Archives of Microbiology

, Volume 189, Issue 2, pp 131–139 | Cite as

Expression of putative virulence factors in the potato pathogen Clavibacter michiganensis subsp. sepedonicus during infection

  • Ingrid Holtsmark
  • Gunnhild W. Takle
  • May Bente BrurbergEmail author
Original Paper


The Gram-positive bacterium Clavibacter michiganensis subsp. sepedonicus is the causal agent of bacterial wilt and ring rot of potato. So far, only two proteins have been shown to be essential for virulence, namely a plasmid-encoded cellulase CelA and a hypersensitive response-inducing protein. We have examined the relative expression of CelA and eight putative virulence factors during infection of potato and in liquid culture, using quantitative real-time PCR. The examined putative virulence genes were celB, a cellulase-encoding gene and genes encoding a pectate lyase, a xylanase and five homologues of the Clavibacter michiganensis subsp. michiganensis pathogenicity factor Pat-1 thought to encode a serine protease. Six of the nine assayed genes were up-regulated during infection of potato, including celA, celB, the xylanase gene, and two of the pat genes. The pectate lyase gene showed only slightly elevated expression, whereas three of the five examined pat genes were down-regulated during infection in potato. Interestingly, the two up-regulated pat genes showed a noticeable sequence difference compared to the three down-regulated pat genes. These results reveal several new proteins that are likely to be involved in Clavibacter michiganensis subsp. sepedonicus pathogenicity.


Clavibacter Virulence factor Infection Gene expression 



Clavibacter michiganensis subsp. michiganensis


Clavibacter michiganensis subsp. sepedonicus



This work was supported by a grant from the Research Council of Norway. Thanks to Arild Sletten and Juliana S. Perminow for discussions and for kindly providing the bacterial strain.


  1. Abramovitch RB, Martin GB (2004) Strategies used by bacterial pathogens to suppress plant defenses. Curr Opin Plant Biol 7:356–364PubMedCrossRefGoogle Scholar
  2. Abramovitch RB, Anderson JC, Martin GB (2006) Bacterial elicitation and evasion of plant innate immunity. Nat Rev Mol Cell Biol 7:601–611PubMedCrossRefGoogle Scholar
  3. Ajin M, Kumar VD, Matt A, John B (2003) Microarray and differential display identify genes involved in jasmonate-dependent anther development. Plant Mol Biol 52:775–786CrossRefGoogle Scholar
  4. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucl Acids Res 25:3389–3402PubMedCrossRefGoogle Scholar
  5. Bacon DJ, Alm RA, Burr DH, Hu L, Kopecko DJ, Ewing CP, Trust TJ, Guerry P (2000) Involvement of a plasmid in virulence of Campylobacter jejuni 81–176. Infect Immun 68:4384–4390PubMedCrossRefGoogle Scholar
  6. Barras F, Vangijsegem F, Chatterjee AK (1994) Extracellular enzymes and pathogenesis of soft-rot Erwinia. Annu Rev Phytopathol 32:201–234Google Scholar
  7. Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340:783–795PubMedCrossRefGoogle Scholar
  8. Brito N, Espino JJ, Gonzalez C (2006) The endo-beta-1,4-xylanase Xyn11A is required for virulence in Botrytis cinerea. Mol Plant Microbe Interact 19:25–32PubMedCrossRefGoogle Scholar
  9. Burger A, Grafen I, Engemann J, Niermann E, Pieper M, Kirchner O, Gartemann KH, Eichenlaub R (2005) Identification of homologues to the pathogenicity factor Pat-1, a putative serine protease of Clavibacter michiganensis subsp. michiganensis. Microbiol Res 160:417–427PubMedCrossRefGoogle Scholar
  10. Bustin SA, Benes V, Nolan T, Pfaffl MW (2005) Quantitative real-time RT-PCR—a perspective. J Mol Endocrinol 34:597–601PubMedCrossRefGoogle Scholar
  11. Carter P, Wells JA (1988) Dissecting the catalytic triad of a serine protease. Nature 332:564–568PubMedCrossRefGoogle Scholar
  12. Citti C, Marechaldrouard L, Saillard C, Weil JH, Bove JM (1992) Spiroplasma-citri UGG and UGA tryptophan codons—sequence of the 2 tryptophanyl-transfer RNAs and organization of the corresponding genes. J Bacteriol 174:6471–6478PubMedGoogle Scholar
  13. Cosgrove DJ (2000) Loosening of plant cell walls by expansins. Nature 407:321–326PubMedCrossRefGoogle Scholar
  14. Czelleng A, Bozso Z, Ott PG, Besenyei E, Varga GJ, Szatmari A, Kiraly L, Klement Z (2006) Identification of virulence-associated genes of Pseudomonas viridiflava activated during infection by use of a novel IVET promoter probing plasmid. Curr Microbiol 52:282–286PubMedCrossRefGoogle Scholar
  15. D’Haeze W, Holsters M (2004) Surface polysaccharides enable bacteria to evade plant immunity. Trends Microbiol 12:555–561PubMedCrossRefGoogle Scholar
  16. Dangl JL, Jones JDG (2001) Plant pathogens and integrated defence responses to infection. Nature 411:826–833PubMedCrossRefGoogle Scholar
  17. De Jong AJ, Hoeberichts FA, Yakimova ET, Maximova E, Woltering EJ (2000) Chemical-induced apoptotic cell death in tomato cells: involvement of caspase-like proteases. Planta 211:656–662PubMedCrossRefGoogle Scholar
  18. Diatchenko L, Lau YFC, Campbell AP, Chenchik A, Moqadam F, Huang B, Lukyanov S, Lukyanov K, Gurskaya N, Sverdlov ED, Siebert PD (1996) Suppression subtractive hybridization: A method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci 93:6025–6030PubMedCrossRefGoogle Scholar
  19. Dreier J, Meletzus D, Eichenlaub R (1997) Characterization of the plasmid encoded virulence region pat-1 of phytopathogenic Clavibacter michiganensis subsp michiganensis. Mol Plant Microbe Interact 10:195–206PubMedCrossRefGoogle Scholar
  20. Egger L, Schneider J, Rheme C, Tapernoux M, Hacki J, Borner C (2003) Serine proteases mediate apoptosis-like cell death and phagocytosis under caspase-inhibiting conditions. Cell Death Differ 10:1188–1203PubMedCrossRefGoogle Scholar
  21. Engel LS, Hill JM, Caballero AR, Green LC, O’Callaghan RJ (1998) Protease IV, a unique extracellular protease and virulence factor from Pseudomonas aeruginosa. J Biol Chem 273:16792–16797PubMedCrossRefGoogle Scholar
  22. Espinosa A, Alfano JR (2004) Disabling surveillance: bacterial type III secretion system effectors that suppress innate immunity. Cell Microbiol 6:1027–1040PubMedCrossRefGoogle Scholar
  23. Ferreira AO, Myers CR, Gordon JS, Martin GB, Vencato M, Collmer A, Wehling MD, Alfano JR, Moreno-Hagelsieb G, Lamboy WF, DeClerck G, Schneider DJ, Cartinhour SW (2006) Whole-genome expression profiling defines the HrpL regulon of Pseudomonas syringae pv. tomato DC3000, allows de novo reconstruction of the Hrp cis clement, and identifies novel coregulated genes. Mol Plant Microbe Int 19:1167–1179CrossRefGoogle Scholar
  24. Gartemann KH, Kirchner O, Engemann J, Grafen I, Eichenlaub R, Burger A (2003) Clavibacter michiganensis subsp. michiganensis: first steps in the understanding of virulence of a Gram-positive phytopathogenic bacterium. J Biotechnol 106:179–191PubMedCrossRefGoogle Scholar
  25. Glazebrook J, Rogers EE, Ausubel FM (1997) Use of Arabidopsis for genetic dissection of plant defense responses. Annu Rev Genet 31:547–569PubMedCrossRefGoogle Scholar
  26. Heid CA, Stevens J, Livak KJ, Williams PM (1996) Real time quantitative PCR. Genome Res 6:986–994PubMedCrossRefGoogle Scholar
  27. Henrissat B (1991) A classification of glycosyl hydrolases based on amino-acid-sequence similarities. Biochem J 280:309–316PubMedGoogle Scholar
  28. Hernandez A, Figueroa A, Rivas LA, Parro V, Mellado RP (2000) RT-PCR as a tool for systematic transcriptional analysis of large regions of the Bacillus subtilis genome. Microbiology 146:823–828PubMedGoogle Scholar
  29. Holmes DE, Nevin KP, O’Neil RA, Ward JE, Adams LA, Woodard TL, Vrionis HA, Lovley DR (2005) Potential for quantifying expression of the Geobacteraceae citrate synthase gene to assess the activity of Geobacteraceae in the subsurface and on current-harvesting electrodes. Appl Environ Microbiol 71:6870–6877PubMedCrossRefGoogle Scholar
  30. Hughes KJD, Tomlinson JA, Griffin RL, Boonham N, Inman AJ, Lane CR (2006) Development of a one-step real-time polymerase chain reaction assay for diagnosis of Phytophthora ramorum. Phytopathology 96:975–981CrossRefGoogle Scholar
  31. Jahr H, Dreier J, Meletzus D, Bahro R, Eichenlaub R (2000) The endo-beta-1,4-glucanase CelA of Clavibacter michiganensis subsp michiganensis is a pathogenicity determinant required for induction of bacterial wilt of tomato. Mol Plant Microbe Interact 13:703–714PubMedCrossRefGoogle Scholar
  32. Jha AK, Bais HP, Vivanco JM (2005) Enterococcus faecalis mammalian virulence-related factors exhibit potent pathogenicity in the Arabidopsis thaliana plant model. Infect Immun 73:464–475PubMedCrossRefGoogle Scholar
  33. Juge N (2006) Plant protein inhibitors of cell wall degrading enzymes. Trends Plant Sci 11:359–367PubMedCrossRefGoogle Scholar
  34. Kita N, Boyd CM, Garrett MR, Jurnak F, Keen NT (1996) Differential effect of site-directed mutations in pelC on pectate lyase activity, plant tissue maceration, and elicitor activity. J Biol Chem 271:26529–26535PubMedCrossRefGoogle Scholar
  35. Laine MJ, Haapalainen M, Wahlroos T, Kankare K, Nissinen R, Kassuwi S, Metzler MC (2000) The cellulase encoded by the native plasmid of Clavibacter michiganensis ssp sepedonicus plays a role in virulence and contains an expansin-like domain. Physiol Mol Plant Pathol 57:221–233CrossRefGoogle Scholar
  36. Lam E, Kato N, Lawton M (2001) Programmed cell death, mitochondria and the plant hypersensitive response. Nature 411:848–853PubMedCrossRefGoogle Scholar
  37. Liu HL, Zhang SP, Schell MA, Denny TP (2005) Pyramiding, unmarked deletions in Ralstonia solanacearum shows that secreted proteins in addition to plant cell-wall-degrading enzymes contribute to virulence. Mol Plant Microbe Interact 18:1296–1305PubMedCrossRefGoogle Scholar
  38. Meletzus D, Bermphol A, Dreier J, Eichenlaub R (1993) Evidence for plasmid-encoded virulence factors in the phytopathogenic bacterium Clavibacter michiganensis subsp. michiganensis NCPPB382. J Bacteriol 175:2131–2136PubMedGoogle Scholar
  39. Mocellin S, Rossi CR, Pilatil P, Nitti D, Marincola FM (2003) Quantitative real-time PCR: a powerful ally in cancer research. Trends Mol Med 9:189–195PubMedCrossRefGoogle Scholar
  40. Monod M, Capoccia S, Lechenne B, Zaugg C, Holdom M, Jousson O (2002) Secreted proteases from pathogenic fungi. Int J Med Microbiol 292:405–419PubMedCrossRefGoogle Scholar
  41. Monteiro-Vitorello CB, Camargo LE, Van Sluys MA, Kitajima JP, Truffi D, do Amaral AM, Harakava R, de Oliveira JC, Wood D, de Oliveira MC, Miyaki C, Takita MA, da Silva AC, Furlan LR, Carraro DM, Camarotte G, Almeida NF Jr, Carrer H, Coutinho LL, El-Dorry HA, Ferro MI, Gagliardi PR, Giglioti E, Goldman MH, Goldman GH, Kimura ET, Ferro ES, Kuramae EE, Lemos EG, Lemos MV, Mauro SM, Machado MA, Marino CL, Menck CF, Nunes LR, Oliveira RC, Pereira GG, Siqueira W, de Souza AA, Tsai SM, Zanca AS, Simpson AJ, Brumbley SM, Setubal JC (2004) The genome sequence of the gram-positive sugarcane pathogen Leifsonia xyli subsp xyli. Mol Plant Microbe Interact 17:827–836PubMedCrossRefGoogle Scholar
  42. Mudgett MB (2005) New insights to the function of phytopathogenic bacterial type III effectors in plants. Annu Rev Plant Biol 56:509–531PubMedCrossRefGoogle Scholar
  43. Mysore KS, Ryu CM (2004) Nonhost resistance: how much do we know? Trends Plant Sci 9:97–104PubMedCrossRefGoogle Scholar
  44. Nissinen R, Lai FM, Laine MJ, Bauer PJ, Reilley AA, Li X, De Boer SH, Ishimaru CA, Metzler MC (1997) Clavibacter michiganensis subsp. sepedonicus elicits a hypersensitive response in tobacco and secretes hypersensitive response-inducing protein(s). Phytopathology 87:678–684CrossRefGoogle Scholar
  45. Nissinen R, Kassuwi S, Peltola R, Metzler MC (2001) In planta-complementation of Clavibacter michiganensis subsp sepedonicus strains deficient in cellulase production or HR induction restores virulence. Eur J Plant Pathol 107:175–182CrossRefGoogle Scholar
  46. Nomura K, Nasser W, Kawagishi H, Tsuyumu S (1998) The pir gene of Erwinia chrysanthemi EC16 regulates hyperinduction of pectate lyase virulence genes in response to plant signals. Proc Natl Acad Sci 95:14034–14039PubMedCrossRefGoogle Scholar
  47. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucl Acids Res 29:e45PubMedCrossRefGoogle Scholar
  48. Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucl Acids Res 30:e36PubMedCrossRefGoogle Scholar
  49. Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper—excel-based tool using pair-wise correlations. Biotechnol Lett 26:509–515PubMedCrossRefGoogle Scholar
  50. Ried JL, Collmer A (1986) Comparison of pectic enzymes produced by Erwinia chrysanthemi, Erwinia carotovora subsp. carotovora, and Erwinia carotovora subsp. atroseptica. Appl Environ Microbiol 52:305–310PubMedGoogle Scholar
  51. Rypniewski WR, Perrakis A, Vorgias CE, Wilson KS (1994) Evolutionary divergence and conservation of trypsin. Protein Eng 7:57–64PubMedCrossRefGoogle Scholar
  52. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  53. Sampedro J, Cosgrove DJ (2005) The expansin superfamily. Genome Biol 6:242.1–242.11CrossRefGoogle Scholar
  54. Shimkets RA (2004) Gene expression: profiling methods and protocols. Humana Press, TotowaGoogle Scholar
  55. Tarchevsky IA (2001) Pathogen-induced plant proteins. Appl Biochem Microbiol 37:441–455 ReviewCrossRefGoogle Scholar
  56. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL-W—improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl Acids Res 22:4673–4680PubMedCrossRefGoogle Scholar
  57. Toth IK, Bell KS, Holeva MC, Birch PRJ (2003) Soft rot erwiniae: from genes to genomes. Mol Plant Pathol 4:17–30CrossRefGoogle Scholar
  58. Watson B, Currier TC, Gordon MP, Chilton MD, Nester EW (1975) Plasmid required for virulence of Agrobacterium tumefaciens. J Bacteriol 123:255–264PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Ingrid Holtsmark
    • 1
    • 2
  • Gunnhild W. Takle
    • 1
    • 2
  • May Bente Brurberg
    • 1
    Email author
  1. 1.Plant Health and Plant Protection DivisionNorwegian Institute for Agricultural and Environmental ResearchÅsNorway
  2. 2.Department of Chemistry, Biotechnology and Food ScienceNorwegian University of Life SciencesÅsNorway

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