Plant Cell Reports

, Volume 28, Issue 4, pp 639–647 | Cite as

Quantitative evaluation of six different viral suppressors of silencing using image analysis of transient GFP expression

  • Taniya Dhillon
  • Joseph M. Chiera
  • John A. Lindbo
  • John J. FinerEmail author
Genetic Transformation and Hybridization


The effects of six different plant viral suppressors of gene silencing were compared using an automated image collection and analysis system developed for continual monitoring of GFP expression. Suppressors were introduced into lima bean cotyledonary tissues either as 3′-GFP translational fusions or as co-introductions with the GFP gene on a separate plasmid. The resultant transient expression profiles for each suppressor depended on whether the suppressor was introduced as a fusion or co-introduced on separate plasmids. As co-introductions, the silencing suppressors HCPro (from Tobacco etch virus), p19 (from Tomato bushy stunt virus), γb (from Barley stripe mosaic virus) and p21 (from Beet yellows virus) led to an almost twofold increase in initial GFP expression levels, followed by a rapid decline. In contrast, fusions of HCPro, p19, and γb to the 3′-end of GFP resulted in slightly lower but more prolonged GFP expression. Compared with the co-introductions, all GFP::Suppressor translational fusions gave reduced GFP fluorescence levels, suggesting interference of the fusion partner with GFP fluorescence. Regardless of the configuration, introductions of the silencing suppressors AL2 (from Tomato golden mosaic virus) and 126-kDa protein (from Tobacco mosaic virus) resulted in very low GFP fluorescence. This is the first report that directly compares the effects of a large number of viral suppressors of silencing on transient transgene expression using both translational fusions and co-introductions.


GFP Lima bean cotyledons Particle bombardment RNA silencing Viral gene-silencing suppressors Gene silencing 



Helper component-proteinase


Cauliflower mosaic virus


Cucumber mosaic virus


Tobacco etch virus


Tomato bushy stunt virus


Tomato golden mosaic virus


Beet yellows virus


Barley stripe mosaic virus


Tobacco mosaic virus


Potato virus X



The authors acknowledge Dr. Robert A. Bouchard for offering guidance in construct design and providing unique insight on the results of suppressor introductions. The authors also wish to thank Drs. Eric J. Stockinger and David M. Bisaro for their helpful suggestions during the course of this work. Salaries and research support were provided by the United Soybean Board and by State and Federal funds appropriated to The Ohio State University/Ohio Agricultural Research and Development Center. Mention of trademark or proprietary products does not constitute a guarantee or warranty of the product by OSU/OARDC and also does not imply approval to the exclusion of other products that may also be suitable.

Supplementary material

ESM1 (MOV 6216 kb)

ESM2 (MOV 5711 kb)

299_2009_675_MOESM3_ESM.doc (32 kb)
Supplemental Table 1 (DOC 33 kb)
299_2009_675_MOESM4_ESM.doc (31 kb)
Supplemental Table 2 (DOC 31 kb)


  1. Altpeter F, Baisakh N, Beachy R, Bock R, Capell T, Christou P, Daniell H, Datta K, Datta S, Dix PJ, Fauquet C, Huang N, Kohli A, Mooibroek H, Nicholson L, Nguyen TT, Nugent G, Raemakers K, Romano A, Somers DA, Stoger E, Taylor N, Visser R (2005) Particle bombardment and the genetic enhancement of crops: myths and realities. Mol Biol 5:305–327Google Scholar
  2. Anandalakshmi R, Pruss GJ, Ge X, Marathe R, Mallory AC, Smith TH, Vance VB (1998) A viral suppressor of gene silencing in plants. Proc Natl Acad Sci USA 95:13079–13084PubMedCrossRefGoogle Scholar
  3. Baulcombe DC (2004) RNA silencing in plants. Nature 431:356–363PubMedCrossRefGoogle Scholar
  4. Bisaro DM (2006) Silencing suppression by geminivirus proteins. Virology 344:158–168PubMedCrossRefGoogle Scholar
  5. Buenrostro-Nava MT, Ling PP, Finer JJ (2005) Development of an automated image acquisition system for monitoring gene expression and tissue growth. Trans Am Soc for Agric Eng 48:841–847Google Scholar
  6. Butaye KMJ, Camune BPA, Delaure SL, De Bolle MFC (2005) Approaches to minimize variation in transgene expression in plants. Mol Breed 16:79–91CrossRefGoogle Scholar
  7. Chapman EJ, Prokhnevsky AI, Gopinath K, Dolja VV, Carrington JC (2004) Viral RNA silencing suppressors inhibit the microRNA pathway at an intermediate step. Gene Dev 18:1179–1186PubMedCrossRefGoogle Scholar
  8. Chiera JM, Bouchard RA, Dorsey SL, Park E, Buenrostro-Nava MT, Ling PP, Finer JJ (2007) Isolation of two highly active soybean (Glycine max (L.) Merr.) promoters and their characterization using a new automated image collection and analysis system. Plant Cell Rep 26:1501–1509PubMedCrossRefGoogle Scholar
  9. Chiera JM, Lindbo JA, Finer JJ (2008) Quantification and extension of transient GFP expression by the co-introduction of a suppressor of silencing. Transgenic Res 17:1143–1154PubMedCrossRefGoogle Scholar
  10. DeBuck S, Jacobs A, VanMontagu M, Depicker A (1998) Agrobacterium tumefaciens transformation and cotransformation frequencies of Arabidopsis thaliana root explants and tobacco protoplasts. Mol Plant Microbe Interact 6:449–457CrossRefGoogle Scholar
  11. Ding S-W, Voinnet O (2007) Antiviral immunity directed by small RNAs. Cell 130:413–426PubMedCrossRefGoogle Scholar
  12. Ding XS, Liu J, Cheng N-H, Folimonov A, Hou Y-M, Bao Y, Katagi C, Carter SA, Nelson RS (2004) The Tobacco mosaic virus 126-kDa protein associated with virus replication and movement suppresses RNA silencing. Mol Plant Microb Interact 17:583–592CrossRefGoogle Scholar
  13. Dunoyer P, Lecellier C-H, Parizotto EA, Himber C, Voinnet O (2004) Probing the microRNA and small interfering RNA pathways with virus-encoded suppressors of RNA silencing. Plant Cell 16:1235–1250PubMedCrossRefGoogle Scholar
  14. Finer JJ, Dhillon T (2008) Transgenic plant production. In: Stewart CN (ed) Plant biotechnology and genetics: principles, techniques and applications. Wiley, New York, pp 245–272CrossRefGoogle Scholar
  15. Finer JE, Finer JJ (2007) A simple method for reducing moisture condensation on Petri dish lids. Plant Cell Tiss Org Cult 91:299–304CrossRefGoogle Scholar
  16. Finer JJ, Vain P, Jones MW, McMullen MD (1992) Development of the particle inflow gun for DNA delivery to plant cells. Plant Cell Rep 11:323–328CrossRefGoogle Scholar
  17. Finer JJ, Finer KR, Santarém ER (1996) Plant cell transformation, physical methods for. In: Meyers RA (ed) Encyclopedia of molecular biology and molecular medicine. VCH Publishers, Weinheim, pp 458–465Google Scholar
  18. Finer JJ, Finer KR, Ponappa T (1999) Particle bombardment-mediated transformation. In: Hammond J, McGarvey PB, Yusibov V (eds) Current topics in microbiology and immunology, plant biotechnology: new products and applications, vol 240. Springer, Heidelberg, pp 59–80Google Scholar
  19. Finer JJ, Beck SL, Buenrostro-Nava MT, Chi YT, Ling PP (2006) Monitoring gene expression in plant tissues; using green fluorescent protein with automated image collection and analysis. In: Dutta Gupta S, Ibaraki Y (eds) Plant tissue culture engineering; focus in biotechnology. Springer, Dordrecht, pp 31–46Google Scholar
  20. Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158PubMedCrossRefGoogle Scholar
  21. Hunold R, Bronner R, Hahne G (1994) Early events in microprojectile bombardment: cell viability and particle location. Plant J 5:593–604CrossRefGoogle Scholar
  22. Johansen LK, Carrington JC (2001) Silencing on the spot. Induction and suppression of RNA silencing in the Agrobacterium-mediated transient expression system. Plant Physiol 126:930–938PubMedCrossRefGoogle Scholar
  23. Jones JDG, Dangl JL (2006) The plant immune system. Nature 444:323–329PubMedCrossRefGoogle Scholar
  24. Kasschau KD, Carrington JS (1998) A counter-defensive strategy of plant viruses: suppression of posttranscriptional gene silencing. Cell 95:461–470PubMedCrossRefGoogle Scholar
  25. Kasschau KD, Xie Z, Allen E, Llave C, Chapman EJ, Krizan KA, Carrington JC (2003) P1/HCPro, a viral suppressor of RNA silencing, interferes with Arabidopsis development and miRNA function. Dev Cell 4:205–217PubMedCrossRefGoogle Scholar
  26. Klein TM, Gradziel T, Fromm ME, Sanford JC (1988) Factors influencing gene delivery into Zea mays cells by high-velocity microprojectiles. BioTechnol 6:559–563CrossRefGoogle Scholar
  27. Kubota K, Tsuda S, Tamai A, Meshi T (2003) Tomato mosaic virus replication protein suppresses virus-targeted posttranscriptional gene silencing. J Virol 77:11016–11026PubMedCrossRefGoogle Scholar
  28. Lakatos L, Szittya G, Silhavy D, Burgyán J (2004) Molecular mechanism of RNA silencing suppression mediated by p19 protein of tombusviruses. EMBO J 23:876–884PubMedCrossRefGoogle Scholar
  29. Lakatos L, Csorba T, Pantaleo V, Chapman EJ, Carrington JC, Liu Y-P, Dolja VV, Calvino LF, Lόpez-Moya JJ, Burgyán J (2006) Small RNA binding is a common strategy to suppress RNA silencing by several viral suppressors. EMBO J 25:2768–2780PubMedCrossRefGoogle Scholar
  30. Li F, Ding S-W (2006) Virus counterdefense: diverse strategies for evading the RNA silencing immunity. Ann Rev Microbiol 60:503–531CrossRefGoogle Scholar
  31. Li H, Li WX, Ding S-W (2002) Induction and suppression of RNA silencing by an animal virus. Science 296:1319–1321PubMedCrossRefGoogle Scholar
  32. Lu R, Folimonov A, Shintaku M, Li W-X, Falk BW, Dawson WO, Ding S-W (2004) Three distinct suppressors of RNA silencing encoded by a 20-kb viral RNA genome. Proc Natl Acad Sci USA 101:15742–15747PubMedCrossRefGoogle Scholar
  33. Mallory AC, Ely L, Smith TH, Marathe R, Anandalakshmi R, Fagard M, Vaucheret H, Pruss G, Bowman L, Vance VB (2001) HC-Pro suppression of transgene silencing eliminates the small RNAs but not transgene methylation or the mobile signal. Plant Cell 13:571–583PubMedCrossRefGoogle Scholar
  34. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioasssays with tobacco tissue culture. Physiol Plant 15:473–497CrossRefGoogle Scholar
  35. Pawlowski WP, Somers DA (1998) Transgenic DNA integrated into the oat genome is frequently interspersed by host DNA. Proc Natl Acad Sci USA 95:12106–12110PubMedCrossRefGoogle Scholar
  36. Ponappa T, Brzozowski AE, Finer JJ (1999) Transient expression and stable transformation of soybean using the jellyfish green fluorescent protein. Plant Cell Rep 19:6–12CrossRefGoogle Scholar
  37. Prols F, Meyer P (1992) The methylation patterns of chromosomal integration regions influence gene activity of transferred DNA in Petunia hybrida. Plant J 2:465–475PubMedGoogle Scholar
  38. Rasband WS (1997–2008) ImageJ, US National Institutes of Health, Bethesda, Maryland, USA.
  39. Reed JC, Kasschau KD, Prokhnevsky AI, Gopinath K, Pogue GP, Carrington JC, Dolja VV (2003) Suppressor of RNA silencing encoded by Beet yellows virus. Virology 306:203–209PubMedCrossRefGoogle Scholar
  40. Richards HA, Halfhill MD, Millwood RJ, Stewart CN (2003) Quantitative GFP fluorescence as an indicator of recombinant protein synthesis in transgenic plants. Plant Cell Rep 22:117–121PubMedCrossRefGoogle Scholar
  41. Roth BM, Pruss GJ, Vance VB (2004) Plant viral suppressors of RNA silencing. Virus Res 102:97–108PubMedCrossRefGoogle Scholar
  42. Roudet-Tavert G, German-Retana S, Delaunay T, Delécolle B, Candresse T, LeGall O (2002) Interaction between potyvirus helper component-proteinase and capsid protein in infected plants. J Gen Vir 83:1765–1770Google Scholar
  43. Sahoo PK, Soltani S, Wong AKC (1988) A survey of thresholding techniques. Comput Vision Graph 41:233–260CrossRefGoogle Scholar
  44. Silhavy D, Molnár A, Lucioli A, Szittya G, Honryik C, Tavazza M, Burgyán J (2002) A viral protein suppresses RNA silencing and binds silencing-generated, 21- to 25-nucleotide double-stranded RNAs. EMBO J 21:3070–3080PubMedCrossRefGoogle Scholar
  45. Svitashev SK, Pawlowski WP, Makarevitch I, Plank DW, Somers DA (2002) Complex transgene locus structures implicate multiple mechanisms for plant transgene rearrangement. Plant J 32:433–445PubMedCrossRefGoogle Scholar
  46. Thévanz P, Ruttimann UE, Unser M (1998) A pyramid approach to subpixel registration based on intensity. IEEE Trans Image Process 7:27–41CrossRefGoogle Scholar
  47. Voinnet O (2001) RNA silencing as a plant immune system against viruses. Trends Genet 17:449–459PubMedCrossRefGoogle Scholar
  48. Voinnet O, Pinto YM, Baulcombe DC (1999) Suppression of gene silencing: a general strategy used by diverse DNA and RNA viruses of plants. Proc Natl Acad Sci USA 96:14147–14152PubMedCrossRefGoogle Scholar
  49. Wang H, Buckley KJ, Yang X, Buchmann RC, Bisaro DM (2005) Adenosine kinase inhibition and suppression of RNA silencing by Geminivirus AL2 and L2 Proteins. J Virol 79:7410–7418PubMedCrossRefGoogle Scholar
  50. Weld R, Heinemann J, Eady C (2001) Transient GFP expression in Nicotiana plumbaginifolia suspension cells: the role of gene silencing, cell death, and T-DNA loss. Plant Mol Biol 45:377–385PubMedCrossRefGoogle Scholar
  51. Wu C, Chiera JM, Ling PP, Finer JJ (2008) Isoxaflutole treatment leads to reversible tissue bleaching and allows for more effective detection of GFP in transgenic soybean tissues. In Vitro Cell Develop Biol Plant 6:540–547Google Scholar
  52. Ye K, Patel DJ (2005) RNA silencing suppressor p21 of Beet yellows virus forms an RNA binding octameric ring structure. Structure 13:1375–1384PubMedCrossRefGoogle Scholar
  53. Yelina NE, Savenkov NI, Solovyev AG, Morozov SY, Valkonen JPT (2002) Long-distance movement, virulence and RNA silencing suppression controlled by a single protein in hordei- and potyviruses: complementary functions between virus families. J Virol 76:12981–12991PubMedCrossRefGoogle Scholar
  54. Yoo S-D, Cho Y-H, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protocols 2:1565–1572CrossRefGoogle Scholar
  55. Yu B, Chapman EJ, Yang Z, Carrington JC, Chen X (2006) Transgenically expressed viral RNA silencing suppressors interfere with microRNA methylation in Arabidopsis. FEBS Lett 580:3117–3120PubMedCrossRefGoogle Scholar
  56. Zhou X, Carranco R, Vitha S, Hall TC (2005) The dark side of green fluorescent protein. New Phytol 168:313–322PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Taniya Dhillon
    • 1
  • Joseph M. Chiera
    • 1
  • John A. Lindbo
    • 2
    • 3
  • John J. Finer
    • 1
    Email author
  1. 1.Department of Horticulture and Crop ScienceOARDC/The Ohio State UniversityWoosterUSA
  2. 2.Department of Plant PathologyOARDC/The Ohio State UniversityWoosterUSA
  3. 3.Campbell’s SeedsDavisUSA

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