1887

Abstract

Genetic relatedness of phytoplasmas is commonly defined on the basis of differences in the highly conserved 16S rRNA gene, which may not resolve closely related phytoplasmas. An example of this is the closely related tomato big bud (TBB) and sweet potato little leaf strain V4 (SPLL-V4) phytoplasmas, which cannot easily be differentiated by their 16S rRNA gene sequences. This study aimed to identify genes on the TBB phytoplasma chromosome which could be used to examine genetic variation between these two closely related phytoplasmas. Random clones generated from TBB phytoplasma genomic DNA were sequenced and characterized by database analysis. Twenty-three genes were identified within 19 random clones, which contained approximately 18·0 kbp of TBB phytoplasma genomic DNA. Half of the TBB phytoplasma genes identified were involved in DNA replication, transcription and translation. The remaining TBB phytoplasma genes were involved in protein secretion, cellular processes and energy metabolism. Phylogenetic analysis of representative genes showed that the TBB phytoplasma grouped with the mycoplasmas with the exception of the TBB phytoplasma gene, which grouped with the onion yellows phytoplasma. PCR primers were designed based on the new genes and tested on isolates of the TBB and SPLL-V4 phytoplasmas. Most primers amplified a product from TBB and SPLL-V4 phytoplasma samples. When amplified products were subjected to RFLP analysis, the restriction patterns were the same as the respective original clones. This result confirmed that the same sequence had been amplified by PCR and showed that these isolates were indistinguishable using the new genes. This study showed that in fact the TBB and SPLL-V4 phytoplasmas are closely related even with the analysis of new genes. These new genes have, however, provided insight into the biology of the TBB and SPLL-V4 phytoplasmas.

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2003-07-01
2024-11-03
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References

  1. Ahrens U., Seemüller E. 1992; Detection of DNA of plant pathogenic mycoplasma like organisms by a polymerase chain reaction that amplifies a sequence of the 16S rRNA gene. Phytopathology 82:828–832
    [Google Scholar]
  2. Altschul S. F., Madden T. L., Schäffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. 1997; Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402
    [Google Scholar]
  3. Barbara D. J., Morton A., Clark M. F., Davies D. L. 2002; Immunodominant membrane proteins from two phytoplasmas in the aster yellows clade (chlorante aster yellows and clover phyllody) are highly divergent in the major hydrophilic region. Microbiology 148:157–167
    [Google Scholar]
  4. Berg M., Seemüller E. 1999; Chromosomal organisation and nucleotide sequence of the genes encoding for the elongation factors G and Tu of the apple proliferation phytoplasma. Gene 226:103–109
    [Google Scholar]
  5. 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
    [Google Scholar]
  6. Blomquist C. L., Barbara D. J., Davies D. L., Clark M. F., Kirkpatrick B. C. 2001; An immunodominant membrane protein gene from the western X-disease phytoplasma is distinct from those of other phytoplasmas. Microbiology 147:571–580
    [Google Scholar]
  7. Dandekar T., Huynen M., Regula J. T. 9 other authors 2000; Re-annotating the Mycoplasma pneumoniae genome sequence: adding value, function and reading frames. Nucleic Acids Res 28:3278–3288
    [Google Scholar]
  8. Davis R. I., Schneider B., Gibb K. S. 1997; Detection and differentiation of phytoplasmas in Australia. Aust J Agric Res 48:535–544
    [Google Scholar]
  9. Doyle J. J., Doyle J. L. 1990; Isolation of plant DNA from fresh tissue. Focus 12:13–15
    [Google Scholar]
  10. Felsenstein J. 1989; phylip – Phylogeny Inference Package (version 3.2). Cladistics 5:164–166
    [Google Scholar]
  11. Fox G. E., Wisotzkey J. D., Jurtshuk P. 1992; How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity. Int J Syst Bacteriol 42:166–170
    [Google Scholar]
  12. Fraser C. M., Gocayne J. D., White O. 25 other authors 1995; The minimal gene complement of Mycoplasma genitalium . Science 270:397–403
    [Google Scholar]
  13. Galperin M. Y., Koonin E. V. 1999; Functional genomics and enzyme evolution. Genetics 106:159–170
    [Google Scholar]
  14. Gibb K. S., Padovan A. C., Mogen B. D. 1995; Studies on sweet potato little-leaf phytoplasma detected in sweet potato and other plant species growing in Northern Australia. Phytopathology 85:169–174
    [Google Scholar]
  15. Gundersen D. E., Lee I.-M., Rehner S. A., Davis R. E., Kingsbury D. T. 1994; Phylogeny of mycoplasma like organisms (phytoplasmas) – a basis for their classification. J Bacteriol 176:5244–5254
    [Google Scholar]
  16. 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
    [Google Scholar]
  17. Henikoff S., Henikoff J. G. 1994; Protein family classification based on searching a database of blocks. Genomics 19:97–107
    [Google Scholar]
  18. Himmelreich R., Hilbert H., Plagens H., Pirkl E., Li B. C., Herrmann R. 1996; Complete sequence analysis of the genome of the bacterium Mycoplasma pneumoniae . Nucleic Acids Res 24:4420–4449
    [Google Scholar]
  19. Hofmann K., Stoffel W. 1993; TMbase – A database of membrane spanning protein segments. J Biol Chem 374:166
    [Google Scholar]
  20. Jarausch W., Saillard C., Dosba F., Bové J.-M. 1994; Differentiation of mycoplasma like organisms (MLOs) in European fruit trees by PCR using specific primers derived from the sequence of a chromosomal fragment of the apple proliferation MLO. Appl Environ Microbiol 60:2916–2923
    [Google Scholar]
  21. Kakizawa S., Oshima K., Kuboyama T. 7 other authors 2001; Cloning and expression analysis of phytoplasma protein translocation genes. Mol Plant Microbe Interact 14:1043–1050
    [Google Scholar]
  22. Kihara A., Akiyama Y., Ito K. 1998; Different pathways for protein degradation by the FtsH/HflKC membrane-embedded protease complex: An implication from the interference by a mutant form of a new substrate protein. YccA. J Mol Biol 279:175–188
    [Google Scholar]
  23. Lehninger A. L., Nelson D. L., Cox M. M. 1993 Principles of Biochemistry , 2nd edn. New York: Worth Publishers;
    [Google Scholar]
  24. 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]
  25. Madiraju M. V. V. S., Qin M.-H., Yamamoto K., Atkinson M. A. L., Rajagopalan M. 1999; The dnaA gene region of Mycobacterium avium and the autonomous replication activities of its 5′ and 3′ flanking regions. Microbiology 145:2913–2921
    [Google Scholar]
  26. McCoy R. E., Caudwell A., Chang C. J. 16 other authors 1989; Plant diseases associated with mycoplasma-like organisms. In The Mycoplasmas , vol. V pp  545–640 Edited by Whitcomb R. F., Tully J. G. New York: Academic Press;
    [Google Scholar]
  27. Ogawa T., Okazaki T. 1994; Cell cycle dependent transcription from the gid and mioC promoters of Escherichia coli . J Bacteriol 176:1609–1615
    [Google Scholar]
  28. Padovan A. C., Firrao G., Schneider B., Gibb K. S. 2000; Chromosome mapping of the sweet potato little leaf phytoplasma reveals genome heterogeneity within the phytoplasmas. Microbiology 146:893–902
    [Google Scholar]
  29. Pearson W. R., Lipman D. J. 1988; Improved tools for biological sequence analysis. Proc Natl Acad Sci U S A 85:2444–2448
    [Google Scholar]
  30. Pollack J. D., Williams M. V., McElhaney R. N. 1997; The comparative metabolism of mollicutes (mycoplasma) – utility for taxonomic classification and the relationship of putative gene annotation and phylogeny to enzymatic function in the smallest free-living cells. Crit Rev Microbiol 23:269–354
    [Google Scholar]
  31. Razin S., Yogev D., Naot Y. 1998; Molecular biology and pathogenicity of mycoplasmas. Microbiol Mol Biol Rev 62:1094–1156
    [Google Scholar]
  32. Sano K.-I., Miyata M. 1994; The gyrB gene lies opposite from the replication origin on the circular chromosome of Mycoplasma capricolum . Gene 151:181–183
    [Google Scholar]
  33. Schneider B., Gibb K. S. 1997; Detection of phytoplasma in declining pears in southern Australia. Plant Dis 81:254–258
    [Google Scholar]
  34. Schneider B., Gibb K. S., Seemüller E. 1997; Sequence and RFLP analysis of the elongation factor Tu gene used in differentiation and classification of phytoplasmas. Microbiology 143:3381–3389
    [Google Scholar]
  35. Schneider B., Gibb K. S., Padovan A. C., Davis R. I., De La Rue S. 1999; Comparison and characterisation of tomato big bud and sweet potato little leaf-group phytoplasmas. J Phytopathol 147:31–40
    [Google Scholar]
  36. Seemüller E., Marcone C., Lauer U., Ragozzino A., Goschl M. 1998; Current status of molecular classification of the phytoplasmas. J Plant Pathol 80:3–26
    [Google Scholar]
  37. Simmons M. P. 2000; A fundamental problem with amino-acid-sequence characters for phylogenetic analyses. Cladistics 16:274–282
    [Google Scholar]
  38. Thompson J. D., Higgins D. G., Gibson T. J. 1994; clustal w: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680
    [Google Scholar]
  39. Wagner M., Fingerhut C., Gross H. J., Schon A. 2001; The first phytoplasma RNase P RNA provides new insights into the sequence requirements of this ribozyme. Nucleic Acids Res 29:2661–2665
    [Google Scholar]
  40. Yu Y.-L., Ye K.-W., Lin C.-P. 1998; An antigenic protein gene of a phytoplasma associated with sweet potato witches' broom. Microbiology 144:1257–1262
    [Google Scholar]
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