Transgenic Research

, Volume 24, Issue 5, pp 859–873 | Cite as

Ectopic expression of GroEL from Xenorhabdus nematophila in tomato enhances resistance against Helicoverpa armigera and salt and thermal stress

  • Punam Kumari
  • Gagan Kumar Mahapatro
  • Nirupama BanerjeeEmail author
  • Neera Bhalla SarinEmail author
Original Paper


The GroEL homolog XnGroEL protein of Xenorhabdus nematophila belongs to a highly conserved family of molecular chaperones/heat shock proteins (Hsps). XnGroEL was shown to possess oral insecticidal activity against a major crop pest Helicoverpa armigera. Under normal conditions, the Hsps/chaperones facilitate folding, assembly, and translocation of cellular proteins, while in stress conditions they protect proteins from denaturation. In this study, we describe generation of transgenic tomato plants overexpressing insecticidal XnGroEL protein and their tolerance to biotic and abiotic stresses. Presence of XnGroEL in the transgenic tomato lines conferred resistance against H. armigera showing 100 % (p ≤ 0.001) mortality of neonates. In addition, XnGroEL provided thermotolerance and protection against high salt concentration to the tomato plants. Expression of XnGroEL minimized photo-oxidation of chlorophyll and reduced oxidative damage of cell membrane system of the plants under heat and salt stress. The enhanced tolerance to abiotic stresses correlated with increase in the anti-oxidative enzyme activity and reduced H2O2 accumulation in transgenic tomato plants. The variety of beneficial properties displayed by XnGroEL protein provides an opportunity for value addition and improvement of crop productivity.


Helicoverpa armigera Heat stress Insect resistance Insecticidal XnGroEL Salt stress Xenorhabdus nematophila 



The Grant from the Department of Biotechnology (No. BT/PR11260/PBD/16/819/2008) to Prof. Neera Bhalla Sarin and Dr. Nirupama Banerjee is gratefully acknowledged. P. K. acknowledges the financial support from the Council for Scientific and Industrial Research. Partial funds from Department of Science and Technology (D.S.T.-PURSE, D.S.T.-F.I.S.T.), U.G.C.-C.A.S., U.G.C.-R.N.W., and J.N.U are gratefully acknowledged.

Supplementary material

11248_2015_9881_MOESM1_ESM.ppt (53 kb)
Supplementary material 1 (PPT 53 kb)


  1. Aebi H (1984) Catalase in vitro. Methods Enzymol 105:121–126CrossRefPubMedGoogle Scholar
  2. Akhurst RJ, Dunphy GB, Beckage N, Thompson S, Federici B (1993) Parasites and pathogens of insects, vol 2. Academic, New York, pp 1–23Google Scholar
  3. Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55:373–399CrossRefPubMedGoogle Scholar
  4. Arnon DI (1949) Copper enzymes in isolated chloroplasts: polyphenol oxidase in Beta vulgaris. Plant Physiol 24:1–15PubMedCentralCrossRefPubMedGoogle Scholar
  5. Arora S, Kanojia AK, Kumar A, Mogha N, Sahu V (2012) Biopesticide formulation to control tomato lepidopteran pest menace. Curr Sci 102:1051–1057Google Scholar
  6. Arora S, Kanojia AK, Kumar A, Mogha N, Sahu V (2014) Biopesticide formulation to control tomato lepidopteran pest menace. Asian Agri-History 18:283–293Google Scholar
  7. Barr HD, Weatherley PE (1962) A re-examination of the relative turgidity technique for estimating water deficit in leaves. Aust J Biol Sci 15:413–428Google Scholar
  8. Bosl B, Grimminger V, Walter S (2006) The molecular chaperone Hsp104—a molecular machine for protein disaggregation. J Struct Biol 156:139–148CrossRefPubMedGoogle Scholar
  9. Boston RS, Viitanen PV, Vierling E (1996) Molecular chaperones and protein folding in plants. Plant Mol Biol 32:191–222CrossRefPubMedGoogle Scholar
  10. Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  11. Chen GX, Asada K (1989) Ascorbate peroxidase in tea leaves: occurrence of two isozymes and the differences in their enzymatic and molecular properties. Plant Cell Physiol 30:987–998Google Scholar
  12. Dhandapani N, Umeshchandra SR, Murugan M (2003) Bio-intensive pest management (BIPM) in major vegetable crops: an Indian perspective. Food Agric Environ 1:333–339Google Scholar
  13. Ferry N, Edwards MG, Gatehouse JA, Gatehouse MRA (2004) Plant-insect interactions: molecular approaches to insect resistance. Curr Opin Biotechnol 15:155–161CrossRefPubMedGoogle Scholar
  14. Fink AL (1999) Chaperone mediated protein folding. Physiol Rev 79:425–449PubMedGoogle Scholar
  15. Forreiter C, Kirschner M, Nover L (1997) Stable transformation of an Arabidopsis cell suspension culture with firefly luciferase providing a cellular system for analysis of chaperone activity in vivo. Plant Cell 9:2171–2181PubMedCentralCrossRefPubMedGoogle Scholar
  16. Forst S, Dowds B, Boemare N, Stackebrandt E (1997) Xenorhabdus and Photorhabdus spp.: bugs that kill bugs. Annu Rev Microbiol 51:47–72CrossRefPubMedGoogle Scholar
  17. Frydman J (2001) Folding of newly translated proteins in vivo: the role of molecular chaperones. Annu Rev Biochem 70:603–647CrossRefPubMedGoogle Scholar
  18. Gill SS, Tuteja N (2010) Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Biochem 48:909–939CrossRefPubMedGoogle Scholar
  19. Gill SS, Singh LP, Gill R, Tuteja N (2012) Generation and scavenging of reactive oxygen species in plants under stress. In: Tuteja N, Gill SS, Tiburcio AF, Tuteja R (eds) Improving crop resistance to abiotic stress. Wiley-VCH Verlag GmbH and Co. KGaA, Germany, pp 49–70CrossRefGoogle Scholar
  20. Goloubinoff P, De Los Rios P (2007) The mechanism of Hsp70 chaperones: (entropic) pulling the models together. Trends Biochem Sci 32:372–380CrossRefPubMedGoogle Scholar
  21. Gupta AS, Webb RP, Holaday AS, Allen RD (1993) Overexpression of superoxide dismutase protects plants from oxidative stress. Plant Physiol 103:1067–1073PubMedCentralPubMedGoogle Scholar
  22. Gupta GP, Birah A, Singh B, Mahapatro GK (2010) Methodology and composition of artificial diet for mass rearing of lepidopteran pests (in particular Helicoverpa armigera, Spodoptera litura and Earias vittella), Patent (IPA No. 1618/DEL/2008) The Patent Office Journal, Part I, 16th, pp 15Google Scholar
  23. Hall JL (2002) Cellular mechanisms for heavy metal detoxification and tolerance. J Exp Bot 53:1–11CrossRefPubMedGoogle Scholar
  24. Harbinson J, Genty B, Baker NR (1989) Relationship between the quantum efficiencies of photosystems I and II in pea leaves. Plant Physiol 90:1029–1034PubMedCentralCrossRefPubMedGoogle Scholar
  25. Hartl FU (1996) Molecular chaperones in cellular protein folding. Nature 381:571–580CrossRefPubMedGoogle Scholar
  26. Heath RL, Packer L (1968) Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125:189–198CrossRefPubMedGoogle Scholar
  27. Heckathorn SA, Downs CA, Sharkey TD, Coleman JS (1998) The small, methionine rich chloroplast heat shock protein protects photosystem II electron transport during heat stress. Plant Physiol 116:439–444PubMedCentralCrossRefPubMedGoogle Scholar
  28. Hendrick JP, Hartl FU (1993) Molecular chaperone functions of heat-shock proteins. Annu Rev Biochem 62:349–384CrossRefPubMedGoogle Scholar
  29. Herbert EE, Goodrich-Blair H (2007) Friend and foe: the two faces of Xenorhabdus nematophila. Nat Rev Microbiol 5:634–646CrossRefPubMedGoogle Scholar
  30. Hoagland DR, Arnon DI (1950) The water-culture method for growing plants without soil. Calif Agric Exp Station Circ 347:1–32Google Scholar
  31. Hofgen R, Willmitzer L (1988) Storage of competent cells for Agrobacterium transformation. Nucleic Acids Res 16:9877PubMedCentralCrossRefPubMedGoogle Scholar
  32. Huang XS, Liu JH, Chen XJ (2010) Overexpression of PtrABF gene, a bZIP transcription factor isolated from Poncirus trifoliata, enhances dehydration and drought tolerance in tobacco via scavenging ROS and modulating expression of stress-responsive genes. BMC Plant Biol 10:230PubMedCentralCrossRefPubMedGoogle Scholar
  33. Jiang C, Xu J, Zhang H, Zhang X, Shi J, Li M, Ming F (2009) A cytosolic class I small heat shock protein, RcHSP17.8, of Rosa chinensis confers resistance to a variety of stresses to Escherichia coli, yeast and Arabidopsis thaliana. Plant Cell Environ 32:1046–1059CrossRefPubMedGoogle Scholar
  34. Joshi MC (2007) Characterization of GroEL homolog of Xenorhabdus nematophila and evaluation of its insecticidal potential in crop pests. Ph.D Thesis, School of Biotechnology, Jawaharlal Nehru University, IndiaGoogle Scholar
  35. Joshi MC, Sharma A, Kant S, Birah A, Gupta GP, Khan SR, Bhatnagar R, Banerjee N (2008) An insecticidal GroEL protein with chitin binding activity from Xenorhabdus nematophila. J Biol Chem 283:28287–28296PubMedCentralCrossRefPubMedGoogle Scholar
  36. Kotak S, Vierling E, Baumlein H, Von Koskull-Doring P (2007) A novel transcriptional cascade regulating expression of heat stress proteins during seed development of Arabidopsis. Plant Cell 19:182–195PubMedCentralCrossRefPubMedGoogle Scholar
  37. Kumar D, Yusuf MA, Singh P, Sardar M, Sarin NB (2013) Modulation of antioxidant machinery in α-tocopherol enriched transgenic Brassica juncea plants tolerant to abiotic stress conditions. Protoplasma 250:1079–1089CrossRefPubMedGoogle Scholar
  38. Kumari P, Kant S, Zaman S, Mahapatro GK, Banerjee N, Sarin NB (2014) A novel insecticidal GroEL protein from Xenorhabdus nematophila confers insect resistance in tobacco. Transgenic Res 23:99–107CrossRefPubMedGoogle Scholar
  39. Lee GJ, Roseman AM, Saibil HR, Vierling E (1997) A small heat shock protein stably binds heat denatured model substrates and can maintain a substrate in a folding competent state. EMBO J 16:659–671PubMedCentralCrossRefPubMedGoogle Scholar
  40. Li S, Liu J, Liu Z, Li X, Wu F, He Y (2014) Heat induced TAS1 target1 mediates thermotolerance via heat stress transcription factor A1a-directed pathways in Arabidopsis. Plant Cell 26:1764–1780PubMedCentralCrossRefPubMedGoogle Scholar
  41. Lindquist S (1986) The heat-shock response. Annu Rev Biochem 55:1151–1191CrossRefPubMedGoogle Scholar
  42. Lindquist S, Craig EA (1988) The heat-shock proteins. Annu Rev Genet 22:631–677CrossRefPubMedGoogle Scholar
  43. Low D, Brandle K, Nover L, Forreiter C (2000) Cytosolic heat stress proteins Hsp17.7 class I and Hsp17.3 class II of tomato act as molecular chaperones in vivo. Planta 211:575–582CrossRefPubMedGoogle Scholar
  44. Madhulatha P, Pandey R, Hazarika P, Rajam MV (2007) High transformation frequency in Agrobacterium-mediated genetic transformation of tomato by using polyamines and maltose in shoot regeneration medium. Physiol Mol Biol Plants 13:191–198Google Scholar
  45. Mishra RK, Singhal GS (1992) Function of photosynthetic apparatus of intact wheat leaves under high light and heat stress and its relationship with peroxidation of thylakoid lipids. Plant Physiol 98:1–6PubMedCentralCrossRefPubMedGoogle Scholar
  46. Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7:405–410CrossRefPubMedGoogle Scholar
  47. Mittler R (2006) Abiotic stress, the field environment and stress combination. Trends Plant Sci 11:15–19CrossRefPubMedGoogle Scholar
  48. Mittler R, Vanderauwera S, Gollery M, Van Breusegem F (2004) Reactive oxygen gene network of plants. Trends Plant Sci 9:490–498CrossRefPubMedGoogle Scholar
  49. Mu C, Zhang S, Yu G, Chen N, Li X et al (2013) Overexpression of small heat shock protein LimHSP 16.45 in Arabidopsis enhances tolerance to abiotic stresses. PLoS ONE 8(12):e82264. doi: 10.1371/journal.pone.0082264 PubMedCentralCrossRefPubMedGoogle Scholar
  50. Murakami T, Matsuba S, Funatsuki H, Kawaguchi K, Saruyama H, Tanida M, Sato Y (2004) Over-expression of a small heat shock protein, sHSP17.7, confers both heat tolerance and UV-B resistance to rice plants. Mol Breeding 13:165–175CrossRefGoogle Scholar
  51. Ogawa D, Yamaguchi K, Nishiuchi T (2007) High-level overexpression of the Arabidopsis HsfA2 gene confers not only increased themotolerance but also salt/osmotic stress tolerance and enhanced callus growth. J Exp Bot 58:3373–3383CrossRefPubMedGoogle Scholar
  52. Ouyang SQ, Liu YF, Liu P, Lei G, He SJ, Ma B, Zhang WK, Zhang JS, Chen SY (2010) Receptor-like kinase OsSIK1 improves drought and salt stress tolerance in rice (Oryza sativa) plants. Plant J 62:316–329CrossRefPubMedGoogle Scholar
  53. Padmanaban N, Arora R (2002) Field evaluation of native NPV for the management of tomato fruit borer Helicoverpa armigera. Pestic Res J 14:113–119Google Scholar
  54. Personat JM, Tejedor-Cano J, Prieto-Dapena P, Almoguera C, Jordano J (2014) Co-overexpression of two heat shock factors results in enhanced seed longevity and in synergistic effects on seedling tolerance to severe dehydration and oxidative stress. BMC Plant Biol 14:56PubMedCentralCrossRefPubMedGoogle Scholar
  55. Queitsch C, Hong SW, Vierling E, Lindquist S (2000) Heat shock protein 101 plays a crucial role in thermotolerance in Arabidopsis. Plant Cell 12:479–492PubMedCentralCrossRefPubMedGoogle Scholar
  56. Reed W, Pawar CS (1982) Heliothis: a global problem. In: Proceedings of the international workshop on Heliothis management, Nov 15–20 1981, ICRISAT, Patancheru, pp 9–14Google Scholar
  57. Rogers SO, Bendich AJ (1994) Extraction of total cellular DNA from plants, algae and fungi. Plant Mol. Biol. Manual D1:1–8Google Scholar
  58. Ruibal C, Castro A, Carballo V, Szabados L, Vidal S (2013) Recovery from heat, salt, and osmotic stress in Physcomitrella patens requires a functional small heat shock protein PpHsp16.4. BMC Plant Biol 13:174PubMedCentralCrossRefPubMedGoogle Scholar
  59. Sairam RK, Srivastava GC (2002) Changes in antioxidant activity in subcellular fractions of tolerant and susceptible wheat genotypes in response to long term salt stress. Plant Sci 162:897–904CrossRefGoogle Scholar
  60. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor, New YorkGoogle Scholar
  61. Scharf KD, Siddique M, Vierling E (2001) The expanding family of Arabidopsis thaliana small heat stress proteins and a new family of proteins containing alpha-crystallin domains (Acd proteins). Cell Stress Chaperones 6:225–237PubMedCentralCrossRefPubMedGoogle Scholar
  62. Schramm F, Ganguli A, Kiehlmann E, Englich G, Walch D, Von Koskull-Doring P (2006) The heat stress transcription factor HsfA2 serves as a regulatory amplifier of a subset of genes in the heat stress response in Arabidopsis. Plant Mol Biol 60:759–772CrossRefPubMedGoogle Scholar
  63. Shi WM, Muramoto Y, Ueda A, Takabe T (2001) Cloning of peroxisomal ascorbate peroxidase gene from barley and enhanced thermotolerance by overexpressing in Arabidopsis thaliana. Gene 273:23–27CrossRefPubMedGoogle Scholar
  64. Song NH, Ahn YJ (2011) DcHsp17.7, a small heat shock protein in carrot, is tissue-specifically expressed under salt stress and confers tolerance to salinity. N Biotechnol 28:698–704CrossRefPubMedGoogle Scholar
  65. Swindell WR, Huebner M, Weber AP (2007) Transcriptional profiling of Arabidopsis heat shock proteins and transcription factors reveals extensive overlap between heat and non-heat stress response pathways. BMC Genom 8:125CrossRefGoogle Scholar
  66. Torok Z, Goloubinoff P, Horvath I, Tsvetkova NM, Glatz A, Balogh G, Varvasovszki V, Los DA, Vierling E, Crowe JH, Vigh L (2001) Synechocystis HSP17 is an amphitropic protein that stabilizes heat-stressed membranes and binds denatured proteins for subsequent chaperone-mediated refolding. Proc Natl Acad Sci USA 98:3098–3103PubMedCentralCrossRefPubMedGoogle Scholar
  67. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354PubMedCentralCrossRefPubMedGoogle Scholar
  68. Tripp J, Mishra SK, Scharf KD (2009) Functional dissection of the cytosolic chaperone network in tomato mesophyll protoplasts. Plant, Cell Environ 32:123–133CrossRefGoogle Scholar
  69. Velikova V, Edreva A, Loreto F (2004) Endogenous isoprene protects Phragmites australis leaves against singlet oxygen. Physiol Plant 122:219–225CrossRefGoogle Scholar
  70. Vierling E (1991) The roles of heat shock proteins in plants. Annu Rev Plant Physiol Plant Mol Biol 42:579–620CrossRefGoogle Scholar
  71. Vinocur B, Altman A (2005) Recent advances in engineering plant tolerance toabiotic stress: achievements and limitations. Curr Opin Biotechnol 16:123–132CrossRefPubMedGoogle Scholar
  72. Wang W, Vinocur B, Altman A (2003) Plant responses to drought, salinity, and extreme temperatures: towards genetic engineering for stress tolerance. Planta 218:1–14CrossRefPubMedGoogle Scholar
  73. Wang W, Vinocur B, Shoseyov O, Altman A (2004) Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. Trends Plant Sci 9:244–252CrossRefPubMedGoogle Scholar
  74. Waters ER, Lee GJ, Vierling E (1996) Evolution, structure and function of the small heat shock proteins in plants. J Exp Bot 47:325–338CrossRefGoogle Scholar
  75. Whalon M, Mota-sanchez D, Hollongworth L, Duynslager L (2004) Arthopod pesticide resistance database, (Michigan State University, East Lansing, MI). Accessed 21 April 2009
  76. Xiong L, Schumaker KS, Zhu JK (2002) Cell signaling during cold, drought, and salt stress. Plant Cell 14:165–183CrossRefGoogle Scholar
  77. Yeh CH, Chen YM, Lin CY (2002) Functional regions of rice heat shock protein, Oshsp16.9, required for conferring thermotolerance in Escherichia coli. Plant Physiol 128:661–668PubMedCentralCrossRefPubMedGoogle Scholar
  78. Zhu JK (2001) Plant salt tolerance. Trends Plant Sci 6:66–72CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2015

Authors and Affiliations

  1. 1.School of Life SciencesJawaharlal Nehru UniversityNew DelhiIndia
  2. 2.Division of EntomologyIndian Agricultural Research InstituteNew DelhiIndia

Personalised recommendations