title: The repetitive domain of ScARP3d triggers entry of Spiroplasma citri into cultured cells of the vector Circulifer haematoceps.
abstract: Spiroplasma citri is a plant pathogenic mollicute transmitted by the leafhopper vector Circulifer haematoceps. Successful transmission requires the spiroplasmas to cross the intestinal epithelium and salivary gland barriers through endocytosis mediated by receptor-ligand interactions. To characterize these interactions we studied the adhesion and invasion capabilities of a S. citri mutant using the Ciha-1 leafhopper cell line. S. citri GII3 wild-type contains 7 plasmids, 5 of which (pSci1 to 5) encode 8 related adhesins (ScARPs). As compared to the wild-type strain GII3, the S. citri mutant G/6 lacking pSci1 to 5 was affected in its ability to adhere and enter into the Ciha-1 cells. Proteolysis analyses, Triton X-114 partitioning and agglutination assays showed that the N-terminal part of ScARP3d, consisting of repeated sequences, was exposed to the spiroplasma surface whereas the C-terminal part was anchored into the membrane. Latex beads cytadherence assays showed the ScARP3d repeat domain (Rep3d) to be involved, and internalization of the Rep3d-coated beads to be actin-dependent. These data suggested that ScARP3d, via its Rep3d domain, was implicated in adhesion of S. citri GII3 to insect cells. Inhibition tests using anti-Rep3d antibodies and competitive assays with recombinant Rep3d both resulted in a decrease of insect cells invasion by the spiroplasmas. Unexpectedly, treatment of Ciha-1 cells with the actin polymerisation inhibitor cytochalasin D increased adhesion and consequently entry of S. citri GII3. For the ScARPs-less mutant G/6, only adhesion was enhanced though to a lesser extent following cytochalasin D treatment. All together these results strongly suggest a role of ScARPs, and particularly ScARP3d, in adhesion and invasion of the leafhopper cells by S. citri.
authors.affiliation: INRA, UMR 1332 Biologie du Fruit et Pathologie, Villenave d'Ornon, France.
authors.forename: Laure
authors.initials: L
authors.lastname: Béven
authors.forename: Sybille
authors.initials: S
authors.lastname: Duret
authors.forename: Brigitte
authors.initials: B
authors.lastname: Batailler
authors.forename: Marie-Pierre
authors.initials: MP
authors.lastname: Dubrana
authors.forename: Colette
authors.initials: C
authors.lastname: Saillard
authors.forename: Joël
authors.initials: J
authors.lastname: Renaudin
authors.forename: Nathalie
authors.initials: N
authors.lastname: Arricau-Bouvery
mesh_terms: D018829:Adhesins, Bacterial / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001422:Bacterial Adhesion / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism / Q000382:microbiology / Q000648:ultrastructure; D015638:Cytochalasin D / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis; D006430:Hemiptera / Q000166:cytology / Q000378:metabolism* / Q000382:microbiology; D054884:Host-Pathogen Interactions; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D009154:Mutation; D020449:Repetitive Sequences, Amino Acid / Q000235:genetics; D045642:Spiroplasma citri / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
chemical_list: D018829:Adhesins, Bacterial; D015638:Cytochalasin D