Transgenic Research

, Volume 20, Issue 3, pp 569–581 | Cite as

Expression of artificial microRNAs in tomato confers efficient and stable virus resistance in a cell-autonomous manner

  • Xiaohui Zhang
  • Hanxia Li
  • Junhong Zhang
  • Chanjuan Zhang
  • Pengjuan Gong
  • Khurram Ziaf
  • Fangming Xiao
  • Zhibiao YeEmail author
Original Paper


Expression of artificial microRNAs (amiRNAs) in plants can target and degrade the invading viral RNA, consequently conferring virus resistance. Two amiRNAs, targeting the coding sequence shared by the 2a and 2b genes and the highly conserved 3′ untranslated region (UTR) of Cucumber mosaic virus (CMV), respectively, were generated and introduced into the susceptible tomato. The transgenic tomato plants expressing amiRNAs displayed effective resistance to CMV infection and CMV mixed with non-targeted viruses, including tobacco mosaic virus and tomato yellow leaf curl virus. A series of grafting assays indicate scions originated from the transgenic tomato plant maintain stable resistance to CMV infection after grafted onto a CMV-infected rootstock. However, the grafting assay also suggests that the amiRNA-mediated resistance acts in a cell-autonomous manner and the amiRNA signal cannot be transmitted over long distances through the vascular system. Moreover, transgenic plants expressing amiRNA targeting the 2a and 2b viral genes displayed slightly more effective to repress CMV RNA accumulation than transgenic plants expressing amiRNA targeting the 3′ UTR of viral genome did. Our work provides new evidence of the use of amiRNAs as an effective approach to engineer viral resistance in the tomato and possibly in other crops.


Artificial miRNA Anti-virus Cucumber mosaic virus Tomato Cell-autonomous Mixed infection 



We thank to Prof. Rongxiang Fang (State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China) for providing CMV-SD for plant viral challenge and Miss Maria Nussbaum for critical reading of the manuscript. This work was supported by grants from the 973 Program (No. 2009CB119000) and NSFC (No. 30671417), and 863 Program (No. 2007AA10Z131).

Supplementary material

11248_2010_9440_MOESM1_ESM.pdf (575 kb)
Supplementary material 1 (PDF 574 kb)


  1. Alvarez JP, Pekker I, Goldshmidt A, Blum E, Amsellem Z, Eshed Y (2006) Endogenous and synthetic microRNAs stimulate simultaneous, efficient, and localized regulation of multiple targets in diverse species. Plant Cell 18:1134–1151PubMedCrossRefGoogle Scholar
  2. Brodersen P, Sakvarelidze-Achard L, Bruun-Rasmussen M, Dunoyer P, Yamamoto YY, Sieburth L, Voinnet O (2008) Widespread translational inhibition by plant miRNAs and siRNAs. Science 320:1185–1190PubMedCrossRefGoogle Scholar
  3. Cillo F, Mascia T, Pasciuto MM, Gallitelli D (2009) Differential effects of mild and severe Cucumber mosaic virus strains in the perturbation of MicroRNA-regulated gene expression in tomato map to the 3′ sequence of RNA 2. Mol Plant Microbe Interact 22:1239–1249PubMedCrossRefGoogle Scholar
  4. Ding SW, Voinnet O (2007) Antiviral immunity directed by small RNAs. Cell 130:413–426PubMedCrossRefGoogle Scholar
  5. Du ZY, Chen FF, Zhao ZJ, Liao QS, Palukaitis P, Chen JS (2008) The 2b protein and the C-terminus of the 2a protein of cucumber mosaic virus subgroup I strains both play a role in viral RNA accumulation and induction of symptoms. Virology 380:363–370PubMedCrossRefGoogle Scholar
  6. Duan CG, Wang CH, Fang RX, Guo HS (2008) Artificial microRNAs highly accessible to targets confer efficient virus resistance in plants. J Virol 82:11084–11095PubMedCrossRefGoogle Scholar
  7. Fillatti J, Kiser J, Rose R, Comai L (1987) Efficient transfer of a glyphosate tolerance gene into tomato using a binary Agrobacterium tumefaciens vector. Nat Biotechnol 5:726–730CrossRefGoogle Scholar
  8. Fuchs M, Provvidenti R, Slightom JL, Gonsalves D (1996) Evaluation of transgenic tomato plants expressing the coat protein gene of Cucumber mosaic virus strain WL under field conditions. Plant Dis 80:270–275CrossRefGoogle Scholar
  9. Gallitelli D (2000) The ecology of Cucumber mosaic virus and sustainable agriculture. Virus Res 71:9–21PubMedCrossRefGoogle Scholar
  10. Gal-On A, Wolf D, Wang Y, Faure J, Pilowsky M, Zelcer A (1998) Transgenic resistance to Cucumber mosaic virus in tomato: Blocking of long-distance movement of the virus in lines harboring adefective viral replicase gene. Phytopathology 88:1101–1107PubMedCrossRefGoogle Scholar
  11. Gielen J, Ultzen T, Bontems S, Loots W, van Schepen A (1996) Coat protein-mediated protection to Cucumber mosaic virus infections in cultivated tomato. Euphytica 88:139–149CrossRefGoogle Scholar
  12. Glick E, Zrachya A, Levy Y, Mett A, Gidoni D, Belausov E, Citovsky V, Gafni Y (2008) Interaction with host SGS3 is required for suppression of RNA silencing by tomato yellow leaf curl virus V2 protein. Proc Natl Acad Sci USA 105:157–161PubMedCrossRefGoogle Scholar
  13. Harries PA, Palanichelvam K, Bhat S, Nelson RS (2008) Tobacco mosaic virus 126-kDa protein increases the susceptibility of Nicotiana tabacum to other viruses and its dosage affects virus-induced gene silencing. Mol Plant Microbe Interact 21:1539–1548PubMedCrossRefGoogle Scholar
  14. Hwang MS, Kim KN, Lee JH, Park YI (2007) Identification of amino acid sequences determining interaction between the cucumber mosaic virus-encoded 2a polymerase and 3a movement proteins. J Gen Virol 88:3445–3451PubMedCrossRefGoogle Scholar
  15. Kaniewski W, Ilardi V, Tomassoli L, Mitsky T, Layton J, Barba M (1999) Extreme resistance to Cucumber mosaic virus (CMV) in transgenic tomato expressing one or two viral coat proteins. Mol Breed 5:111–119CrossRefGoogle Scholar
  16. Kehr J, Buhtz A (2008) Long distance transport and movement of RNA through the phloem. J Exp Bot 59:85–92PubMedCrossRefGoogle Scholar
  17. Khraiwesh B, Ossowski S, Weigel D, Reski R, Frank W (2008) Specific gene silencing by artificial MicroRNAs in Physcomitrella patens: an alternative to targeted gene knockouts. Plant Physiol 148:684–693PubMedCrossRefGoogle Scholar
  18. Levy A, Dafny-Yelin M, Tzfira T (2008) Attacking the defenders: plant viruses fight back. Trends Microbiol 16:194–197PubMedCrossRefGoogle Scholar
  19. Lewsey M, Surette M, Robertson FC, Ziebell H, Choi SH, Ryu KH, Canto T, Palukaitis P, Payne T, Walsh JA, Carr JP (2009) The role of the Cucumber mosaic virus 2b protein in viral movement and symptom induction. Mol Plant Microbe Interact 22:642–654PubMedCrossRefGoogle Scholar
  20. Li F, Ding SW (2006) Virus counterdefense: diverse strategies for evading the RNA-silencing immunity. Annu Rev Microbiol 60:503–531PubMedCrossRefGoogle Scholar
  21. Lucioli A, Sallustio DE, Barboni D, Berardi A, Papacchioli V, Tavazza R, Tavazza M (2008) A cautionary note on pathogen-derived sequences. Nat Biotechnol 26:617–619PubMedCrossRefGoogle Scholar
  22. Molnar A, Bassett A, Thuenemann E, Schwach F, Karkare S, Ossowski S, Weigel D, Baulcombe D (2009) Highly specific gene silencing by artificial microRNAs in the unicellular alga Chlamydomonas reinhardtii. Plant J 58:165–174CrossRefGoogle Scholar
  23. Morroni M, Thompson JR, Tepfer M (2008) Twenty years of transgenic plants resistant to Cucumber mosaic virus. Mol Plant Microbe Interact 21:675–684PubMedCrossRefGoogle Scholar
  24. Naqvi AR, Choudhury NR, Haq QM, Mukherjee SK (2008) MicroRNAs as biomarkers in tomato leaf curl virus (ToLCV) disease. Nucleic Acids Symp Ser (Oxf) 52:507–508CrossRefGoogle Scholar
  25. Niu QW, Lin SS, Reyes JL, Chen KC, Wu HW, Yeh SD, Chua NH (2006) Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance. Nat Biotechnol 24:1420–1428PubMedCrossRefGoogle Scholar
  26. Ossowski S, Schwab R, Weigel D (2008) Gene silencing in plants using artificial microRNAs and other small RNAs. Plant J 53:674–690PubMedCrossRefGoogle Scholar
  27. Pant BD, Buhtz A, Kehr J, Scheible WR (2008) MicroRNA339 is a long-distance signal for the regulation of plant phosphate homeostasis. Plant J 53:731–738PubMedCrossRefGoogle Scholar
  28. Qu J, Ye J, Fang RX (2007) Artificial microRNA-mediated virus resistance in plants. J Virol 81:6690–6699PubMedCrossRefGoogle Scholar
  29. Ruiz-Ferrer V, Voinnet O (2009) Roles of plant small RNAs in biotic stress responses. Annu Rev Plant Biol 60:485–510PubMedCrossRefGoogle Scholar
  30. Schwab R, Ossowski S, Riester M, Warthmann N, Weigel D (2006) Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell 18:1121–1133PubMedCrossRefGoogle Scholar
  31. Seo JK, Kwon SJ, Choi HS, Kim KH (2009) Evidence for alternate states of Cucumber mosaic virus replicase assembly in positive- and negative-strand RNA synthesis. Virology 383:248–260PubMedCrossRefGoogle Scholar
  32. Soards AJ, Murphy AM, Palukaitis P, Carr JP (2002) Virulence and differential local and systemic spread of cucumber mosaic virus in tobacco are affected by the CMV 2b protein. Mol Plant Microbe Interact 15:647–653PubMedCrossRefGoogle Scholar
  33. Stommel JR, Tousignant ME, Wai T, Pasini R, Kaper JM (1998) Viral satellite RNA expression in transgenic tomato confers field tolerance to Cucumber mosaic virus. Plant Dis 82:391–396CrossRefGoogle Scholar
  34. Thompson JR, Tepfer M (2009) The 3′ untranslated region of cucumber mosaic virus (CMV) subgroup II RNA3 arose by interspecific recombination between CMV and tomato aspermy virus. J Gen Virol 90:2293–2298PubMedCrossRefGoogle Scholar
  35. Tomassoli L, Ilardi V, Barba M, Kaniewski W (1999) Resistance of transgenic tomato to cucumber mosaic cucumovirus under field conditions. Mol Breed 5:121–130CrossRefGoogle Scholar
  36. Villani ME, Roggero P, Bitti O, Benvenuto E, Franconi R (2005) Immunomodulation of cucumber mosaic virus infection by intrabodies selected in vitro from a stable single-framework phage display library. Plant Mol Biol 58:305–316PubMedCrossRefGoogle Scholar
  37. Vogler H, Akbergenov R, Shivaprasad PV, Dang V, Fasler M, Kwon MO, Zhanybekova S, Hohn T, Heinlein M (2007) Modification of small RNAs associated with suppression of RNA silencing by tobamovirus replicase protein. J Virol 81:10379–10388PubMedCrossRefGoogle Scholar
  38. Voinnet O (2009) Origin, biogenesis, and activity of plant microRNAs. Cell 136:669–687PubMedCrossRefGoogle Scholar
  39. Wang MA D, Waterhouse P (2000) A single copy of a virus-derived transgene encoding hairpin RNA gives immunity to barley yellow dwarf virus. Mol Plant Pathol 1:347–356CrossRefGoogle Scholar
  40. Warthmann N, Chen H, Ossowski S, Weigel D, Herve P (2008) Highly specific gene silencing by artificial miRNAs in rice. PLoS One 3:e1829PubMedCrossRefGoogle Scholar
  41. Xue B, Gonsalves C, Provvidenti R (1994) Development of transgenic tomato expressing a high level of resistance to Cucumber mosaic virus strains of subgroups I and II. Plant Dis 78:1038–1041CrossRefGoogle Scholar
  42. Zhang XR, Yuan YR, Pei Y, Lin SS, Tuschl T, Patel DJ, Chua NH (2006) Cucumber mosaic virus-encoded 2b suppressor inhibits Arabidopsis Argonaute1 cleavage activity to counter plant defense. Gene Dev 20:3255–3268PubMedCrossRefGoogle Scholar
  43. Zhang S, Sun L, Kragler (2009) The phloem-delivered RNA pool contains small noncoding RNAs and interferes with translation. Plant Physiol 150:378–387PubMedCrossRefGoogle Scholar
  44. Zrachya A, Glick E, Levy Y, Arazi T, Citovsky V, Gafni Y (2007) Suppressor of RNA silencing encoded by tomato yellow leaf curl virus-Israel. Virology 358:159–165PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Xiaohui Zhang
    • 1
  • Hanxia Li
    • 2
  • Junhong Zhang
    • 2
  • Chanjuan Zhang
    • 1
  • Pengjuan Gong
    • 1
  • Khurram Ziaf
    • 1
  • Fangming Xiao
    • 3
  • Zhibiao Ye
    • 1
    • 2
    Email author
  1. 1.National Key Laboratory of Crop Genetic ImprovementHuazhong Agricultural UniversityWuhanChina
  2. 2.Key Laboratory of Horticultural Plant Biology, Ministry of EducationHuazhong Agricultural UniversityWuhanChina
  3. 3.Department of Microbiology, Molecular Biology and BiochemistryUniversity of IdahoMoscowUSA

Personalised recommendations