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

Luciana Galetto,Cristina Marzachì,Jacqueline Fletcher,Andrea Genre,Domenico Bosco,Raffaella Balestrini,Dipshikha Chakravortty

doi:10.1371/journal.pone.0022571

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.

Highlights

Phytoplasmas, wall-less plant pathogenic bacteria of the Class Mollicutes, infect a wide variety of herbaceous and woody plants, causing significant economic losses on cultivated crops worldwide [1]
All insect species used in this study were characterized for their ability to acquire and transmit chrysanthemum yellows phytoplasmas (CYP), for the interactions of their proteins with CYP recombinant membrane proteins (CYP antigenic membrane protein, Amp, and arginine transporter, Art) in dot far Western blots and for the capability of their proteins of binding the CYP Amp in affinity chromatography, evaluated by SDS polyacrylamide gel electrophoresis (PAGE) and Western blots of affinity column elution phases (Table 1)
Several lines of evidence support a role for ATP synthase as a receptor of the phytoplasma antigenic membrane protein in the transmission of CYP: i) in vitro interaction between Amp and insect vector proteins; ii) absence of Amp interaction with proteins of non-vector insect species; iii) localization of ATP synthase b subunit on the plasma membrane of midgut and salivary glands of the leafhopper vector E. variegatus; and iv) different features in the amino acid sequence of E. variegatus ATP synthase b compared to that of a non-vector aphid, despite high sequence similarity

Summary

Introduction

Phytoplasmas, wall-less plant pathogenic bacteria of the Class Mollicutes, infect a wide variety of herbaceous and woody plants, causing significant economic losses on cultivated crops worldwide [1]. Because they have yet to be cultured axenically, phytoplasmas are described as ‘‘Candidatus Phytoplasma spp.’’ [2]. Phytoplasmas are usually transmitted by a narrow range of vector species [10], whereas their plant host range is usually broader [12]. Phytoplasma colonization of the vector depends on several biological features, such as insect feeding behavior and plant host range, as well as on molecular interactions between pathogen and vector membrane proteins [11]. Since phytoplasma membrane proteins are in direct contact with host cells they are likely to be involved in specific interactions with them, as is the case for other mollicutes in which adhesion to host cells has been studied

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