title: Heterologous expression and processing of the flavescence dorée phytoplasma variable membrane protein VmpA in Spiroplasma citri.
abstract: BACKGROUND
  Flavescence dorée (FD) of grapevine is a phloem bacterial disease that threatens European vineyards. The disease is associated with a non-cultivable mollicute, a phytoplasma that is transmitted by the grapevine leafhopper Scaphoideus titanus in a persistent, propagative manner. The specificity of insect transmission is presumably mediated through interactions between the host tissues and phytoplasma surface proteins comprising the so-called variable membrane proteins (Vmps). Plant spiroplasmas and phytoplasmas share the same ecological niches, the phloem sieve elements of host plants and the hemocoel of insect vectors. Unlike phytoplasmas, however, spiroplasmas, and Spiroplasma citri in particular, can be grown in cell-free media and genetically engineered. As a new approach for studying phytoplasmas-insect cell interactions, we sought to mimic phytoplasmas through the construction of recombinant spiroplasmas exhibiting FD phytoplasma Vmps at the cell surface.
  
  
  RESULTS
  Here, we report the expression of the FD phytoplasma VmpA in S. citri. Transformation of S. citri with plasmid vectors in which the vmpA coding sequence was under the control of the S. citri tuf gene promoter resulted in higher accumulation of VmpA than with the native promoter. Expression of VmpA at the spiroplasma surface was achieved by fusing the vmpA coding sequence to the signal peptide sequence of the S. citri adhesin ScARP3d, as revealed by direct colony immunoblotting and immunogold labelling electron microscopy. Anchoring of VmpA to the spiroplasma membrane was further demonstrated by Triton X-114 protein partitioning and Western immunoblotting. Using the same strategy, the secretion of free, functionally active β-lactamase (used as a model protein) into the culture medium by recombinant spiroplasmas was achieved.
  
  
  CONCLUSIONS
  Construction of recombinant spiroplasmas harbouring the FD phytoplasma variable membrane protein VmpA at their surface was achieved, which provides a new biological approach for studying interactions of phytoplasma surface proteins with host cells. Likewise, the secretion of functional β-lactamase by recombinant spiroplasmas established the considerable promise of the S. citri expression system for delivering phytoplasma effector proteins into host cells.
authors.affiliation: INRA, UMR 1332 Biologie du Fruit et Pathologie, Villenave d'Ornon, France. renaudin@bordeaux.inra.fr.|Université de Bordeaux, UMR 1332 Biologie du Fruit et Pathologie, Villenave d'Ornon, France. renaudin@bordeaux.inra.fr.
authors.forename: Joël
authors.initials: J
authors.lastname: Renaudin
authors.affiliation: INRA, UMR 1332 Biologie du Fruit et Pathologie, Villenave d'Ornon, France. laure.beven@bordeaux.inra.fr.|Université de Bordeaux, UMR 1332 Biologie du Fruit et Pathologie, Villenave d'Ornon, France. laure.beven@bordeaux.inra.fr.
authors.forename: Laure
authors.initials: L
authors.lastname: Béven
authors.affiliation: INRA, UMR 1332 Biologie du Fruit et Pathologie, Villenave d'Ornon, France. brigitte.batailler@bordeaux.inra.fr.|Université de Bordeaux, UMR 1332 Biologie du Fruit et Pathologie, Villenave d'Ornon, France. brigitte.batailler@bordeaux.inra.fr.|Université de Bordeaux, UMS3420, Bordeaux Imaging Center, Bordeaux, France. brigitte.batailler@bordeaux.inra.fr.|CNRS, Bordeaux Imaging Center, UMS 3420, Bordeaux, France. brigitte.batailler@bordeaux.inra.fr.|INSERM, Bordeaux Imaging Center, US 004, Bordeaux, France. brigitte.batailler@bordeaux.inra.fr.
authors.forename: Brigitte
authors.initials: B
authors.lastname: Batailler
authors.affiliation: INRA, UMR 1332 Biologie du Fruit et Pathologie, Villenave d'Ornon, France. duret@bordeaux.inra.fr.|Université de Bordeaux, UMR 1332 Biologie du Fruit et Pathologie, Villenave d'Ornon, France. duret@bordeaux.inra.fr.
authors.forename: Sybille
authors.initials: S
authors.lastname: Duret
authors.affiliation: INRA, UMR 1332 Biologie du Fruit et Pathologie, Villenave d'Ornon, France. ddesque@bordeaux.inra.fr.|Université de Bordeaux, UMR 1332 Biologie du Fruit et Pathologie, Villenave d'Ornon, France. ddesque@bordeaux.inra.fr.
authors.forename: Delphine
authors.initials: D
authors.lastname: Desqué
authors.affiliation: INRA, UMR 1332 Biologie du Fruit et Pathologie, Villenave d'Ornon, France. nbouvery@bordeaux.inra.fr.|Université de Bordeaux, UMR 1332 Biologie du Fruit et Pathologie, Villenave d'Ornon, France. nbouvery@bordeaux.inra.fr.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Arricau-Bouvery
authors.affiliation: INRA, UMR 1332 Biologie du Fruit et Pathologie, Villenave d'Ornon, France. smalembi@bordeaux.inra.fr.|Université de Bordeaux, UMR 1332 Biologie du Fruit et Pathologie, Villenave d'Ornon, France. smalembi@bordeaux.inra.fr.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Malembic-Maher
authors.affiliation: INRA, UMR 1332 Biologie du Fruit et Pathologie, Villenave d'Ornon, France. foissac@bordeaux.inra.fr.|Université de Bordeaux, UMR 1332 Biologie du Fruit et Pathologie, Villenave d'Ornon, France. foissac@bordeaux.inra.fr.
authors.forename: Xavier
authors.initials: X
authors.lastname: Foissac
mesh_terms: D018829:Adhesins, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006430:Hemiptera / Q000382:microbiology*; D007303:Insect Vectors / Q000382:microbiology*; D017830:Octoxynol; D045582:Phytoplasma / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D010935:Plant Diseases / Q000382:microbiology; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry; D011401:Promoter Regions, Genetic; D015202:Protein Engineering; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D045642:Spiroplasma citri / Q000235:genetics* / Q000378:metabolism; D014169:Transformation, Bacterial; D027843:Vitis / Q000382:microbiology; D001618:beta-Lactamases / Q000096:biosynthesis / Q000378:metabolism
chemical_list: D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D001618:beta-Lactamases