Plant Molecular Biology

, Volume 57, Issue 4, pp 541–557 | Cite as

Salicylic acid-inducible Arabidopsis CK2-like activity phosphorylates TGA2

  • Hong-Gu Kang
  • Daniel F. KlessigEmail author


We demonstrate that TGA2, TGA5 and TGA6, and TGA3 to a lesser extent, are phosphorylated by an activity in rabbit reticulocytes. Using deletion and point mutagenesis of TGA2, three amino acid (aa) residues, 11 Ser, 12 Thr and 16 Thr, were found to be critical for efficient phosphorylation by a kinase(s) in rabbit reticulocytes. These three residues also were important for phosphorylation by recombinant human Casein Kinase II (CK2) and by a CK2-like kinase in Arabidopsis leaf extracts. Salicylic acid (SA) treatment enhanced the phosphorylation of recombinant TGA2 in vitro; it also enhanced phosphorylation of a TGA2-GFP fusion protein in vivo. By contrast, in vivo phosphorylation of a TGA2-A-GFP fusion protein, in which the 11 Ser, 12 Thr and 16 Thr residues were mutated to non-phosphorylable alanine, was only poorly if at all stimulated by SA treatment. Mutation of the putative CK2 phosphorylation motif did not affect nuclear localization of TGA2. However, the DNA binding activity of TGA2 was reduced by CK2 treatment, whereas that of TGA2-A was unaffected; TGA2’s DNA binding activity after incubation in a rabbit reticulocyte lysate also was substantially lower than that of comparably treated TGA2-A. Taken together, these results suggest that phosphorylation at the putative CK2 phosphorylation site negatively regulates the DNA binding activity of TGA2. Analysis of transgenic Arabidopsis overexpressing TGA2-GFP or TGA2-A-GFP, in the absence of SA treatment, revealed that they accumulated similarly elevated levels of PR-1 gene transcripts. Possible reasons why mutations in the putative CK2 phosphorylation site had little effect on PR-1 induction by TGA2 are discussed.


Arabidopsis thaliana bZIP CK2 Phosphorylation Salicylic acid 



Activation Sequence Factor-1


Amino acid


Basic region/leucine zipper


Casein Kinase II


Electrophoretic mobility shift assay


Green Fluorescent Protein


Polyacrylamide gel electrophoresis


Transcription Factor


Salicylic Acid Response Protein


Wild type


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  1. Bechtold, N., Ellis, J., Pelletier, G. 1993In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plantsC R l’Acad Sci Ser III Sci Vie31611941199Google Scholar
  2. Brivanlou, AH., Darnell, JE.,Jr 2002Signal transduction and the control of gene expressionScience295813818Google Scholar
  3. Cao, H., Bowling, SA., Gordon, AS., Dong, X. 1994Characterization of an Arabidopsis mutant that is nonresponsive to inducers of systemic acquired resistancePlant Cell615831592Google Scholar
  4. Cao, H., Glazebrook, J., Clarke, JD., Volko, S., Dong, X. 1997The Arabidopsis NPR1 gene that controls systemic acquired resistance encodes a novel protein containing ankyrin repeatsCell885763Google Scholar
  5. Chen, W., Chao, G., Singh, KB. 1996The promoter of a H2O2-inducible, Arabidopsis glutathione S-transferase gene contains closely linked OBF- and OBP1-binding sitesPlant J10955966Google Scholar
  6. Chen, W., Singh, KB. 1999The auxin, hydrogen peroxide and salicylic acid induced expression of the Arabidopsis GST6 promoter is mediated in part by an ocs elementPlant J19667677Google Scholar
  7. Ciceri, P., Gianazza, E., Lazzari, B., Lippoli, G., Genga, A., Hoscheck, G., Schmidt, R.J., Viotti, A.. 1997Phosphorylation of Opaque2 changes diurnally and impacts its DNA binding activityPlant Cell997108Google Scholar
  8. Clarke, JD., Volko, SM., Ledford, H., Ausubel, FM., Dong, X. 2000Roles of salicylic acid, jasmonic acid, and ethylene in cpr-induced resistance in ArabidopsisPlant Cell1221752190CrossRefGoogle Scholar
  9. Daniel, X., Sugano, S., Tobin, EM. 2004CK2 phosphorylation of CCA1 is necessary for its circadian oscillator function in ArabidopsisProc Natl Acad Sci USA10132923297Google Scholar
  10. David, SJ., Viestra, RD. 1996Soluble derivatives of green fluorescent protein (GFP) for use in Arabidopsis thalianaWeeds World34348Google Scholar
  11. Delaney, T-P., Uknes, S., Vernooij, B., Friedrich, L., Weymann, K., Negrotto, D., Gaffney, T., Gut-Rella, M., Kessmann, H., Ward, E., Ryals, J. 1994A central role of salicylic acid in plant disease resistanceScience26612471250Google Scholar
  12. Després, C., DeLong, C., Glaze, S., Liu, E., Fobert, PR. 2000The Arabidopsis NPR1/NIM1 protein enhances the DNA binding activity of a subgroup of the TGA family of bZIP transcription factorsPlant Cell12279290Google Scholar
  13. Dong, X. 2001Genetic dissection of systemic acquired resistanceCurr Opin Plant Biol4309314Google Scholar
  14. Droog, F., Spek, A., Kooy, A., Ruyter, A., Hoge, H., Libbenga, K., Hooykaas, P., Zaal, B. 1995Promoter analysis of the auxin-regulated tobacco glutathione S-transferase genes Nt103–1 and Nt103–35 PlantMol Biol29413429Google Scholar
  15. Fan, W., Dong, X. 2002In vivo interaction between NPR1 and transcription factor TGA2 leads to salicylic acid-mediated gene activation in ArabidopsisPlant Cell1413771389Google Scholar
  16. Foster, R., Izawa, T., Chua, NH. 1994Plant bZIP proteins gather at ACGT elementsFASEB J8192200PubMedGoogle Scholar
  17. Gaffney, T., Friedrich, L., Vernooij, B., Negrotto, D., Nye, G., Uknes, S., Ward, E., Kessmann, H., Ryals, J. 1993Requirement of salicylic acid for the induction systemic acquired resistanceScience261754756Google Scholar
  18. Glazebrook, J., Rogers, EE., Ausubel, FM. 1996Isolation of Arabidopsis mutants with enhanced disease susceptibility by direct screeningGenetics143973982PubMedGoogle Scholar
  19. Hardtke, CS., Gohda, K., Osterlund, MT., Oyama, T., Okada, K., Deng, XW. 2000HY5 stability and activity in Arabidopsis is regulated by phosphorylation in its COP1 binding domainEMBO J1949975006CrossRefPubMedGoogle Scholar
  20. Hidalgo, P., Garreton, VV., Berrios, CG., Ojeda, H., Jordana, X., Holuigue, L. 2001A nuclear casein kinase 2 activity is involved in early events of transcriptional activation induced by salicylic acid in tobaccoPlant Physiol125396405Google Scholar
  21. Hunter, T., Karin, M. 1992The regulation of transcription by phosphorylationCell70375387Google Scholar
  22. Izawa, T., Foster, R., Chua, NH. 1993Plant bZIP protein DNA binding specificityJ Mol Biol23011311144CrossRefGoogle Scholar
  23. Jakoby, M., Weisshaar, B., Droge-Laser, W., Vicente-Carbajosa, J., Tiedemann, J., Kroj, T., Parcy, F. 2002bZIP transcription factors in ArabidopsisTrends Plant Sci7106111CrossRefPubMedGoogle Scholar
  24. Janssens, V., Goris, J. 2001Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signallingBiochem J353417439Google Scholar
  25. Johnson, C., Boden, E., Arias, J. 2003Salicylic acid and NPR1 induce the recruitment of trans-activating TGA factors to a defense gene promoter in ArabidopsisPlant Cell1518461858Google Scholar
  26. Jupin, I., Chua, NH. 1996Activation of the CaMV as-1 cis-element by salicylic acid: differential DNA-binding of a factor related to TGA1aEMBO J1556795689Google Scholar
  27. Kachroo, P., Leong, SA., Chattoo, BB. 1995Mg-SINE: a short interspersed nuclear element from the rice blast fungus, Magnaporthe griseaProc Natl Acad Sci USA921112511129Google Scholar
  28. Kang, HG., Foley, RC., Onate-Sanchez, L., Lin, C., Singh, KB. 2003Target genes for OBP3, a Dof transcription factor, include novel basic helix-loop-helix domain proteins inducible by salicylic acidPlant J35362372Google Scholar
  29. Katagiri, F., Lam, E., Chua, NH. 1989Two tobacco DNA-binding proteins with homology to the nuclear factor CREBNature340727730Google Scholar
  30. Kim, H.S., Delaney, TP. 2002Overexpression of TGA5, which encodes a bZIP transcription factor that interacts with NIM1/NPR1, confers SAR-independent resistance in Arabidopsis thaliana to Peronospora parasiticaPlant J32151163Google Scholar
  31. Kinkema, M., Fan, W., Dong, X. 2000Nuclear localization of NPR1 is required for activation of PR gene expressionPlant Cell1223392350CrossRefGoogle Scholar
  32. Klimczak, LJ., Schindler, U., Cashmore, AR. 1992DNA binding activity of the Arabidopsis G-box binding factor GBF1 is stimulated by phosphorylation by casein kinase II from broccoliPlant Cell48798Google Scholar
  33. Kumar, D., Klessig, DF. 2003High-affinity salicylic acid-binding protein 2 is required for plant innate immunity and has salicylic acid-stimulated lipase activityProc Natl Acad Sci USA1001610116106PubMedGoogle Scholar
  34. Lam, E., Lam, YK. 1995Binding site requirements and differential representation of TGF factors in nuclear ASF-1 activityNucleic Acids Res.2337783785Google Scholar
  35. Lebel, E., Heifetz, P., Thorne, L., Uknes, S., Ryals, J., Ward, E. 1998Functional analysis of regulatory sequences controlling PR-1 gene expression in ArabidopsisPlant J16223233Google Scholar
  36. Lin, C., Ahmad, M., Gordon, D., Cashmore, AR. 1995Expression of an Arabidopsis cryptochrome gene in transgenic tobacco results in hypersensitivity to blue, UV-A, and green lightProc Natl Acad. Sci USA9284238427Google Scholar
  37. MacKintosh, C. 1993Assay and purification of protein (serine/threonine) phosphatasesHardie, DG. eds. Protein Phosphorylation: A Practical Approach.Oxford University PressOxford197230Google Scholar
  38. Metraux, J-P., Nawrath, C., Genoud, T. 2002Systemic acquired resistanceEuphytica124237243Google Scholar
  39. Nawrath, C., Metraux, JP. 1999Salicylic acid induction-deficient mutants of Arabidopsis express PR-2 and PR-5 and accumulate high levels of camalexin after pathogen inoculationPlant Cell1113931404Google Scholar
  40. Niggeweg, R., Thurow, C., Kegler, C., Gatz, C. 2000Tobacco transcription factor TGA22 is the main component of as-1-binding factor ASF-1 and is involved in salicylic acid- and auxin-inducible expression of as-1-containing target promotersJ Biol Chem2751989719905Google Scholar
  41. Niggeweg, R., Thurow, C., Weigel, R., Pfitzner, U., Gatz, C. 2000Tobacco TGA factors differ with respect to interaction with NPR1, activation potential and DNA-binding propertiesPlant Mol Biol42775788Google Scholar
  42. Park, JM., Park, CJ., Lee, SB., Ham, BK., Shin, R., Paek, KH. 2001Overexpression of the tobacco Tsi1 gene encoding an EREBP/AP2-type transcription factor enhances resistance against pathogen attack and osmotic stress in tobaccoPlant Cell1310351046CrossRefPubMedGoogle Scholar
  43. Pontier, D., Miao, ZH., Lam, E. 2001Trans-dominant suppression of plant TGA factors reveals their negative and positive roles in plant defense responsesPlant J27529538Google Scholar
  44. Pontier, D., Privat, I., Trifa, Y., Zhou, JM., Klessig, DF., Lam, E. 2002Differential regulation of TGA transcription factors by post-transcriptional controlPlant J32641653Google Scholar
  45. Riera, M., Peracchia, G., Pages, M. 2001Distinctive features of plant protein kinase CK2Mol Cell Biochem227119127Google Scholar
  46. Ryals, J., Weymann, K., Lawton, K., Friedrich, L., Ellis, D., Steiner, HY., Johnson, J., Delaney, TP., Jesse, T., Vos, P., Uknes, S. 1997The Arabidopsis NIM1 protein shows homology to the mammalian transcription factor inhibitor I kappa BPlant Cell9425439Google Scholar
  47. Sambrook, J., Fritsch, EF., Maniatis, T. 1989Molecular Cloning a Laboratory Manual, 2nd ednCold Spring Harbor Laboratory PressPlainviewGoogle Scholar
  48. Schindler, U., Beckmann, H., Cashmore, AR. 1992TGA1 and G-box binding factors: two distinct classes of Arabidopsis leucine zipper proteins compete for the G-box-like element TGACGTGGPlant Cell413091319CrossRefPubMedGoogle Scholar
  49. Schwechheimer, C., Zourelidou, M., Bevan, MW. 1998Plant transcription factor studiesAnnu Rev Plant Physiol Plant Mol Biol49127150Google Scholar
  50. Shah, J., Kachroo, P., Klessig, DF. 1999The Arabidopsis ssi1 mutation restores pathogenesis-related gene expression in npr1 plants and renders defensin gene expression salicylic acid dependentPlant Cell11191206Google Scholar
  51. Shah, J., Tsui, F., Klessig, DF. 1997Characterization of a salicylic acid-insensitive mutant (sai1) of Arabidopsis thaliana, identified in a selective screen utilizing the SA-inducible expression of the tms2 geneMol Plant Microbe Interact.106978Google Scholar
  52. Singh, K., Tokuhisa, JG., Dennis, ES., Peacock, WJ. 1989Saturation mutagenesis of the octopine synthase enhancer: correlation of mutant phenotypes with binding of a nuclear protein factorProc Natl Acad Sci USA8637333737Google Scholar
  53. Stange, C., Ramirez, I., Gomez, I., Jordana, X., Holuigue, L. 1997Phosphorylation of nuclear proteins directs binding to salicylic acid-responsive elementsPlant J1113151324Google Scholar
  54. Strompen, G., Gruner, R., Pfitzner, UM. 1998An as-1-like motif controls the level of expression of the gene for the pathogenesis-related protein 1a from tobaccoPlant Mol Biol37871883Google Scholar
  55. Subramaniam, R., Desveaux, D., Spickler, C., Michnick, SW., Brisson, N. 2001Direct visualization of protein interactions in plant cellsNat Biotechnol19769772Google Scholar
  56. Sugano, , Sandronis, C., Ong, MS., Green, RM., Tobin, EM. 1999The protein kinase CK2 is involved in regulation of circadian rhythms in ArabidopsisProc Natl Acad Sci USA961236212366Google Scholar
  57. Tabata, T., Nakayama, T., Mikami, K., Iwabuchi, M. 1991HBP-1a and HBP-1b: leucine zipper-type transcription factors of wheatEMBO J1014591467Google Scholar
  58. The Arabidopsis Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408:796–815Google Scholar
  59. Tuazon, PT., Traugh, JA. 1991Casein kinase I and II – multipotential serine protein kinases: structure, function, and regulationAdv Second Messenger Phosphoprotein Res23123164Google Scholar
  60. Uknes, S., Mauch-Mani, B., Moyer, M., Potter, S., Williams, S., Dincher, S., Chandler, D., Slusarenko, A., Ward, E., Ryals, J. 1992Acquired resistance in ArabidopsisPlant Cell4645656CrossRefPubMedGoogle Scholar
  61. Ulmasov, T., Hagen, G., Guilfoyle, T. 1994The ocs element in the soybean GH2/4 promoter is activated by both active and inactive auxin and salicylic acid analoguesPlant Mol. Biol.2610551064Google Scholar
  62. Zaal, BJ., Droog, FN., Pieterse, FJ., Hooykaas, PJ. 1996Auxin-sensitive elements from promoters of tobacco GST genes and a consensus as-1-like element differ only in relative strengthPlant Physiol1107988Google Scholar
  63. Whitmarsh, A.J., Davis, RJ. 2000Regulation of transcription factor function by phosphorylationCell Mol Life Sci5711721183Google Scholar
  64. Xiang, C., Miao, ZH., Lam, E. 1996Coordinated activation of as-1-type elements and a tobacco glutathione S-transferase gene by auxins, salicylic acid, methyl-jasmonate and hydrogen peroxidePlant Mol Biol32415426PubMedGoogle Scholar
  65. Xiang, C., Miao, Z., Lam, E. 1997DNA-binding properties, genomic organization and expression pattern of TGA6, a new member of the TGA family of bZIP transcription factors in Arabidopsis thalianaPlant Mol Biol34403415Google Scholar
  66. Yoshioka, K., Kachroo, P., Tsui, F., Sharma, SB., Shah, J., Klessig, DF. 2001Environmentally sensitive, SA-dependent defense responses in the cpr22 mutant of ArabidopsisPlant J26447459Google Scholar
  67. Zhang, Y., Fan, W., Kinkema, M., Li, X., Dong, X. 1999Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR-1 geneProc Natl Acad Sci USA9665236528Google Scholar
  68. Zhang, Y., Tessaro, MJ., Lassner, M., Li, X. 2003Knockout analysis of Arabidopsis transcription factors TGA2, TGA5, and TGA6 reveals their redundant and essential roles in systemic acquired resistancePlant Cell1526472653Google Scholar
  69. Zhou, JM., Trifa, Y., Silva, H., Pontier, D., Lam, E., Shah, J., Klessig, DF. 2000NPR1 differentially interacts with members of the TGA/OBF family of transcription factors that bind an element of the PR-1 gene required for induction by salicylic acidMol Plant Microbe Interact13191202Google Scholar
  70. Zuo, J., Niu, QW., Chua, NH. 2000Technical advance: an estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plantsPlant J24265273CrossRefGoogle Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  1. 1.Boyce Thompson Institute for Plant ResearchIthaca NYUSA

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