Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Intron–hairpin RNA Derived from Replication Associated Protein C1 Gene Confers Immunity to Tomato Yellow Leaf Curl Virus Infection in Transgenic Tomato Plants


The whitefly-transmitted Tomato Yellow Leaf Curl Virus (TYLCV) is the major pathogen of tomato crop in Cuba and one of the most outstanding viral diseases of plants worldwide. In this work, we have developed transgenic tomato plants, transformed with an intron–hairpin genetic construction to induce post- transcriptional gene silencing against the early TYLCV replication associated protein gene (C1). The intron–hairpin RNA produced involves 726 nts of the 3′ end of the TYLCV C1 gene as the arms of the hairpin, and the castor bean catalase intron. Transgenic tomato plants belonging to line 126, which harbor a single transgene copy, showed immunity to TYLCV, even in extreme conditions of infection (4-leaf-stage plants and 300 to many hundreds viruliferous whiteflies per plant during 60 days). Dot blot hybridization of these plants showed no TYLCV DNA presence 60 days after inoculation. Small interfering RNA molecules were detected in both inoculated and non-inoculated plants from line 126. These transgenic tomato plants of the otherwise very TYLCV-susceptible Campbell-28 tomato cultivar, are the first report of resistance to a plant DNA virus obtained by the use of the intron–hairpin RNA approach.

This is a preview of subscription content, log in to check access.


  1. Y Antignus R Vunsh O Lachman M Pearlsman L Maslenis U Hananya A Rosner (2004) ArticleTitleTruncated Rep gene originated from tomato yellow leaf curl virus – Israel [Mild] confers strain-specific resistance in transgenic tomato Ann Appl Biol 144 39–44

  2. D Baulcombe (2004) ArticleTitleRNA silencing in plants Nature 431 356–363 Occurrence Handle15372043 Occurrence Handle1:CAS:528:DC%2BD2cXnsFaiu7c%3D Occurrence Handle10.1038/nature02874

  3. RN Beachy (1997) ArticleTitleMechanisms and applications of pathogen-derived resistance in transgenic plants Curr Opin Biotechnol 8 215–220 Occurrence Handle9079736 Occurrence Handle1:CAS:528:DyaK2sXisVyqurY%3D Occurrence Handle10.1016/S0958-1669(97)80105-X

  4. M Bendahmane B Gronenborn (1997) ArticleTitleEngineering resistance against tomato yellow leaf curl virus (TYLCV) using antisense RNA Plant Mol Biol 33 351–357 Occurrence Handle9037152 Occurrence Handle1:CAS:528:DyaK2sXhsFaktLs%3D Occurrence Handle10.1023/A:1005715805000

  5. JK Brown J Bird (1992) ArticleTitleWhitefly-transmitted geminiviruses and associated disorders in the Americas and the Caribbean Basin Plant Dis 76 220–225 Occurrence Handle10.1094/PD-76-0220

  6. A Brunetti M Tavazza E Noris R Tavazza P Caciagli G Ancora S Crespi GP Accotto (1997) ArticleTitleHigh expression of truncated viral Rep protein confers resistance to tomato yellow leaf curl virus in transgenic tomato plants Mol Plant Microbe Interact 10 571–579 Occurrence Handle1:CAS:528:DyaK2sXjvFOktrk%3D

  7. A Coego R Vázques R Alfonso Y Coll M Pujol E Ménendez M López P Molina L Hernández B Bencomo G Riva Particlede la G Selman-Housein (1996) ArticleTitleEffect of promoter-stimulatory element combination on transient reporter gene expression in tobacco protoplast using PEG-treatment Biotecnología Aplicada 13 147 Occurrence Handle1:CAS:528:DyaK28XltVSgtrg%3D

  8. M Cordero PL Ramos L Hernandez AI Fernandez AL Echemedia R Peral G Gonzalez D Garcia S Valdes A Estevez K Hernandez (2003) ArticleTitleIdentification of tomato mottle Taino begomovirus strains in Cuban potato fields Phytoparasitica 31 478–489 Occurrence Handle1:CAS:528:DC%2BD2cXhtFSitL8%3D

  9. S Dellaporta J Wood JB Hicks (1983) ArticleTitleA plant DNA minipreparation: version II Plant Mol Biol 1 19–21 Occurrence Handle1:CAS:528:DyaL2cXksFWhtrk%3D

  10. M Domínguez PL Ramos AL Echemedia R Peral J Crespo V Andino M Pujol C Borroto (2002) ArticleTitleMolecular characterization of tobacco leaf rugose virus, a new begomovirus infecting tobacco in Cuba Plant Dis 86 1050

  11. AL Echemendía PL Ramos L Díaz A Fuentes M Pujol G González (2004) ArticleTitleFirst report of Sida golden yellow vein virus infecting Sida species in Cuba Plant Pathol 53 234 Occurrence Handle10.1111/j.0032-0862.2004.00965.x

  12. AL Echemendía PL Ramos R Peral A Fuentes G Gonzalez J Sanpedro FJ Morales (2001) ArticleTitleCuban isolate of Bean golden yellow mosaic virus is a member of the Mesomaerican BGYMV group Plant Dis 85 1030

  13. AL Echemendía PL Ramos R Peral A Fuentes M Pujol G Gonzalez (2003) ArticleTitleFirst report of Dicliptera yellow mottle virus (DiYMoV) infecting Dicliptera vahliana in Cuba Plant Pathol 52 787 Occurrence Handle10.1111/j.1365-3059.2003.00896.x

  14. CM Fauquet DP Maxwell B Gronenborn J Stanley (2000) ArticleTitleRevised proposal for naming geminiviruses Arch Virol 145 1743–1761 Occurrence Handle11003482 Occurrence Handle1:CAS:528:DC%2BD3cXnt1WhtL8%3D Occurrence Handle10.1007/s007050070089

  15. A Fire S Xu MK Montgomery SA Kostas SE Driver CC Mello (1998) ArticleTitlePotent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans Nature 391 806–811 Occurrence Handle9486653 Occurrence Handle1:CAS:528:DyaK1cXhtlCju74%3D Occurrence Handle10.1038/35888

  16. M Friedmann M Lapidot S Cohen M Pilowsky (1998) ArticleTitleA novel source of resistance to tomato yellow leaf curl virus exhibiting a symptomless reaction to viral Infection J Am Soc Hortic Sci 123 1004–1007

  17. A Fuentes N Soto D Alfonso P Oramas (1998) ArticleTitleEstudio de las condiciones de cultivo para la regeneración de plantas de tomate (Lycopersicon esculentum Mill), a partir de cotiledones y hojas de la variedad Campbell 28 Biotecnología Aplicada 15 242–245 Occurrence Handle1:CAS:528:DyaK1MXksVyq

  18. DR Gallie (1998) ArticleTitleControlling gene expression in transgenics Curr Opin Plant Biol 1 166–172 Occurrence Handle10066570 Occurrence Handle1:CAS:528:DyaK1cXivVynsb0%3D Occurrence Handle10.1016/S1369-5266(98)80020-4

  19. O Gómez G González (1993) ArticleTitleGenetic approach for resistance to the Cuban geminivirus transmitted by whitefly Tomato Leaf Curl Newslett 3 3

  20. O Gómez M Piñon Y Martínez M Quinones D Fonseca H Laterrot (2004) ArticleTitleBreeding for resistance to begomovirus in tropic-adapted tomato genotypes Plant Breeding 123 275–279 Occurrence Handle10.1111/j.1439-0523.2004.00959.x

  21. L Hanley-Bowdoin SB Settlage BM Orozco S Nagar D Robertson (2000) ArticleTitleGeminiviruses: models for plant DNA replication, transcription, and cell cycle regulation Crit Rev Biochem Mol Biol 35 105–140 Occurrence Handle10821479 Occurrence Handle1:CAS:528:DC%2BD3cXjsFChsb4%3D

  22. YM Hou RL Gilbertson (1996) ArticleTitleIncreased pathogenicity in a pseudorecombinant bipartite geminivirus correlates with intermolecular recombination J Virol 70 5430–5436 Occurrence Handle8764054 Occurrence Handle1:CAS:528:DyaK28Xktleisbg%3D

  23. T Kunik R Salomon D Zamir N Navot M Zeidan I Michelson Y Gafni H Czosnek (1994) ArticleTitleTransgenic tomato plants expressing the tomato yellow leaf curl virus capsid protein are resistant to the virus Biotechnology 15 500–504

  24. M Lapidot M Friedmann (2002) ArticleTitleBreeding for resistance to whitefly-transmitted geminiviruses Ann Appl Biol 140 109–127

  25. ZY Martínez C Blas Particlede M Quiñones C Castellanos EL Peralta J Romero (1998) ArticleTitleHavana tomato virus, a new bipartite geminivirus infecting tomatoes in Cuba Arch Virol 143 1757–1772 Occurrence Handle10.1007/s007050050414

  26. MA Maztke AJM Maztke (1995) ArticleTitleHow and why do plants lnactivate homologous (trans)genes? Plant Physiol 107 679–685

  27. McCormick S (1991) Plant Tissue Culture Manual Kluwer Academic Publisher.

  28. F Monci S Sanchez-Campos J Navas-Castillo E Moriones (2002) ArticleTitleA natural recombinant between the geminiviruses tomato yellow leaf curl Sardinia virus and tomato yellow leaf curl virus exhibits a novel pathogenic phenotype and is becoming prevalent in Spanish populations Virology 303 317–326 Occurrence Handle12490393 Occurrence Handle1:CAS:528:DC%2BD38XpsFyhurw%3D Occurrence Handle10.1006/viro.2002.1633

  29. FJ Morales PK Anderson (2001) ArticleTitleThe emergence and dissemination of whitefly-transmitted geminiviruses in Latin America Arch Virol 146 415–441 Occurrence Handle11338381 Occurrence Handle1:CAS:528:DC%2BD3MXktlWjtro%3D Occurrence Handle10.1007/s007050170153

  30. E Moriones J Navas-Castillo (2000) ArticleTitleTomato yellow leaf curl virus, an emerging virus complex causing epidemics worldwide Virus Res 71 123–134 Occurrence Handle11137167 Occurrence Handle1:CAS:528:DC%2BD3MXptVSnuw%3D%3D Occurrence Handle10.1016/S0168-1702(00)00193-3

  31. T Murashige F Skoog (1962) ArticleTitleA revised medium for rapid growth and bioassays with tobacco tissue culture Physiol Plant 15 473–497 Occurrence Handle1:CAS:528:DyaF3sXksFKm Occurrence Handle10.1111/j.1399-3054.1962.tb08052.x

  32. J Navas-Castillo S Sanchez-Campos E Noris D Louro GP Accotto E Moriones (2000) ArticleTitleNatural recombination between tomato yellow leaf curl virus-is and tomato leaf curl virus J Gen Virol 81 2797–2801 Occurrence Handle11038394 Occurrence Handle1:CAS:528:DC%2BD3cXnvFWnsr4%3D

  33. E Noris A Lucioli R Tavazza P Caciagli GP Accotto M Tavazza (2004) ArticleTitleTomato yellow leaf curl Sardinia virus can overcome transgene-mediated RNA silencing of two essential viral genes J Gen Virol 85 1745–1749 Occurrence Handle15166460 Occurrence Handle1:CAS:528:DC%2BD2cXltlGhtLg%3D Occurrence Handle10.1099/vir.0.79944-0

  34. T Pandolfini B Molesini L Avesani A Spena A Polverari (2003) ArticleTitleExpression of self-complementary hairpin RNA under the control of the rolC promoter confers systemic disease resistance to plum pox virus without preventing local infection BMC Biotechnol 3 7 Occurrence Handle12823862 Occurrence Handle10.1186/1472-6750-3-7

  35. M Pilowsky S Cohen (1990) ArticleTitleTolerance to tomato yellow leaf curl virus derived from Lycopersicon peruvianum Plant Dis 74 248–250

  36. JS Pita VN Fondong A Sangare GW Otim-Nape S Ogwal CM Fauquet (2001) ArticleTitleRecombination, pseudorecombination and synergism of geminiviruses are determinant keys to the epidemic of severe cassava mosaic disease in Uganda J Gen Virol 82 655–665 Occurrence Handle11172108 Occurrence Handle1:CAS:528:DC%2BD3MXhs1Sjtrs%3D

  37. JE Polston PK Anderson (1997) ArticleTitleThe emergence of whitefly-transmitted geminiviruses in tomato in the western hemisphere Plant Dis 81 1358–1369

  38. M Pooggin T Hohn (2004) ArticleTitleFighting geminiviruses by RNAi and vice versa Plant Mol Biol 55 149–152 Occurrence Handle15604671 Occurrence Handle1:CAS:528:DC%2BD2MXhsFajtg%3D%3D Occurrence Handle10.1007/s11103-004-0116-3

  39. PL Ramos AI Fernandez G Castrillo L Diaz AL Echemedia A Fuentes R Peral M Pujol JT Ascencio-Ibanez R Rivera-Bustamante G Arguello-Astorga (2002) ArticleTitleMacroptilium yellow mosaic virus, a new begomovirus infecting Macroptilium lathyroides in Cuba Plant Dis 86 1049

  40. PL Ramos O Guerra V Dorestes N Ramírez R Rivera- Bustamante P Oramas (1996) ArticleTitleDetection of TYLCV in Cuba Plant Dis 80 1208 Occurrence Handle10.1094/PD-80-1208C

  41. PL Ramos O Guerra R Peral R Rivera-Bustamante P Oramas (1997) ArticleTitleTaino tomato mottle virus, a new bipartite geminivirus from Cuba Plant Dis 81 1095

  42. PL Ramos RG Guevara-Gonzalez R Peral JT Ascencio-Ibanez JE Polston GR Arguello-Astorga JC Vega-Arreguin RF Rivera-Bustamante (2003) ArticleTitleTomato mottle Taino virus pseudorecombines with PYMV but not with ToMoV: implications for the delimitation of cis- and trans-acting replication specificity determinants Arch Virol 148 1697–1712 Occurrence Handle14505083 Occurrence Handle1:CAS:528:DC%2BD3sXntVKgsb8%3D Occurrence Handle10.1007/s00705-003-0136-3

  43. M Rom Y Antignus D Gidoni M Pilowsky S Cohen (1993) ArticleTitleAccumulation of tomato yellow leaf curl virus DNA in tolerant and susceptible tomato lines Plant Dis 77 253–257 Occurrence Handle1:CAS:528:DyaK3sXksVGgtbo%3D Occurrence Handle10.1094/PD-77-0253

  44. AI Sanz A Fraile F Garcia-Arenal X Zhou DJ Robinson S Khalid T Butt BD Harrison (2000) ArticleTitleMultiple infection, recombination and genome relationships among begomovirus isolates found in cotton and other plants in Pakistan J Gen Virol 81 1839–1849 Occurrence Handle10859391 Occurrence Handle1:CAS:528:DC%2BD3cXks12hsrc%3D

  45. NA Smith SP Singh MB Wang PA Stoutjesdijk AG Green PM Waterhouse (2000) ArticleTitleTotal silencing by intron-spliced hairpin RNAs Nature 407 319–320 Occurrence Handle11014180 Occurrence Handle1:CAS:528:DC%2BD3cXnsVKls7Y%3D Occurrence Handle10.1038/35036500

  46. R Wezel Particlevan X Dong H Liu P Tien J Stanley Y Hong (2002) ArticleTitleMutation of three cysteine residues in tomato yellow leaf curl virus-China C2 protein causes dysfunction in pathogenesis and posttranscriptional gene-silencing suppression Mol Plant Microbe Interact 15 203–208

  47. R Vanitharani P Chellappan CM Fauquet (2003) ArticleTitleShort interfering RNA-mediated interference of gene expression and viral DNA accumulation in cultured plant cells Proc Natl Acad Sci USA 100 IssueID16 9632–9636 Occurrence Handle12886005 Occurrence Handle1:CAS:528:DC%2BD3sXmtlyrs74%3D Occurrence Handle10.1073/pnas.1733874100

  48. TC Verwoerd BM Dekker A Hoekema (1989) ArticleTitleA small scale procedure for the rapid isolation of plant RNAs Nucleic Acids Res 17 IssueID6 2362 Occurrence Handle2468132 Occurrence Handle1:CAS:528:DyaL1MXitVyrurs%3D

  49. F Vidavsky H Czosnek (1998) ArticleTitleTomato breeding lines immune and tolerant to tomato yellow leaf curl virus (TYLCV) issued from Lycopersicon hirsutum Phytopathology 88 910–914 Occurrence Handle1:STN:280:DC%2BD1cjjvVWltw%3D%3D Occurrence Handle18944868

  50. MB Wang D Abbott PM Waterhouse (2000) ArticleTitleA single copy of virus-derived transgene encoding hairping RNA gives imminuty to barley yellow dwarf virus Mol plant Pathol 1 347–356 Occurrence Handle1:CAS:528:DC%2BD3MXisF2hs74%3D Occurrence Handle10.1046/j.1364-3703.2000.00038.x Occurrence Handle20572982

  51. PM Waterhouse MB Wang T Lough (2001) ArticleTitleGene silencing as an adaptive defence against viruses Nature 411 834–842 Occurrence Handle11459066 Occurrence Handle1:CAS:528:DC%2BD3MXksF2gu7Y%3D Occurrence Handle10.1038/35081168

  52. Y Yang TA Sherwood CP Patte E Hiebert JE Polston (2004) ArticleTitleUse of tomato yellow leaf curl virus Rep gene sequences to engineer TYLCV resistance in tomato Phytopathology 94 490–496 Occurrence Handle1:CAS:528:DC%2BD2cXkt1Sitrs%3D Occurrence Handle18943768

  53. Z Zakay N Navot M Zeidan N Kedar HD Rabinowitch H Czosnek D Zamir (1991) ArticleTitleScreening of Lycopersicon accessions for resistance to tomato yellow leaf curl virus: presence of viral DNA and symptom development Plant Dis 75 279–281 Occurrence Handle10.1094/PD-75-0279

  54. D Zamir I Ekstein-Michelson Z Zakay N Navot M Zeidan M Sarfatti Y Eshed E Harel T Pleban H van-Oss N Kedar HD Rabinowitch H Czosnek (1994) ArticleTitleMapping and introgression of a tomato yellow leaf curl virus tolerance gene, TY1 Theor Appl Genet 88 141–146 Occurrence Handle1:CAS:528:DyaK2MXht1ahu7w%3D Occurrence Handle10.1007/BF00225889

Download references

Author information

Correspondence to Merardo Pujol.

Additional information

Alejandro Fuentes, Pedro L. Ramos: These authors contributed equally to this work

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Fuentes, A., Ramos, P.L., Fiallo, E. et al. Intron–hairpin RNA Derived from Replication Associated Protein C1 Gene Confers Immunity to Tomato Yellow Leaf Curl Virus Infection in Transgenic Tomato Plants. Transgenic Res 15, 291–304 (2006).

Download citation


  • begomoviruses
  • pathogen-derived resistance
  • post-transcriptional gene silencing
  • tomato
  • transgenic plants