Molecular Genetics and Genomics

, Volume 282, Issue 1, pp 65–81

A mitogen-activated protein kinase gene, AhMPK3 of peanut: molecular cloning, genomic organization, and heterologous expression conferring resistance against Spodoptera litura in tobacco

  • Koppolu Raja Rajesh Kumar
  • Tantravahi Srinivasan
  • Pulugurtha Bharadwaja Kirti
Original Paper


Mitogen-activated protein kinase cascade plays a very important role in plant signal transduction mechanism. A full length cDNA of 1,514 bp length, corresponding to a mitogen-activated protein kinase gene was cloned from peanut (Arachis hypogaea). Based on its high homology with ArabidopsisAtMPK3, the cDNA was designated as AhMPK3. It carried an open reading frame of 1,113 bp encoding a 371 amino acid polypeptide. AhMPK3 bears TEY motif in its activation loop and belongs to the A1 subgroup of MAPK family. Southern blot analysis revealed that AhMPK3 exists in two copies in peanut genome and its structural organization revealed well-conserved nature of these signaling components across different species. AhMPK3 when transiently expressed in tobacco leaves was found to localize in both nucleus and cytoplasm. Transgenic tobacco plants ectopically expressing AhMPK3 exhibited enhanced resistance to first and second instar larvae of Spodoptera litura and constitutively higher transcript levels of defense response genes like PR1a, PR1b, LOX1, PIII etc. Apart from this when wounded, transgenic plants accumulated high levels of PIII and PR1b transcripts rapidly compared to wild type indicating the occurrence of a priming phenomenon.


Arachis hypogaea MAPK Transgenic tobacco Herbivore resistance Protease inhibitor-II 

Supplementary material

438_2009_446_MOESM1_ESM.pdf (1.2 mb)
Supplementary material 1 (PDF 1.23 MB)


  1. Agrawal GK, Iwahashi H, Rakwal R (2003) Rice MAPKs. Biochem Biophys Res Commun 302:171–180PubMedCrossRefGoogle Scholar
  2. Ahlfors R, Macioszek V, Rudd J, Brosché M, Schlichting R, Scheel D, Kangasjärvi J (2004) Stress hormone-independent activation and nuclear translocation of mitogen-activated protein kinases in Arabidopsis thaliana during ozone exposure. Plant J 40:512–522PubMedCrossRefGoogle Scholar
  3. Balandin T, Van Der Does C, Albert JM, Bol JF, Linthorst HJ (1995) Structure and induction pattern of a novel proteinase inhibitor class II gene of tobacco. Plant Mol Biol 27:1197–1204PubMedCrossRefGoogle Scholar
  4. Beckers GJ, Conrath U (2007) Priming for stress resistance: from the lab to the field. Curr Opin Plant Biol 10:425–431PubMedCrossRefGoogle Scholar
  5. Bögre L, Ligterink W, Meskiene I, Barker PJ, Heberle-Bors E, Huskisson NS, Hirt H (1997) Wounding induces the rapid and transient activation of a specific MAP kinase pathway. Plant Cell 9:75–83PubMedCrossRefGoogle Scholar
  6. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  7. Brunet A, Roux D, Lenormand P, Dowd S, Keyse S, Pouysségur J (1999) Nuclear translocation of p42/p44 mitogen-activated protein kinase is required for growth factor-induced gene expression and cell cycle entry. EMBO J 18:664–674PubMedCrossRefGoogle Scholar
  8. Catinot J, Buchala A, Abou-Mansour E, Me’traux JP (2008) Salicylic acid production in response to biotic and abiotic stress depends on isochorismate in Nicotiana benthamiana. FEBS Lett doi:10.1016/j.febslet.2007.12.039
  9. Cheong YH, Moon BC, Kim JK, Kim CY, Kim MC, Kim IH, Park CY, Kim JC, Park BO, Koo SC, Yoon HW, Chung WS, Lim CO, Lee SY, Cho MJ (2003) BWMK1, a rice mitogen-activated protein kinase, locates in the nucleus and mediates pathogenesis-related gene expression by activation of a transcription factor. Plant Physiol 132:1961–1972PubMedCrossRefGoogle Scholar
  10. Cobb MH, Goldsmith EJ (2000) Dimerization in MAP-kinase signaling. Trends Biochem Sci 25:7–9PubMedCrossRefGoogle Scholar
  11. Colcombet J, Hirt H (2008) Arabidopsis MAPKs: a complex signalling network involved in multiple biological processes. Biochem J 413:217–226PubMedCrossRefGoogle Scholar
  12. Cutler SR, Ehrhardt DW, Griffitts JS, Somerville CR (2000) Random GFP::cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency. Proc Natl Acad Sci USA 97:3718–3723PubMedCrossRefGoogle Scholar
  13. Datla RSS, Hammerlindl JK, Panchuk B, Pelcher LE, Keller WA (1992) Modified binary plant transformation vectors with the wild-type gene encoding NPTII. Gene 21:383–384Google Scholar
  14. Davis R (2000) Signal transduction by the JNK group of MAP kinases. Cell 103:239–252PubMedCrossRefGoogle Scholar
  15. Daxberger A, Nemak A, Mithöfer A, Fliegmann J, Ligterink W, Hirt H, Ebel J (2007) Activation of members of a MAPK module in β-glucan elicitor-mediated non-host resistance of soybean. Planta 225:1559–1571PubMedCrossRefGoogle Scholar
  16. Djamei A, Pitzschke A, Nakagami H, Rajh I, Hirt H (2007) Trojan horse strategy in Agrobacterium transformation: abusing MAPK defense signaling. Science 318:453–456PubMedCrossRefGoogle Scholar
  17. Frost CJ, Mescher MC, Carlson JE, De Moraes CM (2008) Plant defense priming against herbivores: getting ready for a different battle. Plant Physiology 146:818–824PubMedCrossRefGoogle Scholar
  18. Guo B, Chen X, Dang P, Scully BT, Liang X, Holbrook CC, Yu J and Culbreath AK (2008) Peanut gene expression profiling in developing seeds at different reproduction stages during Aspergillus parasiticus infection. BMC Developmental Biology 8, art. no. 12.
  19. Halitschke R, Baldwin IT (2003) Antisense LOX expression increases herbivore performance by decreasing defense responses and inhibiting growth-related transcriptional reorganization in Nicotiana attenuata. Plant J 36:794–807PubMedCrossRefGoogle Scholar
  20. Hanks SK, Quinn AM, Hunter T (1988) The protein kinase family:conserved features and deduced phylogeny of the catalytic domains. Science 24:42–52CrossRefGoogle Scholar
  21. Holley SR, Yalamanchili RD, Moura DS, Ryan CA, Stratmann JW (2003) Convergence of signaling pathways induced by systemin, oligosaccharide elicitors, and ultraviolet-B radiation at the level of mitogen-activated protein kinases in Lycopersicon peruvianum suspension-cultured cells. Plant Physiol 132:1728–1738PubMedCrossRefGoogle Scholar
  22. Holsters M, De Waele D, Depicker A (1978) Transfection and transformation of Agrobacterium tumefaciens. Mol Gen Genet 163:181–187PubMedCrossRefGoogle Scholar
  23. Hord CLH, Yu-Jin Sun Y-J, Pillitteri LJ, Torii KU, Wang H, Zhang S, and Ma H (2008) Regulation of Arabidopsis early anther development by the mitogen-activated protein kinases, MPK3 and MPK6, and the ERECTA and related receptor-like kinases. Mol Plant, doi:10.1093/mp/ssn029
  24. Horsch RB, Fry JE, Hofmann NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:229–1231Google Scholar
  25. Jonak C, Kiegerl S, Ligterink W, Barker PJ, Huskisson NS, Hirt H (1996) Stress signalling in plants: a mitogen-activated protein kinase pathway is activated by cold and drought. Proc Natl Acad Sci USA 93:11274–11279PubMedCrossRefGoogle Scholar
  26. Jonak C, Okresz L, Bogre L, Hirt H (2002) Complexity, crosstalk and integration of plant MAP kinase signaling. Curr Opin Plant Biol 5:415–424PubMedCrossRefGoogle Scholar
  27. Jonak C, Nakagami H, Hirt H (2004) Heavy metal stress. Activation of distinct mitogen-activated protein kinase pathways by copper and cadmium. Plant Physiol 136:3276–3283PubMedCrossRefGoogle Scholar
  28. Kandoth PK, Ranf S, Pancholi SS, Jayanty S, Walla MD, Miller W, Howe GA, Lincoln DE, Stratmann JW (2007) Tomato MAPKs LeMPK1, LeMPK2, and LeMPK3 function in the systemin-mediated defense response against herbivorous insects. Proc Natl Acad Sci USA 104:12205–12210PubMedCrossRefGoogle Scholar
  29. Kim CY, Liu Y, Thorne ET, Yang H, Fukushige H, Gassmann W, Hildebrand D, Sharp RE, Zhang S (2003) Activation of a stress-responsive mitogen-activated protein kinase cascade induces the biosynthesis of ethylene in plants. Plant cell 15:2707–2718PubMedCrossRefGoogle Scholar
  30. Ko CH, Brendel V, Taylor RD, Walbot V (1998) U-richness is a defining feature of plant introns and may function as an intron recognition signal in maize. Plant Mol Biol 36:573–583PubMedCrossRefGoogle Scholar
  31. Koiwa H, Shade RE, Zhu-Salzman K, D’Urzo MP, Murdock LL, Bressan RA, Hasegawa PM (2000) A plant defensive cystatin (soyacystatin) targets cathepsin 1-like digestive cysteine proteinases (DvCALs) in the larval midgut of western corn rootworm (Diabrotica virgifera virgifera). FEBS Lett 471:67–70PubMedCrossRefGoogle Scholar
  32. Kovtun Y, Chiu WL, Tena G, Sheen J (2000) Functional analysis of oxidative stress activated mitogen-activated protein kinase cascades in plants. Proc Natl Acad Sci USA 97:2940–2945PubMedCrossRefGoogle Scholar
  33. Lee DE, Lee IJ, Han O, Baik MG, Han SS, Back K (2004a) Pathogen resistance of transgenic rice plants expressing mitogen-activated protein kinase 1, MK1, from Capsicum annuum. Mol Cells 17:81–85PubMedGoogle Scholar
  34. Lee J, Rudd JJ, Macioszek VK, Scheel D (2004b) Dynamic changes in the localization of MAP kinase cascade components controlling pathogenesis-related (PR) gene expression during innate immunity in parsley. J Biol Chem 279:22440–22448PubMedCrossRefGoogle Scholar
  35. Ligterink W, Kroj T, zur Nieden U, Hirt H, Scheel D (1997) Receptor-mediated activation of a MAP kinase in pathogen defense of plants. Science 276:2054–2057PubMedCrossRefGoogle Scholar
  36. Liu Y, Jin H, Yang KY, Kim CY, Baker B, Zhang S (2003) Interaction between two mitogen-activated protein kinases during tobacco defense signaling. Plant J 34:149–160PubMedCrossRefGoogle Scholar
  37. Luo M, Dang P, Guo BZ, He G, Holbrook CC, Bausher MG, Lee RD (2005) Generation of expressed sequence tags (ESTs) for gene discovery and marker development in cultivated peanut. Crop Sci 45:346–353CrossRefGoogle Scholar
  38. MAPK group (2002) Mitogen-activated protein kinase cascades in plants: a new nomenclature. Trends Plants Sci 7:301–308CrossRefGoogle Scholar
  39. Mayrose M, Bonshtien A, Sessa G (2004) LeMPK3 is a mitogen-activated protein kinase with dual specificity induced during tomato defense and wounding responses. J Biol Chem 279:14819–14827PubMedCrossRefGoogle Scholar
  40. Mizoguchi T, Irie K, Hirayama T, Hayashida N, Yamaguchi-Shinozaki K, Matsumoto K, Shinozaki K (1996) A gene encoding a mitogen-activated protein kinase kinase kinase is induced simultaneously with genes for a mitogen-activated protein kinase and S6 ribosomal protein kinase by touch, cold, and water stress in Arabidopsis thaliana. Proc Natl Acad Sci USA 93:765–769PubMedCrossRefGoogle Scholar
  41. Moon H, Lee B, Choi G et al (2003) NDP kinase 2 interacts with two oxidative stress-activated MAPKs to regulate cellular redox state and enhances multiple stress tolerance in transgenic plants. Proc Natl Acad Sci USA 100:358–363PubMedCrossRefGoogle Scholar
  42. Morris PC (2001) MAP kinase signal transduction pathways in plants. New Phytol 151:67–89CrossRefGoogle Scholar
  43. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  44. Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucl Acids Res 8:4321–4325PubMedCrossRefGoogle Scholar
  45. Nicole MC, Hamel LP, Morency MJ, Beaudoin N, Ellis BE, Séguin A (2006) MAP-ping genomic organization and organ-specific expression profiles of poplar MAP kinases and MAP kinase kinases. BMC Genomics 7, art. no. 223.
  46. Orr GL, Strickland JA, Walsh TA (1994) Inhibition of Diabrotica larval growth by a multicystatin from potato-tubers. J Insect Physiol 40:893–900CrossRefGoogle Scholar
  47. Pokholok DK, Zeitlinger J, Hannett NM, Reynolds DB, Young RA (2006) Activated signal transduction kinases frequently occupy target genes. Science 313:533–536PubMedCrossRefGoogle Scholar
  48. Qiu JL, Fiil BK, Petersen K, Nielsen HB, Botanga CJ, Thorgrimsen S, Palma K, Petersen M (2008) Arabidopsis MAP kinase 4 regulates gene expression through transcription factor release in the nucleus. EMBO J 27:2214–2221PubMedCrossRefGoogle Scholar
  49. Rayapuram C, Baldwin IT (2006) Using nutritional indices to study LOX3-dependent insect resistance. Plant Cell Environ 29:1585–1594PubMedCrossRefGoogle Scholar
  50. Ren D, Liu Y, Yang KY, Han L, Mao G, Glazebrook J, Zhang S (2008) A fungal-responsive MAPK cascade regulates phytoalexin biosynthesis in Arabidopsis. Proc Natl Acad Sci USA 105:5638–5643PubMedCrossRefGoogle Scholar
  51. Reyna NS, Yang Y (2006) Molecular analysis of the rice MAP kinase gene family in relation to Magnaporthe grisea infection. Mol Plant Microbe Interact 19:530–540PubMedCrossRefGoogle Scholar
  52. Romeis T, Piedras P, Zhang S, Klessig DF, Hirt H, Jones JD (1999) Rapid Avr9- and Cf-9-dependent activation of MAP kinases in tobacco cell cultures and leaves: convergence of resistance gene, elicitor, wound, and salicylate responses. Plant Cell 11:273–287PubMedCrossRefGoogle Scholar
  53. Samuel MA, Miles GP, Ellis BE (2000) Ozone treatment rapidly activates MAP kinase signalling in plants. Plant J 22:367–376PubMedCrossRefGoogle Scholar
  54. Seo S, Okamoto M, Seto H, Ishizuka K, Sano H, Ohashi Y (1995) Tobacco MAP kinase: a possible mediator in wound signal transduction pathway. Science 270:1988–1992PubMedCrossRefGoogle Scholar
  55. Seo S, Sano H, Ohashi Y (1999) Jasmonate-based wound signal transduction requires activation of WIPK, a tobacco mitogen-activated protein kinase. Plant Cell 11:289–298PubMedCrossRefGoogle Scholar
  56. Seo S, Katou S, Seto H, Gomi K, Ohashi Y (2007) The mitogen-activated protein kinases WIPK and SIPK regulate the levels of jasmonic and salicylic acids in wounded tobacco plants. Plant J 49:899–909PubMedCrossRefGoogle Scholar
  57. Sharma PC, Ito A, Shimizu T, Terauchi R, Kamoun S, Saitoh H (2003) Virus-induced silencing of WIPK and SIPK genes reduces resistance to a bacterial pathogen, but has no effect on the INF1-induced hypersensitive response (HR) in Nicotiana benthamiana. Mol Gen Genomics 269:583–591CrossRefGoogle Scholar
  58. Sharma HC, Pampapathy G, Dhillon MK, Ridsdill-Smith JT (2005) Detached leaf assay to screen for host plant resistance to Helicoverpa armigera. J Econ Entomol 98:568–576PubMedCrossRefGoogle Scholar
  59. Shoji T, Nakajima K, Hashimoto T (2000) Ethylene suppresses Jasmonate-induced gene expression in nicotine biosynthesis. Plant Cell Physiol 41:1072–1076PubMedCrossRefGoogle Scholar
  60. Shoresh M, Gal-On A, Leibman D, Chet I (2006) Characterization of a mitogen-activated protein kinase gene from cucumber required for trichoderma-conferred plant resistance. Plant Physiol 142:1169–1179PubMedCrossRefGoogle Scholar
  61. Stulemeijer IJE, Stratmann JW, Joosten MHAJ (2007) The tomato MAP kinases LeMPK1, -2 and -3 are activated during the Cf-4/Avr4-induced HR and have distinct phosphorylation specificities. Plant Physiol 144:1481–1494PubMedCrossRefGoogle Scholar
  62. Töpfer R, Matzeit V, Gronenborn B, Schell J, Steinbiss HH (1987) A set of plant expression vectors for transcriptional and translational fusions. Nucleic Acids Res 15:5890PubMedCrossRefGoogle Scholar
  63. Wan J, Zhang S, Stacey G (2004) Activation of a mitogen-activated protein kinase pathway in Arabidopsis by chitin. Mol Plant Pathol 5:125–135CrossRefPubMedGoogle Scholar
  64. Wang H, Ngwenyama N, Liu Y, Walker JC, Zhang S (2007) Stomatal development and patterning are regulated by environmentally responsive mitogen-activated protein kinases in Arabidopsis. Plant Cell 19:63–73PubMedCrossRefGoogle Scholar
  65. Wang H, Liu Y, Bruffett K, Lee J, Hause G, Walker JC, Zhang S (2008) Haplo-Insufficiency of MPK3 in MPK6 mutant background uncovers a novel function of these two MAPKs in Arabidopsis ovule development. Plant Cell 20:602–613PubMedCrossRefGoogle Scholar
  66. Widmann C, Gibson S, Jarpe MB, Johnson GL (1999) Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human. Physiol Rev 79:143–180PubMedGoogle Scholar
  67. Wildermuth MC, Dewdney J, Wu G, Ausubel FM (2001) Isochorismate synthase isrequired to synthesize salicylic acid for plant defence. Nature 414:562–571PubMedCrossRefGoogle Scholar
  68. Wu J, Hettenhausen C, Meldau S, Baldwin IT (2007) Herbivory rapidly activates MAPK signaling in attacked and unattacked leaf regions but not between leaves of Nicotiana attenuate. Plant Cell 19:1096–1122PubMedCrossRefGoogle Scholar
  69. Yang Y, Li R, Qi M (2000) In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco leaves. Plant J 22:543–551PubMedCrossRefGoogle Scholar
  70. Yap YK, Kodama Y, Waller F, Chung KM, Ueda H, Nakamura K, Oldsen M, Yoda H, Yamaguchi Y, Sano H (2005) Activation of a novel transcription factor through phosphorylation by WIPK, a wound-induced mitogen activated protein kinase in tobacco plants. Plant Physiol 139:127–137PubMedCrossRefGoogle Scholar
  71. Zhang S, Klessig DF (1998) Resistance gene N-mediated de novo synthesis and activation of a tobacco mitogen-activated protein kinase by tobacco mosaic virus infection. Proc Natl Acad Sci USA 95:7433–7438PubMedCrossRefGoogle Scholar
  72. Zhang S, Du H, Klessig DF (1998) Activation of the tobacco SIP kinase by both a cell wall-derived carbohydrate elicitor and purified proteinaceous elicitins from Phytophthora spp. Plant Cell 10:435–450PubMedCrossRefGoogle Scholar
  73. Zhang S, Liu Y, Klessig DF (2000) Multiple levels of tobacco WIPK activation during the induction of cell death by fungal elicitins. Plant J 23:339–347PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Koppolu Raja Rajesh Kumar
    • 1
  • Tantravahi Srinivasan
    • 1
  • Pulugurtha Bharadwaja Kirti
    • 1
  1. 1.Department of Plant SciencesUniversity of HyderabadHyderabadIndia

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