Lab-scale method for plant-mediated delivery of dsRNAs to phloem-feeding leafhoppers

RNA interference (RNAi) regulates gene expression in eukaryotes, and it is an emerging tool in crop protection by exogenous applications of double-stranded RNAs (dsRNAs) to silence the expression of essential pest genes. Nevertheless, delivery of dsRNAs to sap-sucking insects is a major challenge for RNAi applications. The present work aimed at verifying whether in leafhopper species, RNAi can be triggered by plant-mediated delivery, and providing a proof of concept towards field applications. Two phytoplasma vectors species, Euscelidius variegatus and Scaphoideus titanus (Hemiptera: Cicadellidae), were used as case study. Gene silencing can be achieved efficiently in both species through microinjection of dsRNAs, despite the technique being time consuming and inapplicable on large scale. This protocol was set as gold standard for the development of a higher throughput approach. Soaking of nymphs in a solution with co-adjuvant and dsRNAs as well as insect feeding on whole plants or detached leaves immersed in a dsRNA solution were assayed as alternative delivery strategies. Nymph soaking did not induce specific gene silencing, while plant absorption proved to be suitable to deliver both a coloured solution and control dsRNAs targeting green fluorescent protein gene. Insect feeding on detached leaves immersed in dsRNA solution was selected to test silencing of two gut-specific (legumain and natterin) and one ubiquitous (ATP synthase β) genes. The expression of the three genes significantly decreased in E. variegatus insects fed on dsRNA-treated plants. Similarly, a significant reduction of ATP synthase β transcript was measured in S. titanus fed on dsRNA-treated plants.