Chitosan-dsRNA nanoparticles for targeted gene in the model C4 species Setaria viridis: synthesis, characterization, and preliminary assessment of fumarase gene silencing

The development of efficient gene-silencing techniques is essential for advancing plant biotechnology and enabling targeted approaches for crop improvement and weed management. This study investigated the potential of chitosan-based nanoparticles as a delivery system for double-stranded RNA (dsRNA) targeting the fumarase gene in Setaria viridis , a model C4 grass species. Fumarase, a key enzyme in the tricarboxylic acid cycle, was selected due to its critical role in carbon metabolism and potential impact on plant growth. Using ionotropic gelation, we synthesized and characterized chitosan nanoparticles carrying fumarase-specific dsRNA, achieving consistent particle sizes of 246.6 ± 3.0 nm with a positive zeta potential of 15.3 ± 0.10 mV and a polydispersity index of 0.37 ± 0.01. We hypothesized that delivering the dsRNA using the Fumarase gene would lead to decreased levels of Fumarase expression. The nanoparticles demonstrated high dsRNA encapsulation efficiency (99.0%) and maintained colloidal stability for up to 72 hours under storage conditions. Fluorescence microscopy revealed successful uptake and distribution of rhodamine-labeled nanoparticles in S. viridis leaf tissue, with maximum accumulation observed at 48 hours post-application. RT-qPCR analysis showed a 2.6-fold reduction in fumarase transcript levels 48 hours after treatment, though this change did not reach statistical significance (p > 0.05). While the observed gene expression changes were modest, our findings demonstrate the feasibility of nanoparticle-mediated dsRNA delivery in S. viridis and provide insights for optimizing such systems. This study contributes to the developing field of non-transgenic gene regulation in plants and highlights both the potential and current limitations of nanoparticle-based RNA delivery systems for plant biotechnology applications. • Chitosan nanoparticles encapsulate dsRNA with >99% efficiency • Nanoparticles show foliar uptake in S. viridis within 48 h • Nanoparticles formulation remains stable for up to 72 h • Scalable approach for C4 crop biointervention • Doses adjustments might result in significant fumarase gene silencing