle integrity compared to electroporation or sonication. Comparative analysis of production platforms highlights bacterial EV systems as economically viable for continuous manufacturing, while artificial nanovesicles offer batch-to-batch consistency. Encapsulation extends RNA persistence from 2–3 days to 21 days on foliage, substantially improving SIGS field applicability. The recent US EPA registration of Ledprona establishes regulatory precedent, with risk assessment frameworks emphasizing sequence specificity, environmental degradation kinetics, and carrier biosafety. We conclude that engineered EV-mediated RNA delivery represents a paradigm shift in sustainable crop protection, though industrial-scale manufacturing, active pathogen targeting, and multiplexed resistance management strategies require continued development to realize full agronomic potential.