Plant Molecular Biology

, Volume 89, Issue 1–2, pp 157–171 | Cite as

Rice WRKY4 acts as a transcriptional activator mediating defense responses toward Rhizoctonia solani, the causing agent of rice sheath blight

  • Haihua Wang
  • Jiao Meng
  • Xixu Peng
  • Xinke Tang
  • Pinglan Zhou
  • Jianhua Xiang
  • Xiaobo Deng


WRKY transcription factors have been implicated in the regulation of transcriptional reprogramming associated with various plant processes but most notably with plant defense responses to pathogens. Here we demonstrate that expression of rice WRKY4 gene (OsWRKY4) was rapidly and strongly induced upon infection of Rhizoctonia solani, the causing agent of rice sheath blight, and exogenous jasmonic acid (JA) and ethylene (ET). OsWRKY4 is localized to the nucleus of plant cells and possesses transcriptional activation ability. Modulation of OsWRKY4 transcript levels by constitutive overexpression increases resistance to the necrotrophic sheath blight fungus, concomitant with elevated expression of JA- and ET-responsive pathogenesis-related (PR) genes such as PR1a, PR1b, PR5 and PR10/PBZ1. Suppression by RNA interference (RNAi), on the other hand, compromises resistance to the fungal pathogen. Yeast one-hybrid assay and transient expression in tobacco cells reveal that OsWRKY4 specifically binds to the promoter regions of PR1b and PR5 which contain W-box (TTGAC[C/T]), or W-box like (TGAC[C/T]) cis-elements. In conclusion, we propose that OsWRKY4 functions as an important positive regulator that is implicated in the defense responses to rice sheath blight via JA/ET-dependent signal pathway.


Disease resistance Rhizoctonia solani Transcription factor WRKY protein Oryza sativa 



This research was funded by National Natural Science Foundation of China (Grant Nos. 31171803, 31301617) and Project of Hunan Provincial Natural Science Foundation of China (Grant No. 10JJ2030).

Compliance with ethical standards

Conflict of interest

All authors have read and approved this version of the article, and due care has been taken to ensure the integrity of the work. The authors declare that there are no potential conflicts of interest regarding the publication of this paper.

Supplementary material

11103_2015_360_MOESM4_ESM.doc (48 kb)
Supplementary Table S1 Gene-specific primers for quantitative real-time PCR. (DOC 47 kb)
11103_2015_360_MOESM5_ESM.doc (31 kb)
Supplementary Table S2 Specific primers for amplification of the full and 5′-deleted promoters of rice PR1b and PR5. (DOC 31 kb)
11103_2015_360_MOESM1_ESM.tif (282 kb)
Supplementary Fig. S1 Transcription levels of OsWRKY1 and OsWRKY17 in OsWRKY4 RNAi transgenic rice plants. (TIFF 282 kb)
11103_2015_360_MOESM2_ESM.tif (1.7 mb)
Supplementary Fig. S2 Changed disease resistance of OsWRKY4 overexpression and RNAi transgenic rice plants. (TIFF 1708 kb)
11103_2015_360_MOESM3_ESM.jpg (115 kb)
Supplementary Fig. S3 Distribution of W-box or W-box like elements in the promoter of OsWRKY4. (JPEG 114 kb)


  1. Agrawal GK, Jwa NS, Rakwal R (2000a) A novel rice (Oryza sativa L.) acidic PR1 gene highly responsive to cut, phytohormones, and protein phosphatase inhibitors. Biochem Biophys Res Commun 274:157–165CrossRefPubMedGoogle Scholar
  2. Agrawal GK, Rakwal R, Jwa NS (2000b) Rice (Oryza sativa L.) OsPR1b gene is phytohormonally regulated in close interaction with light signals. Biochem Biophys Res Commun 278:290–298CrossRefPubMedGoogle Scholar
  3. Agrawal GK, Rakwal R, Jwa NS (2001a) Differential induction of three pathogenesis-related genes, PR10, PR1b and PR5 by the ethylene generator ethephon under light and dark in rice (Oryza sativa L.) seedlings. J Plant Physiol 158:133–137CrossRefGoogle Scholar
  4. Agrawal GK, Rakwal R, Jwa NS, Agrawal VP (2001b) Signalling molecules and blast pathogen attack activates rice OsPR1a and OsPR1b genes: a model illustrating components participating during defence/stress response. Plant Physiol Biochem 39:1095–1103CrossRefGoogle Scholar
  5. Alexander D, Goodman RM, Gut-Rella M, Glascock C, Weymann K, Friedrich L, Maddox D, Ahl-Goy P, Luntz T, War E, Ryals J (1993) Increased tolerance to 2 oomycete pathogens in transgenic tobacco expressing pathogenesis-related protein 1a. Proc Natl Acad Sci USA 90:7327–7331PubMedCentralCrossRefPubMedGoogle Scholar
  6. Bari R, Jones JD (2009) Role of plant hormones in plant defense responses. Plant Mol Biol 69:473–488CrossRefPubMedGoogle Scholar
  7. Chujo T, Miyamoto K, Shimogawa T, Shimizu T, Otake Y, Yokotani N, Nishizawa Y, Shibuya N, Nojiri H, Yamane H, Minami E, Okada K (2013) OsWRKY28, a PAMP-responsive transrepressor, negatively regulates innate immune responses in rice against rice blast fungus. Plant Mol Biol 82:23–37CrossRefPubMedGoogle Scholar
  8. Cubeta MA, Vilgalys R (1997) Population biology of the Rhizoctonia solani complex. Phytopathology 87:480–484CrossRefPubMedGoogle Scholar
  9. Datta K, Velazhahan R, Oliva N, Ona I, Mew T, Khush GS, Muthukrishnan S, Datta SK (1999) Over-expression of the cloned rice thaumatin-like protein (PR-5) gene in transgenic rice plants enhances environmental friendly resistance to Rhizoctonia solani causing sheath blight disease. Theor Appl Genet 98:1138–1145CrossRefGoogle Scholar
  10. Delteil A, Blein M, Faivre-Rampant O, Guellim A, Estevan J, Hirsch J, Bevitori R, Michel C, Morel JB (2012) Building a mutant resource for the study of disease resistance in rice reveals the pivotal role of several genes involved in defence. Mol Plant Pathol 13:72–82CrossRefPubMedGoogle Scholar
  11. Dong J, Chen C, Chen Z (2003) Expression profiles of the Arabidopsis WRKY gene superfamily during plant defense response. Plant Mol Biol 51:21–37CrossRefPubMedGoogle Scholar
  12. Durrant WE, Dong X (2004) Systemic acquired resistance. Annu Rev Phytopathol 42:185–209CrossRefPubMedGoogle Scholar
  13. Eulgem T (2005) Regulation of the Arabidopsis defense transcriptome. Trends Plant Sci 10:71–78CrossRefPubMedGoogle Scholar
  14. Eulgem T, Somssich IE (2007) Networks of WRKY transcription factors in defense signaling. Curr Opin Plant Biol 10:366–371CrossRefPubMedGoogle Scholar
  15. Eulgem T, Rushton PJ, Robatzek S, Somssich IE (2000) The WRKY superfamily of plant transcription factors. Trends Plant Sci 5:199–206CrossRefPubMedGoogle Scholar
  16. Glazebrook J (2001) Genes controlling expression of defense responses in Arabidopsis: 2001 status. Curr Opin Plant Biol 4:301–308CrossRefPubMedGoogle Scholar
  17. Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annu Rev Phytopathol 43:205–227CrossRefPubMedGoogle Scholar
  18. Hammond-Kosack KE, Parker JE (2003) Deciphering plant pathogen communication: fresh perspectives for molecular resistance breeding. Curr Opin Biotechnol 14:177–193CrossRefPubMedGoogle Scholar
  19. Hellens RP, Allan AC, Friel EN, Bolitho K, Grafton K, Templeton MD, Karunairetnam S, Laing WA (2005) Transient plant expression vectors for functional genomics, quantification of promoter activity and RNA silencing. Plant Methods 1:13PubMedCentralCrossRefPubMedGoogle Scholar
  20. Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6:271–282CrossRefPubMedGoogle Scholar
  21. Hwang SH, Yie SW, Hwang DJ (2011) Heterologous expression of OsWRKY6 gene in Arabidopsis activates the expression of defense related genes and enhances resistance to pathogens. Plant Sci 181:316–323CrossRefPubMedGoogle Scholar
  22. Jing SJ, Zhou X, Song Y, Yu DQ (2009) Heterologous expression of OsWRKY23 gene enhances pathogen defense and dark-induced leaf senescence in Arabidopsis. Plant Growth Regul 58:181–190CrossRefGoogle Scholar
  23. 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 18:3289–3302PubMedCentralCrossRefPubMedGoogle Scholar
  24. Kim KC, Fan B, Chen Z (2006) Pathogen-induced Arabidopsis WRKY7 is a transcriptional repressor and enhances plant susceptibility to Pseudomonas syringae. Plant Physiol 142:1180–1192PubMedCentralCrossRefPubMedGoogle Scholar
  25. Kim KC, Lai Z, Fan B, Chen Z (2008) Arabidopsis WRKY38 and WRKY62 transcription factors interact with histone deacetylase 19 in basal defense. Plant Cell 20:2357–2371PubMedCentralCrossRefPubMedGoogle Scholar
  26. Kloek AP, Verbsky ML, Sharma SB, Schoelz JE, Vogel J, Klessig DF, Kunkel BN (2001) Resistance to Pseudomonas syringae conferred by an Arabidopsis thaliana coronatine-insensitive (coi1) mutation occurs through two distinct mechanisms. Plant J 26:509–522CrossRefPubMedGoogle Scholar
  27. 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
  28. Kunkel BN, Brooks DM (2002) Cross talk between signaling pathways in pathogen defense. Curr Opin Plant Biol 5:325–331CrossRefPubMedGoogle Scholar
  29. Lai ZB, Vinod KM, Zheng ZY, Fan BF, Chen Z (2008) Roles of Arabidopsis WRKY3 and WRKY4 transcription factors in plant responses to pathogens. BMC Plant Biol 8:68PubMedCentralCrossRefPubMedGoogle Scholar
  30. Lee NF, Rush MC (1983) Rice sheath blight: a major rice disease. Plant Dis 67:829–832CrossRefGoogle Scholar
  31. 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 16:319–331PubMedCentralCrossRefPubMedGoogle Scholar
  32. Li J, Brader G, Kariola T, Palva ET (2006) WRKY70 modulates the selection of signaling pathways in plant defense. Plant J 46:477–491CrossRefPubMedGoogle Scholar
  33. Liu XQ, Bai XQ, Qian Q, Wang XJ, Chen MS, Chu CC (2005) OsWRKY03, a rice transcriptional activator that functions in defense signaling pathway upstream of OsNPR1. Cell Res 15:593–603CrossRefPubMedGoogle Scholar
  34. Liu XQ, Bai XQ, Wang XJ, Chu CC (2007) OsWRKY71, a rice transcription factor, is involved in rice defense response. J Plant Physiol 164:969–979CrossRefPubMedGoogle Scholar
  35. Mahmood T, Kakishima M, Komatsu S (2007) Proteomic analysis of jasmonic acid-regulated proteins in rice leaf blades. Protein Pept Lett 14:311–319CrossRefPubMedGoogle Scholar
  36. Maleck K, Levine A, Eulgem T, Morgan A, Schmid J, Lawton KA, Dangl JL, Dietrich RA (2000) The transcriptome of Arabidopsis thaliana during systemic acquired resistance. Nat Genet 26:403–410CrossRefPubMedGoogle Scholar
  37. Mei C, Qi M, Sheng G, Yang Y (2006) Inducible overexpression of a rice allene oxide synthase gene increases the endogenous jasmonic acid level, PR gene expression, and host resistance to fungal infection. Mol Plant Microbe Interact 19:1127–1137CrossRefPubMedGoogle Scholar
  38. Mitsuhara I, Iwai T, Seo S, Yanagawa Y, Kawahigasi H, Hirose S, Ohkawa Y, Ohashi Y (2008) Characteristic expression of twelve rice PR1 family genes in response to pathogen infection, wounding, and defense-related signal compounds. Mol Genet Genom 279:415–427CrossRefGoogle Scholar
  39. Mur LA, Kenton P, Atzorn R, Miersch O, Wasternack C (2006) The outcomes of concentration-specific interactions between salicylate and jasmonate signaling include synergy, antagonism, and oxidative stress leading to cell death. Plant Physiol 140:249–262PubMedCentralCrossRefPubMedGoogle Scholar
  40. Nawrath C, Métraux J-P (1999) Salicylic acid induction deficient mutants of Arabidopsis express PR-2 and PR-5 and accumulate high levels of camalexin after pathogen inoculation. Plant Cell 11:1393–1404PubMedCentralPubMedGoogle Scholar
  41. Niederman T, Genetet I, Bruyere T, Gees R, Stinzi A, Legrand M, Fritig B, Mosinger E (1995) Pathogenesis-related PR-1 proteins are antifungal: isolation and characterization of three 14 kidodalton proteins of tomato and of a basic PR-1 of tobacco with inhibitory activity against Phytophythora infestans. Plant Physiol 108:17–27CrossRefGoogle Scholar
  42. Nimchuk Z, Eulgem T, Holt BF III, Dangl JL (2003) Recognition and response in the plant immune system. Annu Rev Genet 37:579–609CrossRefPubMedGoogle Scholar
  43. Pandey SP, Somssich IE (2009) The role of WRKY transcription factors in plant immunity. Plant Physiol 150:1648–1655PubMedCentralCrossRefPubMedGoogle Scholar
  44. Peng Y, Bartley LE, Chen XW, Dardick C, Chern M, Ruan R, Canlas PE, Ronald PC (2008) OsWRKY62 is a negative regulator of basal and Xa21-mediated defense against Xanthomonas oryzae pv. oryzae in rice. Mol Plant 1:446–458CrossRefPubMedGoogle Scholar
  45. Peng XX, Tang XK, Zhou PL, Hu YJ, Deng XB, He Y, Wang HH (2011) Isolation and expression patterns of rice WRKY82 transcription factor gene responsive to both biotic and abiotic stresses. Agric Sci China 10:891–901CrossRefGoogle Scholar
  46. Peng XX, Hu YJ, Tang XK, Zhou PL, Deng XB, Wang HH, Guo ZJ (2012) Constitutive expression of rice WRKY30 gene increases the endogenous jasmonic acid accumulation, PR gene expression and resistance to fungal pathogens in rice. Planta 236:1485–1498CrossRefPubMedGoogle Scholar
  47. Penninckx IA, Thomma BP, Buchala A, Metraux JP, Broekaert WF (1998) Concomitant activation of jasmonate and ethylene response pathways is required for induction of a plant defensin gene in Arabidopsis. Plant Cell 10:2103–2113PubMedCentralCrossRefPubMedGoogle Scholar
  48. Qiu D, Xiao J, Ding X, Xiong M, Cai M, Cao Y, Li X, Xu C, Wang S (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
  49. Qiu D, Xiao J, Xie W, Liu H, Li X, Xiong L, Wang S (2008) Rice gene network inferred from expression profiling of plants overexpressing OsWRKY13, a positive regulator of disease resistance. Mol Plant 1:538–551PubMedGoogle Scholar
  50. Ross CA, Liu Y, Shen QJ (2007) The WRKY gene family in rice (Oryza sativa). J Integr Plant Biol 49:827–842CrossRefGoogle Scholar
  51. Rush MC, Hoff BJ, Mcllrath WO (1976) A uniform disease rating system for rice disease in the United States. In: Proceedings of the 16th Rice Technical Working Group, Lake Charles, Louisiana, USA, 64Google 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. Sayler RJ, Yang Y (2007) Detection and quantification of Rhizoctonia solani AG-1 IA, the rice sheath blight pathogen, in rice using real-time PCR. Plant Dis 91:1663–1668CrossRefGoogle Scholar
  54. Shimono M, Sugano S, Nakayama A, Jiang CJ, Ono K, Toki S, Takatsuji H (2007) Rice WRKY45 plays a crucial role in benzothiadiazole-inducible blast resistance. Plant Cell 19:2064–2076PubMedCentralCrossRefPubMedGoogle Scholar
  55. Shimono M, Koga H, Akagi A, Hayashi N, Goto S, Sawada M, Kurihara T, Matsushita A, Sugano S, Jiang CJ, Kaku H, Inoue H, Takatsuji H (2012) Rice WRKY45 plays important roles in fungal and bacterial disease resistance. Mol Plant Pathol 13:83–94CrossRefPubMedGoogle Scholar
  56. Tao Z, Liu H, Qiu D, Zhou Y, Li X, Xu C, Wang S (2009) A pair of allelic WRKY genes play opposite roles in rice-bacteria interactions. Plant Physiol 151:936–948PubMedCentralCrossRefPubMedGoogle Scholar
  57. Thomma BPHJ, Eggermont K, Penninckx IAMA, Mauch-Mani B, Vogelsang R, Cammue BPA, Broekaert WF (1998) Separate jasmonate-dependent and salicylate-dependent defense-response pathways in Arabidopsis are essential for resistance to distinct microbial pathogens. Proc Natl Acad Sci USA 95:15107–15111PubMedCentralCrossRefPubMedGoogle Scholar
  58. Triezenberg SJ (1995) Structure and function of transcriptional activation domains. Curr Opin Genet Dev 5:190–196CrossRefPubMedGoogle Scholar
  59. Turck F, Zhou A, Somssich IE (2004) Stimulus-dependent, promoter-specific binding of transcription factor WRKY1 to its native promoter and the defense-related gene PcPR1-1 in parsley. Plant Cell 16:2573–2585PubMedCentralCrossRefPubMedGoogle Scholar
  60. Turner JG, Ellis C, Devoto A (2012) The jasmonate signal pathway. Plant Cell 14:S153–S164Google Scholar
  61. Ülker B, Somssich IE (2004) WRKY transcription factors: from DNA binding towards biological function. Curr Opin Plant Biol 7:491–498CrossRefPubMedGoogle Scholar
  62. van Loon LC, Rep M, Pieterse CMJ (2006) Significance of inducible defense-related proteins in infected plants. Annu Rev Phytopathol 44:135–162CrossRefPubMedGoogle Scholar
  63. Wang J, Jiang J, Oard JH (2000) Structure, expression and promoter activity of two polyubiquitin genes from rice (Oryza sativa L.). Plant Sci 156:201–211CrossRefPubMedGoogle Scholar
  64. Wang HH, Hao JJ, Chen XJ, Hao ZZ, Wang X, Lou YG, Peng YL, Guo ZJ (2007) Overexpression of rice WRKY89 enhances ultraviolet B tolerance and disease resistance in rice plants. Plant Mol Biol 65:799–815CrossRefPubMedGoogle Scholar
  65. Wang ZB, Zuo SM, Li G, Chen XJ, Chen ZX, Zhang YF, Pan XB (2009) Rapid identification technology of resistance to rice sheath blight in seedling stage. Acta Phytopathol Sin 39:174–182Google Scholar
  66. Wang Y, Dang F, Liu Z, Wang X, Eulgem T, Lai Y, Yu L, She J, Shi Y, Lin J, Chen C, Guan D, Qiu A, He S (2013) CaWRKY58, encoding a group I WRKY transcription factor of Capsicum annuum, negatively regulates resistance to Ralstonia solanacearum infection. Mol Plant Pathol 14:131–144CrossRefPubMedGoogle Scholar
  67. Wu KL, Guo ZJ, Wang HH, Li J (2005) The WRKY Family of transcription factors in rice and Arabidopsis and their origins. DNA Res 12:9–26CrossRefPubMedGoogle Scholar
  68. Xie C, Zhang JS, Zhou HL, Li J, Zhang ZG, Wang DW, Chen SY (2003) Serine/threonine kinase activity in the putative histidine kinase-like ethylene receptor NTHK1 from tobacco. Plant J 33:385–393CrossRefPubMedGoogle Scholar
  69. Xu Y, Zhu Q, Panbangred W, Shirasu K, Lamb C (1996) Regulation, expression and function of a new basic chitinase gene in rice (Oryza sativa L.). Plant Mol Biol 30:340–387CrossRefGoogle Scholar
  70. Yang Y, Li R, Qi M (2000) In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco leaves. Plant J 22:543–551CrossRefPubMedGoogle Scholar
  71. Yokotani N, Sato Y, Tanabe S, Chujo T, Shimizu T, Okada K, Yamane H, Shimono M, Sugano S, Takatsuji H, Kaku H, Minami E, Nishizawa Y (2013) WRKY76 is a rice transcriptional repressor playing opposite roles in blast disease resistance and cold stress tolerance. J Exp Bot 64:5085–5097PubMedCentralCrossRefPubMedGoogle Scholar
  72. Yu D, Chen C, Chen Z (2001) Evidence for an important role of WRKY DNA binding proteins in the regulation of NPR1 gene expression. Plant Cell 13:1527–1540PubMedCentralCrossRefPubMedGoogle Scholar
  73. Zhang J, Peng YL, Guo ZJ (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
  74. Zhao CJ, Lu GD, Du XY, Liu L, Lin Y, Shi YJ, Wang ZH (2006) PR1 and PBZ1 expression through the infection progress of rice sheath blight. Acta Phytopathol Sin 36:317–321Google Scholar
  75. Zhao CJ, Wang AR, Shi YJ, Wang LQ, Liu WD, Wang ZH, Lu GD (2008) Identification of defense-related genes in rice responding to challenge by Rhizoctonia solani. Theor Appl Genet 116:501–516CrossRefPubMedGoogle Scholar
  76. Zheng Z, Qamar SA, Chen Z, Mengiste T (2006) Arabidopsis WRKY33 transcription factor is required for resistance to necrotrophic fungal pathogens. Plant J 48:592–605CrossRefPubMedGoogle Scholar
  77. Zhu Q, Dabi T, Beeche A, Yamamoto R, Lawton MA, Lamb C (1995) Cloning and properties of a rice gene encoding phenylalanine ammonia-lyase. Plant Mol Biol 29:535–550CrossRefPubMedGoogle Scholar
  78. Zou JH, Pan XB, Chen ZX, Xu JY, Lu JF, Zhai WX, Zhu LH (2000) Mapping quantitative trait loci controlling sheath blight resistance in two rice cultivars (Oryza sativa L.). Theor Appl Genet 101:569–573CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Haihua Wang
    • 1
    • 2
    • 3
  • Jiao Meng
    • 1
  • Xixu Peng
    • 1
    • 3
  • Xinke Tang
    • 1
    • 2
  • Pinglan Zhou
    • 1
    • 2
  • Jianhua Xiang
    • 1
  • Xiaobo Deng
    • 1
  1. 1.School of Life ScienceHunan University of Science and TechnologyXiangtanChina
  2. 2.Key Laboratory of Integrated Management of the Pests and Diseases on Horticultural Crops in Hunan ProvinceXiangtanChina
  3. 3.Key Laboratory of Ecological Remediation and Safe Utilization of Heavy Metal-Polluted SoilsCollege of Hunan ProvinceXiangtanChina

Personalised recommendations