Root hairs are essential for dsRNA nanoparticle uptake

Nanotechnology offers innovative agricultural solutions, with nanoparticles (NPs) showing promise for targeted delivery of double-stranded RNA (dsRNA). Exogenous dsRNA-mediated RNA interference (RNAi) is a promising gene silencing approach to combat viral pathogens or suppress specific plant endogenous genes without transgenic modification. The effective implementation of RNAi depends on the interactions of NPs with plants, including particle size and charge, uptake, translocation, physical barriers, and physiological and environmental conditions. The difficulties in achieving silencing phenotypes in field conditions remain a barrier to widespread agricultural adoption. Despite advancements, gaps remain in understanding NP uptake mechanisms and transport. Recently, clathrin-mediated endocytosis was found to be a driver of RNA root uptake, which we are also able to confirm with experimental observations. This study focuses on the role of root hairs as key mediators of dsRNA nanoparticle uptake. Inhibition of root hairs reduces RNA uptake, while increased root hair density enhances uptake. Lateral roots can bypass the casparian strip, and higher root hair density leads to increased RNA internalization beyond this barrier. We also propose that caveolar-mediated endocytosis can play a role in cationic polymer complexed-dsRNA NP uptake, and that the integration of RNA into cell walls across the plant allows for a slow release into the cell and sustained silencing phenotype. Validation of the microscopy results was performed by targeting phytoene desaturase (PDS) with dsRNA NPs by irrigation, where bleaching was achieved. Further research is needed to determine how these effects are influenced by plant metabolism, membrane transporters, and cell wall properties.