Table 1.
Insect vector status and results of interaction assays between insects and chrysanthemum yellows phytoplasma recombinant membrane proteins.
Figure 1.
In vitro interaction of phytoplasma antigenic membrane protein with vector and non-vector insect proteins analysed by far Western blots.
(A) Dot far Western blot: total native proteins from vector and non-vector insect species were spotted on membranes with chrysanthemum yellows phytoplasma (CYP) antigenic membrane protein (Amp) as a positive control (K+), and probed with His-tagged recombinant CYP Amp and arginine transporter (Art), histidine tag (His), and buffer devoid of protein bait (K–). Anti-HisG monoclonal antibody was used to detect bound phytoplasma recombinant proteins, and horseradish peroxidase conjugated to rabbit antimouse secondary antibody was used for chemiluminescent detection. (B, C) One dimensional far Western: binding of His-tagged recombinant CYP Amp to insect total (B) or membrane (C) proteins separated in SDS-PAGE. Secondary antibody and chemiluminescent detection were performed as in dot far Western Blot. K–: incubation in buffer devoid of protein bait. Mq: Macrosteles quadripunctulatus, Ev: Euscelidius variegatus, Ed: Empoasca decipiens, St: Scaphoideus titanus, Zp: Zyginidia pullula, Ac: Aphis craccivora, Mp: Myzus persicae.
Figure 2.
Partial purification and identification of insect vector proteins interacting with phytoplasma antigenic membrane protein.
(A) Total vector and non-vector proteins, extracted under native conditions, were loaded on an affinity chromatography column covalently linked to a recombinant chrysanthemum yellows phytoplasma antigenic membrane protein. After washing, interacting insect proteins were eluted, separated by SDS-PAGE, and stained with colloidal Coomassie blue. (B) Western blots of interacting insect proteins from the affinity chromatography with antibodies against actin and ATP synthase β. Mq: Macrosteles quadripunctulatus, Ev: Euscelidius variegatus, Ed: Empoasca decipiens, St: Scaphoideus titanus, Zp: Zyginidia pullula, Ac: Aphis craccivora, Mp: Myzus persicae.
Table 2.
Mass spectrometry identification of proteins of the leafhopper vector Euscelidius variegatus interacting with chrysanthemum yellows phytoplasma antigenic membrane protein.
Table 3.
Mass spectrometry analysis of the three proteins of the leafhopper vector Euscelidius variegatus interacting with chrysanthemum yellows phytoplasma antigenic membrane protein.
Figure 3.
Prediction of phosphorylation and protein-protein binding sites of vector and non-vector ATP synthase β.
The complete sequence of ATP synthase β subunit of the non-vector aphid Acyrthosiphon pisum (NP_001119645) was trimmed to the corresponding partial deduced amino acid sequence of the leafhopper vector Euscelidius variegatus (HQ451985), aligned with ClustalW2, and a consensus was generated and numbered on the complete A. pisum protein. Protein-protein binding sites (P) were predicted for each sequence (E. variegatus and A. pisum: above and below consensus line, respectively) with PredictProtein software. The same software predicted different phosphorylation sites, and these are depicted in red (protein kinase C type), blue (casein kinase II type) and green (cAMP- and cGMP-dependent protein kinase type). Overlapping phosphorylation sites are indicated in italics. *: identical amino acid;/: conserved amino acid substitution;.: semi-conserved amino acid substitution; #: non conserved amino acid substitution.
Figure 4.
Presence of ATP synthase β in plasma membranes and mitochondrial subcellular fractions of Euscelidius variegatus.
(A) Colloidal Coomassie blue-stained SDS-PAGE of proteins from plasma membrane (P) and mitochondrial (M) fractions from whole E. variegatus, or from excised salivary glands and midgut. (B) Western blots of proteins from plasma membrane (P) and mitochondrial (M) fractions of entire E. variegatus as well as of excised salivary glands and midgut with antibodies against ATP synthase β, flotillin 1, a plasma membrane marker, and cytochrome C, a mitochondrial marker.
Figure 5.
Localization of ATP synthase β on the external membranes and in the cytoplasm of salivary gland cells of the vector Euscelidius variegatus.
Whole permeabilised salivary glands, as observed in light microscopy (A), show a strong cytoplasmic labelling of ATP synthase in immunofluorescence (B). The signal is absent in preimmune serum treatment (C). Sections of permeabilized salivary glands (D, E) also show a labelling of mitochondrial ATP synthase in the cytoplasm (arrows). In sections of not permeabilized glands (F), ATP synthase labelling is present on the cell surface. Double-labelling of ATP synthase β (G) and flotillin 1 (H) in not permeabilised sections of salivary glands reveal colocalization of the two signals, as highlighted by the resulting yellow colour in the merged image (I). n: nucleus.
Figure 6.
Localization of ATP synthase β on the external membranes and in the cytoplasm of midgut cells of the vector Euscelidius variegatus.
Whole permeabilised guts, as observed in light microscopy (A), are strongly labelled by the antibody against ATP synthase β (B), while preimmune serum treated samples show no signal (C). Sections of permeabilized guts (D, E) reveal an intense labelling of mitochondrial ATP synthase β in the cytoplasm (arrows). In sections of not permeabilised guts (F), ATP synthase labelling is present on the cell surface. Double immunofluorescence labelling of both ATP synthase β (G) and flotillin 1 (H) shows the coincidence of the respective signals, resulting in the yellow colour of the merged image (I). n: nucleus; gl: gut lumen. Panel E is a magnification of panel D.