Cap analog and Potato virus A HC-Pro silencing suppressor improve GFP transient expression using an infectious virus vector in Nicotiana benthamiana

Document Type : Original article

Authors

Plant Virology Research Center, Shiraz University, Shiraz, Iran

Abstract

Transient expression of proteins in plants has become a choice to facilitate recombinant protein production with its fast and easy application. On the other hand, host defensive mechanisms have been reported to reduce the efficiency of transient expression in plants. Hence, this study was designed to evaluate the effect of cap analog and Potato virus A helper component proteinase (PVA HC-Pro) on green fluorescent protein (GFP) expression efficiency.N. benthamiana leaves were inoculated with capped or un-capped RNA transcripts of a Turnip crinkle virus (TCV) construct containing a green fluorescent protein reporter gene (TCV-sGFP) in place of its coat protein (CP) ORF. PVA HC-Pro as a viral suppressor of RNA silencing was infiltrated in trans by Agrobacterium tumefaciens, increased the GFP foci diameter to six and even more cells in both capped and un capped treatments. The expression level of GFP in inoculated plants with TCV-sGFP transcript pre-infiltrated with PVA HC-Pro was 12.97-fold higher than the GFP accumulation level in pre-infiltrated leaves with empty plasmid (EP) control. Also, the yield of GFP in inoculated N. benthamiana plants with capped TCV-sGFP transcript pre-infiltrated with EP and PVA HC-Pro was 1.54 and 1.2-fold respectively, greater than the level of GFP expressed without cap analog application at 5 days post inoculation (dpi). In addition, the movement of TCV-sGFP was increased in some cells of inoculated leaves with capped transcripts. Results of this study indicated that PVA HC-Pro and mRNA capping can increase GFP expression and its cell to cell movement in N. benthamiana.

Keywords

References

1. Lico CQC, Santi L. Viral vectors for production of recombinant proteins in plants. J Cell Physiol 2008;216:366-377.
2. Pogue GP, Lindbo JA, Garger SJ, Fitzmaurice WP. Making an ally from an enemy: plant virology and the new agriculture. Annu Rev Phytopathol 2002;40:45-74.
3. Fischer RE, Stoger S, Schillberg P, Twyman RM. Plant-based production of biopharmaceuticals. Curr Opin Plant Biol 2004;7:152-158.
4. Varsani AAL, Williamson DS, Rybicki EP. Transient expression of human papillomavirus type 16 L1 protein in Nicotiana benthamiana using an infectious tobamovirus vector. Virus Res 2006;120:91-96.
5. Yusibov VS, Rabindran U, Commandeur RMT, Fischer R. The potential of plant virus vectors for vaccine production. Drugs R&D 2006;7:203-217. 
6. Hull R. Matthews’ Plant Virology. 2002, 4th edn, New York, Academic Press.
7. Boyer JC, Haenni AL. Infectious transcripts and cDNA clones of RNA viruses. Virology 1994;198:415-426.
8. Qu F, Morris TJ. Carmovirus. In: Mahy BWJ, van Regenmortel MHV (ed) Encyclopedia of virology, 2008, 3rd edn. Academic Press, Oxford, England, pp 453-457.
9. King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ. Virus taxonomy: Ninth report of the international committee on taxonomy of viruses. 2012, Elsevier academic press, Amsterdam. 1327 pp. 
10. Cohen Y, Gisel A, Zambryski PC. Cell-to-cell and systemic movement of recombinant green fluorescent protein-tagged turnip crinkle viruses. Virology 2000; 273:258-266.
11. Hacker DL, Petty IT, Wei N, Morris TJ. Turnip crinkle virus genes required for RNA replication and virus movement. Virology 1992;186:1-8.
12. Li WZ, Qu F, Morris TJ. Cell-to-cell movement of turnip crinkle virus is controlled by two small open reading frames that function in trans. Virology 1998;244:405-416.
13. Powers JG, Tim L, Sit TL, Qu F, Morris TJ, Kim KH, Lommel SA. A versatile assay for the identification of RNA silencing suppressors based on complementation of viral movement. Mol Plant Microbe Interact 2008;21:879-890.
14. Qu F, Ren T, Morris TJ. The coat protein of turnip crinkle virus suppresses posttranscriptional gene silencing at an early initiation step. J Virol 2003;77:511-522.
15. Voinnet O. Origin, biogenesis, and activity of plant microRNAs. Cell 2009;136: 669-687.
16. Li F, Ding SW. Virus counter defense: Diverse strategies for evading the RNA silencing immunity. Annu Rev Microbiol 2006;60:503-531.
17. Kasschau KD, Carrington JC. A counter defensive strategy of plant viruses: suppression of posttranscriptional gene silencing. Cell 1998;95:461-70.
18. Brigneti G, Voinnet O, Li WX, Ji LH, Ding SW, Baulcombe DC. Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana. EMBO Journal 1998;17:6739-6746.
19. Baulcombe DC. RNA silencing. Curr Biology 2002;12:82-84.
20. Johansen LK, Carrington JC. Silencing on the spot: induction and suppression of RNA silencing in the Agrobacterium- mediated transient expression system. Plant Physiol 2001;126:930-938.
21. Voinnet O, Rivas S, Mestre P, Baulcombe DC. An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J 2003;33:949-956.
22. Urcuqui-Inchima S, Haenni AL, Bernardi F. Potyvirus proteins: A wealth of functions. Virus Res 2001;74:157-175.
23. Rajamäki ML, Mäki-Valkama T, Mäkinen K, Valkonen JPT. Infection with potyviruses. In: Talbot N (ed) Plant-Pathogen Interactions, 2004, Blackwell Publishing, Oxford, pp 68–91.
24. Gross JD, Moerke NJ, von der Haar T, Lugovskoy AA, Sachs AB, McCarthy JE, Wagner G. Ribosome loading onto the mRNA cap is driven by conformational coupling between eIF4G and eIF4E. Cell 2003;115:739-750.
25. Jani B, Fuchs R. In vitro transcription and capping of Gaussia Luciferase mRNA followed by HeLa cell transfection. J Vis Exp 2012;10:61-67.
26. Gu M, Lima CD. Processing the message: structural insights into capping and decapping mRNA. Curr Opin Struct Biol 2005;15:99-106.
27. Fechter P, Brownlee GG. Recognition of mRNA cap structures by viral and cellular proteins. J Gen Virol 2005;86:1239-1249.
28. Savenkov EI, Valkonen JP. Potyviral helper-component proteinase expressed in transgenic plants enhances titers of Potato leaf roll virus but does not alleviate its phloem limitation. Virology 2001;283:285-293.
29. Ruiz MT, Voinnet O, Baulcombe DC. Initiation and maintenance of virus-induced gene silencing. Plant Cell 1998;10(6):937-946.
30. Petty IT, Hunter BG, Wei N, Jackson AO. Infectious barley stripe mosaic virus RNA transcribed in vitro from full-length genomic cDNA clones. Virology 1989; 171:341. 
31. Zamyatnin AA, Solovyev AG, Bozhkov PV, Valkonen JP, Morozov SY, Savenkov EI. Assessment of the integral membrane protein topology in living cells. Plant J 2006;46:145-154.
32. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2- DDCT method. Methods 2001;25:402-408.
33. Sambrook J, Russel DW. Molecular cloning: A laboratory Manual. 3rd edn, 2001, cold spring harbor laboratory press, cold spring harbor, NY
34. Duncan DB. A significance test for differences between ranked treatments in an analysis of variance. Va J Sci 1951;2:171-189.
35. Knippenberg IV, Goldbach R, Kormelink R. In vitro transcription of Tomato spotted wilt virus is independent of translation. J Gen Virol 2004;85:1335-1338.
36. Meshi T, Ishikawa M, Motoyoshi F, Semba K, Okada Y. In vitro transcription of infectious RNAs from full-length cDNAs of tobacco mosaic virus. Proc Natl Acad Sci USA 1986;83:5043-5047.
37. Beck DL, Forster RLS, Bevan MW, Boxen KA, Lowe SC. Infectious transcripts and nucleotide sequence of cloned cDNA of the potexvirus white clover mosaic virus. Virology 1990;177:152-158.
38. Burgyan J, Nagy PD, Russo M. Synthesis of infectious RNA from full-length cloned cDNA to RNA of cymbidium ringspot tombusvirus. J Gen Virol 1990;71:1857-1860.
39. Hayes RJ, Buck KW. Infectious cucumber mosaic virus RNA transcribed in vitro from clones obtained from cDNA amplified using the polymerase chain reaction. J Gen Virol 1990;71:2503-2508.
40. Green MR, Maniatis T, Melton DA. Human beta-globin pre-mRNA synthesized in vitro is accurately spliced in Xenopus oocyte nuclei. Cell Mar 1983;32(3):681-694.
41. Furuichi Y, Laflandra A, Shatkin AJ. Y-terminal structure and mRNA stability. Nature 1977;266:235-239.
42. Kim KIG, Sunter DM, Chung IS. Improved expression of recombinant GFP using a replicating vector based on Beet curly top virus in leaf-disks and infiltrated Nicotiana benthamiana leaves. Plant Mol Biol 2007;64:103-112.

Files

Share

How to cite

Statistics