Abstract

Plant viral co-infections pose a significant threat to global agricultural productivity. In this study, a novel multivalent attenuated vaccine (CMV-R2TP) was developed using the cucumber mosaic virus (CMV) as a backbone, together with a ROS-responsive vaccine nanodelivery system (pCMV-R2TP@PTP-TAT), offering a comprehensive and environmentally friendly strategy for managing plant viruses. Through the modification of the CMV 2b protein, a stable base vector (pR2-2bIII) capable of accommodating up to 400 bp foreign fragments was constructed. The resulting CMV-R2TP vaccine demonstrated protective efficacy ranging from 41.28% to 53.78% against co-infections caused by CMV, TMV, and PVY. To address the constraints of conventional Agrobacterium -mediated delivery, a novel nanodelivery platform, pCMV-R2TP@PTP-TAT, was synthesized through synergistic modification of PLGA with PEI, TK, and TAT. This system demonstrated excellent physicochemical characteristics, achieving 87.1% ROS-responsive release within just 4 h. It was also found to be capable of efficiently delivering nucleic acids into plant cells, resulting in gene expression levels comparable to those achieved by Agrobacterium transformation within 120 h. Notably, the pCMV-R2TP@PTP-TAT system showed exceptional biocompatibility, neither harming plant tissues nor inducing oxidative stress responses. Overall, a comprehensive technical platform was established, integrating multivalent vaccine design, nanodelivery optimization, and safety assessment. This system combines measurable protective efficacy with favorable environmental safety, offering a scalable approach for sustainable management of plant viral diseases and paving the way for novel applications in agricultural nanobiotechnology. Graphical abstract