Protein-based spherical nanoparticles for dsRNA delivery to nematodes – A platform technology for RNA silencing

Phytoparasitic nematodes are widespread agricultural pests that cause severe damage to roots, resulting in significant crop losses. Chemical control with nematicides is the conventional pest management strategy, but this is a threat to beneficial species and human health. Furthermore, indiscriminate use leads to the emergence of resistant pest populations. Phytoparasitic nematodes can also be controlled by RNA interference (RNAi), a eukaryote defense mechanism against invasive nucleic acids that is triggered by double stranded RNA (dsRNA) and causes the specific cleavage or translational repression of the corresponding mRNA. More than 75 genes in phytoparasitic nematodes have been targeted by RNAi under laboratory conditions, but the application of RNAi in the field is limited by delivery barriers such as inefficient cellular uptake and RNA degradation. The latter is particularly important when targeting soil-dwelling nematodes because free RNA is not stable in soil. We therefore encapsulated dsRNA in proteinaceous spherical nanoparticles (SNPs) formed by the thermal annealing of coat proteins from tobacco mild green mosaic virus (TMGMV). We optimized loading of dsRNA into SNPs by charge neutralization and condensation of dsRNA with Mg²⁺ at pH < 3.0, allowing us to encapsulate up to 0.2 mg dsRNA per 1.0 mg of SNPs, 100–200 nm in diameter. This was a 10-fold improvement over the non-optimized dsRNA-SNP formulation (i.e. encapsulation of dsRNA without charge neutralization and condensation). A transgenic Caenorhabditis elegans line constitutively expressing mCherry was used as a model to confirm that dsRNA remains functional and triggers RNAi following the ingestion of dsRNA-laden SNPs. The silencing effect lasted ∼180 h and reduced mCherry fluorescence by 76.2 ± 13.6 %. We confirmed that dsRNA-loaded SNPs retain their silencing activity when passed through a soil column, indicating that the RNAi-based control of phytoparasitic nematodes using SNPs should be possible in the field.