Entry of Spiroplasma citri into Circulifer haematoceps Cells Involves Interaction between Spiroplasma Phosphoglycerate Kinase and Leafhopper Actin

Abstract

Transmission of the phytopathogenic mollicutes, spiroplasmas, and phytoplasmas by their insect vectors mainly depends on their ability to pass through gut cells, to multiply in various tissues, and to traverse the salivary gland cells. The passage of these different barriers suggests molecular interactions between the plant mollicute and the insect vector that regulate transmission. In the present study, we focused on the interaction between Spiroplasma citri and its leafhopper vector, Circulifer haematoceps. An in vitro protein overlay assay identified five significant binding activities between S. citri proteins and insect host proteins from salivary glands. One insect protein involved in one binding activity was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as actin. Confocal microscopy observations of infected salivary glands revealed that spiroplasmas colocated with the host actin filaments. An S. citri actin-binding protein of 44 kDa was isolated by affinity chromatography and identified by LC-MS/MS as phosphoglycerate kinase (PGK). To investigate the role of the PGK-actin interaction, we performed competitive binding and internalization assays on leafhopper cultured cell lines (Ciha-1) in which His 6 -tagged PGK from S. citri or purified PGK from Saccharomyces cerevisiae was added prior to the addition of S. citri inoculum. The results suggested that exogenous PGK has no effect on spiroplasmal attachment to leafhopper cell surfaces but inhibits S. citri internalization, demonstrating that the process leading to internalization of S. citri in eukaryotic cells requires the presence of PGK. PGK, regardless of origin, reduced the entry of spiroplasmas into Ciha-1 cells in a dose-dependent manner.

Sequences Essential for Transmission of Spiroplasma citri by Its Leafhopper Vector, Circulifer haematoceps , Revealed by Plasmid Curing and Replacement Based on Incompatibility

“…In the plant-pathogenic mollicute "Candidatus Phytoplasma asteris," insect vector specificity is determined by the interaction between the cell surface membrane protein Amp and the insect microfilament complex (39). In S. citri also, membrane-associated proteins were shown to interact with insect microfilaments, as suggested by colocalization of spiroplasmas with the actin filaments of the leafhopper salivary glands (25). In these studies, protein overlay assays revealed that two membrane-associated proteins, a lipoprotein and the glycolytic enzyme phosphoglycerate kinase (PGK), interacted with actin in vitro.…”

“…In these studies, protein overlay assays revealed that two membrane-associated proteins, a lipoprotein and the glycolytic enzyme phosphoglycerate kinase (PGK), interacted with actin in vitro. By use of the Ciha-1 leafhopper cell culture system (11), it was shown that PGK, by specifically binding to actin, was involved in internalization of spiroplasmas into insect cells (25). In contrast to these membraneassociated proteins, the pSci6_06 protein was predicted to be cytosolic, making a direct interaction with leafhopper cells unlikely.…”

Spiroplasma spp.: A Plant, Arthropod, Animal and Human Pathogen

“…Receptors on leafhopper gut epithelial cells likely recognize specific spiroplasma membrane proteins. Several candidate S. citri attachment protein genes have been studied, including spiralin (immunodominant membrane protein) [41], P58 [42], SARP1 [43], P32 of pSci6 plasmid [44][45][46], and phosphoglycerate kinase (PGK) [47]. It has…”

“…It was also shown that S. citri phosphoglycerate kinase (PGK), a glycolytic enzyme, could bind to actin of its leafhopper vector for the internalization of S. citri into leafhopper cells [47]. In addition, the PGK protein or partial PGK peptides were shown to inhibit spiroplasma transmission by leafhoppers [49].…”

“…A relationship between plasmids of S. citri and insect-transmissibility has been demonstrated for spiroplasmas [44][45][46][47][48][49][50][51]. The plasmid pSci6 confers insect transmissibility to a nontransmissible strain of S. citri [44] encoding protein P32; however, when only the p32 gene was introduced into the nontransmissible strain of S. citri, its insect transmissibility was not restored; therefore, pSci6-encoded determinants other than P32 might be essential for insect transmission [46].…”