Plant Cell Reports

, Volume 26, Issue 6, pp 791–804 | Cite as

Pyramiding transgenic resistance in elite indica rice cultivars against the sheath blight and bacterial blight

  • S. Maruthasalam
  • K. Kalpana
  • K. K. Kumar
  • M. Loganathan
  • K. Poovannan
  • J. A. J. Raja
  • E. Kokiladevi
  • R. Samiyappan
  • D. Sudhakar
  • P. Balasubramanian
Genetic Transformation and Hybridization


Elite indica rice cultivars were cotransformed with genes expressing a rice chitinase (chi11) and a thaumatin-like protein (tlp) conferring resistance to fungal pathogens and a serine-threonine kinase (Xa21) conferring bacterial blight resistance, through particle bombardment, with a view to pyramiding sheath blight and bacterial blight resistance. Molecular analyses of putative transgenic lines by polymerase chain reaction, Southern Blot hybridization, and Western Blotting revealed stable integration and expression of the transgenes in a few independent transgenic lines. Progeny analyses showed the stable inheritance of transgenes to their progeny. Coexpression of chitinase and thaumatin-like protein in the progenies of a transgenic Pusa Basmati1 line revealed an enhanced resistance to the sheath blight pathogen, Rhizoctonia solani, as compared to that in the lines expressing the individual genes. A transgenic Pusa Basmati1 line pyramided with chi11, tlp, and Xa21 showed an enhanced resistance to both sheath blight and bacterial blight.


Bacterial blight Chitinase Disease resistance Sheath blight Thaumatin-like protein 



bacterial blight


functional resistance/susceptibility index


highest relative lesion height %


Pusa Basmati1


sheath blight



The authors are grateful to The Rockefeller Foundation, USA [# 99001/225 (PB) and # 99001/227 (DS)] and the Department of Biotechnology, New Delhi, India, for providing the financial support. We greatly acknowledge the help provided by Dr. Legrand, Strasbourg, Cedex, France and Dr. S. Muthukrishnan, Kansas State University, USA.


  1. Adhikari TB, Vera Cruz CM, Zhang Q, Nelson RJ, Skinner DZ, Mew TW, Leach JE (1995) Genetic diversity of Xanthomonas oryzae pv. oryzae in Asia. Appl Environ Microbiol 61:966–971PubMedGoogle Scholar
  2. Anand A, Zhou T, Trick HN, Gill BS, Bockus WW, Muthukrishnan S (2003) Greenhouse and field testing of transgenic wheat plants stably expressing genes for thaumatin-like protein, chitinase and glucanase against Fusarium graminearum. J Exp Bot 54:1101–1111PubMedCrossRefGoogle Scholar
  3. 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 254:1194–1197CrossRefPubMedGoogle Scholar
  4. Chen L, Marmey P, Taylor NJ, Brizard JP, Espinoza C, Cruz P, Huet H, Zhang S, de Kochko A, Beachy RN, Fauquet CM (1998) Expression and inheritance of multiple transgenes in rice plants. Nat Biotechnol 16:1060–1064PubMedCrossRefGoogle Scholar
  5. Christou P (1997) Rice transformation: bombardment. Plant Mol Biol 35:197–203PubMedCrossRefGoogle Scholar
  6. Christou P, Ford TL, Kofron M (1991) Production of transgenic rice (Oryza sativa L.) plants from agronomically important indica and japonica varieties via electric discharge particle acceleration of exogenous DNA into immature embryos. Biotechnology 9:957–962CrossRefGoogle Scholar
  7. Datta K, Baisakh N, Thet KM, Tu J, Datta SK (2002) Pyramiding transgenes for multiple resistance in rice against bacterial blight, yellow stem borer and sheath blight. Theor Appl Genet 106:1–8PubMedGoogle Scholar
  8. Datta K, Tu J, Oliva N, Ona I, Velazhahan R, Mew TW, Muthukrishnan S, Datta SK (2001) Enhanced resistance to sheath blight by constitutive expression of infection-related rice chitinase in transgenic elite indica rice cultivars. Plant Sci 60:405–414CrossRefGoogle 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. Dellaporta ST, Wood J, Hicks JB (1983) A plant DNA minipreparation: Version II. Plant Mol Biol Rep 1:19–21Google Scholar
  11. Fu X, Kohli A, Twyman RM, Christou P (2000) Alternative silencing effects involve distinct types of non-spreading cytosine methylation at a three-gene single-copy transgenic locus in rice. Mol Gen Genet 263:106–118PubMedCrossRefGoogle Scholar
  12. Gahakwa D, Maqbool SB, Fu X, Sudhakar D, Christou P, Kohli A (2000) Transgenic rice as a system to study the stability of transgene expression: multiple heterologous transgenes show similar behaviour in diverse genetic backgrounds. Theor Appl Genet 101:388–309CrossRefGoogle Scholar
  13. Gallagher S, Winston SE, Fuller SH, Hurre JGR (1995) Immunodetection. In: Auubel F, Bernt R, Kingston EE, DMoore D, Seidman JD, Smith JA, John S (eds) Short protocols in molecular biology. Wiley, New York, pp 1040–1048Google Scholar
  14. Gomez KK, Gomez AA (1984) Statistical procedure for agricultural research. Wiley, New YorkGoogle Scholar
  15. Grenier J, Potvin C, Trudel J, Asselin A (1999) Some thaumatin-like proteins hydrolyse polymeric β-1,3-glucan. Plant J 19:473–480PubMedCrossRefGoogle Scholar
  16. Groth DE, Nowick EM (1992) Selection for resistance to rice sheath blight through number of infection cushions and lesion type. Plant Dis 76:721–723CrossRefGoogle Scholar
  17. Heath MC (2000) Hypersensitive response-related death. Plant Mol Biol 44:321–334PubMedCrossRefGoogle Scholar
  18. Hejgaard J, Jacobsen S, Svendsen I (1991) Two antifungal thaumatin-like proteins from barley grain. FEBS Lett 291:127–131PubMedCrossRefGoogle Scholar
  19. Hiilovaara-Teijo M, Hannukkala A, Griffith M, Yu XM, Pihakaski-Maunsbach K (1999) Snow mold-induced apoplastic proteins in winter rye leaves lack antifreeze activity. Plant Physiol 121:665–674PubMedCrossRefGoogle Scholar
  20. Huang N, Angeles ER, Domingo J, Magpantay G, Singh S, Zhang G, Kumaravadivel N, Bennet T, Khush GS (1997) Pyramiding of bacterial blight resistance genes in rice: marker assisted selection using RFLP and PCR. Theor Appl Genet 95:313–320CrossRefGoogle Scholar
  21. Huang J, Wen L, Swegle M, Tian H, Thin TH, Naylor HM, Muthukrishnan S, Reeck GR (1991) Nucleotide sequence of a rice genomic clone that encodes class II endo chitinase. Plant Mol Biol 16:479–480PubMedCrossRefGoogle Scholar
  22. Jacobs AK, Dry IB, Robinson SP (1999) Induction of different pathogenesis-related cDNAs in grapevine infected with powdery mildew and treated with ethephon. Plant Pathol 48:325–336CrossRefGoogle Scholar
  23. Jongedijk E, Tigelaar H, van Roekel JSC, Bres-Vloemans SA, Dekker I, Van Den Elzen PJM, Cornelissen BJC, Melchers LS (1995) Synergistic activity of β-1,3-glucanases and chitinases enhances fungal resistance in transgenic tomato plants. Euphytica 85:173–180CrossRefGoogle Scholar
  24. Kalpana K, Maruthasalam S, Rajesh T, Poovannan K, Kumar KK, Kokiladevi E, Raja JAJ, Sudhakar D, Velazhahan R, Samiyappan R, Balasubramanian P (2006) Engineering sheath blight resistance in elite indica rice cultivars using genes encoding defense proteins. Plant Sci 170:203–215CrossRefGoogle Scholar
  25. Kameswara Rao K, Lakshminarasu M, Jena KK (2002) DNA markers and marker-assisted breeding for durable resistance to bacterial blight disease in rice. Biotech Adv 20:33–47CrossRefGoogle Scholar
  26. Kauffman HE, Reddy APK, Hsieh SPY, Merca SD (1973) An improved technique for evaluating resistance of rice varieties to Xanthomonas oryzae. Plant Dis Rep 57:537–541Google Scholar
  27. Khush GS, Bacalangco E, Ogawa T (1990) A new gene for resistance to bacterial blight from Oryza longistaminata. Rice Genet News L 7:121–122Google Scholar
  28. Kim JK, Jang IC, Wu R, Zuo WN, Boston RS, Lee YH, Ahn IP, Nahm BH (2003) Co-expression of a modified maize ribosome-inactivating protein and a rice basic chitinase gene in transgenic rice plants confers enhanced resistance to sheath blight. Transgenic Res 12:475–484PubMedCrossRefGoogle Scholar
  29. Krishnamurthy J, Samiyappan R, Vidhyasekaran P, Nakkeeran S, Rajeswari E, Raja JAJ, Balasubramanian P (1999) Efficacy of Trichoderma chitinases against Rhizoctonia solani, the rice sheath blight pathogen. J Biosci 24:207–213CrossRefGoogle Scholar
  30. Kumar KK, Poovannan K, Nandakumar R, Thamilarasi K, Geetha C, Jayashree N, Kokiladevi E, Raja JAJ, Samiyappan R, Sudhakar D, Balasubramanian P (2003) A high throughput functional expression assay system for a defence gene conferring transgenic resistance on rice against the sheath blight pathogen, Rhizoctonia solani. Plant Sci 165:969–976CrossRefGoogle Scholar
  31. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–683PubMedCrossRefGoogle Scholar
  32. Leah R, Tommerup H, Svendsen I, Mundy J (1991) Biochemical and molecular characterization of three barley seed proteins with antifungal properties. J Biol Chem 266:1564–1573PubMedGoogle Scholar
  33. Lin W, Anuratha CS, Datta K, Potrykus I, Muthukrishnan S, Datta SK (1995) Genetic engineering of rice for resistance to sheath blight. Biotechnology 13:686–691CrossRefGoogle Scholar
  34. Mew TW, Vera Cruz CM (1979) Variability of Xanthomonas oryzae: specificity in infection of rice differentials. Phytopathology 69:152–155Google Scholar
  35. Mew TW, Vera Cruz CM, Medalla ES (1992) Changes in the race frequency of Xanthomonas oryzae pv. oryzae in response to the planting of rice cultivars in the Philippines. Plant Dis 76:1029–1032CrossRefGoogle Scholar
  36. Murashige T, Skoog FA (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  37. Muthukrishnan S, Liang GH, Trick HN, Gill BS (2001) Pathogenesis-related proteins and their genes in cereals. Plant Cell Tissue Organ Cult 64:93–114CrossRefGoogle Scholar
  38. Narayanan NN, Baisakh N, Oliva NP, VeraCruz CM, Gnanamanickam SS, Datta K, Datta SK (2004) Molecular breeding: marker-assisted selection combined with biolistic transformation for blast and bacterial blight resistance in indica rice (cv. CO39). Mol Breed 14:61–71CrossRefGoogle Scholar
  39. Neuhans JM, Ahl-Goy P, Hinz U, Flores S, Meins FJ (1991) High-level expression of a tobacco chitinase gene in Nicotiana sylvestris: susceptibility of transgenic plants to Cercospora nicotianae infection. Plant Mol Biol 16:141–151CrossRefGoogle Scholar
  40. Osmond RIW, Hrmova M, Fontaine F, Imberty A, Fincher GB (2001) Barley thaumatin-like proteins: ligand specificity, kinetic and structural analyses and biological implications. Eur J Biochem 268:4190–4199PubMedCrossRefGoogle Scholar
  41. Peng JY, Wen FJ, Lister RL, Hodges TK (1995) Inheritance of gusA and neo genes in transgenic rice. Plant Mol Biol 27:91–104PubMedCrossRefGoogle Scholar
  42. Peumans WJ, Proost P, Swennen RL, Van Damme EJM (2002) The abundant class III chitinase homolog in young developing banana fruits behaves as a transient vegetative storage protein and most probably serves as an important supply of amino acids for the synthesis of ripening-associated proteins. Plant Physiol 130:1063–1072PubMedCrossRefGoogle Scholar
  43. Potrykus I, Harms CT, Lorz H (1979) Callus formation from cell culture protoplasts of corn (Zea mays L.). Theor Appl Genet 54:209–214CrossRefGoogle Scholar
  44. Ramesh R, Kumar, KK, Kalpana K, Maruthasalam S, Sudhakar D, Balasubramanian P (2001) Engineering Xa21-mediated resistance in elite indica rice cultivars to Xanthomonas oryzae pv. oryzae. In: Proceedings of the 8th National Rice Biotechnology Network (NRBN) Meeting, Aurangabad, India, p. 89Google Scholar
  45. Ronald PC (1997) The molecular basis of disease resistance in rice. Plant Mol Biol 35:179–186PubMedCrossRefGoogle Scholar
  46. Ronald PC, Alabani B, Tabien R, Abenes L, Wu KS, McCouch S, Tanksley SD (1992) Genetic and physical analysis of rice bacterial blight disease resistance locus, Xa21. Mol Gen Genet 236:113–120PubMedGoogle Scholar
  47. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  48. Savary S, Willocquet L, Elazegui FA, Castilla NP, Teng PS (2000) Rice pest constraints in tropical asia: quantification of yield losses due to rice pests in a range of production situations. Plant Dis 84:357–369CrossRefGoogle Scholar
  49. Singh S, Sidhu JS, Huang N, Vikal Y, Li Z, Brar DS, Dhaliwal HS, Khush GS (2001) Pyramiding three bacterial blight resistance genes (xa5, xa13 and Xa21) using marker-assisted selection into indica rice cultivar PR106. Theor Appl Genet 102:1011–1015CrossRefGoogle Scholar
  50. Sivamani E, Shen P, Opalka N, Beachy RN, Fauquet C (1996) Selection of large quantities of embryogenic calli from indica rice seeds for production of fertile transgenic plants using the biolistic method. Plant Cell Rep 15:322–327CrossRefGoogle Scholar
  51. Song WY, Wang GL, Chen LL, Kim HS, Pi LY, Hosten T, Gardner J, Wang B, Zhai WX, Zhu LH, Fauquet C, Ronald PC (1995) A receptor kinase-like protein encoded by the rice disease resistance gene Xa21. Science 270:1804–1806PubMedCrossRefGoogle Scholar
  52. Takakura Y, Ito T, Saito H, Inoue T, Komari T, Kuwata S (2000) Flower-predominant expression of a gene encoding a novel class I chitinase in rice (Oryza sativa L.). Plant Mol Biol 42:883–897PubMedCrossRefGoogle Scholar
  53. Tang K, Tinjuangjun P, Xu Y, Sun X, Gatehouse JA, Ronald PC, Qi H, Lu X, Christou P, Kohli A (1999) Particle bombardment-mediated co-transformation of elite chinese rice cultivars with genes conferring resistance to bacterial blight and sap sucking insects. Planta 208:552–563CrossRefGoogle Scholar
  54. Tu J, Datta K, Khush GS, Zhang Q, Datta SK (2000) Field performance of Xa21 transgenic indica rice (Oryza sativa L.), IR72. Theor Appl Genet 101:15–20CrossRefGoogle Scholar
  55. Tu J, Ona I, Zhang Q, Mew TW, Khush GS, Datta SK (1998) Transgenic rice variety IR72 with Xa21 is resistant to bacterial blight. Theor Appl Genet 97:31–36CrossRefGoogle Scholar
  56. Van Loon LC, Van Strien EA (1999) The families of pathogenesis-related proteins, their activities, and comparative analysis of PR-1 type proteins. Physiol Mol Plant Pathol 55:85–97CrossRefGoogle Scholar
  57. Velazhahan R, Chen-Cole K, Anuratha CS, Muthukrishnan S (1998) Induction of thaumatin-like proteins (TLPs) in Rhizoctonia solani infected rice and characterization of two new cDNA clones. Physiol Plant 102:21–28CrossRefGoogle Scholar
  58. Wang GL, Song WY, Ruan DL, Sideris S, Ronald PC (1996) The cloned gene Xa21 confers resistance to multiple Xanthomonas oryzae pv. oryzae isolates in transgenic plants. Mol Plant Microbe Interact 9:850–855PubMedGoogle Scholar
  59. Yamamato T, Iketani H, Ieki H, Nishizawa Y, Notsuka K, Hayashi T, Matsuta N (2000) Transgenic grapevine plants expressing a rice chitinase with enhanced resistance to fungal pathogens. Plant Cell Rep 19:639–646CrossRefGoogle Scholar
  60. Yoshimura S, Yoshimura A, Iwata N, McCouch SR, Abenes MN, Baraoidan MR, Mew TW, Nelson RJ (1995) Tagging and combining bacterial blight resistance genes using RAPD and RFLP markers. Mol Breed 1:375–87CrossRefGoogle Scholar
  61. Yun DJ, Ibeas JI, Lee H, Coca MA, Narasimhan ML, Uesono Y, Hasegawa PM, Pardo JM, Bressan RA (1998) Osmotin, a plant antifungal protein, subverts signal transduction to enhance fungal cell susceptibility. Mol Cell 1:807–812PubMedCrossRefGoogle Scholar
  62. Zhang S, Song WY, Chen L, Ruan D, Taylor N, Ronald PC, Beachy RN, Fauquet CM (1998) Transgenic elite indica rice varieties, resistant to Xanthomonas oryzae pv.oryzae. Mol Breed 4:551–558CrossRefGoogle Scholar
  63. Zhu B, Chen THH, Li PH (1996) Analysis of late-blight disease resistance and freezing tolerance in transgenic potato plants expressing sense and antisense genes for an osmotin-like protein. Planta 198:70–77PubMedCrossRefGoogle Scholar
  64. Zhu Q, Maher EA, Masoud S, Dixon RA, Lamb CJ (1994) Enhanced protection against fungal attack by constitutive co-expression of chitinase and glucanase genes in transgenic tobacco. Biotechnology 12:807–812CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • S. Maruthasalam
    • 1
  • K. Kalpana
    • 1
  • K. K. Kumar
    • 1
  • M. Loganathan
    • 1
  • K. Poovannan
    • 1
  • J. A. J. Raja
    • 1
  • E. Kokiladevi
    • 1
  • R. Samiyappan
    • 2
  • D. Sudhakar
    • 1
  • P. Balasubramanian
    • 1
  1. 1.Rice Transformation Laboratory, Center for Plant Molecular Biology, Department of Plant Molecular Biology and BiotechnologyTamil Nadu Agricultural UniversityCoimbatoreIndia
  2. 2.Center for Plant Protection Studies, Department of Plant PathologyTamil Nadu Agricultural UniversityCoimbatoreIndia

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