Living in an insect world: molecular aspects of phytoplasma-vector interac

Phytoplasma diseases of economically important crops are spread worldwide, and specific control measures rely mainly on the use of healthy propagation material and the application of insecticide treatments to control vector population. This latter measure has detrimental effects on the environment and it is inefficient for organic agriculture. Understanding the molecular mechanisms involved in the specific phytoplasma-vector interaction is crucial to develop new control measures. Phytoplasmas are transmitted in a persistent and propagative manner by several hemipteran species, planthoppers, leafhoppers, and psyllids. Due to their wall-less nature, phytoplasma cell membrane is in direct contact with insect proteins, and therefore phytoplasma membrane proteins with extra-cellular predicted domains are good candidates as partners in the specific interaction with the vector. The major antigenic membrane protein (Amp) of 'Candidatus Phytoplasma asteris', chrysanthemum yellows strain (CY), was selected due to its abundance on the phytoplasma membrane, to the fact that its gene sequence is subjected to positive selection, and it is actively transcribed at different stages of insect colonization. A recombinant CY Amp was used in affinity chromatography to elute interacting vector proteins. Under these conditions, CYP Amp interacted with different proteins of vector species only, and a membrane expressed ATP-synthase of the leafhopper Euscelidius variegatus was identified by MS/MS and de novo sequencing as a partner of the CY Amp interactome. The identification was confirmed by dot and 1D Far Western experiments. To confirm a biological role of the interaction in vivo, the two possible barriers to phytoplasma colonization of the vector body (gut epithelium and salivary glands) were separately addressed by artificial feeding and abdominal microinjection experiments, respectively. Masking of CY Amp by its antibody resulted in reduction of acquisition and consequently transmission efficiencies of different vector species, therefore proving that native Amp is involved in vivo in specific crossing of the gut epithelium during the early phases of vector infection, and in specific crossing of the salivary gland barrier during vector colonization. The developed protocols, together with a recent approach for the heterologous expression of phytoplasma membrane proteins, are unique tools to assess the role of phytoplasma membrane proteins in the transmission by vectors.