RNAi-mediated silencing of a regulatory subunit of the 26S proteasome induces mortality and female sterility in leafhopper vectors of grapevine Flavescence dorée phytoplasmas

Abstract RNA interference (RNAi) is a promising strategy for pest control due to its high target specificity, which minimizes lethal effects on non-target arthropods. A critical step in developing RNAi-based approaches is the selection of an appropriate and effective target gene. In this study, we evaluated the impact of RNAi-mediated silencing of the Regulatory Particle non-ATPase 6 ( Rpn6 ) subunit of the 26S proteasome on insect fitness in Scaphoideus titanus and Euscelidius variegatus , the natural and laboratory vectors of grapevine Flavescence dorée phytoplasmas, respectively. To minimize potential off-target effects, RNAi-triggering molecules were designed to target the least conserved regions of the Rpn6 coding sequence, using Apis mellifera as a reference for pollinator species. Efficient Rpn6 knockdown was achieved through abdominal microinjection, resulting in significantly reduced survival and fertility in both vector species. In S. titanus female’s ovary morphology was notably altered, while treated E. variegatus almost failed to reproduce. Sex-cross-injection experiments further indicated that the observed reproductive disruption was primarily due to effects on females. Overall, our results highlight Rpn6 as a promising RNAi-target for the control of S. titanus populations in the vineyards, by impairing both survival and reproduction in a long-term perspective. This work paves the way for the development of more sustainable and species-specific pest management tools.