Silencing of ATP synthase beta in phytoplasma vectors: effects on pathogen multiplication and egg development

Phytoplasmas are wall-less bacteria associated with severe diseases in hundreds of plant species, producing in many cases major yield loss in several crops. Phytoplasmas inhabit the phloem elements of infected plants and are transmitted from plant to plant by hemipteran insect species, belonging to the suborders Auchenorrhyncha (Cicadellidae and Cixiidae are the families with the largest number of vector species) and Sternorrhynca (Psyllidae family). Control of phytoplasma diseases is so far mainly based on insecticide treatments against vector populations. Taking into account the impacts of insecticides on agro-economy, public health, and environment, the development of innovative and more sustainable management strategies is becoming urgent. The leafhoppers Scaphoideus titanus and Euscelidius variegatus are known phytoplasma vectors. The former species is the natural vector of the phytoplasma associated with the grapevine flavescence dorée (FD), 16SrV-C and -D; the latter is the natural vector of the 'Candidatus Phytoplasma asteris' Chrysanthemum yellows strain, 16SrI-B, and a laboratory vector of FD phytoplasma. Previous studies indicated a crucial role of the insect ATP synthase during phytoplasma infection process. In the present work, the silencing of ATP synthase ? was obtained by injection of specific dsRNAs (dsATP) in both S. titanus and E. variegatus. After the injection of dsRNAs in E. variegatus and successive phytoplasma acquisition on infected plants, silenced E. variegatus showed significant reduction of the target protein and lower phytoplasma loads compared to the not-silenced controls. An in vivo role for insect ATP synthase ? could be hypothesized during phytoplasma infection process. Complete lack of progeny was also observed in several repeated experiments for E. variegatus insects following silencing of ATP synthase ?. Consistently, altered morphology of ovaries as well as deregulation of genes involved in egg development were observed in ATP synthase ? silenced E. variegatus adult females in comparison with specimen injected with dsGFP, used as negative controls. Our results are in line with the known role of ATP synthase in promoting germ cell differentiation, as already described in the fruit fly Drosophila melanogaster. The strong sterilizing effect obtained by silencing ATP synthase ? could pave the way towards the development of innovative control approaches to limit phytoplasma vector population, although many issues still need to be addressed, such as specificity, to avoid undesired off-target effects, and efficient strategies to deliver dsRNAs to phloem-sucking insects.