Skip to main content

Advertisement

SpringerLink
Log in

Characterization of resistance mechanism in transgenic Nicotiana benthamiana containing Turnip crinkle virus coat protein

  • Genetic Transformation and Hybridization
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

Two transgenic lines, of Nicotiana benthamiana expressing Turnip crinkle virus (TCV)-coat protein (CP) gene with contrasting phenotype, the highest (#3) and the lowest (#18) CP expressers, were selected and challenged with the homologous TCV. The former, the highest expresser, showed nearly five times more CP expression than the latter. Progenies of #3 and #18 lines showed 30 and 100% infection rates, respectively. The infected progenies of #3 line showed mild and delayed symptom with TCV. This is a coat protein-mediated resistance (CP-MR), and its resistance level is directly proportional to CP transgene expression. However, CP-MR of the transgenic plants was specific only for TCV but not for heterologous viruses. Newly growing leaves of those infected progenies of #3 line did not show any visible symptoms at 4-week post-inoculation (wpi) with TCV, suggesting a reversal from infection. This was confirmed by RT-PCR analysis with the disappearance of the target at 4 wpi. This is a case of RNA-mediated resistance, and a threshold level of transgene expression may be needed to achieve the silent state. To confirm the RNA silencing, we infiltrated Agrobacterium carrying TCV-CP into leaves of progenies of #3 and performed RT-PCR analysis. The results indicate that TCV-CP’s suppressor activity against RNA silencing itself can be silenced by the homologous expression of TCV-CP in the transgenic plants. The transgenic plants containing TCV-CP seem to be a model system to study viral protection mediated by a combination of protein and RNA silencing.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Abel PP, Nelson RS, De B, Hoffmann N, Rogers SG, Fraley RT, Beachy RN (1986) Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene. Science 232:738–743

    Article  PubMed  CAS  Google Scholar 

  • Baulcombe DC (1996) Mechanisms of pathogen-derived resistance to viruses in transgenic plants. Plant Cell 8:1833–1844

    Article  PubMed  CAS  Google Scholar 

  • Baulcombe D (2004) RNA silencing in plants. Nature 431:356–363

    Article  PubMed  CAS  Google Scholar 

  • Beachy RN (1999) Coat-protein-mediated resistance to tobacco mosaic virus: discovery mechanisms and exploitation. Philos Trans R Soc Lond B 354:659–664

    Article  CAS  Google Scholar 

  • Bendahmane M, Fitchen JH, Zhang G, Beachy RN (1997) Studies of coat protein-mediated resistance to tobamo mosaic tobamovirus: correlation between assembly of mutant coat proteins and resistance. J Virol 71:7942–7950

    PubMed  CAS  Google Scholar 

  • Brigneti G, Voinnet O, Li WX, Ji LH, Ding SW, Baulcombe DC (1998) Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana. EMBO J 17:6739–6746

    Article  PubMed  CAS  Google Scholar 

  • Bucher E, Lohuis D, van Poppel PMJA, Greerts-Dimitriadou C, Goldbach R, Prins M (2006) Multilpe virus resistance at a high frequency using a single transgene construct. J Gen Virol 87:3697–3701

    Article  PubMed  CAS  Google Scholar 

  • Choi CW, Qu F, Ren T, Ye X, Morris TJ (2004) RNA silencing-suppressor function of Turnip crinkle virus coat protein cannot be attributed to its interaction with the Arabidopsis protein TIP. J Gen Virol 85:3415–3420

    Article  PubMed  CAS  Google Scholar 

  • Clark MF, Lister RM, Bar-Joseph M (1986) ELISA techniques. Methods Enzymol 118:742–766

    Article  CAS  Google Scholar 

  • Clark WG, Fitchen JH, Beachy RN (1995) Studies of coat-protein mediated resistance to TMV using mutant CP. I. The PM2 assembly defective mutant. Virology 208:485–491

    Article  PubMed  CAS  Google Scholar 

  • Cooley MB, Pathirana S, Wu HJ, Kachroo P, Klessig DF (2000) Members of the Arabidopsis HRT/RPP8 family of resistance genes confer resistance to both viral and oomycete pathogens. Plant Cell 12:663–676

    Article  PubMed  CAS  Google Scholar 

  • de Hann P, Gielen JJL, Prins M, Wijkamp IG, van Schepen A, Peters D, van Grinsven MQJM, Goldbach R (1992) Characterization of RNA-mediated resistance to tomato spotted wilt virus in transgenic tobacco plants. Bio/Technology 10:1133–1137

    Article  Google Scholar 

  • Ding SW (2000) RNA silencing. Curr Opin Biotechnol 11:152–156

    Article  PubMed  CAS  Google Scholar 

  • Ding SW, Li H, Lu R, Li F, Li WX (2004) RNA silencing: a conserved antiviral immunity of plants and animals. Virus Res 102:109–115

    Article  PubMed  CAS  Google Scholar 

  • Dougherty WG, Lindbo JA, Smith HA, Parks TD, Swaney S, Proebsting WM (1994) RNA-mediated virus resistance in transgenic plants: exploitation of a cellular pathway possibly involved in RNA degradation. Mol Plant Microbe Interact 7:544–552

    PubMed  CAS  Google Scholar 

  • English JJ, Mueller E, Baulcombe DC (1996) Suppression of virus accumulation in transgenic plants exhibiting silencing of nuclear genes. Plant Cell 8:179–188

    Article  PubMed  CAS  Google Scholar 

  • Fitchen JH, Beachy RN (1993) Genetically engineered protection against viruses in transgenic plants. Annu Rev Microbiol 4:739–763

    Article  Google Scholar 

  • Garcia S, Billecocq A, Crance J-M, Prins M, Garin D, Bouloy M (2006) Viral suppressors of RNA interference impair RNA silencing induced by a Semliki Forest virus replicon in tick cells. J Gen Virol 87:1985–1989

    Article  PubMed  CAS  Google Scholar 

  • Goldbach R, Bucher E, Prins M (2003) Resistance mechanisms to plant viruses: an overview. Virus Res 92:207–212

    Article  PubMed  CAS  Google Scholar 

  • Goodwin J, Chapman K, Swaney S, Parks TD, Wernsman EA, Dougherty WG (1996) Genetic and biochemical dissection of transgenic RNA-mediated virus resistance. Plant Cell 8:95–105

    Article  PubMed  CAS  Google Scholar 

  • Hacker DL, Petty ITD, Wei N, Morris TJ (1992) Turnip crinkle virus genes required for RNA replication and virus movement. Virology 186:1–8

    Article  PubMed  CAS  Google Scholar 

  • Hammond J, Kamo KK (1993) Transgenic coat protein and antisense RNA resistance to bean yellow mosaic potyvirus. Acta Hortic 336:171–178

    Google Scholar 

  • Hammond J, Lecoq H, Raccah B (1999) Epidemiological risks from mixed virus infections and transgenic plants expressing viral genes. Adv Virus Res 54:189–314

    Article  PubMed  CAS  Google Scholar 

  • Hefferon KL, Khalilian H, AbouHaidar MG (1997) Expression of the PVYO coat protein (CP) under the control of the PVX CP gene leader sequence: protection under greenhouse and field conditions against PVYO and PVYN infection in three potato cultivars. Theor Appl Genet 94:287–292

    Article  CAS  Google Scholar 

  • Hemmes H, Lakatos L, Goldbach R, Burgyán J, Prins M (2007) The NS3 protein of Rice hoja blanca tenuivirus suppresses RNA silencing in plant and insect hosts by efficiently binding both siRNAs and miRNAs. RNA 13:1079–1089

    Article  PubMed  CAS  Google Scholar 

  • Hollings M, Stone OM (1972) Turnip crinkle virus. In: AAB descriptions of plant viruses 109. Association of Applied Biologists, Wellesbourne

  • Hong Y, Levay K, Murphy JF, Klein PG, Shaw JG, Hunt AG (1996) A potyvirus polymerase interacts with the viral coat protein and VPg in yeast cells. Virology 214:159–166

    Article  Google Scholar 

  • Lawson C, Kaniewski W, Haley L, Rozman R, Newell C, Sanders P, Tumer NE (1990) Engineered resistance to mixed virus infection in a commercial potato cultivar: resistance to potato virus X and potato virus Y in transgenic Russet Burbank. Bio/Technology 8:127–134

    Article  PubMed  CAS  Google Scholar 

  • Li WZ, Qu F, Morris TJ (1998) Cell-to-cell movement of turnip crinkle virus is controlled by two small reading frames that function in trans. Virology 244:405–416

    Article  PubMed  CAS  Google Scholar 

  • Li WX, Li H, Lu R, Li F, Dus M, Atkinson P, Brydon EW, Johnson KL, García-Sastre A, Ball LA, Palese P, Ding SW (2004) Interferon antagonist proteins of influenza and vaccinia viruses are suppressors of RNA silencing. Proc Natl Acad Sci USA 101:1350–1355

    Article  PubMed  CAS  Google Scholar 

  • Lin SS, Henriques R, Wu HW, Niu QW, Yeh SD, Chua NH (2007) Strategy and mechanisms of plant virus resistance. Plant Biotechnol Rep 1:125–134

    Article  Google Scholar 

  • Lindbo JA, Dougherty WG (1992) Untranslatable transcripts of the tobacco etch virus coat protein gene sequence can interfere with tobacco etch virus replication in transgenic plants and protoplasts. Virology 189:725–753

    Article  PubMed  CAS  Google Scholar 

  • Lindbo JA, Silva-Rosales L, Proebsting WM, Dougherty WG (1993) Induction of a highly specific antiviral state in transgenic plants: implications for regulation of gene expression and virus resistance. Plant Cell 5:1749–1759

    Article  PubMed  CAS  Google Scholar 

  • Loesch-Fries LS, Merlo D, Zinnen T, Burhop L, Hill K, Krahn K, Jarvis N, Nelson S, Halk E (1987) Protection against tobacco mosaic virus infection in transgenic plants requires accumulation of coat protein rather than coat protein RNA sequences. EMBO J 6:1845–1851

    PubMed  CAS  Google Scholar 

  • Lomonossoff GP (1995) Pathogen-derived resistance to plant viruses. Annu Rev Phytopathol 33:323–343

    Article  CAS  Google Scholar 

  • Lu S, Cullen BR (2004) Adenovirus VA1 noncoding RNA can inhibit small interfering RNA and microRNA biogenesis. J Virol 78:12868–12876

    Article  PubMed  CAS  Google Scholar 

  • Lu B, Stubbs G, Culver JN (1998) Coat protein interactions involved in tobacco mosaic tobamovirus cross-protection. Virology 248:188–198

    Article  PubMed  CAS  Google Scholar 

  • Malnoe P, Farinelli L, Collet GF, Reust W (1994) Small-scale field tests with transgenic potato, cv. Bintje, to test resistance to primary and secondary infections with potato virus Y. Plant Mol Biol 25:963–975

    Article  PubMed  CAS  Google Scholar 

  • Mueller E, Gilbert JE, Davenport G, Brigneti G, Baulcombe DC (1995) Homology-dependent resistance: transgenic virus resistance in plants related to homology-dependent gene silencing. Plant J 7:1001–1023

    Article  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Namba S, Ling K, Gonsalves C, Slightom JL, Gonsalves D (1992) Protection of transgenic plants expressing the coat protein gene of watermelon mosaic virus II or zucchini yellow mosaic virus against six potyviruses. Phytopathology 82:940–946

    Article  CAS  Google Scholar 

  • Nejidat A, Beachy RN (1990) Transgenic tobacco plants expressing a coat protein gene of tobacco mosaic virus are resistant to some other tobamoviruses. Mol Plant Microbe Interact 3:247–251

    PubMed  CAS  Google Scholar 

  • Pang SZ, Bock JH, Gonsalves C, Slightom JL, Gpnsalves D (1994) Resistance of transgenic Nicotiana benthamiana plants to tomato spotted wilt and impatiens necrotic spot tospoviruses: evidence of involvement of the N protein and N gene RNA in resistance. Phytopathology 84:243–249

    Article  CAS  Google Scholar 

  • Powell PA, Nelson RS, De B, Hoffmann N, Rogers SG, Fraley RT, Beachy RN (1986) Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene. Science 232:738–743

    Article  Google Scholar 

  • Powell PA, Sanders PR, Turner N, Fraley RT, Beachy RN (1990) Protection against tobacco mosaic virus infection in transgenic plants requires accumulation of coat protein rather than coat protein RNA sequences. Virology 175:124–130

    Article  PubMed  CAS  Google Scholar 

  • Qu F, Ren T, Morris TJ (2003) The coat protein of Turnip crinkle virus suppresses posttranscriptional gene silencing at an early initiation step. J Virol 77:511–522

    Article  PubMed  CAS  Google Scholar 

  • Ratcliff F, Harrison BD, Baulcombe DC (1997) A similarity between viral defense and gene silencing in plants. Science 276:1558–1560

    Article  PubMed  CAS  Google Scholar 

  • Ratcliff F, McFarlane SA, Baulcombe DC (1999) Gene silencing without DNA. RNA-mediated cross-protection between viruses. Plant Cell 11:1207–1216

    Article  PubMed  CAS  Google Scholar 

  • Ravelonandro M, Monsion M, Teycheney PY, Delbos PR, Dunez J (1992) Transgenic tobacco plants that contain the plum pox virus (PPV) coat protein gene. Acta Hortic 309:191–196

    Google Scholar 

  • Register JC, Beachy RN (1988) Resistance to TMV in transgenic plants results from interference with an early event in infection. Virology 166:524–532

    Article  PubMed  CAS  Google Scholar 

  • Ren T, Qu F, Morris TJ (2000) HRT gene function requires interaction between a NAC protein and viral capsid protein to confer resistance to Turnip crinkle virus. Plant Cell 12:1917–1925

    Article  PubMed  CAS  Google Scholar 

  • Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  • Sanford JC, Johnston SA (1985) The concept of parasite-derived resistance. Deriving resistance genes from the parasite’s own genome. J Theor Biol 113:395–405

    Article  Google Scholar 

  • Savenkov EI, Valkonen JPT (2002) Silencing of a viral RNA silencing suppressor in transgenic plants. J Gen Virol 83:2325–2335

    PubMed  CAS  Google Scholar 

  • Smith HA, Powers H, Swaney S, Brown C, Dougherty WG (1995) Transgenic potato virus Y resistance in potato: evidence for an RNA-mediated cellular response. Phytopathology 85:864–870

    Article  CAS  Google Scholar 

  • Sontheimer EJ (2005) Assembly and function of RNA silencing complexes. Nat Rev Mol Cell Biol 6:127–138

    Article  PubMed  CAS  Google Scholar 

  • Stark DM, Beachy RN (1989) Protection against potyvirus infection in transgenic plants: evidence for broad spectrum resistance. Bio/Technology 7:1257–1262

    Google Scholar 

  • Takeda A, Tsukuda M, Mizumoto H, Okamoto K, Kaido M, Mise K, Okuno T (2005) A plant virus suppresses RNA silencing through viral RNA replication. EMBO J 24:3147–3157

    Article  PubMed  CAS  Google Scholar 

  • Tenllado F, Garcia-Luque I, Serra MT, Diaz-Ruiz JR (1995) Nicotiana benthamiana plants transformed with the 54-kDa region of the pepper mild mottle tobamovirus replicase gene exhibit two types of resistance responses against viral infection. Virology 211:170–183

    Article  PubMed  CAS  Google Scholar 

  • Tennant PF, Gonsalves C, Ling KS, Fitch M, Manshardt R, Slightom JL, Gonsalves D (1994) Different protection against papaya ringspot virus isolates in coat protein gene transgenic papaya and classically cross-protected papaya. Phytopathology 84:1359–1366

    Article  Google Scholar 

  • Thomas CL, Leh V, Lederer C, Maule A (2003) Turnip crinkle virus coat protein mediates suppression of RNA silencing in Nicotiana benthamiana. Virology 306:33–41

    Article  PubMed  CAS  Google Scholar 

  • van der Vlugt RAA, Ruiter RK, Goldbach R (1992) Evidence for sense RNA-mediated resistance to PVYN in tobacco plants transformed with the viral coat protein cistron. Plant Mol Biol 20:631–639

    Article  PubMed  Google Scholar 

  • Vance V, Vaucheret H (2001) RNA silencing in plants-defense and counterdefense. Science 292:2277–2280

    Article  PubMed  CAS  Google Scholar 

  • Voinnet O (2005) Induction and suppression of RNA silencing: insights from viral infections. Nat Rev Genet 6:206–220

    Article  PubMed  CAS  Google Scholar 

  • Voloudakis AE, Aleman-Verdaguer ME, Padgett HS, Beachy RN (2005) Characterization of resistance in transgenic Nicotiana benthamiana encoding N-terminal deletion and assembly mutants of the Tobacco etch potyvirus coat protein. Arch Virol 150:2567–2582

    Article  PubMed  CAS  Google Scholar 

  • Wang MB, Metzlaff M (2005) RNA silencing and antiviral defense in plants. Curr Opin Plant Biol 8:216–222

    Article  PubMed  Google Scholar 

  • Wilson TM (1993) Strategies to protect crop plants against viruses: pathogen-derived resistance blossoms. Proc Natl Acad Sci USA 90:3134–3141

    Article  PubMed  CAS  Google Scholar 

  • Worrall D, Elias L, Ashford D, Smallwood M, Sidebottom C, Lillford P, Telford J, Holt C, Bowles D (1998) A carrot leucine-rich-repeat protein that inhibits ice recrystallisation. Science 282:115–117

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by a grant (R05-2003-000-10293-0) from the Basic Research Program of the Korea Research Foundation (KRF).

Author information

Authors and Affiliations

  1. Department of Biology and Medicinal Science, Pai Chai University, Daejeon, 302-735, South Korea

    Ayyappan Vasudevan, Tae-Kyun Oh, Jae Sung Park, Sumitra Vijayachandran Lakshmi, Bong Kum Choi, Sue Hoon Kim, Hyun Ju Lee, Jing Ji, Sei Chang Kim & Chang Won Choi

  2. Department of Life Biology, Daejeon University, Daejeon, 300-716, South Korea

    Joo-Hwan Kim

  3. Department of Biotechnology, Bharathidasan University, Tiruchirappalli, 620 024, Tamil Nadu, India

    Andy Ganapathi

Authors
  1. Ayyappan Vasudevan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tae-Kyun Oh
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jae Sung Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sumitra Vijayachandran Lakshmi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Bong Kum Choi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sue Hoon Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hyun Ju Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jing Ji
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Joo-Hwan Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Andy Ganapathi
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Sei Chang Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Chang Won Choi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Chang Won Choi.

Additional information

Communicated by P. Lakshmanan.

Ayyappan Vasudevan and Tae-Kyun Oh have contributed equally in this study.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Vasudevan, A., Oh, TK., Park, J.S. et al. Characterization of resistance mechanism in transgenic Nicotiana benthamiana containing Turnip crinkle virus coat protein. Plant Cell Rep 27, 1731–1740 (2008). https://doi.org/10.1007/s00299-008-0595-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-008-0595-9

Keywords

Search

Navigation