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Silencing of ATP Synthase β Impairs Egg Development in the Leafhopper Scaphoideus titanus, Vector of the Phytoplasma Associated with Grapevine Flavescence Doree.

ABSTRACT: Scaphoideus titanus (Hemiptera: Cicadellidae) is the natural vector of Flavescence dorée phytoplasma, a quarantine pest of grapevine with severe impact on European viticulture. RNA interference (RNAi) machinery components are present in S. titanus transcriptome and injection of ATP synthase β dsRNAs into adults caused gene silencing, starting three days post injection (dpi) up to 20 dpi, leading to decrease cognate protein. Silencing of this gene in the closely related leafhopper Euscelidiusvariegatus previously showed female sterility and lack of mature eggs in ovaries. Here, alteration of developing egg morphology in S. titanus ovaries as well as overexpression of hexamerin transcript (amino acid storage protein) and cathepsin L protein (lysosome proteinase) were observed in dsATP-injected females. To evaluate RNAi-specificity, E.variegatus was used as dsRNA-receiving model-species. Different doses of two sets of dsRNA-constructs targeting distinct portions of ATP synthase β gene of both species induced silencing, lack of egg development, and female sterility in E. variegatus, indicating that off-target effects must be evaluated case by case. The effectiveness of RNAi in S. titanus provides a powerful tool for functional genomics of this non-model species and paves the way toward RNAi-based strategies to limit vector population, despite several technical and regulatory constraints that still need to be overcome to allow open field application.

SUBMITTER: Ripamonti M

PROVIDER: S-EPMC8775575 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Phytoplasmas are insect-borne pathogenic bacteria that cause major economic losses to several crops worldwide. The dynamic microbial community associated with insect vectors influences several aspects of their biology, including their vector competence for pathogens. Unraveling the diversity of the microbiome of phytoplasma insect vectors is gaining increasing importance in the quest to develop novel microbe-based pest control strategies that can minimize the use of insecticides for better environmental quality. The leafhopper Scaphoideus titanus is the primary vector of the Flavescence dorée phytoplasma, a quarantine pest which is dramatically affecting the main grape-growing European countries. In this study, the RNA-Seq data, which were previously used for insect virus discovery, were further explored to assess the composition of the whole microbial community associated with insects caught in the wild in both its native (the United States) and invasive (Europe) areas. The first de novo assembly of the insect transcriptome was used to filter the host sequencing reads. The remaining ones were assembled into contigs and analyzed by blastx to provide the taxonomic identification of the microorganisms associated with S. titanus, including the non-bacterial components. By comparing the transcriptomic libraries, we could differentiate the stable and consistent associations from the more ephemeral and flexible ones. Two species appeared to be universal to the core microbiome of S. titanus: the obligate bacterial symbiont Candidatus Sulcia muelleri and an Ophiocordyceps-allied fungus distantly related to yeast-like symbionts described from other hemipterans. Bacteria of the genus Cardinium have been identified as another dominant member of the microbiome, but only in the European specimens. Although we are yet to witness how the interplay among the microorganisms influences the vector competence of S. titanus, this unbiased in silico characterization of its microbiome is paramount for identifying the naturally occurring targets for new biocontrol strategies to counteract Flavescence dorée spread in Europe.

Project description:The phytoplasma-borne disease flavescence dorée is still a threat to European viticulture, despite mandatory control measures and prophylaxis against the leafhopper vector. Given the economic importance of grapevine, it is essential to find alternative strategies to contain the spread, in order to possibly reduce the current use of harmful insecticides. Further studies of the pathogen, the vector and the mechanisms of phytoplasma-host interactions could improve our understanding of the disease. In this work, RNA-Seq technology followed by three de novo assembly strategies was used to provide the first comprehensive transcriptomics landscape of flavescence dorée phytoplasma (FD) infecting field-grown Vitis vinifera leaves.With an average of 8300 FD-mapped reads per library, we assembled 347 sequences, corresponding to 215 annotated genes, and identified 10 previously unannotated genes, 15 polycistronic transcripts and three genes supposedly localized in the gaps of the FD92 draft genome. Furthermore, we improved the annotation of 44 genes with the addition of 5'/3' untranslated regions. Functional classification revealed that the most expressed genes were either related to translation and protein biosynthesis or hypothetical proteins with unknown function. Some of these hypothetical proteins were predicted to be secreted, so they could be bacterial effectors with a potential role in modulating the interaction with the host plant. Interestingly, qRT-PCR validation of the RNA-Seq expression values confirmed that a group II intron represented the FD genomic region with the highest expression during grapevine infection. This mobile element may contribute to the genomic plasticity that is necessary for the phytoplasma to increase its fitness and endorse host-adaptive strategies.The RNA-Seq technology was successfully applied for the first time to analyse the FD global transcriptome profile during grapevine infection. Our results provided new insights into the transcriptional organization and gene structure of FD. This may represent the starting point for the application of high-throughput sequencing technologies to study differential expression in FD and in other phytoplasmas with an unprecedented resolution.

Project description:BackgroundFlavescence dorée (FD) is a grapevine disease caused by phytoplasma and it is one of the most destructive pathologies in Europe. Nowadays, the only strategies used to control the epidemics are insecticides against vector, but more sustainable techniques are required. Completely resistant Vitis vinifera varieties have not been uncovered yet, but differences in susceptibility among cultivars and spontaneous recovery from FD symptoms have been observed. The grapevine cultivar 'Tocai friulano' shows very low susceptibility to FD but its defence strategy to counteract the phytoplasma spread has not been deciphered yet. In this work, the mechanisms occurring within 'Tocai friulano' FD-infected plants were examined in depth to identify the phytoplasma distribution and the defence pathways involved.ResultsIn 'Tocai friulano' symptoms of FD-infection remained confined near the area where they appeared during all the vegetative season. Analyses of secondary phloem showed a total absence of FD phytoplasma (FDp) in the trunk and its disappearance in 2-year-old arms from July to November, which was different from 'Pinot gris', a highly susceptible variety. Diverse modulations of defence genes and accumulation of metabolites were revealed in 1-year-old canes of 'Tocai friulano' FD-infected plants, depending on the sanitary status. Symptomatic portions showed high activation of both jasmonate- and salicylate-mediated responses, together with a great accumulation of resveratrol. Whereas activation of jasmonate-mediated response and high content of ε-viniferin were identified in asymptomatic 1-year-old cane portions close to the symptomatic ones.ConclusionSuccessful defence mechanisms activated near the symptomatic areas allowed the compartmentation of FD symptoms and phytoplasmas within the infected 'Tocai friulano' plants. These results could suggest specific agronomical practices to be adopted during FD management of this variety, and drive research of resistance genes against FD.

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Silencing of ATP Synthase β Impairs Egg Development in the Leafhopper Scaphoideus titanus, Vector of the Phytoplasma Associated with Grapevine Flavescence Doree.

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