1887

Abstract

The -type III secretion (TTS) system is a key pathogenicity factor of the plant pathogen pv. DC3000 that translocates effector proteins into the cytosol of the eukaryotic host cell. The translocation of a subset of effectors is dependent on specific chaperones. In this study an operon encoding a TTS chaperone (ShcS1) and the truncated effector HopS1′ was characterized. Yeast two-hybrid analysis and pull-down assays demonstrated that these proteins interact. Using protein fusions to AvrRpt2 it was shown that ShcS1 facilitates the translocation of HopS1′, suggesting that ShcS1 is a TTS chaperone for HopS1′ and that amino acids 1 to 118 of HopS1′ are required for translocation. pv. DC3000 carries two homologues, and , which are located in different operons, and both operons include additional putative effector genes. Transcomplementation experiments showed that ShcS1 and ShcO1, but not ShcS2, can facilitate the translocation of HopS1′ : : AvrRpt2. To characterize the specificities of the putative chaperones, yeast two-hybrid interaction studies were performed between the three chaperones and putative target effectors. These experiments showed that both ShcS1 and ShcO1 bind to two different effectors, HopS1′ and HopO1-1, that share only 16 % amino acid sequence identity. Using gel filtration it was shown that ShcS1 forms homodimers, and this was confirmed by yeast two-hybrid experiments. In addition, ShcS1 is also able to form heterodimers with ShcO1. These data demonstrate that ShcS1 and ShcO1 are exceptional class IA TTS chaperones because they can bind more than one target effector.

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2005-01-01
2024-03-28
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References

  1. Abramovitch R. B., Martin G. B. 2004; Strategies used by bacterial pathogens to suppress plant defenses. Curr Opin Plant Biol 7:356–364 [CrossRef]
    [Google Scholar]
  2. Ausubel F. M., Brent R., Kingston R. E., Moore D. D., Seidman J. G., Smith J. A., Struhl K. 1987 Current Protocols in Molecular Biology USA: Wiley;
    [Google Scholar]
  3. Badel J. L., Nomura K., Bandyopadhyay S., Shimizu R., Collmer A., He S. Y. 2003; Pseudomonas syringae pv. tomato DC3000 HopPtoM (CEL ORF3) is important for lesion formation but not growth in tomato and is secreted and translocated by the Hrp type III secretion system in a chaperone-dependent manner. Mol Microbiol 49:1239–1251 [CrossRef]
    [Google Scholar]
  4. Birtalan S. C., Phillips R. M., Ghosh P. 2002; Three-dimensional secretion signals in chaperone-effector complexes of bacterial pathogens. Mol Cell 9:971–980 [CrossRef]
    [Google Scholar]
  5. Boch J., Joardar V., Gao L., Robertson T. L., Lim M., Kunkel B. N. 2002; Identification of Pseudomonas syringae pv. tomato genes induced during infection of Arabidopsis thaliana . Mol Microbiol 44:73–88 [CrossRef]
    [Google Scholar]
  6. Buell C. R., Joardar V., Lindeberg M. 41 other authors 2003; The complete genome sequence of the Arabidopsis and tomato pathogen Pseudomonas syringae pv. tomato DC3000. Proc Natl Acad Sci U S A 100:10181–10186 [CrossRef]
    [Google Scholar]
  7. Büttner D., Bonas U. 2002; Port of entry – the type III secretion translocon. Trends Microbiol 10:186–192 [CrossRef]
    [Google Scholar]
  8. Chang J. H., Goel A. K., Grant S. R., Dangl J. L. 2004; Wake of the flood: ascribing functions to the wave of type III effector proteins of phytopathogenic bacteria. Curr Opin Microbiol 7:11–18 [CrossRef]
    [Google Scholar]
  9. Collmer A., Lindeberg M., Petnicki-Ocwieja T., Schneider D. J., Alfano J. R. 2002; Genomic mining type III secretion system effectors in Pseudomonas syringae yields new picks for all TTSS prospectors. Trends Microbiol 10:462–469 [CrossRef]
    [Google Scholar]
  10. Cornelis G. R., Van Gijsegem F. 2000; Assembly and function of type III secretory systems. Annu Rev Microbiol 54:735–774 [CrossRef]
    [Google Scholar]
  11. Creasey E. A., Delahay R. M., Bishop A. A., Shaw R. K., Kenny B., Knutton S., Frankel G. 2003; CesT is a bivalent enteropathogenic Escherichia coli chaperone required for translocation of both Tir and Map. Mol Microbiol 47:209–221
    [Google Scholar]
  12. Cuppels D. A., Ainsworth T. 1995; Molecular and physiological characterization of Pseudomonas syringae pv. tomato and Pseudomonas syringae pv. maculicola strains that produce the phytotoxin coronatine. Appl Environ Microbiol 61:3530–3536
    [Google Scholar]
  13. Dale C., Young S. A., Haydon D. T., Welburn S. C. 2001; The insect endosymbiont Sodalis glossinidius utilizes a type III secretion system for cell invasion. Proc Natl Acad Sci U S A 98:1883–1888 [CrossRef]
    [Google Scholar]
  14. Daniels M. J., Barber C. E., Turner P. C., Sawczyc M. K., Byrde R. J. W., Fielding A. H. 1984; Cloning of genes involved in pathogenicity of Xanthomonas campestris pv. campestris using the broad host range cosmid pLAFR1. EMBO J 3:3323–3328
    [Google Scholar]
  15. Day J. B., Plano G. V. 1998; A complex composed of SycN and YscB functions as a specific chaperone for YopN in Yersinia pestis . Mol Microbiol 30:777–788 [CrossRef]
    [Google Scholar]
  16. Escolar L., Pieplow S., Rossier O., Bonas U, Van den Ackerveken G. 2001; Type III secretion and in planta recognition of the Xanthomonas avirulence proteins AvrBs1 and AvrBsT. Mol Plant Pathol 2:287–296 [CrossRef]
    [Google Scholar]
  17. Feldman M. F., Cornelis G. R. 2003; The multitalented type III chaperones: all you can do with 15 kDa. FEMS Microbiol Lett 219:151–158 [CrossRef]
    [Google Scholar]
  18. Fellay R., Frey J., Krisch H. 1987; Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of Gram-negative bacteria. Gene 52:147–154 [CrossRef]
    [Google Scholar]
  19. Figurski D., Helinski D. R. 1979; Replication of an origin-containing derivative of plasmid RK2 is dependent on a plasmid function provided in trans . Proc Natl Acad Sci U S A 76:1648–1652 [CrossRef]
    [Google Scholar]
  20. Finley R. L. Jr, Brent R. 1994; Interaction mating reveals binary and ternary connections between Drosophila cell cycle regulators. Proc Natl Acad Sci U S A 91:12980–12984 [CrossRef]
    [Google Scholar]
  21. Galán J. E., Collmer A. 1999; Type III secretion machines: bacterial devices for protein delivery into host cells. Science 284:1322–1328 [CrossRef]
    [Google Scholar]
  22. Gaudriault S., Paulin J.-P., Barny M.-A. 2002; The DspB/F protein of Erwinia amylovora is a type III secretion chaperone ensuring efficient intrabacterial production of the Hrp-secreted DspA/E pathogenicity factor. Mol Plant Pathol 3:313–320 [CrossRef]
    [Google Scholar]
  23. Greenberg J. T., Vinatzer B. A. 2003; Identifying type III effectors of plant pathogens and analyzing their interaction with plant cells. Curr Opin Microbiol 6:20–28 [CrossRef]
    [Google Scholar]
  24. Guttman D. S., Greenberg J. T. 2001; Functional analysis of the type III effectors AvrRpt2 and AvrRpm1 of Pseudomonas syringae with the use of a single-copy genomic integration system. Mol Plant–Microbe Interact 14:145–155 [CrossRef]
    [Google Scholar]
  25. Guttman D. S., Vinatzer B. A., Sarkar S. F., Ranall M. V., Kettler G., Greenberg J. T. 2002; A functional screen for the type III (Hrp) secretome of the plant pathogen Pseudomonas syringae . Science 295:1722–1726 [CrossRef]
    [Google Scholar]
  26. Hotson A., Mudgett M. B. 2004; Cysteine proteases in phytopathogenic bacteria: identification of plant targets and activation of innate immunity. Curr Opin Plant Biol 7:384–390 [CrossRef]
    [Google Scholar]
  27. Hueck C. J. 1998; Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol Mol Biol Rev 62:379–433
    [Google Scholar]
  28. Jackson R. W., Athanassopoulos E., Tsiamis G. 7 other authors 1999; Identification of a pathogenicity island, which contains genes for virulence and avirulence, on a large native plasmid in the bean pathogen Pseudomonas syringae pathovar phaseolicola. Proc Natl Acad Sci 96:10875–10880 [CrossRef]
    [Google Scholar]
  29. Jin Q., Thilmony R., Zwiesler-Vollick J., He S. Y. 2003; Type III protein secretion in Pseudomonas syringae . Microbes Infect 5:301–310 [CrossRef]
    [Google Scholar]
  30. Keen N. T. 1990; Gene-for-gene complementarity in plant–pathogen interactions. Annu Rev Genetic 24:447–463 [CrossRef]
    [Google Scholar]
  31. Klement Z. 1982; Hypersensitivity. In Phytopathogenic Procaryotes pp 149–177 Edited by Mount M. S., Lacy G. H. New York: Academic Press;
    [Google Scholar]
  32. Kolonin M. G., Finley R. L. Jr 1998; Targeting cyclin-dependent kinases in Drosophila with peptide aptamers. Proc Natl Acad Sci U S A 95:14266–14271 [CrossRef]
    [Google Scholar]
  33. Kunkel B. N., Bent A. F., Dahlbeck D., Innes R. W., Staskawicz B. J. 1993; RPS2 , an Arabidopsis disease resistance locus specifying recognition of Pseudomonas syringae strains expressing the avirulence gene avrRpt2 . Plant Cell 5:865–875 [CrossRef]
    [Google Scholar]
  34. Leach J. E., White F. F. 1996; Bacterial avirulence genes. Annu Rev Phytopathol 34:153–179 [CrossRef]
    [Google Scholar]
  35. Michiels T., Cornelis G. R. 1991; Secretion of hybrid proteins by the Yersinia Yop export system. J Bacteriol 173:1677–1685
    [Google Scholar]
  36. Mudgett M. B., Staskawicz B. J. 1999; Characterization of the Pseudomonas syringae pv. tomato AvrRpt2 protein: demonstration of secretion and processing during bacterial pathogenesis. Mol Microbiol 32:927–941 [CrossRef]
    [Google Scholar]
  37. Mudgett M. B., Chesnokova O., Dahlbeck D., Clark E. T., Rossier O., Bonas U., Staskawicz B. J. 2000; Molecular signals required for type III secretion and translocation of the Xanthomonas campestris AvrBs2 protein to pepper plants. Proc Natl Acad Sci 97:13324–13329 [CrossRef]
    [Google Scholar]
  38. Neumayer W., Groll M., Lehmann V., Antoneka U., Kahler S., Heesemann J., Wilharm G. 2004; Yersinia enterocolitica type III secretion chaperone SycH. Recombinant expression, purification, characterisation, and crystallisation. Protein Expr Purif 35:237–247 [CrossRef]
    [Google Scholar]
  39. Page A. L., Parsot C. 2002; Chaperones of the type III secretion pathway: jacks of all trades. Mol Microbiol 46:1–11 [CrossRef]
    [Google Scholar]
  40. Parsot C., Hamiaux C., Page A. L. 2003; The various and varying roles of specific chaperones in type III secretion systems. Curr Opin Microbiol 6:7–14 [CrossRef]
    [Google Scholar]
  41. Penfold R. J., Pemberton J. M. 1992; An improved suicide vector for construction of chromosomal insertion mutations in bacteria. Gene 118:145–146 [CrossRef]
    [Google Scholar]
  42. Petnicki-Ocwieja T., Schneider D. J., Tam V. C. 9 other authors 2002; Genomewide identification of proteins secreted by the Hrp type III protein secretion system of Pseudomonas syringae pv. tomato DC3000. Proc Natl Acad Sci U S A 99:7652–7657 [CrossRef]
    [Google Scholar]
  43. Rimpiläinen M., Forsberg Å., Wolf-Watz H. 1992; A novel protein, LcrQ, involved in the low-calcium response of Yersinia pseudotuberculosis shows extensive homology to YopH. J Bacteriol 174:3355–3363
    [Google Scholar]
  44. Rost B. 1996; PHD: predicting one-dimensional protein structure by profile-based neural networks. Methods Enzymol 266:525–539
    [Google Scholar]
  45. Sambrook J., Fritsch E., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  46. Schechter L. M., Roberts K. A., Jamir Y., Alfano J. R., Collmer A. 2004; Pseudomonas syringae type III secretion system targeting signals and novel effectors studied with a Cya translocation reporter. J Bacteriol 186:543–555 [CrossRef]
    [Google Scholar]
  47. Shan L., Oh H.-S., Chen J., Guo M., Zhou J., Alfano J. R., Collmer A., Jia X., Tang X. 2004; The HopPtoF locus of Pseudomonas syringae pv. tomato DC3000 encodes a type III chaperone and a cognate effector. Mol Plant–Microbe Interact 17:447–455 [CrossRef]
    [Google Scholar]
  48. Singer A. U., Desveaux D., Betts L., Chang J. H., Nimchuk Z., Grant S. R., Dangl J. L., Sondek J. 2004; Crystal structures of the type III effector protein AvrPphF and its chaperone reveal residues required for plant pathogenesis. Structure (Camb) 12:1669–1681 [CrossRef]
    [Google Scholar]
  49. Stainier I., Iriarte M., Cornelis G. R. 1997; YscM1 and YscM2, two Yersinia enterocolitica proteins causing downregulation of yop transcription. Mol Microbiol 26:833–843 [CrossRef]
    [Google Scholar]
  50. Staskawicz B. J., Dahlbeck D., Keen N. T. 1984; Cloned avirulence gene of Pseudomonas syringae pv. glycinea determines race-specific incompatibility on Glycine max (L.) Merr. Proc Natl Acad Sci U S A 81:6024–6028 [CrossRef]
    [Google Scholar]
  51. Stebbins C. E., Galan J. E. 2001; Maintenance of an unfolded polypeptide by a cognate chaperone in bacterial type III secretion. Nature 414:77–81 [CrossRef]
    [Google Scholar]
  52. van Dijk K., Tam V. C., Records A. R., Petnicki-Ocwieja T., Alfano J. R. 2002; The ShcA protein is a molecular chaperone that assists in the secretion of the HopPsyA effector from the type III (Hrp) protein secretion system of Pseudomonas syringae . Mol Microbiol 44:1469–1481 [CrossRef]
    [Google Scholar]
  53. van Eerde A., Hamiaux C., Perez J., Parsot C., Dijkstra B. W. 2004; Structure of Spa15, a type III secretion chaperone from Shigella flexneri with broad specificity. EMBO Rep 5:477–483 [CrossRef]
    [Google Scholar]
  54. Wattiau P., Cornelis G. R. 1993; SycE, a chaperone-like protein of Yersinia enterocolitica involved in the secretion of YopE. Mol Microbiol 8:123–131 [CrossRef]
    [Google Scholar]
  55. Wehling M. D., Guo M., Fu Z. Q., Alfano J. R. 2004; The Pseudomonas syringae HopPtoV protein is secreted in culture and translocated into plant cells via the type III protein secretion system in a manner dependent on the ShcV type III chaperone. J Bacteriol 186:3621–3630 [CrossRef]
    [Google Scholar]
  56. Whalen M. C., Innes R. W., Bent A. F., Staskawicz B. J. 1991; Identification of Pseudomonas syringae pathogens of Arabidopsis and a bacterial gene determining avirulence on both Arabidopsis and soybean. Plant Cell 3:49–59 [CrossRef]
    [Google Scholar]
  57. Xiao Y., Hutcheson S. W. 1994; A single promoter sequence recognized by a newly identified alternative sigma factor directs expression of pathogenicity and host range determinants in Pseudomonas syringae . J Bacteriol 176:3089–3091
    [Google Scholar]
  58. Xiao Y., Heu S., Yi J., Lu Y., Hutcheson S. W. 1994; Identification of a putative alternate sigma factor and characterization of a multicomponent regulatory cascade controlling the expression of Pseudomonas syringae pv. syringae Pss61 hrp and hrmA genes. J Bacteriol 176:1025–1036
    [Google Scholar]
  59. Zwiesler-Vollick J., Plovanich-Jones A. E., Nomura K., Bandyopadhyay S., Joardar V., Kunkel B. N., He S. Y. 2002; Identification of novel hrp -regulated genes through functional genomic analysis of the Pseudomonas syringae pv. tomato DC3000 genome. Mol Microbiol 45:1207–1218 [CrossRef]
    [Google Scholar]
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