DOTAP-functionalized DNA nanocages enable efficient miR168d trafficking to antagonize PVY infection by modulating HSP90-5 homeostasis

Background Plant viruses cause severe agricultural losses. Conventional pesticides have issues such as residues and resistance, while the delivery efficiency of functional microRNAs in RNA interference strategies is low. This study aimed to evaluate the inhibitory effect of miR168d on Potato virus Y in Nicotiana benthamiana and construct a high-efficiency nanodelivery system. Results MicroRNA168d significantly reduces the replication and spread of Potato virus Y by targeting and inhibiting Heat Shock Protein 90-5. For the nanocomplex with a tetrahedral DNA nanostructure as the carrier and the cationic lipid 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) as the coating—characterization showed that it enhances the vascular transport efficiency, nuclease resistance, and cellular permeability of miR168d. Additionally, this nanocomplex exhibits low toxicity and good biocompatibility toward tobacco suspension cells. After foliar application, the nanocomplex group showed higher accumulation of miR168d in the leaves and stems of N.benthamiana compared with that in the control group. Specifically, the accumulation of mRNA and protein of the PVY coat protein in the nanocomplex group decreased by 64.3% and the corresponding percentage (consistent with the reduction at the protein level), respectively. As a result, the disease resistance of the plants was significantly improved . Conclusion This study reveals the antiviral mechanism of the miR168d-HSP90-5 regulatory module, provides a green non-transgenic nanoscale strategy, and is of great significance for agricultural antiviral breeding and sustainable agriculture.