1887

Abstract

With the aim of identifying proteins involved in transmission by the leafhopper , protein maps of four transmissible and four non-transmissible strains were compared. Total cell lysates of strains were analysed by two-dimensional gel electrophoresis using commercially available immobilized pH gradients (IPGs) covering a pH range of 4–7. Approximately 530 protein spots were visualized by silver staining and the resulting protein spot patterns for the eight strains were found to be highly similar. However, comparison using PDQuest 2-D analysis software revealed two trains of protein spots that were present only in the four transmissible strains. Using MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry and a nearly complete protein database, established during the still-ongoing GII-3-3X genome project, the sequences of both proteins were deduced. One of these proteins was identified in the general databases as adhesion-related protein (P89) involved in the attachment of to gut cells of the insect vector. The second protein, with an apparent molecular mass of 32 kDa deduced from the electrophoretic mobility, could not be assigned to a known protein and was named P32. The P32-encoding gene (714 bp) was carried by a large plasmid of 35·3 kbp present in transmissible strains and missing in non-transmissible strains. PCR products with primers designed from the gene were obtained only with genomic DNA isolated from transmissible strains. Therefore, P32 has a putative role in the transmission process and it could be considered as a marker for leafhopper transmissibility. Functional complementation of a non-transmissible strain with the gene did not restore the transmissible phenotype, despite the expression of P32 in the complemented strain. Electron microscopic observations of salivary glands of leafhoppers infected with the complemented strain revealed a close contact between spiroplasmas and the plasmalemma of the insect cells. This further suggests that P32 protein contributes to the association of with host membranes.

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2006-04-01
2020-08-14
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References

  1. Archambaud C, Gouin E, Pizarro-Cerda J, Cossart P, Dussurget O. 2005; Translation elongation factor EF-Tu is a target for Stp, a serine-threonine phosphatase involved in virulence of Listeria monocytogenes . Mol Microbiol56:383–396[CrossRef]
    [Google Scholar]
  2. Barré A, de Daruvar A., Blanchard A. 2004; MolliGen, a database dedicated to the comparative genomics of mollicutes. Nucleic Acids Res32:307–310[CrossRef]
    [Google Scholar]
  3. Berg M, Melcher U, Fletcher J. 2001; Characterization of Spiroplasma citri adhesion related protein SARP1, which contains a domain of a novel family designated sarpin. Gene275:57–64[CrossRef]
    [Google Scholar]
  4. Blum H, Beier H, Gross H. J. 1987; Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis8:93–99[CrossRef]
    [Google Scholar]
  5. Bordier C. 1981; Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem256:1604–1607
    [Google Scholar]
  6. Boutareaud A, Danet J. L, Garnier M, Saillard C. 2004; 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 . Appl Environ Microbiol70:3960–3967[CrossRef]
    [Google Scholar]
  7. Bové J. M. 1995; Cyprus. In Virus and Virus-like Diseases of Citrus in the Near East Region pp 123–135 Rome: FAO;
    [Google Scholar]
  8. Bové J. M, Garnier M. 2003; Phloem- and xylem-restricted plant pathogenic bacteria. Plant Science164:423–438[CrossRef]
    [Google Scholar]
  9. Bové J. M, Carle P, Garnier M, Laigret F, Renaudin J, Saillard C. 1989; Molecular and cellular biology of spiroplasmas. In The Mycoplasmasvol. 5 pp 243–364 Edited by Whitcomb R. F., Tully J. G.. New York: Academic Press;
    [Google Scholar]
  10. Bradford M. M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem72:248–254[CrossRef]
    [Google Scholar]
  11. Burdett V, Inamine J, Rajagopalan S. 1982; Heterogeneity of tetracycline resistance determinants in Streptococcus . J Bacteriol149:995–1004
    [Google Scholar]
  12. Cowley S, Ko M, Pick N.8 other authors 2004; The Mycobacterium tuberculosis protein serine/threonine kinase PknG is linked to cellular glutamate/glutamine levels and is important for growth in vivo. Mol Microbiol52:1691–1702[CrossRef]
    [Google Scholar]
  13. Dallo S. F, Kannan T. R, Blaylock M. W, Baseman J. B. 2002; Elongation factor Tu and E1 beta subunit of pyruvate dehydrogenase complex act as fibronectin binding proteins in Mycoplasma pneumoniae . Mol Microbiol46:1041–1051[CrossRef]
    [Google Scholar]
  14. Duret S, Danet J. L, Garnier M, Renaudin J. 1999; Gene disruption through homologous recombination in Spiroplasma citri : an scm 1-disrupted motility mutant is pathogenic. J Bacteriol181:7449–7456
    [Google Scholar]
  15. Duret S, Berho N, Danet J. L, Garnier M, Renaudin J. 2003; Spiralin is not essential for the helicity, motility, or pathogenicity but is required for efficient transmission of Spiroplasma citri by its leafhopper vector Circulifer haematoceps . Appl Environ Microbiol69:6225–6234[CrossRef]
    [Google Scholar]
  16. Fairbanks G, Steck T. L, Wallach D. F. 1971; Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry10:2606–2617[CrossRef]
    [Google Scholar]
  17. Fletcher J, Wayadande A, Melcher U, Ye F. C. 1998; The phytopathogenic mollicute-insect vector interface: a closer look. Phytopathology88:1351–1358[CrossRef]
    [Google Scholar]
  18. Foissac X, Saillard C, Gandar J, Zreik L, Bové J. M. 1996a; Spiralin polymorphism in strains of Spiroplasma citri is not due to differences in posttranslational palmitoylation. J Bacteriol178:2934–2940
    [Google Scholar]
  19. Foissac X, Danet J. L, Saillard C, Whitcomb R. F, Bové J. M. 1996b; Experimental infections of plant by spiroplasmas. In Molecular and Diagnostic Procedures in Mycoplasmologyvol. 2 pp 385–389 Edited by Razin S., Tully J. G.. New York: Academic Press;
    [Google Scholar]
  20. Fos A, Saillard C, Vignault J. C, Ali Y, Brun P, Vogel R, Bové J. M, Lallemand J. 1986; The leafhopper Neoaliturus haematoceps is a vector of Spiroplasma citri in the Mediterranean area. Ann Inst Pasteur Microbiol137A:97–107
    [Google Scholar]
  21. Hosseini Pour A. 2000; Determination of some molecular and cellular characteristics of Spiroplasma citri, the causal agent of citrus stubborn disease in Kerman, Fars and Mazadaran provinces PhD thesis Tarbiat Modares University Tehran;
    [Google Scholar]
  22. Jacobson G. R, Rosenbusch J. P. 1976; Abundance and membrane association of elongation factor Tu in E. coli . Nature261:23–26[CrossRef]
    [Google Scholar]
  23. Kaloostian G. H, Oldfield G. N, Pierce H. D, Calavan E. C. 1979; Spiroplasma citri and its transmission to citrus and other plants by leafhoppers. In Leafhoppers Vectors and Plant Disease Agents pp 447–450 Edited by Maramorosch K., Harris K. F.. New York: Academic Press;
    [Google Scholar]
  24. Killiny N, Castroviejo M, Saillard C. 2005; Spiroplasma citri spiralin acts in vitro as a lectin binding to glycoproteins from its insect vector Circulifer haematoceps . Phytopathology95:541–548[CrossRef]
    [Google Scholar]
  25. Kwon M. O, Wayadande A. C, Fletcher J. 1999; Spiroplasma citri movement into the intestines and salivary glands of its leafhopper vector, Circulifer tenellus . Phytopathology89:1144–1151[CrossRef]
    [Google Scholar]
  26. Liu H. Y, Gumpf D. J, Oldfield G. N, Calavan E. C. 1983; The relationship of Spiroplasma citri and Circulifer tenellus . Phytopathology73:585–590[CrossRef]
    [Google Scholar]
  27. Markham P. G, Townsend R, Bar-Joseph M, Daniels M. J, Plaskitt A, Meddins B. M. 1974; Spiroplasmas are the causal agent of citrus little-leaf disease. Ann Appl Biol78:49–57[CrossRef]
    [Google Scholar]
  28. Najar A, Bouachem S, Danet J. L, Saillard C, Garnier M, Bove J. M. 1998; Présence en Tunisie de Spiroplasma citri , l'agent causal du stubborn des agrumes et de son vecteur, la cicadelle Circulifer haematoceps .Contamination de C. haematoceps et de C. opacipennis par S. citri . Fruits 53:391–396
    [Google Scholar]
  29. Oldfield G. N, Kaloostian G. H, Pierce H. D, Calavan E. C, Granett A. L, Blue R. L. 1976; Beet leafhopper transmit citrus stubborn disease. Calif Agric30:15
    [Google Scholar]
  30. Özbek E, Miller S. A, Meulia T, Hogenhout S. A. 2003; Infection and replication sites of Spiroplasma kunkelii (Class: Mollicutes) in midgut and Malpighian tubules of leafhopper Dalbulus maidis . J Invertebr Pathol82:167–175[CrossRef]
    [Google Scholar]
  31. Razin S, Jacobs E. 1992; Mycoplasma adhesion. J Gen Microbiol138:407–422[CrossRef]
    [Google Scholar]
  32. Renaudin J. 2002; Extrachromosomal elements and gene transfert. In Molecular and Pathogenicity of Mycoplasmas pp 347–370 Edited by Razin S., Herrmann R.. New York: Kluwer Academic/Plenum;
    [Google Scholar]
  33. Rottem S. 2003; Interaction of mycoplasmas with host cells. Physiol Rev83:417–432[CrossRef]
    [Google Scholar]
  34. Saglio P, Laflèche D, Bonisol C, Bové J. M. 1971; Culture in vitro des mycoplasmes associés au stubborn des agrumes et leur observation au microscope électronique. C R Acad Sci272:1387–1390
    [Google Scholar]
  35. Sambrook J, Fritsch E. F, Maniatis T. 1989; Molecular Cloning: a Laboratory Manual, 2nd edn.. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  36. Stamburski C, Renaudin J, Bové J. M. 1991; First step toward a virus-derived vector for gene cloning and expression in spiroplasmas, organisms which read UGA as a tryptophan codon – synthesis of chloramphenicol acetyltransferase in Spiroplasma citri . J Bacteriol173:2225–2230
    [Google Scholar]
  37. Townsend R, Markham P. G, Plaskitt K. A. 1977; Multiplication and morphology of Spiroplasma citri in the leafhopper Euscelis plebejus . Ann Appl Biol87:307–313[CrossRef]
    [Google Scholar]
  38. Tully J. G, Whitcomb R. F, Clark H. F, Williamson D. L. 1977; Pathogenic mycoplasma: cultivation and vertebrate pathogenicity of a new spiroplasma. Science195:892–894[CrossRef]
    [Google Scholar]
  39. Vignault J. C, Bové J. M, Saillard C. 17 other authors 1980; Mise en culture de spiroplasmes à partir de matériel végétal et d'insectes provenant de pays circum méditerranéens et du Proche Orient. C R Acad Sci290:775–780
    [Google Scholar]
  40. Wang J, Li C, Yang H, Mushegian A, Jin S. 1998; A novel serine/threonine protein kinase homologue of Pseudomonas aeruginosa is specifically inducible within the host infection site and is required for full virulence in neutropenic mice. J Bacteriol180:6764–6768
    [Google Scholar]
  41. Wayadande A. C, Fletcher J. 1995; Transmission of Spiroplasma citri lines and their ability to cross gut and salivary gland barriers within the leafhopper vector Circulifer tenellus . Phytopathology85:1256–1259[CrossRef]
    [Google Scholar]
  42. Ye F. C, Melcher U, Fletcher J. 1997; Molecular characterization of a gene encoding a membrane protein of Spiroplasma citri . Gene189:95–100[CrossRef]
    [Google Scholar]
  43. Yu J, Wayadande A. C, Fletcher J. 2000; Spiroplasma citri surface protein P89 implicated in adhesion to cells of the vector Circulifer tenellus . Phytopathology90:716–722[CrossRef]
    [Google Scholar]
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