“Flavescence dorée” phytoplasma uses its adhesin VmpA, the insect surface protein Uk1_LRR and clathrin to enter into its vector host cell

International audience

English. The “flavescence dorée” (FD) phytoplasma completes a propagative and circulative cycle into its insect vector before to be inoculated to grapevine. To achieve this cycle, the FD phytoplasma invades different types of insect cells, especially midgut and salivary gland cells. Adhesion of bacteria to eukaryotic cells is the first step of signal transduction that leads to endocytosis of the bacteria by the host cell. Adhesion of FD phytoplasma to midgut epithelium cells is partially mediated by the variable membrane protein A (VmpA), an adhesin which shows lectin properties. To decipher the molecular mechanisms implicated in cellular infection by the FD phytoplasma, it was looked for Euscelidius variegatus cell proteins interacting with recombinant VmpA-His6 and different drogues to inhibit various ways of bacterial entry into cells. The E. variegatus proteins interacting with VmpA were identified by far Western blot followed by mass spectrometry analysis. It was selected 13 candidate proteins possessing potential N-glycosylation sites and predicted transmembrane domains. Their impact in VmpA binding was assessed by reducing their expression in E. variegatus cells in culture with dsRNA-mediated RNAi. The reduced expression of an unknown transmembrane protein with leucine rich repeat domains (Uk1_LRR) was correlated with the decreased adhesion of VmpA-His6-coated beads to the insect cells. In non-infected E. variegatus, the uk1_LRR was more expressed in digestive tubes than in salivary glands. In infected E. variegatus, the expression of uk1_LRR tended towards increase in the early steps of the infection and then decreased. By infection assays of Drosophila S2 cells40 using chlorpromazine, cytochalasin D and nystatin that inhibit endocytosis, it was shown that phytoplasma entry into cells is clathrin-dependent. In E. variegatus fed on infected plants, reduced expression of clathrin heavy chain gene induced by RNAi correlated to a decrease of midgut and salivary gland cells colonization by the phytoplasma. In conclusion, the protein uk1_LRR seems to be implicated in the binding with phytoplasmas via VmpA in the early stages of insect infection following phytoplasmas ingestion, and clathrin is important for the FD phytoplasma to enter insect cells and colonize its insect vector.