RNAi-based genomics tool via microinjection and nanoparticles for beet leafhopper control

Beet curly top virus (BCTV) is one of the most serious viral diseases and poses a significant threat to crops, particularly sugar beets, tomatoes, peppers, spinach, hemp, cucurbits, and beans. Transmitted by the beet leafhopper (Circulifer tenellus), the virus circulates through the insect's body without replication. Rapid acquisition of the virus by leafhoppers, coupled with their ability to retain and spread it for extended periods, necessitates innovative control strategies. This study centers on developing an RNAi-based functional genomics tool in the beet leafhopper to address the challenge posed by BCTV. We recently analyzed the transcriptome of BCTV-infected and healthy leafhoppers and identified a suite of genes that are differentially regulated by virus infection. From these, three candidate genes encoding proteins involved in oogenesis (ovum development), DNA repair and metabolism, and toxicity were prioritized for functional analysis. We employed two dsRNA delivery methods for RNAi experiments on 5-day-old, age-synchronized adult female beet leafhoppers: microinjection and nanoparticle-mediated delivery. For microinjection, 80 nL of dsRNA (500 ng/µL) was injected directly into the insects, while the nanoparticle-based method involved spraying insects with 200 µL of dsRNA mixed with star polycation-SPc nanoparticles at the same concentration. Quantitative Reverse transcription polymerase chain reaction (qRT-PCR) analysis assessed the reduction in gene expression. Results showed RNAi successfully silenced genes in beet leafhoppers, suggesting RNAi-based vector control potential. This represents the first evidence of RNAi in beet leafhopper and demonstrates the potential for translational research in vector and disease control.