When a Palearctic bacterium meets a Nearctic insect vector: Genetic and ecological insights into the emergence of the grapevine Flavescence dorée epidemics in Europe.

Flavescence dorée (FD) is a European quarantine grapevine disease transmitted by the Deltocephalinae leafhopper Scaphoideus titanus. Whereas this vector had been introduced from North America, the possible European origin of FD phytoplasma needed to be challenged and correlated with ecological and genetic drivers of FD emergence. For that purpose, a survey of genetic diversity of these phytoplasmas in grapevines, S. titanus, black alders, alder leafhoppers and clematis were conducted in five European countries. Out of 132 map genotypes, only 11 were associated to FD outbreaks, three were detected in clematis, whereas 127 were detected in alder trees, alder leafhoppers or in grapevines out of FD outbreaks. Most of the alder trees were found infected, including 8% with FD genotypes M6, M38 and M50, also present in alders neighboring FD-free vineyards and vineyard-free areas. The Macropsinae Oncopsis alni could transmit genotypes unable to achieve transmission by S. titanus, while the Deltocephalinae Allygus spp. and Orientus ishidae transmitted M38 and M50 that proved to be compatible with S. titanus. Variability of vmpA and vmpB adhesin-like genes clearly discriminated 3 genetic clusters. Cluster Vmp-I grouped genotypes only transmitted by O. alni, while clusters Vmp-II and -III grouped genotypes transmitted by Deltocephalinae leafhoppers. Interestingly, adhesin repeated domains evolved independently in cluster Vmp-I, whereas in clusters Vmp-II and-III showed recent duplications. Latex beads coated with various ratio of VmpA of clusters II and I, showed that cluster II VmpA promoted enhanced adhesion to the Deltocephalinae Euscelidius variegatus epithelial cells and were better retained in both E. variegatus and S. titanus midguts. Our data demonstrate that most FD phytoplasmas are endemic to European alders. Their emergence as grapevine epidemic pathogens appeared restricted to some genetic variants pre-existing in alders, whose compatibility to S. titanus correlates with different vmp gene sequences and VmpA binding properties.

Two Phytoplasmas Elicit Different Responses in the Insect Vector Euscelidius variegatus Kirschbaum.

Phytoplasmas are plant-pathogenic bacteria transmitted by hemipteran insects. The leafhopper Euscelidius variegatus is a natural vector of chrysanthemum yellows phytoplasma (CYp) and a laboratory vector of flavescence dorée phytoplasma (FDp). The two phytoplasmas induce different effects on this species: CYp slightly improves whereas FDp negatively affects insect fitness. To investigate the molecular bases of these different responses, transcriptome sequencing (RNA-seq) analysis of E. variegatus infected with either CYp or FDp was performed. The sequencing provided the first de novo transcriptome assembly for a phytoplasma vector and a starting point for further analyses on differentially regulated genes, mainly related to immune system and energy metabolism. Insect phenoloxidase activity, immunocompetence, and body pigmentation were measured to investigate the immune response, while respiration and movement rates were quantified to confirm the effects on energy metabolism. The activation of the insect immune response upon infection with FDp, which is not naturally transmitted by E. variegatus, confirmed that this bacterium is mostly perceived as a potential pathogen. Conversely, the acquisition of CYp, which is naturally transmitted by E. variegatus, seems to increase the insect fitness by inducing a prompt response to stress. This long-term relationship is likely to improve survival and dispersal of the infected insect, thus enhancing the opportunity of phytoplasma transmission.

Variable Membrane Protein A of Flavescence Dorée Phytoplasma Binds the Midgut Perimicrovillar Membrane of Euscelidius variegatus and Promotes Adhesion to Its Epithelial Cells.

Phytoplasmas are uncultivated plant pathogens and cell wall-less bacteria and are transmitted from plant to plant by hemipteran insects. The phytoplasma's circulative propagative cycle in insects requires the crossing of the midgut and salivary glands, and primary adhesion to cells is an initial step toward the invasion process. The flavescence dorée (FD) phytoplasma possesses a set of variable membrane proteins (Vmps) exposed on its surface, and this pathogen is suspected to interact with insect cells. The results showed that VmpA is expressed by the flavescence dorée phytoplasma present in the midgut and salivary glands. Phytoplasmas cannot be cultivated at present, and no mutant can be produced to investigate the putative role of Vmps in the adhesion of phytoplasma to insect cells. To overcome this difficulty, we engineered the Spiroplasma citri mutant G/6, which lacks the ScARP adhesins, for VmpA expression and used VmpA-coated fluorescent beads to determine if VmpA acts as an adhesin in ex vivo adhesion assays and in vivo ingestion assays. VmpA specifically interacted with Euscelidiusvariegatus insect cells in culture and promoted the retention of VmpA-coated beads to the midgut of E. variegatus In this latest case, VmpA-coated fluorescent beads were localized and embedded in the perimicrovillar membrane of the insect midgut. Thus, VmpA functions as an adhesin that could be essential in the colonization of the insect by the FD phytoplasmas.IMPORTANCE Phytoplasmas infect a wide variety of plants, ranging from wild plants to cultivated species, and are transmitted by different leafhoppers, planthoppers, and psyllids. The specificity of the phytoplasma-insect vector interaction has a major impact on the phytoplasma plant host range. As entry into insect cells is an obligate process for phytoplasma transmission, the bacterial adhesion to insect cells is a key step. Thus, studying surface-exposed proteins of phytoplasma will help to identify the adhesins implicated in the specific recognition of insect vectors. In this study, it is shown that the membrane protein VmpA of the flavescence dorée (FD) phytoplasma acts as an adhesin that is able to interact with cells of Euscelidiusvariegatus, the experimental vector of the FD phytoplasma.

Differential expression of Spiroplasma citri surface protein genes in the plant and insect hosts.

BACKGROUND: Spiroplasma citri is a cell wall-less, plant pathogenic bacteria that colonizes two distinct hosts, the leafhopper vector and the host plant. Given the absence of a cell wall, surface proteins including lipoproteins and transmembrane polypeptides are expected to play key roles in spiroplasma/host interactions. Important functions in spiroplasma/insect interactions have been shown for a few surface proteins such as the major lipoprotein spiralin, the transmembrane S. citri adhesion-related proteins (ScARPs) and the sugar transporter subunit Sc76. S. citri efficient transmission from the insect to the plant is expected to rely on its ability to adapt to the different environments and more specifically to regulate the expression of genes encoding surface-exposed proteins. RESULTS: Genes encoding S. citri lipoproteins and ScARPs were investigated for their expression level in axenic medium, in the leafhopper vector Circulifer haematoceps and in the host plant (periwinkle Catharanthus roseus) either insect-infected or graft-inoculated. The vast majority of the lipoprotein genes tested (25/28) differentially responded to the various host environments. Considering their relative expression levels in the different environments, the possible involvement of the targeted genes in spiroplasma host adaptation was discussed. In addition, two S. citri strains differing notably in their ability to express adhesin ScARP2b and pyruvate dehydrogenase E1 component differed in their capacity to multiply in the two hosts, the plant and the leafhopper vector. CONCLUSIONS: This study provided us with a list of genes differentially expressed in the different hosts, leading to the identification of factors that are thought to be involved in the process of S. citri host adaptation. The identification of such factors is a key step for further understanding of S. citri pathogenesis. Moreover the present work highlights the high capacity of S. citri in tightly regulating the expression level of a large set of surface protein genes, despite the small size of its genome.

Heterologous expression and processing of the flavescence dorée phytoplasma variable membrane protein VmpA in Spiroplasma citri.

BACKGROUND: Flavescence dorée (FD) of grapevine is a phloem bacterial disease that threatens European vineyards. The disease is associated with a non-cultivable mollicute, a phytoplasma that is transmitted by the grapevine leafhopper Scaphoideus titanus in a persistent, propagative manner. The specificity of insect transmission is presumably mediated through interactions between the host tissues and phytoplasma surface proteins comprising the so-called variable membrane proteins (Vmps). Plant spiroplasmas and phytoplasmas share the same ecological niches, the phloem sieve elements of host plants and the hemocoel of insect vectors. Unlike phytoplasmas, however, spiroplasmas, and Spiroplasma citri in particular, can be grown in cell-free media and genetically engineered. As a new approach for studying phytoplasmas-insect cell interactions, we sought to mimic phytoplasmas through the construction of recombinant spiroplasmas exhibiting FD phytoplasma Vmps at the cell surface. RESULTS: Here, we report the expression of the FD phytoplasma VmpA in S. citri. Transformation of S. citri with plasmid vectors in which the vmpA coding sequence was under the control of the S. citri tuf gene promoter resulted in higher accumulation of VmpA than with the native promoter. Expression of VmpA at the spiroplasma surface was achieved by fusing the vmpA coding sequence to the signal peptide sequence of the S. citri adhesin ScARP3d, as revealed by direct colony immunoblotting and immunogold labelling electron microscopy. Anchoring of VmpA to the spiroplasma membrane was further demonstrated by Triton X-114 protein partitioning and Western immunoblotting. Using the same strategy, the secretion of free, functionally active ß-lactamase (used as a model protein) into the culture medium by recombinant spiroplasmas was achieved. CONCLUSIONS: Construction of recombinant spiroplasmas harbouring the FD phytoplasma variable membrane protein VmpA at their surface was achieved, which provides a new biological approach for studying interactions of phytoplasma surface proteins with host cells. Likewise, the secretion of functional ß-lactamase by recombinant spiroplasmas established the considerable promise of the S. citri expression system for delivering phytoplasma effector proteins into host cells.

Invasion of insect cells by Spiroplasma citri involves spiralin relocalization and lectin/glycoconjugate-type interactions.

Spiroplamas are helical, cell wall-less bacteria belonging to the Class Mollicutes, a group of microorganisms phylogenetically related to low G+C, Gram-positive bacteria. Spiroplasma species are all found associated with arthropods and a few, including Spiroplasma citri are pathogenic to plant. Thus S. citri has the ability to colonize cells of two very distinct hosts, the plant and the insect vector. While spiroplasmal factors involved in transmission by the leafhopper Circulifer haematoceps have been identified, their specific contribution to invasion of insect cells is poorly understood. In this study we provide evidence that the lipoprotein spiralin plays a major role in the very early step of cell invasion. Confocal laser scanning immunomicroscopy revealed a relocalization of spiralin at the contact zone of adhering spiroplasmas. The implication of a role for spiralin in adhesion to insect cells was further supported by adhesion assays showing that a spiralin-less mutant was impaired in adhesion and that recombinant spiralin triggered adhesion of latex beads. We also showed that cytochalasin D induced changes in the surface-exposed glycoconjugates, as inferred from the lectin binding patterns, and specifically improved adhesion of S. citri wild-type but not of the spiralin-less mutant. These results indicate that cytochalasin D exposes insect cell receptors of spiralin that are masked in untreated cells. In addition, competitive adhesion assays with lectins strongly suggest spiralin to exhibit glycoconjugate binding properties similar to that of the Vicia villosa agglutinin (VVA) lectin.

Flavescence dorée phytoplasma enters insect cells by a clathrin-mediated endocytosis allowing infection of its insect vector.

To perform its propagative and circulative cycle into its insect vector, the flavescence dorée phytoplasma invades different cell types. Clathrin-mediated endocytosis is used by a wide range of bacteria to infect eukaryote cells. Among the insect proteins interacting with the phytoplasma adhesin VmpA, we identified the adaptor protein complex AP-1 and AP-2 suggesting that phytoplasmas could enter the insect cells via clathrin-mediated endocytosis. By infection assays of insect cells in culture, we showed that phytoplasmas entry into Drosophila S2 cells was more efficient than infection of the Euva cell line developed from the insect vector Euscelidius variegatus. Chlorpromazine, cytochalasin D and knockdown of clathrin heavy chain (chc) gene expression using RNA interference inhibited entry of phytoplasmas into S2 cells. During invasion of S2 cells, phytoplasmas were observed very closed to recombinant GFP-labelled clathrin light chain. To verify the role of clathrin in the insect colonization by phytoplasmas, RNAi was performed via artificial feeding of chc dsRNA by the vector E. variegatus. This decreased the expression of chc gene in the midgut and heads of E. variegatus. The chc lower expression correlated to a decreased of midgut and salivary gland cells colonization after the insects had ingested phytoplasmas from infected plants. In conclusion, results indicate that clathrin is important for the FD phytoplasma to enter insect cells and colonize its insect vector.

A knockdown gene approach identifies an insect vector membrane protein with leucin-rich repeats as one of the receptors for the VmpA adhesin of flavescence dorée phytoplasma.

Introduction: The adhesion of flavescence dorée phytoplasma to the midgut epithelium cells of their insect vectors is partially mediated by the variable membrane protein A (VmpA), an adhesin which shows lectin properties. In order to identify the insect receptor for VmpA, we identified Euscelidius variegatus cell proteins interacting with recombinant VmpA-His6. Methods: The E. variegatus proteins were identified by mass spectrometry analysis of VmpA-E. variegatus protein complexes formed upon in vitro interaction assays. To assess their impact in VmpA binding, we reduced the expression of the candidate genes on E. variegatus cells in culture by dsRNA-mediated RNAi. The effect of candidate gene knockdown on VmpA binding was measured by the capacity of E. variegatus cells to bind VmpA-coated fluorescent beads. Results and discussion: There were 13 candidate proteins possessing potential N-glycosylation sites and predicted transmembrane domains selected. The decrease of expression of an unknown transmembrane protein with leucine-rich repeat domains (uk1_LRR) was correlated with the decreased adhesion of VmpA beads to E. variegatus cells. The uk1_LRR was more expressed in digestive tubes than salivary glands of E. variegatus. The protein uk1_LRR could be implicated in the binding with VmpA in the early stages of insect infection following phytoplasmas ingestion.

Interactions between the flavescence dorée phytoplasma and its insect vector indicate lectin-type adhesion mediated by the adhesin VmpA.

The flavescence dorée phytoplasma undergoes a propagative cycle in its insect vectors by first interacting with the insect cell surfaces, primarily in the midgut lumen and subsequently in the salivary glands. Adhesion of flavescence dorée phytoplasma to insect cells is mediated by the adhesin VmpA. We hypothesize that VmpA may have lectin-like activity, similar to several adhesins of bacteria that invade the insect gut. We first demonstrated that the luminal surface of the midgut and the basal surface of the salivary gland cells of the natural vector Scaphoideus titanus and those of the experimental vector Euscelidius variegatus were differentially glycosylated. Using ELISA, inhibition and competitive adhesion assays, and protein overlay assays in the Euva-6 insect cell line, we showed that the protein VmpA binds insect proteins in a lectin-like manner. In conclusion, the results of this study indicate that N-acetylglucosamine and mannose present on the surfaces of the midgut and salivary glands serve as recognition sites for the phytoplasma adhesin VmpA.

Infection of the Circulifer haematoceps cell line Ciha-1 by Spiroplasma citri: the non-insect-transmissible strain 44 is impaired in invasion.

Successful transmission of Spiroplasma citri by its leafhopper vector requires a specific interaction between the spiroplasma surface and the insect cells. With the aim of studying these interactions at the cellular and molecular levels, a cell line, named Ciha-1, was established using embryonic tissues from the eggs of the S. citri natural vector Circulifer haematoceps. This is the first report, to our knowledge, of a cell line for this leafhopper species and of its successful infection by the insect-transmissible strain S. citri GII3. Adherence of the spiroplasmas to the cultured Ciha-1 cells was studied by c.f.u. counts and by electron microscopy. Entry of the spiroplasmas into the insect cells was analysed quantitatively by gentamicin protection assays and qualitatively by double immunofluorescence microscopy. Spiroplasmas were detected within the cell cytoplasm as early as 1 h after inoculation and survived at least 2 days inside the cells. Comparing the insect-transmissible GII3 and non-insect-transmissible 44 strains revealed that adherence to and entry into Ciha-1 cells of S. citri 44 were significantly less efficient than those of S. citri GII3.

Entry of Spiroplasma citri into Circulifer haematoceps cells involves interaction between spiroplasma phosphoglycerate kinase and leafhopper actin.

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.

Proteolytic Post-Translational Processing of Adhesins in a Pathogenic Bacterium.

Mollicutes, including mycoplasmas and spiroplasmas, have been considered as good representatives of the « minimal cell ¼ concept: these wall-less bacteria are small in size and possess a minimal genome and restricted metabolic capacities. However, the recent discovery of the presence of post-translational modifications unknown so far, such as the targeted processing of membrane proteins of mycoplasma pathogens for human and swine, revealed a part of the hidden complexity of these microorganisms. In this study, we show that in the phytopathogen, insect-vectored Spiroplasma citri GII-3 adhesion-related protein (ScARP) adhesins are post-translationally processed through an ATP-dependent targeted cleavage. The cleavage efficiency could be enhanced in vitro when decreasing the extracellular pH or upon the addition of polyclonal antibodies directed against ScARP repeated units, suggesting that modification of the surface charge and/or ScARP conformational changes could initiate the cleavage. The two major sites for primary cleavage are localized within predicted disordered regions and do not fit any previously reported cleavage motif; in addition, the inhibition profile and the metal ion requirements indicate that this post-translational modification involves at least one non-conventional protease. Such a proteolytic process may play a role in S. citri colonization of cells of the host insect. Furthermore, our work indicates that post-translational cleavage of adhesins represents a common feature to mollicutes colonizing distinct hosts and that processing of surface antigens could represent a way to make the most out of a minimal genome.

Molecular characterization of Coxiella burnetii isolates by infrequent restriction site-PCR and MLVA typing.

BACKGROUND: Coxiella burnetii, the causative agent of Q fever, has a wide host range. Few epidemiological tools are available, and they are often expensive or not easily standardized across laboratories. In this work, C. burnetii isolates from livestock and ticks were typed using infrequent restriction site-PCR (IRS-PCR) and multiple loci variable number of tandem repeats (VNTR) analysis (MLVA). RESULTS: By applying IRS-PCR, 14 C. burnetii isolates could be divided into six groups containing up to five different isolates. Clustering as deduced from MLVA typing with 17 markers provided an increased resolution with an excellent agreement to IRS-PCR, and with the plasmid type of each strain. MLVA was then applied to 28 additional C. burnetii isolates of different origin and 36 different genotypes were identified among the 42 isolates investigated. The clustering obtained is in agreement with published Multiple Locus Sequence Typing (MLST) data. Two panels of markers are proposed, panel 1 which can be confidently typed on agarose gel at a lower cost and in any laboratory setting (10 minisatellite markers with a repeat unit larger than 9 bp), and panel 2 which comprises 7 microsatellites and provides a higher discriminatory power. CONCLUSION: Our analyses demonstrate that MLVA is a powerful and promising molecular typing tool with a high resolution and of low costs. The consistency of the results with independent methods suggests that MLVA can be applied for epidemiological studies. The resulting data can be queried on a dedicated MLVA genotyping Web service.

Immune response and survival of Circulifer haematoceps to Spiroplasma citri infection requires expression of the gene hexamerin.

Spiroplasma citri is a cell wall-less bacterium that infects plants. It is transmitted by the leafhopper Circulifer haematoceps, which hosts this bacterium in the haemocel and insect tissues. Bacterial factors involved in spiroplasma colonization of the insect host have been identified, but the immune response of the leafhopper to S. citri infection remains unknown. In this study, we showed that C. haematoceps activates both humoral and cellular immune responses when challenged with bacteria. When infected by S. citri, C. haematoceps displayed a specific immune response, evidenced by activation of phagocytosis and upregulation of a gene encoding the protein hexamerin. S. citri infection also resulted in decreased phenoloxidase-like activity. Inhibition of hexamerin by RNA interference resulted in a significant reduction in phenoloxidase-like activity and increased mortality of infected leafhoppers. Therefore, the gene hexamerin is involved in S. citri control by interfering with insect phenoloxidase activity.

The repetitive domain of ScARP3d triggers entry of Spiroplasma citri into cultured cells of the vector Circulifer haematoceps.

Spiroplasma citri is a plant pathogenic mollicute transmitted by the leafhopper vector Circulifer haematoceps. Successful transmission requires the spiroplasmas to cross the intestinal epithelium and salivary gland barriers through endocytosis mediated by receptor-ligand interactions. To characterize these interactions we studied the adhesion and invasion capabilities of a S. citri mutant using the Ciha-1 leafhopper cell line. S. citri GII3 wild-type contains 7 plasmids, 5 of which (pSci1 to 5) encode 8 related adhesins (ScARPs). As compared to the wild-type strain GII3, the S. citri mutant G/6 lacking pSci1 to 5 was affected in its ability to adhere and enter into the Ciha-1 cells. Proteolysis analyses, Triton X-114 partitioning and agglutination assays showed that the N-terminal part of ScARP3d, consisting of repeated sequences, was exposed to the spiroplasma surface whereas the C-terminal part was anchored into the membrane. Latex beads cytadherence assays showed the ScARP3d repeat domain (Rep3d) to be involved, and internalization of the Rep3d-coated beads to be actin-dependent. These data suggested that ScARP3d, via its Rep3d domain, was implicated in adhesion of S. citri GII3 to insect cells. Inhibition tests using anti-Rep3d antibodies and competitive assays with recombinant Rep3d both resulted in a decrease of insect cells invasion by the spiroplasmas. Unexpectedly, treatment of Ciha-1 cells with the actin polymerisation inhibitor cytochalasin D increased adhesion and consequently entry of S. citri GII3. For the ScARPs-less mutant G/6, only adhesion was enhanced though to a lesser extent following cytochalasin D treatment. All together these results strongly suggest a role of ScARPs, and particularly ScARP3d, in adhesion and invasion of the leafhopper cells by S. citri.

Involvement of a minimal actin-binding region of Spiroplasma citri phosphoglycerate kinase in spiroplasma transmission by its leafhopper vector.

BACKGROUND: Spiroplasma citri is a wall-less bacterium that colonizes phloem vessels of a large number of host plants. Leafhopper vectors transmit S. citri in a propagative and circulative manner, involving colonization and multiplication of bacteria in various insect organs. Previously we reported that phosphoglycerate kinase (PGK), the well-known glycolytic enzyme, bound to leafhopper actin and was unexpectedly implicated in the internalization process of S. citri into Circulifer haematoceps cells. METHODOLOGY/PRINCIPAL FINDINGS: In an attempt to identify the actin-interacting regions of PGK, several overlapping PGK truncations were generated. Binding assays, using the truncations as probes on insect protein blots, revealed that the actin-binding region of PGK was located on the truncated peptide designated PGK-FL5 containing amino acids 49-154. To investigate the role of PGK-FL5-actin interaction, competitive spiroplasma attachment and internalization assays, in which His(6)-tagged PGK-FL5 was added to Ciha-1 cells prior to infection with S. citri, were performed. No effect on the efficiency of attachment of S. citri to leafhopper cells was observed while internalization was drastically reduced. The in vivo effect of PGK-FL5 was confirmed by competitive experimental transmission assays as injection of PGK-FL5 into S. citri infected leafhoppers significantly affected spiroplasmal transmission. CONCLUSION: These results suggest that S. citri transmission by its insect vector is correlated to PGK ability to bind actin.

Characterizing the replication and stability regions of Spiroplasma citri plasmids identifies a novel replication protein and expands the genetic toolbox for plant-pathogenic spiroplasmas.

Spiroplasma citri strain GII3 contains seven plasmids, pSciA and pSci1-6, that share extensive regions of sequence homology and display a mosaic gene organization. Plasmid pSci2 comprises 12 coding sequences (CDS), three of which encode polypeptides homologous to proteins Soj/ParA, involved in chromosome partitioning, and TrsE and Mob/TraG, implicated in the type IV secretion pathway. One CDS encodes the adhesin-like protein ScARP3d whereas the other eight encode polypeptides with no homology to known proteins. The pSci2 CDS pE and soj have counterparts in all seven plasmids. Through successive deletions, various pSci2 derivatives were constructed and assessed for their ability to replicate by transformation of S. citri 44, a strain which has no plasmid. The smallest functional replicon was found to contain a single CDS (pE) and its flanking intergenic regions. Shuttle (S. citri/Escherichia coli) plasmids, in which CDS pE was disrupted, failed to replicate in S. citri, suggesting that PE is the replication protein of the S. citri plasmids. Successive propagations of pSci2-derived transformed spiroplasmas, in the absence of selection pressure, revealed that only pSci2 derivatives having an intact soj gene were stably maintained, indicating that the soj-encoded polypeptide is most likely involved in plasmid partitioning. Upon transformation, pSci2 derivatives, including shuttle (S. citri/E. coli) plasmids, were shown to replicate in all S. citri strains tested regardless of whether the strain possesses endogenous plasmids, such as strain GII3, or not, such as strain R8A2. In addition, the pSci replicons were introduced efficiently into the plant-pathogenic spiroplasmas Spiroplasma kunkelii and Spiroplasma phoeniceum, the transformation of which had never, to our knowledge, been described before. These studies show that, besides their implications for the biology of S. citri, the pSci plasmids hold considerable promise as vectors of general use for genetic studies of plant-pathogenic spiroplasmas. As an example, a HA-tagged S. citri protein was expressed in S. kunkelii. Detection of pE-hybridizing sequences in various group I spiroplasma species indicated that pE replicating plasmids were not restricted to the three plant-pathogenic spiroplasmas.