Plant Molecular Biology

, Volume 62, Issue 4–5, pp 735–752 | Cite as

A novel approach for developing resistance in rice against phloem limited viruses by antagonizing the phloem feeding hemipteran vectors

  • Prasenjit Saha
  • Indranil Dasgupta
  • Sampa DasEmail author


Rice production is known to be severely affected by virus transmitting rice pests, brown planthopper (BPH) and green leafhopper (GLH) of the order hemiptera, feeding by phloem abstraction. ASAL, a novel lectin from leaves of garlic (Allium sativum) was previously demonstrated to be toxic towards hemipteran pests when administered in artificial diet as well as in ASAL expressing transgenic plants. In this report ASAL was targeted under the control of phloem-specific Agrobacterium rolC and rice sucrose synthase-1 (RSs1) promoters at the insect feeding site into popular rice cultivar, susceptible to hemipteran pests. PCR, Southern blot and C-PRINS analyses of transgenic plants have confirmed stable T-DNA integration and the transgenes were co-segregated among self-fertilized progenies. The T0 and T1 plants, harbouring single copy of intact T-DNA expression cassette, exhibit stable expression of ASAL in northern and western blot analyses. ELISA showed that the level of expressed ASAL was as high as 1.01% of total soluble protein. Immunohistofluorescence localization of ASAL depicted the expected expression patterns regulated by each promoter type. In-planta bioassay studies revealed that transgenic ASAL adversely affect survival, growth and population of BPH and GLH. GLH resistant T1 plants were further evaluated for the incidence of tungro disease, caused by co-infection of GLH vectored Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV), which appeared to be dramatically reduced. The result presented here is the first report of such GLH mediated resistance to infection by RTBV/RTSV in ASAL expressing transgenic rice plant.


ASAL transgenic rice GLH Immunohistofluorescence RTBV resistance 



Allium sativum agglutinin from leaf


benzylamino purine


brown planthopper


bovine serum albumin




cycling-primed in situ labelling


days post inoculation


enzyme linked immunosorbent assay


fluorescein isothiocyanate


green leafhopper




Murashige and Skoog


α-napthaline acetic acid


phosphate buffered saline


rice sucrose synthase-1 promoter


Rice tungro bacilliform virus


Rice tungro spherical virus


sodium dodecyl sulphate-polyacrylamide gel electrophoresis


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Authors are grateful to Council of Scientific and Industrial Research; Government of India for providing fellowships to PS. We are grateful to Prof. S. C. Roy, Centre of Advanced Study (CAS), Cell and Chromosome Research, Department of Botany, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, India for his help to carry out C-PRINS study in his laboratory. Authors thank the Programme Coordinator, CAS, Dept. Bot. CU for providing above technical opportunities. We are thankful to Regional Rice Research Station, Chinsurah, West Bengal, India for providing nuclear stock seed of Pusa Basmati 1 rice cultivar. For back up service of Mr. Arup Kumar Dey, BI is sincerely acknowledged. Authors are also thankful to Gautam Basu for critically reading the manuscript. The expert technical help of Anand Singh Rana in viral assays and Trilok Singh Rawat in inoculations of DU(SC) are acknowledged.

Supplementary material


  1. Abbo S, Dunford RP, Miller TE, Reader SM, King IP (1993) Primer-mediated in situ detection of the B-hordein gene cluster on barley chromosome1H. Proc Natl Acad Sci USA 90:11821–11824PubMedCrossRefGoogle Scholar
  2. Bandyopadhyay S, Roy A, Das S (2001) Binding of garlic (Allium sativum) leaf lectin to the gut receptors of homopteran pests is correlated to its insecticidal activity. Plant Sci 161:1025–1033CrossRefGoogle Scholar
  3. Banerjee S, Hess D, Majumder P, Roy D, Das S (2004) The interactions of Allium sativum leaf agglutinin with a chaperonin group of unique receptor protein isolated from a bacterial endosymbiont of the mustard aphid. J Biol Chem 279:23782–23789PubMedCrossRefGoogle Scholar
  4. Barre A, Van Damme EJM, Peumaus WJ, Rouge P (1996) Structure-function relationship of monocot mannose-binding lectins. Plant Physiol 112:1531–1540PubMedCrossRefGoogle Scholar
  5. Bennet J (2001) Summing-up: cutting-edge science for rice improvement-breakthroughs and beneficiaries. In: Goode J, Chadwick D (Eds.), Rice biotechnology: improving yield, stress tolerance and grain quality, Novartis Foundation/John Wiley, UK, pp. 242–251Google Scholar
  6. Bradford MM (1976) A rapid and sensitive method for the quantitation of proteins using the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  7. Chang T, Chen L, Chen S, Cai H, Liu X, Xiao G, Zhu Z (2003) Transformation of tobacco with genes encoding Helianthus tuberosus agglutinin (HTA) confers resistance to peach-potato aphid (Myzus persicae). Transgenic Res 12:607–614PubMedCrossRefGoogle Scholar
  8. Dahal G, Hibino H, Aguiero VM (1997) Population characteristics and tungro transmission by Nephotettix virescens (Hemiptera: Cicadellidea) on selected resistant rice cultivars. Bull Entomol Res 87:387–395CrossRefGoogle Scholar
  9. Dai S, Zheng P, Marmey P, Zhang S, Tian W, Chen S, Beachy RN, Fauquet C (2001) Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment. Mol Breed 7:25–33CrossRefGoogle Scholar
  10. Dasgupta I, Das BK, Nath SP, Mukhopadhyay S, Niaiz FR, Varma A (1996) Detection of rice tungro bacilliform virus in field and glasshouse samples from India using the polymerase chain reaction. J Virol Methods 58:53–58PubMedCrossRefGoogle Scholar
  11. Dasgupta I, Hull R, Eastop S, Poggi PC, Blakebrough M, Boulton MI, Davies JW (1991) Rice tungro bacilliform virus DNA independently infects rice after Agrobacterium mediated transfer. J Gen Virol 72:1215–1221PubMedGoogle Scholar
  12. Dong J, Kharb P, Cervera M, Hall CT (2001) The use of FISH in chromosomal localization of transgenes in rice. Methods Cell Sci 23:105–113PubMedCrossRefGoogle Scholar
  13. Dutta I, Saha P, Majumder P, Sarkar A, Chakraborti D, Banerjee S, Das S (2005a) The efficacy of a novel insecticidal protein, Allium sativum leaf lectin (ASAL), against homopteran insects monitored in transgenic tobacco. Plant Biotechnol J 3:601–611CrossRefGoogle Scholar
  14. Dutta I, Majumder I, Saha P, Ray K, Das S (2005b) Constitutive and phloem specific expression of Allium sativum leaf agglutinin (ASAL) to engineer aphid (Lipaphis erysimi) resistance in transgenic Indian mustard (Brassica juncea). Plant Sci 169:996–1007CrossRefGoogle Scholar
  15. Eamens AL, Blanchard CL, Dennis ES, Upadhyaya NM (2004) A bidirectional gene trap construct for tDNA and Ds-mediated insertional mutagenesis in rice (Oryza sativa L.). Plant Biotechnol J 2:367–380CrossRefPubMedGoogle Scholar
  16. Eisemann CH, Donaldson RA, Pearson RD, Cadagon LC, Vuocolo T, Pellam RL (1994) Larvicidal action of lectins on Lucilia cuprina; mechanism of action. Entomol Exp Appl 72:1–11CrossRefGoogle Scholar
  17. Fagard M, Vaucheret H (2000) (Trans)gene siliencing in plants: How many mechanisms? Annu Rev Plant Physiol Plant Mol Biol 51:167–194PubMedCrossRefGoogle Scholar
  18. Fleet GH (1991) Cell walls. In: Harrison AHR, Harrison JS (Eds.), Yeast organelles, Academic Press, London, pp 199–277Google Scholar
  19. Filichkin SA, Brumfield S, Filichkin TP, Young MJ (1997) In vitro interactions of the aphid endosymbiotic SymL chaperonin with barley yellow dwarf virus. J Virol 71:569–577PubMedGoogle Scholar
  20. Finnegan J, McElory D (1994) Transgene inactivation: plant fight back! Bio/Technol 12:883–888CrossRefGoogle Scholar
  21. Fitches E, Woodhouse SD, Edwards JP, Gatehoush JA (2001) In vitro and in vivo binding of snowdrop lectin (Galanthus nivalis agglutinin; GNA) and jackbean (Canavalia ensiformis; Con A) lectins within tomato moth (Lacanobia oleraceae) larvae; mechanisms of insecticidal action. J Insect Physiol 47:777–787PubMedCrossRefGoogle Scholar
  22. Foissac X, Loc NT, Christou P, Gatehouse AMR, Gatehouse JA (2000) Resistance to green leafhopper (Nephotettix virescens) and brown planthopper (Nilaparvata lugens) in transgenic rice expressing snowdrop lectin (Galanthus nivalis agglutinin; GNA). J Insect Physiol 46:573–583PubMedCrossRefGoogle Scholar
  23. Froissart R, Michalakis Y, Blanc S (2002) Helper component-transcomplementation in the vector transmission of plant viruses. Phytopathol 92:576–579Google Scholar
  24. Gatehouse AM, Davison GM, Stewart JN, Gatehouse LN, Kumar A, Geoghegan IE, Birch ANE, Gatehouse JA (1999) Concanavalin A inhibits development of tomato moth (Lacanobia oleracea) and peach-potato aphid (Myzus persicae) when expressed in transgenic potato plants. Mol Breed 5:153–165CrossRefGoogle Scholar
  25. Graham MW, Craig S, Waterhouse PM (1997) Expression patterns of vascular-specific promoter RolC and Sh in transgenic potatoes and their use in engineering PLRV-resistant plants. Plant Mol Biol 33:729–735PubMedCrossRefGoogle Scholar
  26. Hogenhout SA, van den Wilk F, Verbeek M, Goldbach RW, van den Heuvel JFJM (1998) Potato leafroll virus binds to the equatorial domain of the aphid endosymbiotic GroEL homolog. J Virol 72:358–365PubMedGoogle Scholar
  27. Huet H, Mahendra S, Wang J, Sivamani E, Ong CA, Chen L, de Kochko A, Beachy RN, Fauquet C (1999) Near immunity to rice tungro spherical virus achieved in rice by a replicase-mediated resistance strategy. Phytopathol 89:1022–1027Google Scholar
  28. Jacob SS, Valuthambi K (2003) A cointegrate Ti plasmid vector for Agrobacterium tumefaciens-mediated transformation of indiac rice cv Pusa Basmati1. J Plant Biochem Biotech 12:1–9Google Scholar
  29. Jain RK, Jain S (2000) Transgenic strategies for genetic improvement of Basmati rice. Indian J Exp Biol 38:6–17PubMedGoogle Scholar
  30. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusion: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907PubMedGoogle Scholar
  31. Jiang J, Gill BS, Wang G-L, Ronald CP, Ward DC (1995) Metaphase and interphase fluorescence in situ hybridization mapping of the rice genome with bacterial artificial chromosomes. Proc Natl Acad Sci USA 92:4487–4491PubMedCrossRefGoogle Scholar
  32. Jin WW, Li ZY, Fang Q, Altosaar I, Lui LH, Song YC (2002) Fluorescence in situ hybridization analysis of alien genes in Agrobacterium-mediated Cry1A (b)- transformed rice. Ann Bot 90:31–36PubMedCrossRefGoogle Scholar
  33. Jones MC, Gough K, Dasgupta I, Subba-Rao BL, Cliffe J, Qu R, Shen P, Kaniewska M, Blakebrough M, Davies JW, Beachy RN, Hull R (1991) Rice tungro disease is caused by an RNA and a DNA virus. J Gen Virol 72:757–761PubMedCrossRefGoogle Scholar
  34. Kim SR, Lee J, Jun SH, Park S, Kang HG, Kwon S, An G (2003) Transgene structures in T-DNA-inserted rice plants. Plant Mol Biol 52:761–773PubMedCrossRefGoogle Scholar
  35. Kononov ME, Bassuner B, Gelvin SB (1997) Integration of T-DNA binary vector ‘backbone’ sequences into the tobacco genome: evidence for multiple complex patterns of integration. Plant J 11:945–957PubMedCrossRefGoogle Scholar
  36. Kubaláková M, Vrana J, Číhalíková J, Lysák MA, Doležel J (2001) Localization of DNA sequences on plant chromosomes using PRINS and C-PRINS. Methods Cell Sci 23:71–82PubMedCrossRefGoogle Scholar
  37. Lee SI, Lee S-H, Koo JC, Chun HJ, Lim CO, Mun JH, Song YH, Cho MJ (1999) Soybean Kunitz trypsin inhibitor (SKTI) confers resistance to the brown planthoppe (Nilaparvata lugens Stal) in transgenic rice. Mol Breed 5:1–9Google Scholar
  38. Majumder P, Banerjee S, Das S (2004) Identification of receptors responsible for binding of the mannose specific lectin to the gut epithelial membrane of the target insects. Glycoconjugate J 20:525–530CrossRefGoogle Scholar
  39. Matsuki R, Onodera H, Yamauchi T, Uchimiya H (1989) Tissue-specific expression of the rolC promoter of the Ri plasmid in transgenic rice plants. Mol Gen Genet 220:12–16CrossRefGoogle Scholar
  40. Mohanty A, Sarma NP, Tyagi AK (1999) Agrobacterium-mediated high frequency transformation of an elite indica rice variety Pusa Basmati 1 and transformation of the transgene to R2 progey. Plant Sci 147:127–137CrossRefGoogle Scholar
  41. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco cultures. Plant Physiol 15:473–497CrossRefGoogle Scholar
  42. Muskens MWM, Vissers APA, Mol JNM, Kooter JM (2000) Role of inverted DNA repeats in transcriptional and post-transcriptional gene silencing. Plant Mol Biol 43:243–260PubMedCrossRefGoogle Scholar
  43. Nagadhara D, Ramesh S, Pasalu IC, Rao YK, Krishnaiah NV, Sarma NP, Brown DP, Gatehouse JA, Reddy VD, Rao KV (2003) Transgenic indica rice resistant to sap-sucking insects. Plant Biotechnol J 1:231–240CrossRefPubMedGoogle Scholar
  44. Nagadhara D, Ramesh S, Pasalu IC, Rao YK, Sarma NP, Reddy VD, Rao KV (2004) Transgenic rice plants expressing the snowdrop lectin (gna) exhibit high-level resistance to the whitebacked planthopper (Sogetella furcifera). Theor Appl Genet 109:1399–1405PubMedCrossRefGoogle Scholar
  45. Powell KS, Spence J, Bharathi M, Gatehouse AJ, Gatehouse AMR (1998) Immunohistochemical and developmental studies to elucidate the mechanism of action of the snowdrop lectin on the rice brown planthopper, Nilaparvata lugens (Stal). J Insect Physiol 44:529–539PubMedCrossRefGoogle Scholar
  46. Rao KV, Rathore KS, Hodges TK, Fu X, Stoger E, Sudhakar S, Williams P, Christou P, Bharathi M, Bown DP, Powell KS, Spence J, Gatehouse A, Gatehouse JA (1998) Expression of snowdrop lectin (GNA) in transgenic plants confers resistance to rice brown planthopper. Plant J 15:469– 477PubMedCrossRefGoogle Scholar
  47. Schmulling T, Schell J, Spena A (1989) Promoters of the rolA, B, and C genes of Agrobacterium rhizogenes are differentially regulated in transgenic plants. Plant Cell 1:665–670PubMedCrossRefGoogle Scholar
  48. Shi Y, Wang MB, Powell KS, Damme EV, Hilder VA, Gatehouse AMR, Boulter D, Gatehouse JA (1994) Use of the rice sucrose synthase-1 promoter to direct phloem-specific expression of β-glucuronidase and snowdrop lectin genes in transgenic tobacco plants. J Exp Bot 45:623– 631Google Scholar
  49. Shing RK, Shing US, Khush GS, Rohilla R (2000) Genetics and biotechnology of quality traits in aromatic rices In: Shing RK, Shing US, Khush GS (Eds.), Aromatic rices, Science Publishers Inc, Enfield, NH, USA, pp 47–69Google Scholar
  50. Shou H, Frame BR, Whitham SAWK (2004) Assessment of transgenic maize events produced by particle bombardment or Agrobacterium-mediated transformation. Mol Breed 13:201–208CrossRefGoogle Scholar
  51. Sivamani E, Huet H, Shen P, Ong CA, de-Kochko A, Fauquet C, Beachy RN (1999) Rice plants (Oryza sativa L.) containing Rice tungro spherical virus (RTSV) coat protein transgenes are resistant to virus infection. Mol Breed 5:177–185CrossRefGoogle Scholar
  52. Sudhakar D, Fu X, Stoger E, Williams S, Spence J, Brown DP, Bharathi M, Gatehouse JA, Christou P (1998) Expression and immunolocalization of the snowdrop lectin, GNA in transgenic rice plants. Transgenic Res 7:371–378PubMedCrossRefGoogle Scholar
  53. Sugaya S, Hayakawa K, Handa K, Uchimiya H (1989) Cell-specific expression of the rolC gene of the TL-DNA of Ri plasmid in transgenic tobacco plants. Plant Cell Physiol 30:649–653Google Scholar
  54. Suh S-O, Noda H, Blackwell M (2001) Insect symbiosis: derivation of yeast-like endosymbionts within an entomopathogenic filamentous lineage. Mol Biol Evol 18:995–1000PubMedGoogle Scholar
  55. Travella S, Ross SM, Harden J, Everett C, Snape JW, Harwood WA (2004) A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques. Plant Cell Rep 23:780–789PubMedCrossRefGoogle Scholar
  56. Vaucheret H, Fagard M (2001) Transcriptional gene silencing in plant: target, inducers and regulators. Trends Genet 17:29–35PubMedCrossRefGoogle Scholar
  57. Wang M, Boulter D, Gatehouse JA (1992) A complete sequence of the rice sucrose synthase-1 (RSs1) gene. Plant Mol Biol 19:881–885PubMedCrossRefGoogle Scholar
  58. Wenck A, Czako M, Kanevski I, Marton L (1997) Frequent collinear long transfer of DNA inclusive of the whole binary vector during Agrobacterium-mediated transformation. Plant Mol Biol 34:913–922PubMedCrossRefGoogle Scholar
  59. Weis WI, Drickaner K (1996) Structural basis of lectin-carbohydrate recognition. Annu Rev Biotechnol 65:441–473CrossRefGoogle Scholar
  60. Yin Y, Zhu Q, Dai S, Lamb C, Beachy RN (1997) RF2a, a bZIP transcriptional activator of the phloem-specific rice tungro bacilliform virus promoter, functions in vascular development. EMBO J 16:5247–5259PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  1. 1.Plant Molecular and Cellular GeneticsBose InstituteKolkataIndia
  2. 2.Department of Plant Molecular BiologyUniversity Delhi South CampusNew DelhiIndia

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