Spiralin polymorphism in strains of Spiroplasma citri is not due to differences in posttranslational palmitoylation.

Spiralin is defined as the major membrane protein of the helical mollicute Spiroplasma citri. According to the S. citri strain used, spiralin shows polymorphism in its electrophoretic mobility. The spiralin gene sequences of eight S. citri strains were determined by direct sequencing of the PCR-amplified genes. All spiralins were found to be 241 amino acids long, except for the spiralin of strain Palmyre, which is 242 amino acids long. The molecular masses calculated from these sequences did not explain the differences observed in the electrophoretic mobilities. In all of the spiralins examined, the first 24 N-terminal amino acids were conserved, including a cysteine at position 24, and had the features of typical signal peptides of procaryotic lipoproteins. When S. citri strains were grown in the presence of [3H]palmitic acid, at least 10 proteins, including spiralin, became labeled. In the presence of globomycin, a lipoprotein signal peptidase inhibitor in eubacteria, apparently unprocessed spiralin could be detected. Formic acid hydrolysis of the [3H]palmitic acid-labeled spiralins of four representative S. citri strains yielded two peptide fragments for each spiralin, as expected from the gene sequence. On fragment was [3H]palmitic acid labeled, and it had almost the same electrophoretic mobility irrespective of the spiralins used. Samples of the unlabeled peptide fragments from the four representative strains had slightly different electrophoretic mobilities (delta Da approximately equal to 800 Da); however, these were much smaller than those of the whole spiralins before formic acid hydrolysis (delta Da approximately equal to 8,000 Da). These results suggest that spiralin polymorphism in S. citri is not due to differences in posttranslational modification by palmitic acid and is certainly a structural property of the whole protein or could result from an unidentified posttranslational modification of spiralin.     

Spiralin: Major membrane protein specific for subgroup I-1 Spiroplasmas

Preparations of spiralin from membranes ofSpiroplasma citri, strain C189, purified by sequential solubilization with detergents followed by agarose-suspension electrophoresis induced rabbit antibodies that were largely specific forSpiroplasma citri Group I-1 spiroplasmas, as demonstrated by metabolic inhibition (MI), growth inhibition (GI), and deformation (DF) tests. By contrast, antibodies againstS. citri whole-membrane protein preparations reacted broadly with representative type cultures of seven subgroups of theS. citri complex. Neither antimembrane nor antispiralin sera reacted withS. floricola, S. mirum, or Group IV, (VI), (VII), or (VIII) spiroplasmas. Minor cross-reactions in MI and DF tests between antispiralin serum and Subgroup I-2 and I-3 antigens may have represented shared epitopes in a set of homologous membrane proteins of the three spiroplasmas, or antibodies against highly antigenic traces of other common membrane proteins in the purified spiralin preparations. The unique antigenic properties of spiralin, the most abundant protein in theS. citri membrane, explain in part the unique profiles shown by this spiroplasma species in comparative taxonomic serological tests.     

Sequence Analysis of Spiroplasma phoeniceum and Spiroplasma kunkelii Spiralin Genes and Comparison with Other Spiralin Genes

The spiralin genes from two phytopathogenic spiroplasmas, Spiroplasma phoeniceum and Spiroplasma kunkelii, were amplified by PCR, cloned, and sequenced. Comparison of the amino acid sequences of the five spiralins analyzed to date confirm that the spiralins have a general amphiphilic character and possess a conserved lipoprotein signal peptide. It also shows that a conserved central region and an amino acid repetition, including a VTKXE consensus sequence, are present in all spiralins analyzed. Received: 11 March 1997 / Accepted: 14 April 1997     

Spiralin Is Not Essential for Helicity, Motility, or Pathogenicity but Is Required for Efficient Transmission of Spiroplasma citri by Its Leafhopper Vector Circulifer haematoceps

Spiralin is the most abundant protein at the surface of the plant pathogenic mollicute Spiroplasma citri and hence might play a role in the interactions of the spiroplasma with its host plant and/or its insect vector. To study spiralin function, mutants were produced by inactivating the spiralin gene through homologous recombination. A spiralin-green fluorescent protein (GFP) translational fusion was engineered and introduced into S. citri by using an oriC-based targeting vector. According to the strategy used, integration of the plasmid by a single-crossover recombination at the spiralin gene resulted in the expression of the spiralin-GFP fusion protein. Two distinct mutants were isolated. Western and colony immunoblot analyses showed that one mutant (GII3-9a5) did produce the spiralin-GFP fusion protein, which was found not to fluoresce, whereas the other (GII3-9a2) produced neither the fusion protein nor the wild-type spiralin. Both mutants displayed helical morphology and motility, similarly to the wild-type strain GII-3. Genomic DNA analyses revealed that GII3-9a5 was unstable and that GII3-9a2 was probably derived from GII3-9a5 by a double-crossover recombination between plasmid sequences integrated into the GII3-9a5 chromosome and free plasmid. When injected into the leafhopper vector Circulifer haematoceps, the spiralinless mutant GII3-9a2 multiplied to high titers in the insects (1.1 × 10⁶ to 2.8 × 10⁶ CFU/insect) but was transmitted to the host plant 100 times less efficiently than the wild-type strain. As a result, not all plants were infected, and symptom production in these plants was delayed for 2 to 4 weeks compared to that in the wild-type strain. In the infected plants however, the mutant multiplied to high titers (1.2 × 10⁶ to 1.4 × 10⁷ CFU/g of midribs) and produced the typical symptoms of the disease. These results indicate that spiralin is not essential for pathogenicity but is required for efficient transmission of S. citri by its insect vector.     

Transmission characteristics of Spiroplasma citri and its effect on leafhopper vectors from the Circulifer tenellus complex

Several leafhopper variants of the Circulifer tenellus complex were collected in “citrus stubborn” affected areas in Israel. Two of these variants transmitted the Spiroplasma citri to Matthiola incana after being injected with the disease agent. The variant from Atriplex halimus was designated Circulifer tenellus-A (CTA) and the variant from Portulaca oleracea was designated Circulifer tenellus-? (CTP). Transmission characteristics were determined for both leafhoppers. A high rate of transmission (43.3%) was obtained by single CTA leafhoppers that were injected with the Amiad S. citri isolate from the Upper Galilee, compared with 7% transmission obtained with the CTP leafhoppers. The Gilgal S. citri isolate from the Jordan Valley, was not transmitted by either. Injection was more effective than acquisition access feeding to render the leafhopper infective for both CTA and CTP. The minimum acquisition access period needed for the CTA variant to transmit the Amiad isolate was 1 h. Longer AAPs did not necessarily result in a higher rate of transmission. The minimum incubation period was 6 days and the maximum was 32 days. The LP₅₀ calculated from the logarithmic curve y = 45.74Ln(x)–53.68 was 9.64 days. The minimum inoculation access period (IAP) was lh. The same transmission parameters for the CTP variant could not be determined, as no transmission was obtained even when groups of five-six insects were placed on a single plant.     

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.     

Identification of surface proteins ofSpiroplasma citri with protease and antibody probes

Spiroplasma citri, a helical, wall-less prokaryote, is an insect-borne phytopathogen. Though proteins having domains on the surface ofS. citri cells may be important in pathogenicity or transmissibility, only one surface protein, spiralin (29 KDa), has previously been identified. Intact cells of strain BR3 were treated with chymotrypsin, proteinase K, or trypsin, and the surviving proteins were analyzed by SDS-PAGE. Seven proteins, in addition to spiralin, were degraded, indicative of surface exposure of those polypeptides. Surface immunoprecipitation (SIP) was used to test accessibility of the proteins to anti-S. citri membrane serum, another indication of surface exposure. With unlabeled cells, five such proteins were identified. Four of these have sizes that correspond to those seen with protease treatments. When¹²⁵I surfacelabeled spiroplasmas were used for SIP, twelve surface proteins were detected, eight of which correspond to bands identified by the other methods. A protein of 89 KDa in strain BR3 was not universally detected in otherS. citri strains and spiroplasma species.     

Effect of Polyclonal, Monoclonal, and Recombinant (Single-Chain Variable Fragment) Antibodies on In Vitro Morphology, Growth, and Metabolism of the Phytopathogenic Mollicute Spiroplasma citri

Antibodies are known to affect the morphology, growth, and metabolism of mollicutes and thus may serve as candidate molecules for a plantibody-based control strategy for plant-pathogenic spiroplasmas and phytoplasmas. Recombinant single-chain variable fragment (scFv) antibodies are easy to engineer and express in plants, but their inhibitory effects on mollicutes have never been evaluated and compared with those of polyclonal and monoclonal antibodies. We describe the morphology, growth, and glucose metabolism of Spiroplasma citri in the presence of polyclonal, monoclonal, and recombinant antibodies directed against the immunodominant membrane protein spiralin. We showed that the scFv antibodies had no effect on S. citri glucose metabolism but were as efficient as polyclonal antibodies in inhibiting S. citri growth in liquid medium. Inhibition of motility was also observed.     

I-SceI-mediated plasmid deletion and intra-molecular recombination in Spiroplasma citri

S. citri wild-type strain GII3 carries six plasmids (pSci1 to -6) that are thought to encode determinants involved in the transmission of the spiroplasma by its leafhopper vector. In this study we report the use of meganuclease I-SceI for plasmid deletion in S. citri. Plasmids pSci1NT-I and pSci6PT-I, pSci1 and pSci6 derivatives that contain the tetM selection marker and a unique I-SceI recognition site were first introduced into S. citri strains 44 (having no plasmid) and GII3 (carrying pSci1-6), respectively. Due to incompatibility of homologous replication regions, propagation of the S. citri GII3 transformant in selective medium resulted in the replacement of the natural pSci6 by pSci6PT-I. The spiroplasmal transformants were further transformed by an oriC plasmid carrying the I-SceI gene under the control of the spiralin gene promoter. In the S. citri 44 transformant, expression of I-SceI resulted in rapid loss of pSciNT-I showing that expression of I-SceI can be used as a counter-selection tool in spiroplasmas. In the case of the S. citri GII3 transformant carrying pSci6PT-I, expression of I-SceI resulted in the deletion of plasmid fragments comprising the I-SceI site and the tetM marker. Delineating the I-SceI generated deletions proved they had occurred though recombination between homologous sequences. To our knowledge this is the first report of I-SceI mediated intra-molecular recombination in mollicutes.     

Disruption of a Gene Predicted To Encode a Solute Binding Protein of an ABC Transporter Reduces Transmission of Spiroplasma citri by the Leafhopper Circulifer haematoceps

Spiroplasma citri is transmitted from plant to plant by phloem-feeding leafhoppers. In an attempt to identify mechanisms involved in transmission, mutants of S. citri affected in their transmission must be available. For this purpose, transposon (Tn4001) mutagenesis was used to produce mutants which have been screened for their ability to be transmitted by the leafhopper vector Circulifer haematoceps to periwinkle plants. With one mutant (G76) which multiplied in leafhoppers as efficiently as S. citri wild-type (wt) strain GII-3, the plants showed symptoms 4 to 5 weeks later than those infected with wt GII-3. Thirty to fifty percent of plants exposed to leafhoppers injected with G76 remained symptomless, whereas for wt GII-3, all plants exposed to the transmission showed severe symptoms. This suggests that the mutant G76 was injected into plants by the leafhoppers less efficiently than wt GII-3. To check this possibility, the number of spiroplasma cells injected by a leafhopper through a Parafilm membrane into SP4 medium was determined. Thirty times less mutant G76 than wt GII-3 was transmitted through the membrane. These results suggest that mutant G76 was affected either in its capacity to penetrate the salivary glands and/or to multiply within them. In mutant G76, transposon Tn4001 was shown to be inserted into a gene encoding a putative lipoprotein (Sc76) In the ABCdb database Sc76 protein was noted as a solute binding protein of an ABC transporter of the family S1_b. Functional complementation of the G76 mutant with the Sc76 gene restored the wild phenotype, showing that Sc76 protein is involved in S. citri transmission by the leafhopper vector C. haematoceps.     

Growth inhibition of phytopathogenic spiroplasmas by membrane-interactive antimicrobial peptides Novispirin T7 and Caerin 1.1

Spiroplasma kunkelii and Spiroplasma citri, both helical-shaped cell wall-less bacteria, are the causative agents of corn stunt disease and citrus stubborn disease, respectively. Plants exhibiting natural resistance to these phytopathogenic spiroplasmas are currently lacking. Engineering artificial plant resistance using antimicrobial peptides (AMPs) has been conceived as a new approach to control the agronomically important spiroplasmal diseases. In preparation for such task, the present study focused on screening of AMPs that have potentials to curb the growth of S. kunkelii and S. citri. Four AMPs, including Novispirin T7, Caerin 1.1, Tricholongin and Dhvar4, were selected for in vitro growth inhibition test. A liquid assay method was developed for quick qualitative and quantitative evaluations of the AMPs. Our results demonstrated that Novispirin T7 and Caerin 1.1 were able to inhibit the growth of both phytopathogenic spiroplasmas with the efficacy comparable to that of tetracycline. Cell deformations were observed in spiroplasma cultures treated with these two peptides, indicating interactions of the AMPs with the spiroplasma cell membranes. The minimum inhibitory concentrations (MICs) of the AMPs against S. kunkelii and S. citri were determined.     

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 His₆-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.     

Versatile Use of oriC Plasmids for Functional Genomics of Mycoplasma capricolum subsp. capricolum

Replicative oriC plasmids were recently developed for several mollicutes, including three Mycoplasma species belonging to the mycoides cluster that are responsible for bovine and caprine diseases: Mycoplasma mycoides subsp. mycoides small-colony type, Mycoplasma mycoides subsp. mycoides large-colony type, and Mycoplasma capricolum subsp. capricolum. In this study, oriC plasmids were evaluated in M. capricolum subsp. capricolum as genetic tools for (i) expression of heterologous proteins and (ii) gene inactivation by homologous recombination. The reporter gene lacZ, encoding β-galactosidase, and the gene encoding spiralin, an abundant surface lipoprotein of the related mollicute Spiroplasma citri, were successfully expressed. Functional Escherichia coli β-galactosidase was detected in transformed Mycoplasma capricolum subsp. capricolum cells despite noticeable codon usage differences. The expression of spiralin in M. capricolum subsp. capricolum was assessed by colony and Western blotting. Accessibility of this protein at the cell surface and its partition into the Triton X-114 detergent phase suggest a correct maturation of the spiralin precursor. The expression of a heterologous lipoprotein in a mycoplasma raises potentially interesting applications, e.g., the use of these bacteria as live vaccines. Targeted inactivation of gene lppA encoding lipoprotein A was achieved in M. capricolum subsp. capricolum with plasmids harboring a replication origin derived from S. citri. Our results suggest that the selection of the infrequent events of homologous recombination could be enhanced by the use of oriC plasmids derived from related mollicute species. Mycoplasma gene inactivation opens the way to functional genomics in a group of bacteria for which a large wealth of genome data are already available and steadily growing.     

Biotic and abiotic factors associated with citrus progressive decline in Fars Province,Iran

Citrus decline has become a devastating problem in citrus-growing regions of southern Iran. The affected trees show progressive yellowing of leaves, thinning of the canopy, dieback and total collapse. Despite a number of attempts, the aetiology of the decline has remained uncertain. In the present study, we evaluated the potential association of various biotic and abiotic factors with the decline in the Fars Province of Iran. Biotic agents surveyed included Candidatus Liberibacter asiaticus, Spiroplasma citri, phytoplasmas, Tylenchulus semipenetrans and root infecting fungi and oomycetes. Abiotic factors studied were soil and water salinity, changes in prevailing temperature and other environmental conditions. In our surveys, Ca. L. asiaticus, either alone or in combination with other factors, had the highest frequency of association (92%) with the decline, followed by S. citri (75%). Ca. L. asiaticus was not detected in any of the non-decline trees examined. Pythium or Phytophthora species and a fungus of theFusarium solani species complex were also isolated from roots of many declining trees. Phytoplasmas were found only in few cases, and populations of citrus nematode were often below the threshold of economic loss. Soil/water salinity were in the suitable range for citrus cultivation in most cases. It is suggested that the decline is initiated by Ca. L. asiaticus infection, which is known to weaken the root system and make it vulnerable to infection by opportunistic soil fungi and oomycetes. S. citri, summer temperatures, low air humidity and overbearing of the trees seem to be other potential factors contributing to the intensity of the disease.     

In Planta Horizontal Transfer of a Major Pathogenicity Effector Gene

Xanthomonas citri pv. citri is a clonal group of strains that causes citrus canker disease and appears to have originated in Asia. A phylogenetically distinct clonal group that causes identical disease symptoms on susceptible citrus, X. citri pv. aurantifolii, arose more recently in South America. Genomes of X. citri pv. aurantifolii strains carry two DNA fragments that hybridize to pthA, an X. citri pv. citri gene which encodes a major type III pathogenicity effector protein that is absolutely required to cause citrus canker. Marker interruption mutagenesis and complementation revealed that X. citri pv. aurantifolii strain B69 carried one functional pthA homolog, designated pthB, that was required to cause cankers on citrus. Gene pthB was found among 38 open reading frames on a 37,106-bp plasmid, designated pXcB, which was sequenced and annotated. No additional pathogenicity effectors were found on pXcB, but 11 out of 38 open reading frames appeared to encode a type IV transfer system. pXcB transferred horizontally in planta, without added selection, from B69 to a nonpathogenic X. citri pv. citri (pthA::Tn5) mutant strain, fully restoring canker. In planta transfer efficiencies were very high (>0.1%/recipient) and equivalent to those observed for agar medium with antibiotic selection, indicating that pthB conferred a strong selective advantage to the recipient strain. A single pathogenicity effector that can confer a distinct selective advantage in planta may both facilitate plasmid survival following horizontal gene transfer and account for the origination of phylogenetically distinct groups of strains causing identical disease symptoms.     

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 His₆-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.Phloem-feeding leafhoppers transmit plant pathogenic mollicutes, spiroplasmas, and phytoplasmas from plant to plant in a persistent propagative manner (26, 43). These phytopathogenic mollicutes are restricted to phloem and to certain vector tissues; thus, their vectors are phloem sap-feeding specialists. After being ingested from plant phloem by their insect vectors, they traverse the insect gut wall, move into the hemolymph, where they multiply, and invade the salivary glands (20, 33, 34, 36). During their movements in the insect vector until its transmission to a new host plant, spiroplasmas and phytoplasmas must traverse two major physical barriers, namely, the insect intestine and the salivary gland (35, 53). Until now, little was known about the molecular and cellular interactions contributing to the crossing of these physical barriers. Several lines of evidence suggest that host-pathogen interactions could be a prerequisite for invasion and colonization of insect vector organs (2, 48, 53). For human and animal pathogenic mollicutes, it is well established that successful colonization of the host cells requires adhesion as the first step. This event is mediated by surface proteins, and among these proteins adhesins play an important role (8, 44). Recently, it was reported that an antigenic membrane protein (Amp) of onion yellow phytoplasma interacts with the insect microfilament complex and that interaction plays an important role in determining the insect vector specificity (48). Several other immunodominant membrane proteins from various phytoplasmas have been mentioned in the literature as candidates for involvement in host-phytoplasma interactions (29, 30).Spiroplasma citri, the first phytopathogenic mollicute available in culture (45), has emerged as an outstanding model for studying spiroplasma interactions with its two hosts: the periwinkle plant and the insect vector Circulifer haematoceps. Following observations of membrane-bound cytoplasmic vesicles of midgut epithelium and salivary gland cells, S. citri was hypothesized to cross these physical barriers by receptor-mediated cell endocytosis (3, 33, 39). Several S. citri protein candidates have been identified as involved in transmission and, for a few of them, in an interaction with leafhopper vector proteins. Spiralin, the most abundant membrane protein, was suspected to be involved in the transmission for two reasons: (i) a S. citri spiralinless mutant was less effective in its transmissibility (19); (ii) spiralin acted in vitro as a lectin able to bind to glycoproteins of insect vectors and therefore might function as a ligand able to interact with leafhopper receptors (32). In addition, the ability of S. citri to be transmitted by C. haematoceps is clearly affected by disruption of a gene predicted to encode a lipoprotein with homology to a solute-binding protein of an ABC transporter (14). The proteome of nontransmissible S. citri strains specifically lacks adhesion-related proteins (ScARPs) and the membrane-associated protein P32 present in the proteome of transmissible strains (12, 13, 31). These proteins are encoded by plasmids pSci1 to -6 (46), which are present only in transmissible strains, and ScARPs share strong similarities with the adhesion-related protein SARP1 of S. citri strain BR3, in which the presence has been correlated to the ability for the spiroplasma to adhere to insect cells in vitro (9, 55). The specific interactions of S. citri with eukaryotic cells remain to be elucidated, but a combination of the effects of several proteins or a complex would be necessary to explain the invasion of a variety of host cell types by S. citri (33).Nevertheless, in the last sequence of events involved in insect vector transmission, the first contact and recognition for the efficient penetration of the salivary gland cells represents an essential step. In the present study, confocal images of infected salivary glands show the localization of S. citri cells along the actin filaments. We report the results of the first attempt to decipher the role of the spiroplasma''s phosphoglycerate kinase (PGK) in the internalization of S. citri in its insect vector''s cells.     

Partial Chromosome Sequence of Spiroplasma citri Reveals Extensive Viral Invasion and Important Gene Decay

The assembly of 20,000 sequencing reads obtained from shotgun and chromosome-specific libraries of the Spiroplasma citri genome yielded 77 chromosomal contigs totaling 1,674 kbp (92%) of the 1,820-kbp chromosome. The largest chromosomal contigs were positioned on the physical and genetic maps constructed from pulsed-field gel electrophoresis and Southern blot hybridizations. Thirty-eight contigs were annotated, resulting in 1,908 predicted coding sequences (CDS) representing an overall coding density of only 74%. Cellular processes, cell metabolism, and structural-element CDS account for 29% of the coding capacity, CDS of external origin such as viruses and mobile elements account for 24% of the coding capacity, and CDS of unknown function account for 47% of the coding capacity. Among these, 21% of the CDS group into 63 paralog families. The organization of these paralogs into conserved blocks suggests that they represent potential mobile units. Phage-related sequences were particularly abundant and include plectrovirus SpV1 and SVGII3 and lambda-like SpV2 sequences. Sixty-nine copies of transposases belonging to four insertion sequence (IS) families (IS30, IS481, IS3, and ISNCY) were detected. Similarity analyses showed that 21% of chromosomal CDS were truncated compared to their bacterial orthologs. Transmembrane domains, including signal peptides, were predicted for 599 CDS, of which 58 were putative lipoproteins. S. citri has a Sec-dependent protein export pathway. Eighty-four CDS were assigned to transport, such as phosphoenolpyruvate phosphotransferase systems (PTS), the ATP binding cassette (ABC), and other transporters. Besides glycolytic and ATP synthesis pathways, it is noteworthy that S. citri possesses a nearly complete pathway for the biosynthesis of a terpenoid.Spiroplasmas are arthropod-associated bacteria belonging to the class Mollicutes, a group of wall-less microorganisms phylogenetically related to low-G+C-content, Gram-positive bacteria (51). Spiroplasma citri is a helical plant-pathogenic mollicute responsible for the “stubborn” disease of citrus (39). It inhabits the phloem sap of infected plants to which it is transmitted by sap-sucking hemipteran insect in a circulative and propagative manner (31, 32). S. citri can infect a wide range of plant species, including crop and wild plants, as it is transmitted by polyphagous leafhoppers (13). Spiroplasmas are available in pure culture, and their study has therefore benefited from the use of molecular genetics. In particular, the relationships of spiroplasmas with their two hosts, the plant and the leafhopper vector, have been extensively studied (11, 22). In S. citri, the inactivation of genes and functional complementation of mutants have shown that (i) fructose consumption by the spiroplasma is a major cause for symptom production in plants, (ii) the solute binding protein of a putative ABC-type transporter is involved in the insect transmission process, and (iii) spiralin, the major membrane protein, is not essential for helicity, motility, and pathogenicity but is required for efficient transmission by the leafhopper vector (10, 19, 23, 24, 28). To characterize other spiroplasma genes potentially involved in insect transmission and pathogenicity, the genome of S. citri strain GII3-3X is currently being deciphered.The S. citri genome is characterized by an abundance of extrachromosomal elements, including seven plasmids, pSciA and pSci1 to pSci6, present as 10 to 14 copies per cell. These plasmids are vertically inherited, but some of them could also be horizontally transferred, as they encode proteins involved in partitioning and the cell-to-cell transfer of DNA molecules (12, 40). Plasmids pSci1 to pSci5 encode surface proteins of the S. citri adhesion-related protein (ScARP) family, and pSci6 was previously shown to confer insect transmissibility (9). Therefore, it is likely that the abundance and diversity of plasmids could provide S. citri strain GII3-3X with the ability to quickly adapt to various vector insects and, hence, to be transmitted to diverse host plants. However, chromosome-encoded determinants are also expected to play a role in spiroplasma biology. In S. citri, the chromosome sizes vary from 1.6 to 1.9 Mbp among strains (53, 54), and part of the size variation is thought to result from different amounts of prophage sequences (35). Many S. citri strains are infected by single-stranded DNA-containing filamentous phages (Plectrovirus), whose sequences also occur as partial or full-length prophages integrated into the spiroplasma chromosome (7, 35, 38). Here we report the partial chromosome sequence of S. citri strain GII3-3X and the functional assignment of the predicted coding sequences.