title: Role of the major antigenic membrane protein in phytoplasma transmission by two insect vector species.
abstract: BACKGROUND
  Phytoplasmas are bacterial plant pathogens (class Mollicutes), transmitted by phloem feeding leafhoppers, planthoppers and psyllids in a persistent/propagative manner. Transmission of phytoplasmas is under the control of behavioral, environmental and geographical factors, but molecular interactions between membrane proteins of phytoplasma and vectors may also be involved. The aim of the work was to provide experimental evidence that in vivo interaction between phytoplasma antigenic membrane protein (Amp) and vector proteins has a role in the transmission process. In doing so, we also investigated the topology of the interaction at the gut epithelium and at the salivary glands, the two barriers encountered by the phytoplasma during vector colonization.
  
  
  METHODS
  Experiments were performed on the 'Candidatus Phytoplasma asteris' chrysanthemum yellows strain (CYP), and the two leafhopper vectors Macrosteles quadripunctulatus Kirschbaum and Euscelidius variegatus Kirschbaum. To specifically address the interaction of CYP Amp at the gut epithelium barrier, insects were artificially fed with media containing either the recombinant phytoplasma protein Amp, or the antibody (A416) or both, and transmission, acquisition and inoculation efficiencies were measured. An abdominal microinjection protocol was employed to specifically address the interaction of CYP Amp at the salivary gland barrier. Phytoplasma suspension was added with Amp or A416 or both, injected into healthy E. variegatus adults and then infection and inoculation efficiencies were measured. An internalization assay was developed, consisting of dissected salivary glands from healthy E. variegatus exposed to phytoplasma suspension alone or together with A416 antibody. The organs were then either observed in confocal microscopy or subjected to DNA extraction and phytoplasma quantification by qPCR, to visualize and quantify possible differences among treatments in localization/presence/number of CYP cells.
  
  
  RESULTS
  Artificial feeding and abdominal microinjection protocols were developed to address the two barriers separately. The in vivo interactions between Amp of 'Candidatus Phytoplasma asteris' Chrysanthemum yellows strain (CYP) and vector proteins were studied by evaluating their effects on phytoplasma transmission by Euscelidius variegatus and Macrosteles quadripunctulatus leafhoppers. An internalization assay was developed, consisting of dissected salivary glands from healthy E. variegatus exposed to phytoplasma suspension alone or together with anti-Amp antibody. To visualize possible differences among treatments in localization/presence of CYP cells, the organs were observed in confocal microscopy. Pre-feeding of E. variegatus and M. quadripunctulatus on anti-Amp antibody resulted in a significant decrease of acquisition efficiencies in both species. Inoculation efficiency of microinjected E. variegatus with CYP suspension and anti-Amp antibody was significantly reduced compared to that of the control with phytoplasma suspension only. The possibility that this was due to reduced infection efficiency or antibody-mediated inhibition of phytoplasma multiplication was ruled out. These results provided the first indirect proof of the role of Amp in the transmission process.
  
  
  CONCLUSIONS
  Protocols were developed to assess the in vivo role of the phytoplasma native major antigenic membrane protein in two phases of the vector transmission process: movement through the midgut epithelium and colonization of the salivary glands. These methods will be useful also to characterize other phytoplasma-vector combinations. Results indicated for the first time that native CYP Amp is involved in vivo in specific crossing of the gut epithelium and salivary gland colonization during early phases of vector infection.
authors.affiliation: Istituto per la Protezione Sostenibile delle Piante, CNR, Torino, Italy. rashidii.mahnaz@gmail.com.|DISAFA, Università degli Studi di Torino, Grugliasco, TO, Italy. rashidii.mahnaz@gmail.com.|Present address: University of Idaho, College of Agricultural and Life Sciences, Aberdeen, ID, USA. rashidii.mahnaz@gmail.com.
authors.forename: Mahnaz
authors.initials: M
authors.lastname: Rashidi
authors.affiliation: Istituto per la Protezione Sostenibile delle Piante, CNR, Torino, Italy. luciana.galetto@ipsp.cnr.it.
authors.forename: Luciana
authors.initials: L
authors.lastname: Galetto
authors.affiliation: Istituto per la Protezione Sostenibile delle Piante, CNR, Torino, Italy. domenico.bosco@unito.it.|DISAFA, Università degli Studi di Torino, Grugliasco, TO, Italy. domenico.bosco@unito.it.
authors.forename: Domenico
authors.initials: D
authors.lastname: Bosco
authors.affiliation: DISAFA, Università degli Studi di Torino, Grugliasco, TO, Italy. andrea.bulgarelli@edu.unito.it.
authors.forename: Andrea
authors.initials: A
authors.lastname: Bulgarelli
authors.affiliation: Istituto per la Protezione Sostenibile delle Piante, CNR, Torino, Italy. marta.vallino@ipsp.cnr.it.
authors.forename: Marta
authors.initials: M
authors.lastname: Vallino
authors.affiliation: Istituto per la Protezione Sostenibile delle Piante, CNR, Torino, Italy. flavio.veratti@ipsp.cnr.it.
authors.forename: Flavio
authors.initials: F
authors.lastname: Veratti
authors.affiliation: Istituto per la Protezione Sostenibile delle Piante, CNR, Torino, Italy. cristina.marzachi@ipsp.cnr.it.
authors.forename: Cristina
authors.initials: C
authors.lastname: Marzachì
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000378:metabolism*; D004772:Entomology / Q000379:methods; D041981:Gastrointestinal Tract / Q000382:microbiology; D006430:Hemiptera / Q000382:microbiology*; D019476:Insect Proteins / Q000378:metabolism*; D007303:Insect Vectors / Q000382:microbiology*; D008565:Membrane Proteins / Q000378:metabolism*; D008828:Microbiological Techniques; D045582:Phytoplasma / Q000502:physiology*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D012469:Salivary Glands / Q000382:microbiology
chemical_list: D000942:Antigens, Bacterial; D019476:Insect Proteins; D008565:Membrane Proteins