Transgenic Research

, Volume 16, Issue 3, pp 385–398 | Cite as

Production of siRNA targeted against TYLCV coat protein transcripts leads to silencing of its expression and resistance to the virus

  • Avi Zrachya
  • Pravin P. Kumar
  • Usha Ramakrishnan
  • Yael Levy
  • Abraham Loyter
  • Tzahi Arazi
  • Moshe Lapidot
  • Yedidya Gafni
Original Paper


The coat protein (CP) of Tomato yellow leaf curl virus (TYLCV), encoded by the v1 gene, is the only known component of the viral capsid. In addition, the CP plays a role in the virus transport into the host cell nucleus where viral genes are replicated and transcribed. In this study, we analyzed the effect of small interfering double-stranded RNAs (siRNAs), derived from an intron-hairpin RNA (ihpRNA) construct and targeting the v1 gene product, on CP accumulation. Transient assays involving agroinfiltration of the CP-silencing construct followed by infiltration of a fused GFP-CP (green fluorescent protein-coat protein) gene showed down-regulation of GFP expression in Nicotianabenthamiana. Some of the transgenic tomato plants (cv. Micro-Tom), expressing the siRNA targeted against the TYLCV CP gene, did not show disease symptoms 7 weeks post-inoculation with the virus, while non-transgenic control plants were infected within 2 weeks post inoculation. The present study demonstrates, for the first time, that siRNA targeted against the CP of TYLCV can confer resistance to the virus in transgenic tomato plants, thereby enabling flowering and fruit production.


Geminiviruses PTGS Virus resistance 


  1. Baulcombe D (2004) RNA silencing in plants. Nature 431:356–363PubMedCrossRefGoogle Scholar
  2. Bendahmane A, Querci M, Kanyuka K, Baulcombe DC (2000) Agrobacterium transient expression system as a tool for the isolation of disease resistance genes: application to the R × 2 locus in potato. Plant J 21:73–81PubMedCrossRefGoogle Scholar
  3. Boulton MI, Steinkellner H, Donson J, Markham PG, King DI, Davies JW (1989) Mutational analysis of the virion-sense genes of maize streak virus. J Gen Virol 70(Pt 9):2309–2323PubMedCrossRefGoogle Scholar
  4. Briddon RW, Watts J, Markham PG, Stanley J (1989) The coat protein of beet curly top virus is essential for infectivity. Virology 172:628–633PubMedCrossRefGoogle Scholar
  5. Chellappan P, Vanitharani R, Fauquet CM (2004) Short interfering RNA accumulation correlates with host recovery in DNA virus-infected hosts, and gene silencing targets specific viral sequences. J Virol 78: 7465–7477PubMedCrossRefGoogle Scholar
  6. Chen H, Nelson RS, Sherwood JL (1994) Enhanced recovery of transformants of Agrobacterium tumefaciens after freeze-thaw transformation and drug selection. BioTechniques 16:664–670PubMedGoogle Scholar
  7. Cohen S, Antignus Y (1994) Tomato yellow leaf curl virus (TYLCV), a whitefly-borne geminivirus of tomatoes. In: Harris KF (eds) Vol 10. Springer-Verlag, New York, pp 259–288Google Scholar
  8. Ding SW, Li H, Lu R, Li F, Li W-X (2004) RNA silencing: a conserved antiviral immunity of plants and animals. Virus Res. 102: 109–115PubMedCrossRefGoogle Scholar
  9. Dong X, van Wezel R, Stanley J, Hong Y (2003) Functional characterization of the nuclear localization signal for a suppressor of posttranscriptional gene silencing. J Virol 77: 7026–7033PubMedCrossRefGoogle Scholar
  10. Fillati JJ, Kiser J, Rose R, Comai L (1987) Efficient transfer of a glyphosate tolerance gene into tomato using a binary Agrobacterium tumefaciens vector. Biotechnology 5: 726–730CrossRefGoogle Scholar
  11. Friedmann M, Lapidot M, Cohen S, Pilowsky M (1998) A novel source of resistance to tomato yellow leaf curl virus exhibiting a symptomless reaction to viral infection. J Am Soc Hort Sci 123:1004–1007Google Scholar
  12. Gafni Y (1998) Nucleocytoplasmic trafficking of geminiviral proteins. Current Sci 75:1148–1152Google Scholar
  13. Gafni Y (2003) Tomato yellow leaf curl virus, the intracellular dynamics of a plant DNA virus. Mol Plant Pathol 4:9–15CrossRefGoogle Scholar
  14. Gafni Y, Epel BL (2002) The role of host and viral proteins in intra- and inter-cellular trafficking of geminiviruses. Physiol Mol Plant Pathol 60:231–241CrossRefGoogle Scholar
  15. Gal-On A, Wolf D, Antignus Y, Patlis L, Hyun Ryu K, Eun Min B, Pearlsman M, Lachman O, Gaba V, Wang Y, Moshe Shibolet Y, Yang J, Zelcer A (2005) Transgenic cucumbers harboring the 54-kDa putative gene of Cucumber fruit mottle mosaic tobamovirus are highly resistant to viral infection and protect non-transgenic scions from soil infection. Trans Res 14:81–93CrossRefGoogle Scholar
  16. Hallan V, Gafni Y (2001) Tomato yellow leaf curl virus (TYLCV) capsid protein (CP) subunit interactions: implications for viral assembly. Arch Virol 146:1765–1773PubMedCrossRefGoogle Scholar
  17. Hannon GJ (2002) RNA interference. Nature 418:244–251PubMedCrossRefGoogle Scholar
  18. Hood EE, Gelvin SB, Melchers LS, Hoekema A (1993) New Agrobacterium helperplasmids for gene transfer to plants. Trans Res 2:208–218CrossRefGoogle Scholar
  19. Kheyr-Pour A, Bendahmane M, Matzeit V, Accotto GP, Crespi S, Gronenborn B (1991) Tomato yellow leaf curl virus from Sardinia is a whitefly-transmitted monopartite geminivirus. Nucleic Acids Res 19:6763–6769PubMedCrossRefGoogle Scholar
  20. Koncz C, Schell J (1986) The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet 204:383–396CrossRefGoogle Scholar
  21. Kunik T, Palanichelvam K, Czosnek H, Citovsky V, Gafni Y (1998) Nuclear import of the capsid protein of tomato yellow leaf curl virus (TYLCV) in plant and insect cells. Plant J 13:393–399PubMedCrossRefGoogle Scholar
  22. Kunik T, Salomon R, Zamir D, Navot N, Zeidan M, Michelson I, Gafni Y, Czosnek H (1994) Transgenic tomato plants expressing the tomato yellow leaf curl virus capsid protein are resistant to the virus. Biotechnology (NY) 12:500–504CrossRefGoogle Scholar
  23. Lapidot M, Friedmann M (2002) Breeding for resistance to whitefly-transmitted geminiviruses. Annals of Applied Biology 140:109–127CrossRefGoogle Scholar
  24. Lapidot M, Friedmann M, Lachman O, Yehezkel A, Nahon S, Cohen S, Pilowsky M (1997) Comparison of resistance level to Tomato yellow leaf curl virus among commercial cultivars and breeding lines. Plant Disease 81:1425–1428Google Scholar
  25. Lazarowitz SG, Pinder AJ, Damsteegt VD, Rogers SG (1989) Maize streak virus genes essential for systemic spread and symptom development. EMBO J 8:1023–1032PubMedGoogle Scholar
  26. Mansoor S, Briddon RW, Zafar Y, Stanley J (2003) Geminivirus disease complexes: an emerging threat. Trends Plant Sci 8:128–134PubMedCrossRefGoogle Scholar
  27. Martins CR, Johnson JA, Lawrence DM, Choi TJ, Pisi AM, Tobin SL, Lapidus D, Wagner JD, Ruzin S, McDonald K, Jackson AO (1998) Sonchus yellow net rhabdovirus nuclear viroplasms contain polymerase-associated proteins. J Virol 72:5669–5679PubMedGoogle Scholar
  28. Mathieu O, Bender J (2004) RNA-directed DNA methylation. J Cell Sci 117:4881–4888PubMedCrossRefGoogle Scholar
  29. Meissner R, Jacobson Y, Melamed S, Levyatuv S, Shalev G, Ashri A, Elkind YAL (1997) A new model system for tomato genetics. The Plant J 12:1465–1472CrossRefGoogle Scholar
  30. Mello CC, Conte D Jr (2004) Revealing the world of RNA interference. Nature 431:338–342PubMedCrossRefGoogle Scholar
  31. Moriones E, Navas-Castillo J (2000) Tomato yellow leaf curl virus, an emerging virus complex causing epidemics worldwide. Virus Res 71:123–134PubMedCrossRefGoogle Scholar
  32. Nakhla MK, Maxwell DP (1998) Epidemiology and management of tomato yellow leaf curl disease. In: Hadidi A, Khetarpal RK, Koganezawa H (eds) Plant virus disease control. APS Press, St. Paul, Minnesota, USA, pp 565–578Google Scholar
  33. Navot N, Pichersky E, Zeidan M, Zamir D, Czosnek H (1991) Tomato yellow leaf curl virus: a whitefly-transmitted geminivirus with a single genomic component. Virology 185:151–161PubMedCrossRefGoogle Scholar
  34. Noris E, Accotto GP, Tavazza R, Brunetti A, Crespi S, Tavazza M (1996) Resistance to tomato yellow leaf curl geminivirus in Nicotiana benthamiana plants transformed with a truncated viral C1 gene. Virology 224:130–138PubMedCrossRefGoogle Scholar
  35. Noris E, Lucioli A, Tavazza R, Caciagli P, Accotto GP, Tavazza M (2004) Tomato yellow leaf curl Sardinia virus can overcome transgene-mediated RNA silencing of two essential viral genes. J Gen Virol 85:1745–1749PubMedCrossRefGoogle Scholar
  36. Noris E, Vaira AM, Caciagli P, Masenga V, Gronenborn B, Accotto GP (1998) Amino acids in the capsid protein of tomato yellow leaf curl virus that are crucial for systemic infection, particle formation, and insect transmission. J Virol 72:10050–10057PubMedGoogle Scholar
  37. Palanichelvam K, Kunik T, Citovsky V, Gafni Y (1998) The capsid protein of tomato yellow leaf curl virus binds cooperatively to single-stranded DNA. J Gen Virol 79(Pt 11):2829–2833PubMedGoogle Scholar
  38. Park SH, Morris JL, Park JE, Hirschi KD, Smith RH (2003) Efficient and genotype-independent Agrobacterium–mediated tomato transformation. J Plant Physiol 160:1253–1257PubMedCrossRefGoogle Scholar
  39. Saghai-Maroof MA, Soliman KM, Jorgensen RA, Allard RW (1984) Ribosomal DNA spacer-length polymorphisms in barley: mendelian inheritance, chromosomal location and population dynamics. Proc Nation Acad Sci USA 81:8014–8018CrossRefGoogle Scholar
  40. Sagi G, Katz A, Guenoune-Gelbart D, Epel BL (2005) Class 1 reversibly glycosylated polypeptides are plasmodesmal-associated proteins delivered to plasmodesmata via the golgi apparatus. Plant Cell 17:1788–1800PubMedCrossRefGoogle Scholar
  41. Sambrook J, Russell DW (2001) Molecular Cloning – a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory PressGoogle Scholar
  42. Smith NA, Singh SP, Wang M-B, Stoutjesdijk PA, Green AG, Waterhouse PM (2000) Gene expression: Total silencing by intron-spliced hairpin. Nature 407:319–320PubMedCrossRefGoogle Scholar
  43. Vanitharani R, Chellappan P, Fauquet CM (2005) Geminiviruses and RNA silencing. Trends Plant Sci 10:144–151PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Avi Zrachya
    • 1
    • 3
  • Pravin P. Kumar
    • 2
  • Usha Ramakrishnan
    • 2
  • Yael Levy
    • 1
  • Abraham Loyter
    • 3
  • Tzahi Arazi
    • 4
  • Moshe Lapidot
    • 1
  • Yedidya Gafni
    • 1
  1. 1.Department of Genetics, Agricultural Research OrganizationThe Volcani CenterBet DaganIsrael
  2. 2.Department of Plant Biotechnology, School of BiotechnologyMadurai Kamaraj UniversityMaduraiIndia
  3. 3.Department of Biological Chemistry, The Alexander Silberman Institute of Life SciencesThe Hebrew University of JerusalemJerusalemIsrael
  4. 4.Department of Ornamental HorticultureThe Volcani CenterBet DaganIsrael

Personalised recommendations