Plant Cell Reports

, Volume 34, Issue 6, pp 1063–1074 | Cite as

The NPR1 homolog GhNPR1 plays an important role in the defense response of Gladiolus hybridus

  • Xionghui Zhong
  • Lin Xi
  • Qinglong Lian
  • Xian Luo
  • Ze Wu
  • Shanshan Seng
  • Xue Yuan
  • Mingfang YiEmail author
Original Paper


Key message

GhNPR1 shares similar functions as Arabidopsis NPR1 . Silencing of GhNPR1 in Gladiolus results in an enhanced susceptibility to Curvularia gladioli. We propose that GhNPR1 plays important roles in plant immunity.


Gladiolus plants and corms are susceptible to various types of pathogens including fungi, bacteria and viruses. Understanding the innate defense mechanism in Gladiolus is a prerequisite for the development of new protection strategies. The non-expressor of pathogenesis-related gene 1 (NPR1) and bzip transcription factor TGA2 play a key role in regulating salicylic acid (SA)-mediated systemic acquired resistance (SAR). In this study, the homologous genes, GhNPR1 and GhTGA2, were isolated from Gladiolus and functionally characterized. Expression of GhNPR1 exhibited a 3.8-fold increase in Gladiolus leaves following salicylic acid treatment. A 1332 bp fragment of the GhNPR1 promoter from Gladiolus hybridus was identified. Inducibility of the GhNPR1 promoter by SA was demonstrated using transient expression assays in the leaves of Nicotiana benthamiana. The GhNPR1 protein is located in the nucleus and cytomembrane. GhNPR1 interacts with GhTGA2, as observed using the bimolecular fluorescence complementation system. Overexpression of GhNPR1 in an Arabidopsis npr1 mutant can restore its basal resistance to Pseudomonas syringae pv. tomato DC3000. Silencing of GhNPR1, using a tobacco rattle virus-based silencing vector, resulted in an enhanced susceptibility to Curvularia gladioli. In conclusion, these results suggest that GhNPR1 plays a pivotal role in the SA-dependent systemic acquired resistance in Gladiolus.


Disease resistance Fungal diseases Gladiolus Salicylic acid Systemic acquired resistance 



This study has been supported by the Science and Technology Specific Project Foundation of Ministry of Agriculture, PR China (No. 200903020), National Natural Science Foundation of China (No. 31171991) and Chinese Universities Scientific Fund (No. 15053201 and No. 15054201).

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

299_2015_1765_MOESM1_ESM.pdf (4.2 mb)
Supplementary material 1 (PDF 4291 kb) Supplementary Fig. 1 Alignment of Gladiolus NPR1 homologous proteins from various species. Supplementary Fig. 2 Phylogenetic relationships between GhNPR1 and homologous proteins. Supplementary Fig. 3 Alignment of Gladiolus TGA2 homologous proteins from various species. Supplementary Fig. 4 Phylogenetic analysis of GhTGA2 and seven other TGAs from Arabidopsis. Supplementary Fig. 5 The genomic sequence of the GhNPR1 gene. Supplementary Fig. 6 Isolation and identification of Curvularia gladioli causing leaf blight of Gladiolus Supplementary Fig. 7 Pathogenicity was tested by inoculating the leaves with a mycelium agar plug (a, b) and a conidial suspension (c) of Curvularia gladioli
299_2015_1765_MOESM2_ESM.doc (39 kb)
Supplementary material 2 (DOC 39 kb)


  1. Baiswar P, Chandra S, Kumar R (2007) Status of gladiolus diseases and their management in India-A review. J Ornam Hortic 10(4):209–214Google Scholar
  2. Barsalobres Cavallari C, Petitot A, Severino F, Maia I, Fernandez D (2013) Host response profiling to fungal infection: molecular cloning, characterization and expression analysis of NPR1 gene from coffee (Coffea arabica). Microbial pathogens and strategies for combating them: science, technology and education, vol 1. Formatex Research Center, BadajozGoogle Scholar
  3. Benschop M, Kamenetsky R, Le Nard M, Okubo H, De Hertogh A (2010) The global flower bulb industry: production, utilization, research. Acta Physiol Plant 36:1Google Scholar
  4. Boyle P, Le Su E, Rochon A, Shearer HL, Murmu J, Chu JY, Fobert PR, Després C (2009) The BTB/POZ domain of the Arabidopsis disease resistance protein NPR1 interacts with the repression domain of TGA2 to negate its function. Plant Cell 21(11):3700–3713CrossRefPubMedCentralPubMedGoogle Scholar
  5. Burch-Smith TM, Anderson JC, Martin GB, Dinesh-Kumar SP (2004) Applications and advantages of virus-induced gene silencing for gene function studies in plants. Plant J 39(5):734–746CrossRefPubMedGoogle Scholar
  6. Cao H, Bowling SA, Gordon AS, Dong X (1994) Characterization of an Arabidopsis mutant that is nonresponsive to inducers of systemic acquired resistance. Plant Cell 6(11):1583–1592CrossRefPubMedCentralPubMedGoogle Scholar
  7. Cao H, Glazebrook J, Clarke JD, Volko S, Dong XN (1997) The Arabidopsis NPR1 gene that controls systemic acquired resistance encodes a novel protein containing ankyrin repeats. Cell 88(1):57–63CrossRefPubMedGoogle Scholar
  8. Cao H, Li X, Dong XN (1998) Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene in systemic acquired resistance. P Natl Acad Sci USA 95(11):6531–6536CrossRefGoogle Scholar
  9. Chen W, Yin X, Wang L, Tian J, Yang RY, Liu DF, Yu ZH, Ma N, Gao JP (2013) Involvement of rose aquaporin RhPIP1; 1 in ethylene-regulated petal expansion through interaction with RhPIP2; 1. Plant Mol Biol 83(3):219–233CrossRefPubMedGoogle Scholar
  10. Chern M, Fitzgerald HA, Canlas PE, Navarre DA, Ronald PC (2005) Overexpression of a rice NPR1 homolog leads to constitutive activation of defense response and hypersensitivity to light. Mol Plant Microbe Interact 18(6):511–520CrossRefPubMedGoogle Scholar
  11. Després C, Chubak C, Rochon A, Clark R, Bethune T, Desveaux D, Fobert PR (2003) The Arabidopsis NPR1 disease resistance protein is a novel cofactor that confers redox regulation of DNA binding activity to the basic domain/leucine zipper transcription factor TGA1. Plant Cell 15(9):2181–2191CrossRefPubMedCentralPubMedGoogle Scholar
  12. Dong XN (2004) NPR1, all things considered. Curr Opin Plant Biol 7(5):547–552CrossRefPubMedGoogle Scholar
  13. Ekengren SK, Liu Y, Schiff M, Dinesh Kumar S, Martin GB (2003) Two MAPK cascades, NPR1, and TGA transcription factors play a role in Pto-mediated disease resistance in tomato. Plant J 36(6):905–917CrossRefPubMedGoogle Scholar
  14. Fan WH, Dong XN (2002) In vivo interaction between NPR1 and transcription factor TGA2 leads to salicylic acid-mediated gene activation in Arabidopsis. Plant Cell 14(6):1377–1389CrossRefPubMedCentralPubMedGoogle Scholar
  15. Fu ZQ, Dong XN (2013) Systemic acquired resistance: turning local infection into global defense. Annu Rev Plant Biol 64:839–863CrossRefPubMedGoogle Scholar
  16. Giri MK, Swain S, Gautam JK, Singh S, Singh N, Bhattacharjee L, Nandi AK (2014) The Arabidopsis thaliana At4g13040 gene, a unique member of the AP2/EREBP family, is a positive regulator for salicylic acid accumulation and basal defense against bacterial pathogens. J Plant Physiol 171(10):860–867CrossRefPubMedGoogle Scholar
  17. Haan D, Doorn V (2000) PCR detection of Fusarium oxysporum f. sp. gladioli race 1, causal agent of Gladiolus yellows disease, from infected corms. Plant Pathol 49(1):89–100CrossRefGoogle Scholar
  18. Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Res 27(1):297–300CrossRefPubMedCentralPubMedGoogle Scholar
  19. Hwang SH, Hwang DJ (2010) Isolation and characterization of the rice NPR1 promoter. Plant Biotechnol Rep 4(1):29–35CrossRefGoogle Scholar
  20. Hwang SH, Lee IA, Yie SW, Hwang DJ (2008) Identification of an OsPR10a promoter region responsive to salicylic acid. Planta 227(5):1141–1150CrossRefPubMedCentralPubMedGoogle Scholar
  21. Iavicoli A, Boutet E, Buchala A, Métraux JP (2003) Induced systemic resistance in Arabidopsis thaliana in response to root inoculation with Pseudomonas fluorescens CHA0. Mol Plant Microbe Interact 16(10):851–858CrossRefPubMedGoogle Scholar
  22. Jakoby M, Weisshaar B, Dröge Laser W, Vicente Carbajosa J, Tiedemann J, Kroj T, Parcy F (2002) bZIP transcription factors in Arabidopsis. Trends Plant Sci 7(3):106–111CrossRefPubMedGoogle Scholar
  23. Johnson C, Boden E, Arias J (2003) Salicylic acid and NPR1 induce the recruitment of trans-activating TGA factors to a defense gene promoter in Arabidopsis. Plant Cell 15(8):1846–1858CrossRefPubMedCentralPubMedGoogle Scholar
  24. Kagaya Y, Ohmiya K, Hattori T (1999) RAV1, a novel DNA-binding protein, binds to bipartite recognition sequence through two distinct DNA-binding domains uniquely found in higher plants. Nucleic Acids Res 27(2):470–478CrossRefPubMedCentralPubMedGoogle Scholar
  25. Kammerer S, Burpee L, Harmon P (2011) Identification of a new Waitea circinata variety causing basal leaf blight of seashore paspalum. Plant Dis 95(5):515–522CrossRefGoogle Scholar
  26. Kerppola TK (2006) Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells. Nat Protoc 1(3):1278–1286CrossRefPubMedCentralPubMedGoogle Scholar
  27. Kinkema M, Fan WH, Dong XN (2000) Nuclear localization of NPR1 is required for activation of PR gene expression. Plant Cell 12(12):2339–2350CrossRefPubMedCentralPubMedGoogle Scholar
  28. Koschmann J, Machens F, Becker M, Niemeyer J, Schulze J, Bülow L, Stahl DJ, Hehl R (2012) Integration of bioinformatics and synthetic promoters leads to the discovery of novel elicitor-responsive cis-regulatory sequences in Arabidopsis. Plant Physiol 160(1):178–191CrossRefPubMedCentralPubMedGoogle Scholar
  29. Lawton KA, Friedrich L, Hunt M, Weymann K, Delaney T, Kessmann H, Staub T, Ryals J (1996) Benzothiadiazole induces disease resistance in Arabidopsis by activation of the systemic acquired resistance signal transduction pathway. Plant J 10(1):71–82CrossRefPubMedGoogle Scholar
  30. Le Henanff G, Heitz T, Mestre P, Mutterer J, Walter B, Chong J (2009) Characterization of Vitis vinifera NPR1 homologs involved in the regulation of pathogenesis-related gene expression. BMC Plant Biol 9(1):54CrossRefPubMedCentralPubMedGoogle Scholar
  31. Le Henanff G, Farine S, Kieffer Mazet F, Miclot AS, Heitz T, Mestre P, Bertsch C, Chong JL (2011) Vitis vinifera VvNPR1. 1 is the functional ortholog of AtNPR1 and its overexpression in grapevine triggers constitutive activation of PR genes and enhanced resistance to powdery mildew. Planta 234(2):405–417CrossRefPubMedGoogle Scholar
  32. Lescot M, Déhais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, Rouzé P, Rombauts S (2002) PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res 30(1):325–327CrossRefPubMedCentralPubMedGoogle Scholar
  33. Lian QL, Xin HB, Zhong XH, Zhang ZY, Li XX, Yuan X, Han HJ, He XL, Yi MF (2011) Cloning, characterization and expression analysis of a 9-lipoxygenase gene in Gladiolus hybridus. Sci Hortic 130(2):468–475CrossRefGoogle Scholar
  34. Liu YG, Chen YL (2007) High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences. Biotechniques 43:649–656CrossRefPubMedGoogle Scholar
  35. Liu Y, Schiff M, Marathe R, Dinesh Kumar S (2002) Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus. Plant J 30(4):415–429CrossRefPubMedGoogle Scholar
  36. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT Method. Methods 25(4):402–408CrossRefPubMedGoogle Scholar
  37. Magie R (1948) Curvularia spot, a new disease of gladiolus. Plant Dis Rep 32:11–13Google Scholar
  38. 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(4):403–410CrossRefPubMedGoogle Scholar
  39. Malnoy M, Jin Q, Borejsza Wysocka E, He S, Aldwinckle H (2007) Overexpression of the apple MpNPR1 gene confers increased disease resistance in Malus × domestica. Mol Plant Microbe Interact 20(12):1568–1580CrossRefPubMedGoogle Scholar
  40. Métraux J, Signer H, Ryals J, Ward E, Wyss Benz M, Gaudin J, Raschdorf K, Schmid E, Blum W, Inverardi B (1990) Increase in salicylic acid at the onset of systemic acquired resistance in cucumber. Science 250(4983):1004–1006 (80)CrossRefPubMedGoogle Scholar
  41. Pajerowska Mukhtar KM, Emerine DK, Mukhtar MS (2013) Tell me more: roles of NPRs in plant immunity. Trends Plant Sci 18(7):402–411CrossRefPubMedGoogle Scholar
  42. Parmelee J (1956) The identification of the Curvularia parasite of gladiolus. Mycologia 48(4):558–567CrossRefGoogle Scholar
  43. Pianzzola M, Moscatelli M, Vero S (2004) Characterization of Penicillium isolates associated with blue mold on apple in Uruguay. Plant Dis 88(1):23–28CrossRefGoogle Scholar
  44. Pieterse C, Van Wees S, Hoffland E, Van Pelt JA, Van Loon LC (1996) Systemic resistance in Arabidopsis induced by biocontrol bacteria is independent of salicylic acid accumulation and pathogenesis-related gene expression. Plant Cell 8(8):1225–1237CrossRefPubMedCentralPubMedGoogle Scholar
  45. Prestridge DS (1991) SIGNAL SCAN: a computer program that scans DNA sequences for eukaryotic transcriptional elements. Comput Appl Biosci CABIOS 7(2):203–206Google Scholar
  46. Sandhu D, Tasma IM, Frasch R, Bhattacharyya MK (2009) Systemic acquired resistance in soybean is regulated by two proteins, orthologous to Arabidopsis NPR1. BMC Plant Biol 9(1):105CrossRefPubMedCentralPubMedGoogle Scholar
  47. Shah J, Tsui F, Klessig DF (1997) Characterization of a salicylic acid-insensitive mutant (sai1) of Arabidopsis thaliana, identified in a selective screen utilizing the SA-inducible expression of the tms2 gene. Mol Plant Microbe Interact 10(1):69–78CrossRefPubMedGoogle Scholar
  48. Shi Z, Maximova S, Liu Y, Verica J, Guiltinan M (2010) Functional analysis of the Theobroma cacao NPR1 gene in Arabidopsis. BMC Plant Biol 10(1):248CrossRefPubMedCentralPubMedGoogle Scholar
  49. Shi Z, Maximova S, Liu Y, Verica J, Guiltinan MJ (2013) The salicylic acid receptor NPR3 is a negative regulator of the transcriptional defense response during early flower development in Arabidopsis. Mol Plant 6(3):802–816CrossRefPubMedGoogle Scholar
  50. Spoel SH, Dong XN (2012) How do plants achieve immunity? Defence without specialized immune cells. Nat Rev Immunol 12(2):89–100CrossRefPubMedGoogle Scholar
  51. Timmermans A (1942) Botrytis gladiolorum n. sp., the agent of the Botrytis rot of Gladioli. Nederlandsch kruidkd Archief 52:59–64Google Scholar
  52. Torres DP, Silva MA, Furtado GQ (2013) Infection of Curvularia gladioli on different gladiolus genotypes. Trop Plant Pathol 38(6):543–546CrossRefGoogle Scholar
  53. Vernooij B, Friedrich L, Ahl Goy P, Staub T, Kessmann H, Ryals J (1995) 2, 6-Dichloroisonicotinic acid-induced resistance to pathogens without the accumulation of salicylic acid. Mol Plant Microbe Interact 8(2):228–234CrossRefGoogle Scholar
  54. Walter M, Chaban C, Schütze K, Batistic O, Weckermann K, Näke C, Blazevic D, Grefen C, Schumacher K, Oecking C (2004) Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J 40(3):428–438CrossRefPubMedGoogle Scholar
  55. Wege S, Scholz A, Gleissberg S, Becker A (2007) Highly efficient virus-induced gene silencing (VIGS) in California poppy (Eschscholzia californica): an evaluation of VIGS as a strategy to obtain functional data from non-model plants. Ann Bot 100(3):641–649CrossRefPubMedCentralPubMedGoogle Scholar
  56. Yang Y, Li R, Qi M (2000) In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco leaves. Plant J 22(6):543–551CrossRefPubMedGoogle Scholar
  57. Yu DQ, Chen CH, Chen ZX (2001) Evidence for an important role of WRKY DNA binding proteins in the regulation of NPR1 gene expression. Plant Cell 13(7):1527–1540CrossRefPubMedCentralPubMedGoogle Scholar
  58. Zhang M, Wu HY, Tian ZQ, Zhang TY (2006) Three new records of Curvularia in China. J Henan Agric Univ 40(5):524–526Google Scholar
  59. Zhang JY, Qiao YS, Lv D, Gao ZH, Qu SC, Zhang Z (2012) Malus hupehensis NPR1 induces pathogenesis-related protein gene expression in transgenic tobacco. Plant Biol 14(s1):46–56CrossRefPubMedGoogle Scholar
  60. Zhong XH, Yuan X, Wu Z, Khan MA, Chen J, Li XX, Gong BH, Zhao Y, Wu J, Wu CY (2014) Virus-induced gene silencing for comparative functional studies in Gladiolus hybridus. Plant Cell Rep 33(2):301–312CrossRefPubMedGoogle Scholar
  61. Zhou JM, Trifa Y, Silva H, Pontier D, Lam E, Shah J, Klessig DF (2000) NPR1 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 acid. Mol Plant Microbe Interact 13(2):191–202CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Xionghui Zhong
    • 1
  • Lin Xi
    • 1
  • Qinglong Lian
    • 2
  • Xian Luo
    • 3
  • Ze Wu
    • 1
  • Shanshan Seng
    • 1
  • Xue Yuan
    • 4
  • Mingfang Yi
    • 1
    Email author
  1. 1.Department of Ornamental Horticulture and Landscape ArchitectureChina Agricultural UniversityBeijingChina
  2. 2.Institute of Facility AgricultureChinese Academy of Agricultural EngineeringBeijingChina
  3. 3.College of HorticultureSichuan Agricultural UniversityYa’anChina
  4. 4.National Library of ChinaBeijingChina

Personalised recommendations