The Major Antigenic Membrane Protein of “Candidatus Phytoplasma asteris” Selectively Interacts with ATP Synthase and Actin of Leafhopper Vectors

Abstract

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.

A Plant Bacterial Pathogen Manipulates Its Insect Vector's Energy Metabolism

“…However, it is still not clear which of them (ATP synthase or ATP/ADP translocase) contributes more to CLas energy. CLas could be like phytoplasmas, as it depends partly on its host for energy (17). Consequently, extracellular ATP in the gut lumen and hemocoel may also be required for CLas survival (17).…”

“…Interestingly, it has been shown that the antigenic membrane protein of "Candidatus Phytoplasma asteris" interacts with the ATP synthase of its leafhopper vectors (17). Because the phytoplasmas lack the ATP synthetic pathway and depend partly on their host for energy, it has been suggested that host extracellular ATP in the gut lumen and hemocoel may be required for the survival of phytoplasmas (17).…”

“…Because the phytoplasmas lack the ATP synthetic pathway and depend partly on their host for energy, it has been suggested that host extracellular ATP in the gut lumen and hemocoel may be required for the survival of phytoplasmas (17). Vahling et al (16) also suggested that the addition of external ATP to the culture medium might facilitate the growth of the CLas bacterium in vitro.…”

Decreasing Global Transcript Levels over Time Suggest that Phytoplasma Cells Enter Stationary Phase during Plant and Insect Colonization

“…The amp transcripts were the most abundant of all the analyzed gene transcripts in all three host species, and the amp transcript levels decreased in A. thaliana and M. quadripunctulatus during the CYP infection cycle. Amp interacts specifically with the M. striifrons actin vector (10), possibly to enable phytoplasma motility (11). Indeed, amp transcript levels were stable during infection of E. variegatus, possibly implying a continuous need of the gene product to reach stable colonization of the insect body.…”

“…In vitro studies have also shown that phytoplasma immunodominant membrane proteins interact with vector proteins (10,11) and plant proteins (12) and are subjected to strong positive selection (13)(14)(15). Moreover, phytoplasmas can modulate their genome expression according to the infection stage and the infected host species, as suggested by microarray analysis (16) and gene expression study of pathogen transcription factors (17) and of genes lying within potential mobile units (18).…”