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Francesca CANUTO

Thesis presented to obtain the degree of Doctor of the University of Bordeaux
Specialization: Microbiology - Immunology

Team Mollicutes

Thursday, December 7, 2023 - 2:00 pm - Amphithéâtre Colette et Josy Bové - Campus INRAE Villenave d'Ornon

Abstract : Flavescence dorée is an epidemic grapevine disease associated with a phytoplasma infection and affecting viticultural areas throughout Southern Europe. The phytoplasma is transmitted among Vitis by its leafhopper vector Scaphoideus titanus. A better understanding of the interaction between the phytoplasma and its vector could help in the development of new strategies to reduce disease spread in the vineyard. The flavescence dorée phytoplasma (FDp) adhesion to the midgut epithelium cells of its insect vector is partly mediated by the Variable Membrane Protein A (VmpA), an adhesin which shows lectin properties. Morevover, VmpA sequence variation is correlated to the vector competence towards different phytoplasmas genotypes. The aim of this work was to identify proteins of the FDp experimental vector E. variegatus interacting with VmpA.
For that purpose, we identified E. variegatus cells proteins interacting with recombinant VmpA-His6 by mass spectrometry analysis of VmpA-E. variegatus protein complexes formed upon in vitro interaction assays. Thirteen candidate proteins possessing potential N-glycosylation sites and predicted transmembrane domains were selected. To assess their impact in VmpA binding, we reduced the expression of the candidate genes on E. variegatus cells in culture by dsRNA-mediated RNAi. We optimised RNAi protocol to simultaneously inhibit twelve genes in E. variegatus cultured cells with a satisfying efficiency. The effect of candidate gene knockdown on VmpA binding was measured by the capacity of VmpA-coated fluorescent beads to bind E. variegatus cells. The decrease in the expression of an unknown transmembrane protein with Leucine Rich Repeat domains (uk1_LRR) was correlated with the decreased adhesion of VmpA-beads to E. variegatus cells. Uk1_LRR expression levels evaluated in infected insects throughout the infection slightly increased following ingestion of phytoplasmas meanwhile it was stable in non-infected insects. Moreover, the uk1_LRR was more expressed in digestive tubes than in salivary glands of E. variegatus, suggesting the possible implication of this protein in early stages of the infection by the phytoplasma.
A double yeast hybrid assay was set up in order to explore the potential protein-protein interactions occurring between VmpA and E. variegatus through heterologous expression in yeast cells. No interaction was evidenced through this technique.
In conclusion, our results suggest the implication of E. variegatus uk1_LRR in VmpA mediated adhesion of the phytoplasma to its insect vector cells. The role of uk1_LRR as VmpA receptor still need to be confirmed, as well as its implication in phytoplasma transmission and vector specificity.

Jury Members:

Mme BÉBÉAR Cécile, Professeur-PH à l’Université de Bordeaux - Examinateur
Mme BRAULT Véronique, Directrice de Recherche, INRAE Colmar - Rapporteur
Mme MARZACHì Cristina, Directrice de Recherche, IPSP-CNR Turin – Rapporteur
M. SAUVION Nicolas, Ingénieur de Recherche, INRAE PHI-CIRAD Montpellier – Examinateur
M. FOISSAC Xavier, Directeur de Recherche, INRAE Bordeaux - Directeur de thèse