Chitosan–Tripolyphosphate Nanoparticles Enhance Foliar dsRNA Delivery and Suppress Turnip Crinkle Virus in Arabidopsis

Topically applied double-stranded RNA (dsRNA) can trigger antiviral RNA interference in plants, but naked dsRNA is unstable and poorly taken up after foliar application. Here, we developed an eco-friendly chitosan- tripolyphosphate (CS-TPP) nanocarrier to enhance dsRNA delivery using turnip crinkle virus (TCV) as a model and tested it in Arabidopsis thaliana. A dsRNA targeting the TCV-coat protein (CP) gene was produced in Escherichia coli, and complexation with CS-TPP was induced by ionic gelation. Complete complexation occurred at a 1:1 CS-TPP:dsRNA ratio, producing cationic nanoparticles that protected dsRNA from RNase digestion and maintained its integrity during incubation at 37°C. Using Cy3 fluorescence imaging, we observed stronger epidermis-associated fluorescence in plants treated with the CS-TPP@dsRNA complex than in those treated with naked dsRNA, and RT-qPCR confirmed higher CP-target RNA fragment accumulation in planta. Small RNA sequencing revealed increased levels of TCV-derived, 21-24 nt siRNA molecules targeting the CP region, suggesting the applied dsRNA was processed in planta. Importantly, a CS-TPP@ TCV-dsRNA foliar spray application reduced systemic CP RNA levels at 7 and 14 days post-treatment and alleviated disease symptoms more effectively than naked dsRNA. These results demonstrate that CS-TPP nanocomplexation improves dsRNA stability and delivery, enabling improved and more durable antiviral protection.