Role of the major antigenic membrane protein in phytoplasma transmission by two insect vector species
Articolo
Data di Pubblicazione:
2015
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.
Conclusion: 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 epitheliu
Tipologia CRIS:
03A-Articolo su Rivista
Keywords:
“Candidatus Phytoplasma asteris”, Chrysanthemum yellows phytoplasma, Macrosteles quadripunctulatus,
Euscelidius variegatus, Chrysanthemum carinatum
Elenco autori:
Rashidi M.; Galetto L.; Bosco D.; Bulgarelli A.; Vallino M.; Veratti F.; Marzachì C.
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