Interactional Endocytosis and Transmembrane Transport Promote Cellular Internalization of Nano-Delivered RNA Drugs...

Botrytis cinerea is a widespread plant pathogenic ascomycete that causes grey mould in over 1400 species and impacts global crop yields. Double-stranded RNA (dsRNA)-induced gene silencing is a promising technology for pest control, but efficient delivery remains a major challenge. This work presents a star polycation (SPc)-based nano-delivery platform that enhances dsRNA internalization and enables effective control of B. cinerea. SPc assembles with dsRNA via electrostatic interactions, hydrogen bonding and Van der Waals forces, forming stable nanoscale complexes. SPc facilitates dsRNA uptake into fungal mycelia by 2.19-fold, primarily via two activated routes: endocytosis and transmembrane transport, which increase vesicle and particle numbers by 3.74- and 1.87-fold, respectively. Six genes (pkc, ypt10, pil1, mfs1, mfs2 and mfs3) in these two routes play crucial roles in the delivery process. RNA interference, chemical inhibition and mutation experiments demonstrate that the two routes interact to optimize the cellular uptake of SPc-loaded dsRNA. Finally, a high-efficiency RNA fungicide is developed, comparable to commercial fungicide in protecting cucumber leaves and tomato fruits. This study reveals the synergistic mechanism of nanocarrier-mediated gene delivery and identifies key genes, supporting the design and application of RNA pesticides.