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Journal of General Plant Pathology

, Volume 84, Issue 3, pp 189–201 | Cite as

AlgU contributes to the virulence of Pseudomonas syringae pv. tomato DC3000 by regulating production of the phytotoxin coronatine

  • Takako Ishiga
  • Yasuhiro Ishiga
  • Shigeyuki Betsuyaku
  • Nobuhiko Nomura
Bacterial and Phytoplasma Diseases
  • 148 Downloads

Abstract

Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), which causes bacterial speck disease of tomato, has been used as a model pathogen to investigate the molecular basis of plant–pathogen interactions. The function of many potential virulence factors encoded in the Pst DC3000 genome and their modes of action are not fully understood. P. syringae is known to produce the exopolysaccharide alginate. Although AlgU, a sigma factor, is known to regulate the expression of genes such as algD related to alginate biosynthesis, the molecular mechanisms of AlgU in the virulence of Pst DC3000 is still unclear. To investigate the function of AlgU and alginate in plant–bacterial pathogen interactions, we generated ΔalgU and ΔalgD mutants. After inoculation with ΔalgU but not ΔalgD, host plants of Pst DC3000 including tomato and Arabidopsis had milder disease symptoms and reduced bacterial populations. Expression profiles of Pst DC3000 genes revealed that AlgU can regulate not only the expression of genes encoding alginate biosynthesis, but also the expression of genes related to type III effectors and the phytotoxin coronatine (COR). We also demonstrated that the ΔalgU mutant showed full virulence in the Arabidopsis fls2 efr1 double mutant, which is compromised in the recognition of PAMPs. Further, the application of COR was able to restore the phenotype of the ΔalgU mutant in the stomatal response. These results suggest that AlgU has an important role in the virulence of Pst DC3000 by regulating COR production.

Keywords

Pseudomonas syringae pv. tomato Tomato Arabidopsis thaliana AlgU Coronatine PAMP-triggered immunity Stomatal-based defense 

Notes

Acknowledgements

We thank Dr. Christina Baker for editing the manuscript. This work was supported, in part, by JST ERATO NOMURA Microbial Community Control Project, JST, Japan.

Supplementary material

10327_2018_775_MOESM1_ESM.pptx (83 kb)
Figure S1. Growth curves for Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) strains including the wild-type (WT), the ∆algD mutant (∆algD), and the ∆algU (∆algU) mutant. Pst DC3000 strains were grown at 28°C for 24 h in a. King’s B (KB) or b. mannitol–glutamate (MG) broth. Strains were adjusted to OD600 of 0.1 with the respective medium, and OD600 measured after 24 h. Figure S2. Expression profiles of defense genes in 2-week-old plants of Arabidopsis after inoculation with 5 × 106 colony forming units (CFU)/ml of wild-type (WT) Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) or the ΔalgU mutant (ΔalgU). Total RNA was isolated at 6, 12, 24, and 48 h post inoculation (hpi). Expression of a. AtPR1 and b. AtPR2 was determined using real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) with gene-specific primer sets. Expression was normalized using AtUBQ1 (Supplementary Table S1). Vertical bars indicate the standard error for three biological replicates. Asterisks indicate a significant difference from WT in a t test (*P < 0.05; **P < 0.01). (PPTX 83 KB)
10327_2018_775_MOESM2_ESM.xlsx (10 kb)
Supplementary material 2 (XLSX 9 KB)

References

  1. Ahuja I, Kissen R, Bones AM (2012) Phytoalexins in defense against pathogens. Trends Plant Sci 17:73–90CrossRefPubMedGoogle Scholar
  2. Bednarek P (2012) Chemical warfare or modulators of defence responses—the function of secondary metabolites in plant immunity. Curr Opin Plant Biol 15:407–414CrossRefPubMedGoogle Scholar
  3. Bent AF, Innes RW, Ecker JR, Staskawicz BJ (1992) Disease development in ethylene-insensitive Arabidopsis thaliana infected with virulent and avirulent Pseudomonas and Xanthomonas pathogens. Mol Plant Microbe Interact 5:372–378CrossRefPubMedGoogle Scholar
  4. Berens ML, Berry HM, Mine A, Argueso CT, Tsuda K (2017) Evolution of hormone signaling networks in plant defense. Annu Rev Phytopathol 55:401–425CrossRefPubMedGoogle Scholar
  5. Brooks DM, Bender CL, Kunkel BN (2005) The Pseudomonas syringae phytotoxin coronatine promotes virulence by overcoming salicylic acid-dependent defences in Arabidopsis thaliana. Mol Plant Pathol 6:629–639CrossRefPubMedGoogle Scholar
  6. Buell CR, Joardar V, Lindeberg M, Selengut J, Paulsen IT, Gwinn ML, Dodson RJ, Deboy RT, Durkin AS, Kolonay JF, Madupu R, Daugherty S, Brinkac L, Beanan MJ, Haft DH, Nelson WC, Davidsen T, Zafar N, Zhou L, Liu J, Yuan Q, Khouri H, Fedorova N, Tran B, Russell D, Berry K, Utterback T, Van Aken SE, Feldblyum TV, D’Ascenzo M, Deng WL, Ramos AR, Alfano JR, Cartinhour S, Chatterjee AK, Delaney TP, Lazarowitz SG, Martin GB, Schneider DJ, Tang X, Bender CL, White O, Fraser CM, Collmer A (2003) The complete genome sequence of the Arabidopsis and tomato pathogen Pseudomonas syringae pv. tomato DC3000. Proc Natl Acad Sci USA 100:10181–10186CrossRefPubMedPubMedCentralGoogle Scholar
  7. Chen LQ, Hou BH, Lalonde S, Takanaga H, Hartung ML, Qu XQ, Guo WJ, Kim JG, Underwood W, Chaudhuri B, Chermak D, Antony G, White FF, Somerville SC, Mudgett MB, Frommer WB (2010) Sugar transporters for intercellular exchange and nutrition of pathogens. Nature 468:527–532CrossRefPubMedPubMedCentralGoogle Scholar
  8. Chisholm ST, Coaker G, Day B, Staskawicz BJ (2006) Host–microbe interactions: shaping the evolution of the plant immune response. Cell 124:803–814CrossRefPubMedGoogle Scholar
  9. Chitrakar R, Melotto M (2010) Assessing stomatal response to live bacterial cells using whole leaf imaging. J Vis Exp 44:e2185Google Scholar
  10. Clarke CR, Hayes BW, Runde BJ, Markel E, Swingle BM, Vinatzer BA (2016) Comparative genomics of Pseudomonas syringae pathovar tomato reveals novel chemotaxis pathways associated with motility and plant pathogenicity. PeerJ 4:e2570CrossRefPubMedPubMedCentralGoogle Scholar
  11. Cowan MM (1999) Plant products as antimicrobial agents. Clin Microbiol Rev 12:564–582PubMedPubMedCentralGoogle Scholar
  12. Cunnac S, Lindeberg M, Collmer A (2009) Pseudomonas syringae type III secretion system effectors: repertoires in search of functions. Curr Opin Microbiol 12:53–60CrossRefPubMedGoogle Scholar
  13. Dolph PJ, Majerczak DR, Coplin DL (1988) Characterization of a gene cluster for exopolysaccharide biosynthesis and virulence in Erwinia stewartii. J Bacteriol 170:865–871CrossRefPubMedPubMedCentralGoogle Scholar
  14. Gimenez-Ibanez S, Rathjen JP (2010) The case for the defense: plants versus Pseudomonas syringae. Microbes Infect 12:428–437CrossRefPubMedGoogle Scholar
  15. Gimenez-Ibanez S, Boter M, Fernández-Barbero G, Chini A, Rathjen JP, Solano R (2014) The bacterial effector HopX1 targets JAZ transcriptional repressors to activate jasmonate signaling and promote infection in Arabidopsis. PLoS Biol 12:e1001792CrossRefPubMedPubMedCentralGoogle Scholar
  16. Hacquard S, Spaepen S, Garrido-Oter R, Schulze-Lefert P (2017) Interplay between innate immunity and the plant microbiota. Annu Rev Phytopathol 55:565–589CrossRefPubMedGoogle Scholar
  17. Hurley B, Lee D, Mott A, Wilton M, Liu J, Liu YC, Angers S, Coaker G, Guttman DS, Desveaux D (2014) The Pseudomonas syringae type III effector HopF2 suppresses Arabidopsis stomatal immunity. PLoS ONE 9:e114921CrossRefPubMedPubMedCentralGoogle Scholar
  18. Ishiga Y, Ichinose Y (2016) Pseudomonas syringae pv. tomato OxyR is required for virulence in tomato and Arabidopsis. Mol Plant Microbe Interact 29:119–131CrossRefPubMedGoogle Scholar
  19. Ishiga Y, Ishiga T, Uppalapati SR, Mysore KS (2011) Arabidopsis seedling flood-inoculation technique: a rapid and reliable assay for studying plant–bacterial interactions. Plant Methods 7:32CrossRefPubMedPubMedCentralGoogle Scholar
  20. Ishiga Y, Ishiga T, Wangdi T, Mysore KS, Uppalapati SR (2012) NTRC and chloroplast-generated reactive oxygen species regulate Pseudomonas syringae pv. tomato disease development in tomato and Arabidopsis. Mol Plant Microbe Interact 25:294–306CrossRefPubMedGoogle Scholar
  21. Jiang S, Yao J, Ma KW, Zhou H, Song J, He SY, Ma W (2013) Bacterial effector activates jasmonate signaling by directly targeting JAZ transcriptional repressors. PLoS Pathog 9:e1003715CrossRefPubMedPubMedCentralGoogle Scholar
  22. Jones JD, Dangl JL (2006) The plant immune system. Nature 444:323–329CrossRefPubMedGoogle Scholar
  23. Kadota Y, Sklenar J, Derbyshire P, Stransfeld L, Asai S, Ntoukakis V, Jones JDG, Shirasu K, Menke F, Jones A, Zipfel C (2014) Direct regulation of the NADPH oxidase RBOHD by the PRR-associated kinase BIK1 during plant immunity. Mol Cell 54:43–55CrossRefPubMedGoogle Scholar
  24. Kao CC, Barlow E, Sequeira L (1992) Extracellular polysaccharide is required for wild-type virulence of Pseudomonas solanacearum. J Bacteriol 174:1068–1071CrossRefPubMedPubMedCentralGoogle Scholar
  25. Katagiri F, Thilmony R, He SY (2002) The Arabidopsis thalianaPseudomonas syringae interaction. Arabidopsis Book 1:e0039CrossRefPubMedPubMedCentralGoogle Scholar
  26. Katzen F, Ferreiro DU, Oddo CG, Ielmini MV, Becker A, Pühler A, Ielpi L (1998) Xanthomonas campestris pv. campestris gum mutants: effects on xanthan biosynthesis and plant virulence. J Bacteriol 180:1607–1617PubMedPubMedCentralGoogle Scholar
  27. Keane PJ, Kerr A, New PB (1970) Crown gall of stone fruit. II. Identification and nomenclature of Agrobacterium isolates. Aust J Biol Sci 23:585–595CrossRefGoogle Scholar
  28. Keen NT, Tamaki S, Kobayashi D, Trollinger D (1988) Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria. Gene 70:191–197CrossRefPubMedGoogle Scholar
  29. Keith RC, Keith LMW, Hernández-Guzmán G, Uppalapati SR, Bender CL (2003) Alginate gene expression by Pseudomonas syringae pv. tomato DC3000 in host and non-host plants. Microbiology 149:1127–1138CrossRefPubMedGoogle Scholar
  30. King EO, Ward MK, Raney DE (1954) Two simple media for the demonstration of pyocyanin and fluorescin. J Lab Clin Med 44:301–307PubMedGoogle Scholar
  31. Laue H, Schenk A, Li H, Lambertsen L, Neu TR, Molin S, Ullrich MS (2006) Contribution of alginate and levan production to biofilm formation by Pseudomonas syringae. Microbiology 152:2909–2918CrossRefPubMedGoogle Scholar
  32. Lee S, Ishiga Y, Clermont K, Mysore KS (2013) Coronatine inhibits stomatal closure and delays hypersensitive response cell death induced by nonhost bacterial pathogens. PeerJ 1:e34CrossRefPubMedPubMedCentralGoogle Scholar
  33. Lindeberg M, Cunnac S, Collmer A (2012) Pseudomonas syringae type III effector repertoires: last words in endless arguments. Trends Microbiol 20:199–208CrossRefPubMedGoogle Scholar
  34. Lozano-Durán R, Bourdais G, He SY, Robatzek S (2014) The bacterial effector HopM1 suppresses PAMP-triggered oxidative burst and stomatal immunity. New Phytol 202:259–269CrossRefPubMedGoogle Scholar
  35. Macho AP, Zipfel C (2014) Plant PRRs and the activation of innate immune signaling. Mol Cell 54:263–272CrossRefPubMedGoogle Scholar
  36. Mansfield J, Genin S, Magori S, Citovsky V, Sriariyanum M, Ronald P, Dow M, Verdier V, Beer SV, Machado MA, Toth I, Salmond G, Foster GD (2012) Top 10 plant pathogenic bacteria in molecular plant pathology. Mol Plant Pathol 13:614–629CrossRefPubMedGoogle Scholar
  37. Markel E, Stodghill P, Bao Z, Myers CR, Swingle B (2016) AlgU controls expression of virulence genes in Pseudomonas syringae pv. tomato DC3000. J Bacteriol 198:2330–2344CrossRefPubMedPubMedCentralGoogle Scholar
  38. Melotto M, Underwood W, Koczan J, Nomura K, He SY (2006) Plant stomata function in innate immunity against bacterial invasion. Cell 126:969–980CrossRefPubMedGoogle Scholar
  39. Melotto M, Underwood W, He SY (2008) Role of stomata in plant innate immunity and foliar bacterial diseases. Annu Rev Phytopathol 46:101–122CrossRefPubMedPubMedCentralGoogle Scholar
  40. Melotto M, Zhang L, Oblessuc PR, He SY (2017) Stomatal defense a decade later. Plant Physiol 174:561–571CrossRefPubMedPubMedCentralGoogle Scholar
  41. Nogales J, Vargas P, Farias GA, Olmedilla A, Sanjuán J, Gallegos MT (2015) FleQ coordinates flagellum-dependent and -independent motilities in Pseudomonas syringae pv. tomato DC3000. Appl Environ Microbiol 81:7533–7545CrossRefPubMedPubMedCentralGoogle Scholar
  42. Nomura K, Melotto M, He SY (2005) Suppression of host defense in compatible plant–Pseudomonas syringae interactions. Curr Opin Plant Biol 8:361–368CrossRefPubMedGoogle Scholar
  43. O’Brien JA, Daudi A, Butt VS, Bolwell GP (2012) Reactive oxygen species and their role in plant defence and cell wall metabolism. Planta 236:765–779CrossRefPubMedGoogle Scholar
  44. Okkotsu Y, Little AS, Schurr MJ (2014) The Pseudomonas aeruginosa AlgZR two-component system coordinates multiple phenotypes. Front Cell Infect Microbiol 4:82CrossRefPubMedPubMedCentralGoogle Scholar
  45. Río-Álvarez I, Rodríguez-Herva JJ, Martínez PM, González-Melendi P, García-Casado G, Rodríguez-Palenzuela P, López-Solanilla E (2014) Light regulates motility, attachment and virulence in the plant pathogen Pseudomonas syringae pv. tomato DC3000. Environ Microbiol 16:2072–2085CrossRefPubMedGoogle Scholar
  46. Saile E, McGarvey JA, Schell MA, Denny TP (1997) Role of extracellular polysaccharide and endoglucanase in root invasion and colonization of tomato plants by Ralstonia solanacearum. Phytopathology 87:1264–1271CrossRefPubMedGoogle Scholar
  47. Sawinski K, Mersmann S, Robatzek S, Böhmer M (2013) Guarding the green: pathways to stomatal immunity. Mol Plant Microbe Interact 26:626–632CrossRefPubMedGoogle Scholar
  48. Schäfer A, Tauch A, Jäger W, Kalinowski J, Thierbach G, Pühler A (1994) Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145:69–73CrossRefPubMedGoogle Scholar
  49. Schenk A, Berger M, Keith LM, Bender CL, Muskhelishvili G, Ullrich MS (2006) The algT gene of Pseudomonas syringae pv. glycinea and new insights into the transcriptional organization of the algT-muc gene cluster. J Bacteriol 188:8013–8021CrossRefPubMedPubMedCentralGoogle Scholar
  50. Shimizu R, Taguchi F, Marutani M, Mukaihara T, Inagaki Y, Toyoda K, Shiraishi T, Ichinose Y (2003) The ΔfliD mutant of Pseudomonas syringae pv. tabaci, which secretes flagellin monomers, induces a strong hypersensitive reaction (HR) in non-host tomato cells. Mol Genet Genomics 269:21–30PubMedGoogle Scholar
  51. Sreedharan A, Penaloza-Vazquez A, Kunkel BN, Bender CL (2006) CorR regulates multiple components of virulence in Pseudomonas syringae pv. tomato DC3000. Mol Plant Microbe Interact 19:768–779CrossRefPubMedGoogle Scholar
  52. Tintor N, Ross A, Kanehara K, Yamada K, Fan L, Kemmerling B, Nürnberger T, Tsuda K, Saijo Y (2013) Layered pattern receptor signaling via ethylene and endogenous elicitor peptides during Arabidopsis immunity to bacterial infection. Proc Natl Acad Sci USA 110:6211–6216CrossRefPubMedPubMedCentralGoogle Scholar
  53. Underwood W, Melotto M, He SY (2007) Role of plant stomata in bacterial invasion. Cell Microbiol 9:1621–1629CrossRefPubMedGoogle Scholar
  54. Uppalapati SR, Ishiga Y, Wangdi T, Kunkel BN, Anand A, Mysore KS, Bender CL (2007) The phytotoxin coronatine contributes to pathogen fitness and is required for suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas syringae pv. tomato DC3000. Mol Plant Microbe Interact 20:955–965CrossRefPubMedGoogle Scholar
  55. Wang K, Senthil-Kumar M, Ryu CM, Kang L, Mysore KS (2012) Phytosterols play a key role in plant innate immunity against bacterial pathogens by regulating nutrient efflux into the apoplast. Plant Physiol 158:1789–1802CrossRefPubMedPubMedCentralGoogle Scholar
  56. Wang S, Sun J, Fan F, Tan Z, Zou Y, Lu D (2016) A Xanthomonas oryzae pv. oryzae effector, XopR, associates with receptor-like cytoplasmic kinases and suppresses PAMP-triggered stomatal closure. Sci China Life Sci 59:897–905CrossRefPubMedGoogle Scholar
  57. Wasternack C (2017) The Trojan horse coronatine: the COI1-JAZ2-MYC2,3,4-ANAC019,055,072 module in stomata dynamics upon bacterial infection. New Phytol 213:972–975CrossRefPubMedGoogle Scholar
  58. Xin XF, He SY (2013) Pseudomonas syringae pv. tomato DC3000: a model pathogen for probing disease susceptibility and hormone signaling in plants. Annu Rev Phytopathol 51:473–498CrossRefPubMedGoogle Scholar
  59. Yu J, Peñaloza-Vázquez A, Chakrabarty AM, Bender CL (1999) Involvement of the exopolysaccharide alginate in the virulence and epiphytic fitness of Pseudomonas syringae pv. syringae. Mol Microbiol 33:712–720CrossRefPubMedGoogle Scholar
  60. Yu X, Lund SP, Scott RA, Greenwald JW, Records AH, Nettleton D, Lindow SE, Gross DC, Beattie GA (2013) Transcriptional responses of Pseudomonas syringae to growth in epiphytic versus apoplastic leaf sites. Proc Natl Acad Sci USA 110:E425-434Google Scholar
  61. Yu X, Lund SP, Greenwald JW, Records AH, Scott RA, Nettleton D, Lindow SE, Gross DC, Beattie GA (2014) Transcriptional analysis of the global regulatory networks active in Pseudomonas syringae during leaf colonization. MBio 5:e01683–e01614PubMedPubMedCentralGoogle Scholar
  62. Zeng W, He SY (2010) A prominent role of the flagellin receptor FLAGELLIN-SENSING2 in mediating stomatal response to Pseudomonas syringae pv. tomato DC3000 in Arabidopsis. Plant Physiol 153:1188–1198CrossRefPubMedPubMedCentralGoogle Scholar
  63. Zheng XY, Spivey NW, Zeng W, Liu PP, Fu ZQ, Klessig DF, He SY, Dong X (2012) Coronatine promotes Pseudomonas syringae virulence in plants by activating a signaling cascade that inhibits salicylic acid accumulation. Cell Host Microbe 11:587–596CrossRefPubMedPubMedCentralGoogle Scholar
  64. Zhou Z, Wu Y, Yang Y, Du M, Zhang X, Guo Y, Li C, Zhou JM (2015) An Arabidopsis plasma membrane proton ATPase modulates JA signaling and is exploited by the Pseudomonas syringae effector protein AvrB for stomatal invasion. Plant Cell 27:2032–2041CrossRefPubMedPubMedCentralGoogle Scholar
  65. Zipfel C (2008) Pattern-recognition receptors in plant innate immunity. Curr Opin Immunol 20:10–16CrossRefPubMedGoogle Scholar
  66. Zipfel C, Felix G (2005) Plants and animals: a different taste for microbes? Curr Opin Plant Biol 8:353–360CrossRefPubMedGoogle Scholar
  67. Zipfel C, Robatzek S, Navarro L, Oakeley EJ, Jones JDG, Felix G, Boller T (2004) Bacterial disease resistance in Arabidopsis through flagellin perception. Nature 428:764–767CrossRefPubMedGoogle Scholar
  68. Zipfel C, Kunze G, Chinchilla D, Caniard A, Jones JDG, Boller T, Felix G (2006) Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation. Cell 125:749–760CrossRefPubMedGoogle Scholar

Copyright information

© The Phytopathological Society of Japan and Springer Japan KK, part of Springer Nature 2018

Authors and Affiliations

  • Takako Ishiga
    • 1
  • Yasuhiro Ishiga
    • 1
  • Shigeyuki Betsuyaku
    • 1
  • Nobuhiko Nomura
    • 1
  1. 1.Faculty of Life and Environmental SciencesUniversity of TsukubaTsukubaJapan

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