Phytoplasmas, uncultivable phloem-limited phytopathogenic wall-less bacteria, represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. Phytoplasma membrane proteins are in direct contact with hosts and are presumably involved in determining vector specificity. Such a role has been proposed for phytoplasma transmembrane proteins encoded by circular extrachromosomal elements, at least one of which is a plasmid. Little is known about the interactions between major phytoplasma antigenic membrane protein (Amp) and insect vector proteins. The aims of our work were to identify vector proteins interacting with Amp and to investigate their role in transmission specificity. In controlled transmission experiments, four Hemipteran species were identified as vectors of “Candidatus Phytoplasma asteris”, the chrysanthemum yellows phytoplasmas (CYP) strain, and three others as non-vectors. Interactions between a labelled (recombinant) CYP Amp and insect proteins were analysed by far Western blots and affinity chromatography. Amp interacted specifically with a few proteins from vector species only. Among Amp-binding vector proteins, actin and both the α and β subunits of ATP synthase were identified by mass spectrometry and Western blots. Immunofluorescence confocal microscopy and Western blots of plasma membrane and mitochondrial fractions confirmed the localisation of ATP synthase, generally known as a mitochondrial protein, in plasma membranes of midgut and salivary gland cells in the vector Euscelidius variegatus. The vector-specific interaction between phytoplasma Amp and insect ATP synthase is demonstrated for the first time, and this work also supports the hypothesis that host actin is involved in the internalization and intracellular motility of phytoplasmas within their vectors. Phytoplasma Amp is hypothesized to play a crucial role in insect transmission specificity.

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. 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. 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. 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.

The titer of chrysanthemum yellows phytoplasma (CYP, “Candidatus Phytoplasma asteris”) in the three vector species Euscelis incisus Kirschbaum, Eusce