1887

Abstract

Membrane proteins mediate several important processes, including attachment, in several Mollicute species. Phytoplasmas are non-culturable plant pathogenic mollicutes that are transmitted in a specific manner by certain phloem-feeding insect vectors. Because it is likely that phytoplasma membrane proteins are involved with some aspect of the transmission process, their identification, isolation and characterization are important first steps in understanding phytoplasma transmission. A 32 kDa immunodominant protein (IDP) from the Western X-disease (WX) phytoplasma was purified from infected plants by immunoprecipitation using monoclonal antibodies, and two peptides from a tryptic digest were sequenced. PCR primers designed from these sequences amplified a 145 bp product which hybridized with WX-related phytoplasmas in Southern blots. This PCR product was used to identify a 2·5 kbp RI–dIII fragment that was cloned and sequenced. A complete 864 bp ORF () was identified for which the putative translation product contained both of the tryptic digest peptide sequences that were used to design the PCR primers. Analysis of the predicted IdpA sequence indicated two transmembrane domains but no cleavage point. The amino acid sequence had no significant homology with other known phytoplasma IDP genes. The ORF was cloned into an expression vector and a fusion protein of the predicted size was identified in Western blots using a WX-specific antiserum. A rabbit polyclonal antiserum was prepared to the purified expression protein and this reacted with both the -expressed and native WX phytoplasma proteins. This newly identified WX IDP (IdpA) is distinct from other known mollicute membrane proteins.

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2001-03-01
2019-12-11
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References

  1. Barbara, D. J., Davies, D. L. & Clark, M. F. (1998). Cloning and sequencing of a major membrane protein from chlorante (aster yellows) phytoplasma. In Proceedings of the 12th International Meeting of the International Organization for Mycoplasmology. Abstract G.04, p. 183. Sydney: Australian Society of Microbiology.
  2. Berg, M. & Seemüller, E. ( 1999; ). Chromosomal organization and nucleotide sequence of the genes coding for the elongation factors G and Tu of the apple proliferation phytoplasma. Gene 226, 103-109.[CrossRef]
    [Google Scholar]
  3. Berg, M., Davies, D. L., Clark, M. F., Vetten, H. J., Maier, G., Marcone, C. & Seemüller, E. ( 1999; ). Isolation of the gene encoding an immunodominant membrane protein of the apple proliferation phytoplasma, and expression and characterization of the gene product. Microbiology 145, 1937-1943.[CrossRef]
    [Google Scholar]
  4. Berg, M., Yu, J., Melcher, U. & Fletcher, J. ( 2000; ). Spiroplasma citri putative adhesin P89: development of serological and molecular markers. Phytopathology 90, S6.
    [Google Scholar]
  5. Clark, M. F. ( 1981; ). Immunosorbent assays in plant pathology. Annu Rev Phytopathol 19, 83-106.[CrossRef]
    [Google Scholar]
  6. Clark, M. F., Morton, A. & Buss, S. L. ( 1989; ). Preparation of mycoplasma immunogens from plants and a comparison of polyclonal and monoclonal antibodies made against primula yellows MLO-associated antigens. Ann Appl Biol 114, 111-124.[CrossRef]
    [Google Scholar]
  7. Davies, D. L. & Clark, M. F. ( 1991; ). Production and characterization of polyclonal and monoclonal antibodies against peach yellow leafroll MLO-associated antigens. Acta Hortic 309, 275-283.
    [Google Scholar]
  8. Davies, D. L., Clark, M. F. & Barbara, D. J. (1999). Cloning and sequencing of the genes determining a major membrane protein associated with the chlorante isolate of aster yellows and clover phyllody. First Internet Conference on Phytopathogenic Mollicutes. http://www.uniud.it/phytoplasma/pap/davi7140.html.
  9. Errampalli, D. & Fletcher, J. ( 1993; ). Production of monospecific polyclonal antibodies against aster yellows mycoplasmalike organism-associated antigen. Phytopathology 83, 1279-1282.[CrossRef]
    [Google Scholar]
  10. Feinberg, A. P. & Vogelstein, B. ( 1983; ). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132, 6-13.[CrossRef]
    [Google Scholar]
  11. Freitag, J. H. ( 1964; ). Interaction and mutual suppression among three strains of aster yellows virus. Virology 24, 401-413.[CrossRef]
    [Google Scholar]
  12. Gaidenko, T. A., Yang, X., Lee, Y. M. & Price, C. W. ( 1999; ). Threonine phosphorylation of modulator protein RsbR governs its ability to regulate a serine kinase in the environmental stress signaling pathway of Bacillus subtilis. J Mol Biol 288, 29-39.[CrossRef]
    [Google Scholar]
  13. Gundersen, D. E., Lee, I. M., Rehner, S. A., Davis, R. E. & Kingsbury, D. T. ( 1994; ). Phylogeny of mycoplasmalike organisms (phytoplasmas) – a basis for their classification. J Bacteriol 176, 5244-5254.
    [Google Scholar]
  14. Gundersen, D. E., Lee, I. M., Schaff, D. A., Harrison, N. A., Chang, C. J., Davis, R. E. & Kingsbury, D. T. ( 1996; ). Genomic diversity and differentiation among phytoplasma strains in 16S rRNA groups I (aster yellows and related phytoplasmas) and Iii (X-disease and related phytoplasmas). Int J Syst Bacteriol 46, 64-75.[CrossRef]
    [Google Scholar]
  15. Harlow, E. & Lane, D. (1988). Antibodies: a Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory.
  16. Jensen, D. D. ( 1957; ). Differential transmission of peach yellow leaf roll virus to peach and celery by the leafhopper, Collodonus montanus. Phytopathology 47, 575-578.
    [Google Scholar]
  17. Jiang, Y. P., Lei, J. D. & Chen, T. A. ( 1988; ). Purification of aster yellows agent from diseased lettuce using affinity chromatography. Phytopathology 78, 828-831.[CrossRef]
    [Google Scholar]
  18. Kirkpatrick, B. C. (1986). Characterization of Western X-disease mycoplasma-like organisms. PhD thesis, University of California at Berkeley.
  19. Kirkpatrick, B. C., Stegner, D. C., Morris, T. J. & Purcell, A. H. ( 1987; ). Cloning and detection of DNA from a nonculturable plant pathogenic mycoplasma-like organism. Science 238, 197-200.[CrossRef]
    [Google Scholar]
  20. Kirkpatrick, B. C., Fisher, G. A., Fraser, J. D. & Purcell, A. H. ( 1990; ). Epidemiological and phylogenetic studies on Western X-disease mycoplasma-like organisms. In Recent Advances in Mycoplasmology , pp. 288-296. Edited by G. Stanek, G. H. Cassell, J. G. Tully & R. F. Whitcomb. New York:Gustav Fisher Verlag.
  21. Krause, D. C. ( 1996; ). Mycoplasma pneumoniae cytadherence – unravelling the tie that binds. Mol Microbiol 20, 247-253.[CrossRef]
    [Google Scholar]
  22. Kwon, M. O., Wayadande, A. C. & Fletcher, J. ( 1999; ). Spiroplasma citri movement into the intestines and salivary glands of its leafhopper vector, Circulifer tenellus. Phytopathology 89, 1144-1151.[CrossRef]
    [Google Scholar]
  23. Laemmli, U. K. ( 1970; ). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[CrossRef]
    [Google Scholar]
  24. Lefol, C., Caudwell, A., Lherminier, J. & Larrue, J. ( 1993; ). Attachment of the flavescence doree pathogen (MLO) to leafhopper vectors and other insects. Ann Appl Biol 123, 611-622.[CrossRef]
    [Google Scholar]
  25. Lefol, C., Lherminier, J., Boudon-Padieu, E., Larrue, J., Louis, C. & Caudwell, A. ( 1994; ). Propagation of flavescence doree MLO (mycoplasma-like organism) in the leafhopper vector Euscelidius variegatus Kbm. J Invertebr Pathol 63, 285-293.[CrossRef]
    [Google Scholar]
  26. Lim, P. O. & Sears, B. B. ( 1991; ). DNA sequence of the ribosomal protein genes rp12 and rps19 from a plant-pathogenic mycoplasma-like organism. FEMS Microbiol Lett 84, 71-74.[CrossRef]
    [Google Scholar]
  27. Lim, P. O. & Sears, B. B. ( 1992; ). Evolutionary relationships of a plant-pathogenic mycoplasma-like organism and Acholeplasma laidlawii deduced from two ribosomal protein gene sequences. J Bacteriol 174, 2606-2611.
    [Google Scholar]
  28. McCoy, R. E., Caudwell, A., Chang, C. J. & 16 other authors (1989). Plant diseases associated with mycoplasma-like organisms. In The Mycoplasmas, pp. 545–640. Edited by R. F. Whitcomb & J. G. Tully. New York: Academic Press.
  29. Markham, P. J. & Townsend, R. ( 1979; ). Experimental vectors of spiroplasmas. In Leafhopper Vectors and Plant Disease Agents , pp. 413-445. Edited by K. Maramorosch & K. F. Harris. New York:Academic Press.
  30. Milne, R. G., Masenga, V., Lenzi, R., Ramasso, E. & Sarindu, N. ( 1991; ). Gold immunolabeling and electron microscopy of mycoplasma-like organisms in plant tissues using pre-embedding and post-embedding techniques. Phytoparasitica 19, 263.
    [Google Scholar]
  31. Nakai, K. & Kenehisa, M. ( 1991; ). Expert system for predicting protein localization sites in Gram-negative bacteria. Proteins 11, 95-110.[CrossRef]
    [Google Scholar]
  32. Nielson, M. W. ( 1979; ). Taxonomic relationships of leafhopper vectors of plant pathogens. In Leafhopper Vectors and Plant Disease Agents , pp. 3-27. Edited by K. Maramorosch & K. F. Harris. New York:Academic Press.
  33. Purcell, A. H., Richardson, J. & Finlay, A. ( 1981; ). Multiplication of the agent of X-disease in a non-vector leafhopper Macrosteles fascifrons. Ann Appl Biol 99, 283-289.[CrossRef]
    [Google Scholar]
  34. Purcell, A. H., Suslow, K. G. & Kirkpatrick, B. C. ( 1988; ). Vector transmission of X-disease mycoplasma-like organisms from California. In Stone Fruit Tree Decline, Fourth Workshop Proceedings , pp. 60. Edited by M. V. McKenry. Parlier, CA:US Department of Agriculture.
  35. Razin, S. & Jacobs, E. ( 1992; ). Mycoplasma adhesion. J Gen Microbiol 138, 407-422.[CrossRef]
    [Google Scholar]
  36. Rojas, M. R., Zerbini, F. M., Allison, R. F., Gilbertson, R. L. & Lucas, W. J. ( 1997; ). Capsid protein and helper component proteinase function as potyvirus cell-to-cell movement proteins. Virology 237, 283-295.[CrossRef]
    [Google Scholar]
  37. Rottem, S. & Kahane, I. (1993). Mycoplasma Cell Membranes. New York: Plenum.
  38. Saeed, E., Rage, P. & Cousin, M. T. ( 1992; ). Determination of the antigenic protein size associated with Faba bean phyllody MLO by using (SDS-PAGE) electrophoresis and immunotransfer. J Phytopathol 136, 1-8.[CrossRef]
    [Google Scholar]
  39. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory.
  40. Seddas, A., Meignoz, R., Daire, X., Boudon-Padieu, E. & Caudwell, A. ( 1993; ). Purification of grapevine flavescence doree MLO (mycoplasma-like organism) by immunoaffinity. Curr Microbiol 27, 229-236.[CrossRef]
    [Google Scholar]
  41. Seem ller, E., Schneider, B., M urer, R. & 8 other authors ( 1994; ). Phylogenetic classification of phytopathogenic mollicutes by sequence analysis of 16S ribosomal DNA. Int J Syst Bacteriol 44, 440–446.[CrossRef]
    [Google Scholar]
  42. Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. ( 1996; ). Mass spectrometic sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem 68, 850-858.[CrossRef]
    [Google Scholar]
  43. Smart, C. D., Schneider, B., Blomquist, C. L., Guerra, L. J., Harrison, N. A., Ahrens, U., Lorenz, K. H., Seemüller, E. & Kirkpatrick, B. C. ( 1996; ). Phytoplasma-specific PCR primers based on sequences of the 16S–23S rRNA spacer region. Appl Environ Microbiol 62, 2988-2993.
    [Google Scholar]
  44. Ye, F. C., Melcher, U. & Fletcher, J. ( 1997; ). Molecular characterization of a gene encoding a membrane protein of Spiroplasma citri. Gene 189, 95-100.[CrossRef]
    [Google Scholar]
  45. Yu, Y.-L., Yeh, K.-W. & Lin, C.-P. ( 1998; ). An antigenic protein gene of a phytoplasma associated with sweet potato witches’ broom. Microbiology 144, 1257-1262.[CrossRef]
    [Google Scholar]
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