, Volume 232, Issue 6, pp 1325–1337

Expression and functional analysis of two genes encoding transcription factors, VpWRKY1 and VpWRKY2, isolated from Chinese wild Vitis pseudoreticulata

  • Huie Li
  • Yan Xu
  • Yu Xiao
  • Ziguo Zhu
  • Xiaoqing Xie
  • Heqing Zhao
  • Yuejin Wang
Original Article


In this study, two WRKY genes were isolated from Erysiphe necator-resistant Chinese wild Vitis pseudoreticulata W. T. Wang ‘Baihe-35-1’, and designated as VpWRKY1 (GenBank accession no. GQ884198) and VpWRKY2 (GenBank accession no. GU565706). Nuclear localization of the two proteins was demonstrated in onion epidermal cells, while trans-activation function was confirmed in the leaves of ‘Baihe-35-1’. Expression of VpWRKY1 and VpWRKY2 was induced rapidly by salicylic acid treatment in ‘Baihe-35-1’. Expression of VpWRKY1 and VpWRKY2 was also induced rapidly by E. necator infection in 11 grapevine genotypes; the maximum induction of VpWRKY1 was greater in E. necator-resistant grapevine genotypes than in susceptible ones post E. necator inoculation. Furthermore, ectopic expression of VpWRKY1 or VpWRKY2 in Arabidopsis enhanced resistance to powdery mildew Erysiphe cichoracearum, and enhanced salt tolerance of transgenic plants. VpWRKY2 also enhanced cold tolerance of transgenic plants. In addition, the two proteins were shown to regulate the expression of some defense marker genes in Arabidopsis and grapevine. The data suggest that VpWRKY1 and VpWRKY2 may underlie the resistance in transgenic grapevine to E. necator and tolerance to salt and cold stresses.


Chinese wild Vitis Disease resistance Grapevine Powdery mildew Transcription factor WRKY 



Quantitative reverse transcriptase-polymerase chain reaction


Vitis pseudoreticulataWRKY1


Vitis pseudoreticulataWRKY2


Salicylic acid


Methyl jasmonate




Hours post inoculation


Days post inoculation


Hours post treatment


Expressed sequence tag

Supplementary material

425_2010_1258_MOESM1_ESM.doc (5.7 mb)
Supplementary material 1 (DOC 5850 kb)


  1. Belhadj A, Telef N, Cluzet S, Bouscaut J, Corio MF, Merillon M (2008) Ethephon elicits protection against Erysiphe necator in grapevine. J Agric Food Chem 56:5781–5787CrossRefPubMedGoogle Scholar
  2. Bellin D, Peressotti E, Merdinoglu D, Wiedemann-Merdinoglu S, Adam-Blondon AF, Cipriani G et al (2009) Resistance to Plasmopara viticola in grapevine ‘Bianca’ is controlled by a major dominant gene causing localized necrosis at the infection site. Theor Appl Genet 120:163–176CrossRefPubMedGoogle Scholar
  3. Bisson LF, Waterhouse AL, Ebeler SE, Walker MA, Lapsley JT (2002) The present and future of the international wine industry. Nature 418:696–699CrossRefPubMedGoogle Scholar
  4. Chujo T, Takai R, Akimoto-Tomiyama C, Ando S, Minami E, Nagamura Y et al (2007) Involvement of the elicitor-induced gene OsWRKY53 in the expression of defense related genes in rice. Biochim Biophys Acta 1769:497–505PubMedGoogle Scholar
  5. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743CrossRefPubMedGoogle Scholar
  6. Coleman C, Copetti D, Cipriani G, Hoffmann S, Kozma P, Kovacs L et al (2009) The powdery mildew resistance gene REN1 co-segregates with an NBS-LRR gene cluster in two Central Asian grapevines. BMC Genet 10:89CrossRefPubMedGoogle Scholar
  7. Dong J, Chen C, Chen Z (2003) Expression profiles of Arabidopsis WRKY gene superfamily during plant defense response. Plant Mol Biol 51:21–37CrossRefPubMedGoogle Scholar
  8. Eulgem T (2005) Regulation of the Arabidopsis defense transcriptome. Trends Plant Sci 10:71–78CrossRefPubMedGoogle Scholar
  9. Eulgem T, Somssich IE (2007) Networks of WRKY transcription factors in defense signaling. Curr Opin Plant Biol 10:366–371CrossRefPubMedGoogle Scholar
  10. Eulgem T, Rushton PJ, Robatzek S, Somssich IE (2000) The WRKY superfamily of plant transcription factors. Trends Plant Sci 5:199–206CrossRefPubMedGoogle Scholar
  11. Fung RWM, Gonzalo M, Fekete C, Kovacs LG, He Y, Marsh E et al (2008) Powdery mildew induces defense-oriented reprogramming of the transcription in a susceptible but not in a resistance grapevine. Plant Physiol 146:236–249CrossRefPubMedGoogle Scholar
  12. Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annu Rev Phytopathol 43:205–227CrossRefPubMedGoogle Scholar
  13. Guan X, Zhao HQ, Xu Y, Wang YJ (2010) Transient expression of glyoxal oxidase from the Chinese wild grape Vitis pseudoreticulata can suppress powdery mildew in a susceptible genotype. Protoplasma. doi:10.1007/s00709-010-0162-4
  14. Guillaumie S, Mzid R, Mechin V, Leon C, Hichri I, Destrac-Irvine A et al (2010) The grapevine transcription factor WRKY2 influences the lignin pathway and xylem development in tobacco. Plant Mol Biol 72:215–234CrossRefPubMedGoogle Scholar
  15. Guo AY, Chen X, Gao G, Zhang H, Zhu QH, Liu XC et al (2008) PlantTFDB: a comprehensive plant transcription factor database. Nucleic Acids Res 36:966–969CrossRefGoogle Scholar
  16. Gutterson N, Reuber TL (2004) Regulation of disease resistance pathways by AP2/ERF transcription factors. Curr Opin Plant Biol 7:465–471CrossRefPubMedGoogle Scholar
  17. Jaillon O, Aury JM, Noel B, Policriti A, Clepet C, Casagrande A et al (2007) The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. Nature 449:463–468CrossRefPubMedGoogle Scholar
  18. Jiang W, Yu D (2009) Arabidopsis WRKY2 transcription factor mediates seed germination and postgermination arrest of development by abscisic acid. BMC Plant Biol 9:96CrossRefPubMedGoogle Scholar
  19. Jing S, Zhou X, Song Y, Yu D (2009) Heterologous expression of OsWRKY23 gene enhances pathogen defense and dark-induced leaf senescence in Arabidopsis. Plant Growth Regul 58:181–190CrossRefGoogle Scholar
  20. Journot-Catalino N, Somssich IE, Roby D, Kroj T (2006) The transcription factors WRKY11 and WRKY17 act as negative regulators of basal resistance in Arabidopsis thaliana. Plant Cell 18:3289–3302CrossRefPubMedGoogle Scholar
  21. Knoth C, Ringler J, Dangl JL, Eulgem T (2007) Arabidopsis WRKY70 is required for full RPP4-mediated disease resistance and basal defense against Hyaloperonospora parasitica. Mol Plant Microbe Interact 20:120–128CrossRefPubMedGoogle Scholar
  22. Lai Z, Vinod KM, Zheng Z, Fan B, Chen Z (2008) Roles of Arabidopsis WRKY3 and WRKY4 transcription factors in plant responses to pathogens. BMC Plant Biol 8:68CrossRefPubMedGoogle Scholar
  23. Li J, Brader G, Palva ET (2004) The WRKY70 transcription factor: a node of convergence for jasmonate-mediated and salicylate-mediated signals in plant defense. Plant Cell 16:319–331CrossRefPubMedGoogle Scholar
  24. Liu X, Bai X, Wang X, Chu C (2007) OsWRKY71, a rice transcription factor, is involved in rice defense response. J Plant Physiol 164:969–979CrossRefPubMedGoogle Scholar
  25. Marchive C, Mzid R, Deluc L, Barrieu F, Pirello J, Gauthier A et al (2007) Isolation and characterization of a Vitis vinifera transcription factor, VvWRKY1, and its effect on responses to fungal pathogens in transgenic tobacco plants. J Exp Bot 58:1999–2010CrossRefPubMedGoogle Scholar
  26. Mare C, Mazzucotelli E, Crosatti C, Francia E, Stanca AM, Cattivelli L (2004) Hv-WRKY38: a new transcription factor involved in cold- and drought-response in barley. Plant Mol Biol 55:399–416CrossRefPubMedGoogle Scholar
  27. Mzid R, Marchive C, Blancard D, Deluc L, Barrieu F, Corio-Costet MF et al (2007) Overexpression of VvWRKY2 in tobacco enhances broad resistance to necrotrophic fungal pathogens. Physiol Plant 131:434–447CrossRefPubMedGoogle Scholar
  28. Naoumkina M, He X, Dixon R (2008) Elicitor-induced transcription factors for metabolic reprogramming of secondary metabolism in Medicago truncatula. BMC Plant Biol 8:132CrossRefPubMedGoogle Scholar
  29. Pandey SP, Somssich IE (2009) The role of WRKY transcription factors in plant immunity. Plant Physiol 150:1648–1655CrossRefPubMedGoogle Scholar
  30. Pavlousek P (2007) Evaluation of resistance to powdery mildew in grapevine genetic resources. J Cent Eur Agric 8:105–114Google Scholar
  31. Qiu Y, Yu D (2009) Over-expression of the stress-induced OsWRKY45 enhances disease resistance and drought tolerance in Arabidopsis. Environ Exp Bot 65:35–47CrossRefGoogle Scholar
  32. Qiu D, Xiao J, Ding X, Xiong M, Cai M, Cao Y et al (2007) OsWRKY13 mediates rice disease resistance by regulating defense related genes in salicylate- and jasmonate-dependent signaling. Mol Plant Microbe Interact 20:492–499CrossRefPubMedGoogle Scholar
  33. Repka V, Fischerova I, Silharova K (2004) Methyl jasmonate is a potent elicitor of multiple defense responses in grapevine leaves and cell-suspension culture. Biol Plant 48:273–283CrossRefGoogle Scholar
  34. Rizhsky L, Liang H, Mittler R (2002) The combined effect of drought stress and heat shock on gene expression in tobacco. Plant Physiol 130:1143–1151CrossRefPubMedGoogle Scholar
  35. Savitch LV, Subramaniam R, Allard GC, Singh J (2007) The GLK1 ‘regulon’ encodes disease defense related proteins and confers resistance to Fusarium graminearum in Arabidopsis. Biochem Biophys Res Commun 359:234–238CrossRefPubMedGoogle Scholar
  36. Singh KB, Foley RC, Onate-Sanchez L (2002) Transcription factors in plant defense and stress responses. Curr Opin Plant Biol 5:430–436CrossRefPubMedGoogle Scholar
  37. Ulker B, Somssich IE (2004) WRKY transcription factors: from DNA binding towards biological function. Curr Opin Plant Biol 7:491–498CrossRefPubMedGoogle Scholar
  38. Ulker U, Shahid MM, Somssich IE (2007) The WRKY70 transcription factor of Arabidopsis influences both the plant senescence and defense signaling pathways. Planta 226:125–137CrossRefPubMedGoogle Scholar
  39. Velasco R, Zharkikh A, Troggio M, Cartwright DA, Cestaro A, Pruss D et al (2007) A high quality draft consensus sequence of the genome of a heterozygous grapevine variety. PLoS ONE 2:e1326CrossRefPubMedGoogle Scholar
  40. Wang LJ, Li SH (2006) Thermotolerance and related antioxidant enzyme activities induced by heat acclimation and salicylic acid in grape (vitis vinifera L.) leaves. Plant Growth Regul 48:137–144CrossRefGoogle Scholar
  41. Wang YJ, Liu Y, He PC, Chen J, Lamicanra O, Lu J (1995) Evaluation of foliar resistance to Uncinula necator in Chinese wild Vitis species. Vitis 34:159–164Google Scholar
  42. Wang Z, Zhu Y, Wang L, Liu X, Liu Y, Phillips J et al (2009) A WRKY transcription factor participates in dehydration tolerance in Boea hygrometrica by binding to the W-box elements of the galactinol synthase (BhGolS1) promoter. Planta 230:1155–1166CrossRefPubMedGoogle Scholar
  43. Wesley SV, Helliwell CA, Smith NA et al (2001) Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J 27:581–590CrossRefPubMedGoogle Scholar
  44. Xiao S, Ellwood S, Findlay K, Oliver RP, Turner JG (1997) Characterization of three loci controlling resistance of Arabidopsis thaliana accession Ms-0 to two powdery mildew diseases. Plant J 12:757–768CrossRefPubMedGoogle Scholar
  45. Xiao S, Calis O, Patrick E, Zhang G, Charoenwattana P, Muskett P et al (2005) The atypical resistance gene, RPW8, recruits components of basal defence for powdery mildew resistance in Arabidopsis. Plant J 42:95–110CrossRefPubMedGoogle Scholar
  46. Xu XP, Chen CH, Fan BF, Chen ZX (2006) Physical and functional interactions between pathogen-induced Arabidopsis WRKY18, WRKY40, WRKY60 transcription factors. Plant Cell 18:1310–1326CrossRefPubMedGoogle Scholar
  47. Xu Y, Zhu Z, Xiao Y, Wang Y (2009) Construction of a cDNA library of Vitis pseudoreticulata native to China inoculated with uncinula necator and the analysis of potential defence-related expressed sequence tags (ESTs). Safr J Enol Vitic 30:65–71Google Scholar
  48. Xu W, Yu Y, Ding J, Hua Z, Wang Y (2010) Characterization of a novel stilbene synthase promoter involved in pathogen- and stress-inducible expression from Chinese wild Vitis pseudoreticulata. Planta 231:475–487CrossRefPubMedGoogle Scholar
  49. Yu S, Jing S, Yu D (2009) Overexpression of the stress-induced OsWRKY08 improves osmotic stress tolerance in Arabidopsis. Chin Sci Bull 54:4671–4678CrossRefGoogle Scholar
  50. Zhang JJ, Wang YJ, Wang XP (2003) An improved method for rapidly extracting total RNA from Vitis. J Fruit Sci 20:178–181 (in Chinese with English abstract)Google Scholar
  51. Zhang J, Peng Y, Guo Z (2008) Constitutive expression of pathogen inducible OsWRKY31 enhances disease resistance and affects root growth and auxin response in transgenic rice plants. Cell Res 18:508–521CrossRefPubMedGoogle Scholar
  52. Zhou QY, Tian AG, Zou HF, Xie ZM, Lei G, Huang J et al (2008) Soybean WRKY-type transcription factor genes, GmWRKY13, GmWRKY21, GmWRKY54, confer differential tolerance to abiotic stresses in transgenic Arabidopsis plants. Plant Biotechnol J 6:486–503CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Huie Li
    • 1
    • 2
    • 3
  • Yan Xu
    • 1
    • 2
    • 3
  • Yu Xiao
    • 1
    • 2
    • 3
  • Ziguo Zhu
    • 1
    • 2
    • 3
  • Xiaoqing Xie
    • 1
    • 2
    • 3
  • Heqing Zhao
    • 1
    • 2
    • 3
  • Yuejin Wang
    • 1
    • 2
    • 3
  1. 1.College of HorticultureNorthwest A&F UniversityYanglingPeople’s Republic of China
  2. 2.Key Laboratory of Horticultural Plant Germplasm Resource Utilization in Northwest China, Ministry of Agriculture of the People’s Republic of ChinaNorthwest A&F UniversityYanglingPeople’s Republic of China
  3. 3.Shaanxi Key Laboratory of Molecular Biology of AgricultureNorthwest A&F UniversityYanglingPeople’s Republic of China

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