, Volume 230, Issue 2, pp 277–291 | Cite as

Defense-related gene expression and enzyme activities in transgenic cotton plants expressing an endochitinase gene from Trichoderma virens in response to interaction with Rhizoctonia solani

  • Vinod Kumar
  • Vilas Parkhi
  • Charles M. Kenerley
  • Keerti S. Rathore
Original Article


There are many reports on obtaining disease-resistance trait in plants by overexpressing genes from diverse organisms that encode chitinolytic enzymes. Current study represents an attempt to dissect the mechanism underlying the resistance to Rhizoctonia solani in cotton plants expressing an endochitinase gene from Trichoderma virens. Several assays were developed that provided a powerful demonstration of the disease protection obtained in the transgenic cotton plants. Transgene-dependent endochitinase activity was confirmed in various tissues and in the medium surrounding the roots of transformants. Biochemical and molecular analyses conducted on the transgenic plants showed rapid/greater induction of ROS, expression of several defense-related genes, and activation of some PR enzymes and the terpenoid pathway. Interestingly, even in the absence of a challenge from the pathogen, the basal activities of some of the defense-related genes and enzymes were higher in the endochitinase-expressing cotton plants. This elevated defensive state of the transformants may act synergistically with the potent, transgene-encoded endochitinase activity to confer a strong resistance to R. solani infection.


Chitinase Defense Disease resistance Rhizoctonia solani Transgenic cotton Trichoderma virens 



4-Methylumbelliferyl-β-d-N,N′, N″-triacetylchitotrioside




δ-Cadinene synthase






2,7-Dichlorofluorescin diacetate


Hours post-inoculation








Pathogenesis-related protein 1


Reactive oxygen species





We thank Drs. Robert D. Stipanovic and Lorraine Puckhaber for their help with terpenoid analysis and Drs. Alois Bell and Charles Howell for their valuable suggestions and advice during the course of this investigation. This research was supported by funds from Texas Higher Education Coordinating Board – Advanced Research Program (#000517-0005-2006), Cotton Inc., and Texas AgriLife Research.

Supplementary material

425_2009_937_MOESM1_ESM.pdf (5.4 mb)
Supplementary material 1 (PDF 5479 kb)


  1. Abeles FB, Forrence LE (1970) Temporal and hormonal control of beta-1, 3-glucanase in Phaseolus vulgaris L. Plant Physiol 45:395–400PubMedCrossRefGoogle Scholar
  2. Alvarez ME, Pennell RI, Meijer PJ, Ishikawa A, Dixon RA, Lamb C (1998) Reactive oxygen intermediates mediate a systemic signal network in the establishment of plant immunity. Cell 92:773–784PubMedCrossRefGoogle Scholar
  3. Baek JM, Howell CR, Kenerley CM (1999) The role of an extracellular chitinase from Trichoderma virens Gv29-8 in the biocontrol of Rhizoctonia solani. Curr Genet 35:41–50PubMedCrossRefGoogle Scholar
  4. Baranski R, Klocke E, Nothnagel T (2008) Chitinase CHIT36 from Trichoderma harzianum enhances resistance of transgenic carrot to fungal pathogens. J Phytopathol 156:513–521CrossRefGoogle Scholar
  5. Barber MS, Bertram RE, Ride JP (1989) Chitin oligosaccharides elicit lignification in wounded wheat leaves. Physiol Mol Plant P 34:3–12CrossRefGoogle Scholar
  6. Bell AA (1967) Formation of gossypol in infected or chemically irritated tissues of gossypium species. Phytopathology 57:759–764Google Scholar
  7. Bell AA (1969) Phytoalexin production and Verticillium wilt resistance in cotton. Phytopathology 59:1119–1127Google Scholar
  8. Benhamou N, Asselin A (1989) Attempted localization of a substrate for chitinases in plant cells reveals abundant N-acetyl-d-glucosamine residues in secondary walls. Biol Cell 67:341–350CrossRefGoogle Scholar
  9. Benhamou N, Broglie K, Broglie R, Chet I (1993a) Antifungal effect of bean endochitinase on Rhizoctonia solani: ultrastructural changes and cytochemical aspects of chitin breakdown. Can J Microbiol 39:318–328PubMedCrossRefGoogle Scholar
  10. Benhamou N, Broglie K, Chet I, Broglie R (1993b) Cytology of infection of 35S-bean chitinase transgenic canola plants by Rhizoctonia solani—cytochemical aspects of chitin breakdown in vivo. Plant J 4:295–305CrossRefGoogle Scholar
  11. Bergmeyer HU (1974) Methods of enzymatic analysis. Academic Press, New York, p 495Google Scholar
  12. Bindschedler LV, Dewdney J, Blee KA, Stone JM, Asai T, Plotnikov J, Denoux C, Hayes T, Gerrish C, Davies DR, Ausubel FM, Bolwell GP (2006) Peroxidase-dependent apoplastic oxidative burst in Arabidopsis required for pathogen resistance. Plant J 47:851–863PubMedCrossRefGoogle Scholar
  13. Bolar JP, Norelli JL, Wong KW, Hayes CK, Harman GE, Aldwinckle HS (2000) Expression of endochitinase from Trichoderma harzianum in transgenic apple increases resistance to apple scab and reduces vigor. Phytopathology 90:72–77PubMedCrossRefGoogle Scholar
  14. Bolar JP, Norelli JL, Harman GE, Brown SK, Aldwinckle HS (2001) Synergistic activity of endochitinase and exochitinase from Trichoderma atroviride (T. harzianum) against the pathogenic fungus (Venturia inaequalis) in transgenic apple plants. Transgenic Res 10:533–543PubMedCrossRefGoogle Scholar
  15. Broglie K, Chet I, Holliday M, Cressman R, Biddle P, Knowlton S, Mauvais CJ, Broglie R (1991) Transgenic plants with enhanced resistance to the fungal pathogen Rhizoctonia solani. Science 254:1194–1197PubMedCrossRefGoogle Scholar
  16. Carsolio C, Benhamou N, Haran S, Cortes C, Gutierrez A, Chet I, Herrera-Estrella A (1999) Role of the Trichoderma harzianum endochitinase gene, ech42, in mycoparasitism. Appl Environ Microbiol 65:929–935PubMedGoogle Scholar
  17. Collinge DB, Kragh KM, Mikkelsen JD, Nielsen KK, Rasmussen U, Vad K (1993) Plant chitinases. Plant J 3:31–40PubMedCrossRefGoogle Scholar
  18. Dana MD, Pintor-Toro JA, Cubero B (2006) Transgenic tobacco plants overexpressing chitinases of fungal origin show enhanced resistance to biotic and abiotic stress agents. Plant Physiol 142:722–730CrossRefGoogle Scholar
  19. De Jong AJ, Cordewener J, Lo Schiavo F, Terzi M, Vandekerckhove J, Van Kammen A, De Vries SC (1992) A carrot somatic embryo mutant is rescued by chitinase. Plant Cell 4:425–433PubMedCrossRefGoogle Scholar
  20. De Marco JL, Lima LHC, de Sousa MV, Felix CR (2000) A Trichoderma harzianum chitinase destroys the cell wall of the phytopathogen Crinipellis perniciosa, the causal agent of witches’ broom disease of cocoa. World J Microbiol Biotechnol 16:383–386CrossRefGoogle Scholar
  21. Delannoy E, Jalloul A, Assigbetse K, Marmey P, Geiger JP, Lherminier J, Daniel JF, Martinez C, Nicole M (2003) Activity of class III peroxidases in the defense of cotton to bacterial blight. Mol Plant Microbe Interact 16:1030–1038PubMedCrossRefGoogle Scholar
  22. Distefano G, La Malfa S, Vitale A, Lorito M, Deng Z, Gentile A (2008) Defence-related gene expression in transgenic lemon plants producing an antimicrobial Trichoderma harzianum endochitinase during fungal infection. Transgenic Res 17:873–879PubMedCrossRefGoogle Scholar
  23. Djonovic S, Pozo MJ, Dangott LJ, Howell CR, Kenerley CM (2006) Sm1, a proteinaceous elicitor secreted by the biocontrol fungus Trichoderma virens induces plant defense responses and systemic resistance. Mol Plant Microbe Interact 19:838–853PubMedCrossRefGoogle Scholar
  24. Emani C, Garcia JM, Lopata-Finch E, Pozo MJ, Uribe P, Kim DJ, Sunilkumar G, Cook DR, Kenerley CM, Rathore KS (2003) Enhanced fungal resistance in transgenic cotton expressing an endochitinase gene from Trichoderma virens. Plant Biotechnol J 1:321–336PubMedCrossRefGoogle Scholar
  25. Felix G, Regenass M, Boller T (1993) Specific perception of subnanomolar concentrations of chitin fragments by tomato cells—induction of extracellular alkalinization, changes in protein-phosphorylation, and establishment of a refractory state. Plant J 4:307–316CrossRefGoogle Scholar
  26. Fravel DR (2005) Commercialization and implementation of biocontrol. Annu Rev Phytopathol 43:337–359PubMedCrossRefGoogle Scholar
  27. Fry SC, Aldington S, Hetherington PR, Aitken J (1993) Oligosaccharides as signals and substrates in the plant-cell wall. Plant Physiol 103:1–5PubMedCrossRefGoogle Scholar
  28. Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annu Rev Phytopathol 43:205–227PubMedCrossRefGoogle Scholar
  29. Graham LS, Sticklen MB (1994) Plant chitinases. Can J Bot 72:1057–1083Google Scholar
  30. Howell CR, Stipanovic RD (1995) Mechanisms in the biocontrol of Rhizoctonia solani-induced cotton seedling disease by Gliocladium virens antibiosis. Phytopathology 85:469–472CrossRefGoogle Scholar
  31. Howell CR, Hanson LE, Stipanovic RD, Puckhaber LS (2000) Induction of terpenoid synthesis in cotton roots and control of Rhizoctonia solani by seed treatment with Trichoderma virens. Phytopathology 90:248–252PubMedCrossRefGoogle Scholar
  32. Hunter RE, Halloin JM, Veech JA, Carter WW (1978) Terpenoid accumulation in hypocotyls of cotton seedlings during aging and after infection by Rhizoctonia solani. Phytopathology 68:347–350CrossRefGoogle Scholar
  33. Jalloul A, Montillet JL, Assigbetse K, Agnel JP, Delannoy E, Triantaphylides C, Daniel JF, Marmey P, Geiger JP, Nicole M (2002) Lipid peroxidation in cotton: Xanthomonas interactions and the role of lipoxygenases during the hypersensitive reaction. Plant J 32:1–12PubMedCrossRefGoogle Scholar
  34. Johrde A, Schweizer P (2008) A class III peroxidase specifically expressed in pathogen-attacked barley epidermis contributes to basal resistance. Mol Plant Pathol 9:687–696PubMedCrossRefGoogle Scholar
  35. Kaku H, Shibuya N, Xu PL, Aryan AP, Fincher GB (1997) N-acetylchitooligosaccharides elicit expression of a single (1–3)-beta-glucanase gene in suspension-cultured cells from barley (Hordeum vulgare). Physiol Plant 100:111–118CrossRefGoogle Scholar
  36. Kaku H, Nishizawa Y, Ishii-Minami N, Akimoto-Tomiyama C, Dohmae N, Takio K, Minami E, Shibuya N (2006) Plant cells recognize chitin fragments for defense signaling through a plasma membrane receptor. Proc Natl Acad Sci USA 103:11086–11091PubMedCrossRefGoogle Scholar
  37. Kawano T (2003) Roles of the reactive oxygen species-generating peroxidase reactions in plant defense and growth induction. Plant Cell Rep 21:829–837PubMedGoogle Scholar
  38. Kim DJ, Baek JM, Uribe P, Kenerley CM, Cook DR (2002) Cloning and characterization of multiple glycosyl hydrolase genes from Trichoderma virens. Curr Genet 40:374–384PubMedCrossRefGoogle Scholar
  39. Kloepper JW (1991) Development of invivo assays for prescreening antagonists of Rhizoctonia solani on cotton. Phytopathology 81:1006–1013CrossRefGoogle Scholar
  40. Lee S, Choi H, Suh S, Doo IS, Oh KY, Choi EJ, Taylor ATS, Low PS, Lee Y (1999) Oligogalacturonic acid and chitosan reduce stomatal aperture by inducing the evolution of reactive oxygen species from guard cells of tomato and Commelina communis. Plant Physiol 121:147–152PubMedCrossRefGoogle Scholar
  41. Lin W, Anuratha CS, Datta K, Potrykus I, Muthukrishnan S, Datta SK (1995) Genetic-engineering of rice for resistance to sheath blight. Biotechnol 13:686–691CrossRefGoogle Scholar
  42. Linthorst HJM (1991) Pathogenesis-related proteins of plants. Crit Rev Plant Sci 10:123–150CrossRefGoogle Scholar
  43. Lorito M, Scala F (1999) Microbial genes expressed in transgenic plants to improve disease resistance. J Plant Pathol 81:73–88Google Scholar
  44. Lorito M, Harman GE, Hayes CK, Broadway RM, Tronsmo A, Woo SL, Dipietro A (1993) Chitinolytic enzymes produced by Trichoderma harzianum—antifungal activity of purified endochitinase and chitobiosidase. Phytopathology 83:302–307CrossRefGoogle Scholar
  45. Lorito M, Woo SL, Fernandez IG, Colucci G, Harman GE, Pintor-Toro JA, Filippone E, Muccifora S, Lawrence CB, Zoina A, Tuzun S, Scala F (1998) Genes from mycoparasitic fungi as a source for improving plant resistance to fungal pathogens. Proc Natl Acad Sci USA 95:7860–7865PubMedCrossRefGoogle Scholar
  46. Marmey P, Jalloul A, Alhamdia M, Assigbetse K, Cacas JL, Voloudakis AE, Champion A, Clerivet A, Montillet JL, Nicole M (2007) The 9-lipoxygenase GhLOX1 gene is associated with the hypersensitive reaction of cotton Gossypium hirsutum to Xanthomonas campestris pv malvacearum. Plant Physiol Biochem 45:596–606PubMedCrossRefGoogle Scholar
  47. Martinez C, Montillet JL, Bresson E, Agnel JP, Dai GH, Daniel JF, Geiger JP, Nicole M (1998) Apoplastic peroxidase generates superoxide anions in cells of cotton cotyledons undergoing the hypersensitive reaction to Xanthomonas campestris pv. malvacearum race 18. Mol Plant Microbe Interact 11:1038–1047CrossRefGoogle Scholar
  48. Miya A, Albert P, Shinya T, Desaki Y, Ichimura K, Shirasu K, Narusaka Y, Kawakami N, Kaku H, Shibuya N (2007) CERK1, a LysM receptor kinase, is essential for chitin elicitor signaling in Arabidopsis. Proc Natl Acad Sci USA 104:19613–19618PubMedCrossRefGoogle Scholar
  49. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  50. Murray F, Llewellyn D, McFadden H, Last D, Dennis ES, Peacock WJ (1999) Expression of the Talaromyces flavus glucose oxidase gene in cotton and tobacco reduces fungal infection, but is also phytotoxic. Mol Breed 5:219–232CrossRefGoogle Scholar
  51. Navazio L, Baldan B, Moscatiello R, Zuppini A, Woo SL, Mariani P, Lorito M (2007) Calcium-mediated perception and defense responses activated in plant cells by metabolite mixtures secreted by the biocontrol fungus Trichoderma atroviride. BMC Plant Biol 7:41PubMedCrossRefGoogle Scholar
  52. Ning W, Chen F, Mao BZ, Li Q, Liu ZX, Guo ZJ, He ZH (2004) N-acetylchitooligosaccharides elicit rice defence responses including hypersensitive response-like cell death, oxidative burst and defence gene expression. Physiol Mol Plant Pathol 64:263–271CrossRefGoogle Scholar
  53. Plazek A, Zur I (2003) Cold-induced plant resistance to necrotrophic pathogens and antioxidant enzyme activities and cell membrane permeability. Plant Sci 164:1019–1028CrossRefGoogle Scholar
  54. Powell NT, Melendez PL, Batten CK (1971) Disease complexes in tobacco involving Meloidogyne incognita and certain soil-borne fungi. Phytopathology 61:1332–1337CrossRefGoogle Scholar
  55. Punja ZK (2006) Recent developments toward achieving fungal disease resistance in transgenic plants. Can J Plant Pathol 28:S298–S308Google Scholar
  56. Ren Y, Wee KE, Chang FN (2000) Deficiency of current methods in assaying endochitinase activity. Biochem Biophys Res Commun 268:302–305PubMedCrossRefGoogle Scholar
  57. Roby D, Broglie K, Cressman R, Biddle P, Chet IL, Broglie R (1990) Activation of a bean chitinase promoter in transgenic tobacco plants by phytopathogenic fungi. Plant Cell 2:999–1007PubMedCrossRefGoogle Scholar
  58. Sasaki K, Iwai T, Hiraga S, Kuroda K, Seo S, Mitsuhara I, Miyasaka A, Iwano M, Ito H, Matsui H, Ohashi Y (2004) Ten rice peroxidases redundantly respond to multiple stresses including infection with rice blast fungus. Plant Cell Physiol 45:1442–1452PubMedCrossRefGoogle Scholar
  59. Shibuya N, Minami E (2001) Oligosaccharide signalling for defence responses in plant. Physiol Mol Plant Pathol 59:223–233CrossRefGoogle Scholar
  60. Shrestha CL, Ona I, Muthukrishnan S, Mew TW (2008) Chitinase levels in rice cultivars correlate with resistance to the sheath blight pathogen Rhizoctonia solani. Eur J Plant Pathol 120:69–77CrossRefGoogle Scholar
  61. Stipanovic RD, Bell AA, Benedict CR (1999) Cotton pest resistance: the role of pigment gland constituents. In: Cutler HG, Cutler SJ (eds) Biologically active natural products: agrochemicals. CRC Press, Boca Raton, pp 211–220Google Scholar
  62. Thordal-Christensen H, Zhang ZG, Wei YD, Collinge DB (1997) Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley–powdery mildew interaction. Plant J 11:1187–1194CrossRefGoogle Scholar
  63. van Hengel AJ, Guzzo F, van Kammen A, de Vries SC (1998) Expression pattern of the carrot EP3 endochitinase genes in suspension cultures and in developing seeds. Plant Physiol 117:43–53PubMedCrossRefGoogle Scholar
  64. van Hengel AJ, Tadesse Z, Immerzeel P, Schols H, van Kammen A, de Vries SC (2001) N-acetylglucosamine and glucosamine-containing arabinogalactan proteins control somatic embryogenesis. Plant Physiol 125:1880–1890PubMedCrossRefGoogle Scholar
  65. Wan J, Zhang XC, Neece D, Ramonell KM, Clough S, Kim SY, Stacey MG, Stacey G (2008) A LysM receptor-like kinase plays a critical role in chitin signaling and fungal resistance in Arabidopsis. Plant Cell 20:471–481PubMedCrossRefGoogle Scholar
  66. Wu GS, Shortt BJ, Lawrence EB, Levine EB, Fitzsimmons KC, Shah DM (1995) Disease resistance conferred by expression of a gene encoding H2O2-generating glucose-oxidase in transgenic potato plants. Plant Cell 7:1357–1368PubMedCrossRefGoogle Scholar
  67. Yamada A, Shibuya N, Kodama O, Akatsuka T (1993) Induction of phytoalexin formation in suspension-cultured rice cells by N-acetyl-chitooligosaccharides. Biosci Biotechnol Biochem 57:405–409CrossRefGoogle Scholar
  68. Zhang D, Hrmova M, Wan CH, Wu C, Balzen J, Cai W, Wang J, Densmore LD, Fincher GB, Zhang H, Haigler CH (2004) Members of a new group of chitinase-like genes are expressed preferentially in cotton cells with secondary walls. Plant Mol Biol 54:353–372PubMedCrossRefGoogle Scholar
  69. Zhong R, Kays SJ, Schroeder BP, Ye ZH (2002) Mutation of a chitinase-like gene causes ectopic deposition of lignin, aberrant cell shapes, and overproduction of ethylene. Plant Cell 14:165–179PubMedCrossRefGoogle Scholar
  70. Zhou BY, Wang JH, Guo ZF, Tan HQ, Zhu XC (2006) A simple colorimetric method for determination of hydrogen peroxide in plant tissues. Plant Growth Regul 49:113–118CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Vinod Kumar
    • 1
  • Vilas Parkhi
    • 1
  • Charles M. Kenerley
    • 3
  • Keerti S. Rathore
    • 1
    • 2
  1. 1.Institute for Plant Genomics and BiotechnologyTexas A&M UniversityCollege StationUSA
  2. 2.Department of Soil and Crop SciencesTexas A&M UniversityCollege StationUSA
  3. 3.Department of Plant Pathology and MicrobiologyTexas A&M UniversityCollege StationUSA

Personalised recommendations