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Development and assessment of a Potato virus X-based expression system with improved biosafety

Published online by Cambridge University Press:  15 August 2005

Ulrike Manske
Federal Biological Research Center for Agriculture and Forestry, Institute for Plant Virology, Microbiology and Biosafety, Messeweg 11-12, D-38104 Braunschweig, Germany
Joachim Schiemann
Federal Biological Research Center for Agriculture and Forestry, Institute for Plant Virology, Microbiology and Biosafety, Messeweg 11-12, D-38104 Braunschweig, Germany


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Over the last decade, plant virus-based vectors have been developed and successfully exploited for high-yield production of heterologous proteins in plants. However, widespread application of recombinant viruses raises concerns about possible risks to the environment. One of the primary safety issues that must be considered is the uncontrolled spread of the genetically engineered virus from experimental plants to susceptible weeds or crops. Using a movement-deficient Potato virus X (PVX)-based transient gene expression vector which harbors the β-glucuronidase (gus) gene, we established a plant viral expression system that provides containment of the recombinant virus and allows for safe and efficient protein production. By deletion of the viral 25k movement protein gene, systemic spread of the modified virus in non-transgenic Nicotiana benthamiana plants was successfully inhibited. In transgenic N. benthamiana plants expressing the 25K viral movement protein, this deficiency was complemented, thus resulting in systemic infection with the movement-deficient virus. While no differences in virus spread and accumulation were observed compared to infection caused by wild-type PVX in non-transgenic plants, the movement protein transgenic plants exhibited none of the normal symptoms of viral infection. Several biosafety aspects were investigated including the potential for recombination between the defective virus and the movement protein transgene, as well as complementation effects in non-transgenic plants doubly infected with the defective and the wild-type virus. Furthermore, the applicability of the safety system for the production of heterologous proteins was evaluated with gus as a model gene. With respect to the stability of the gus insert and the expression level of the GUS protein, there were no differences between the novel system developed and the conventional PVX-based expression system.

Research Article
© ISBR, EDP Sciences, 2005


Aaziz, R, Tepfer, M (1999) Recombination in RNA viruses and in virus-resistant transgenic plants. J. Gen. Virol. 80: 13391346 CrossRef
Adair, TL, Kearney, C (2000) Recombination between a 3-kilobase tobacco mosaic virus transgene and a homologous viral construct in the restoration of viral and nonviral genes. Arch. Virol. 145: 18671883 CrossRef
Agranovsky, AA, Folimonov, AS, Folimonova, SYu, Morozov, SYu, Schiemann, J, Lesemann, D, Atabekov, JG (1998) Beet yellows closterovirus HSP70-like protein mediates the cell-to-cell movement of a potexvirus transport-deficient mutant and a hordeivirus-based chimeric virus. J. Gen. Virol. 79: 889895 CrossRef
Angell SM, Davies C, Baulcombe DC (1996) Cell-to-cell movement of potato virus X is associated with a change in the size-exclusion limit of plasmodesmata in trichome cells of Nicotiana clevelandii. Virology 216: 197–201
Ares, X, Calamante, G, Cabral, S, Lodge, J, Hemenway, P, Beachy, RN, Mentaberry, A (1998) Transgenic plants expressing potato virus X ORF2 protein (p24) are resistant to tobacco mosaic virus and Ob tobamoviruses. J. Virol. 72: 731738
Avesani, L, Falorni, A, Tornielli, GB, Marusic, C, Porceddu, A, Polverari, A, Faleri, C, Calcinaro, F, Pezzotti, M (2003) Improved in planta expression of the human islet autoantigen glutamic acid decarboxylase (GAD65). Transgenic Res. 12: 203212 CrossRef
Awram, P, Gardner, RC, Forster, RL, Bellamy, AR (2002) The potential of plant viral vectors and transgenic plants for subunit vaccine production. Adv. Virus Res. 58: 81124 CrossRef
Baulcombe, DC, Chapman, S, Santa Cruz S (1995) Jellyfish green fluorescent protein as a reporter for virus infections. Plant J. 7: 10451053 CrossRef
Borja, M, Rubio, T, Scholthof, HB, Jackson, AO (1999) Restoration of wild-type virus by double recombination of tombusvirus mutants with a host transgene. Mol. Plant Microbe Interact. 12: 153162 CrossRef
Breiteneder, H, Krebitz, M, Wiedermann, U, Wagner, B, Essl, D, Steinkellner, H, Turpen, TH, Ebner, C, Buck, D, Niggemann, B, Scheiner, O (2001) Rapid production of recombinant allergens in Nicotiana benthamiana and their impact on diagnosis and therapy. Int. Arch. Allergy Immunol. 124: 4850 CrossRef
Campbell, RN (1999) Assessing the spread of engineered TMV. Science 286: 903 CrossRef
Chapman, S, Kavanagh, T, Baulcombe, D (1992) Potato virus X as a vector for gene expression in plants. Plant J. 2: 549557
Clark, MF, Adams, AN (1977) Characteristics of microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. J. Gen. Virol. 34: 475483 CrossRef
Dawson, WO, Lewandowski, DJ, Hilf, ME, Bubrick, P, Raffo, AJ, Shaw, JJ, Grantham, GL, Desjardins, PR (1989) A tobacco mosaic virus-hybrid expresses and loses an added gene. Virology 172: 285292 CrossRef
Dietrich, C, Maiss, E (2003) Fluorescent labelling reveals spatial separation of potyvirus populations in mixed infected Nicotiana benthamiana plants. J. Gen. Virol. 84: 28712876 CrossRef
Dirnberger, D, Steinkellner, H, Abdennebi, L, Remy, JJ, van de Wiel D (2001) Secretion of biologically active glycoforms of bovine follicle stimulating hormone in plants. Eur. J. Biochem. 268: 45704579 CrossRef
Donson, J, Kearney, CM, Hilf, ME, Dawson, WO (1991) Systemic expression of a bacterial gene by a tobacco mosaic virus-based vector. Proc. Natl. Acad. Sci. USA 88: 72047208 CrossRef
Escobar, C, Hernandez, LE, Jimenez, A, Creissen, G, Ruiz, MT, Mullineaux, PM (2003) Transient expression of Arabidopsis thaliana ascorbate peroxidase 3 in Nicotiana benthamiana plants infected with recombinant potato virus X. Plant Cell Rep. 21: 699704
Franconi, R, Di Bonito, P, Dibello, F, Accardi, L, Muller, A, Cirilli, A, Simeone, P, Dona, MG, Venuti, A, Giorgi, C (2002) Plant-derived human papillomavirus 16 E7 oncoprotein induces immune response and specific tumor protection. Cancer Res. 62: 36543658
Gleba, Y, Marillonnet, S, Klimyuk, V (2004) Engineering viral expression vectors for plants: the “full virus” and the “deconstructed virus” strategies. Curr. Opin. Plant Biol. 7: 182188 CrossRef
Greene, AE, Allison, RF (1994) Recombination between viral RNA and transgenic plant transcripts. Science 263: 14231425 CrossRef
Greene, AE, Allison, RF (1996) Deletions in the 3' untranslated region of cowpea chlorotic mottle virus transgene reduce recovery of recombinant viruses in transgenic plants. Virology 225: 231234 CrossRef
Hanley, K, Nguyen, LV, Khan, F, Pogue, GP, Vojdani, F, Panda, S, Pinot, F, Oriedo, VB, Rasochova, L, Subramanian, M, Miller, B, White, EL (2003) Development of a plant viral-vector-based gene expression assay for the screening of yeast cytochrome p450 monooxygenases. Assay Drug Dev. Technol. 1: 147160 CrossRef
Hausmann, L, Töpfer, R (1999) Entwicklung von Plasmid-Vektoren. Vortr. Pflanzenzüchtung 45: 155172
Hiriart, JB, Lehto, K, Tyystjarvi, E, Junttila, T, Aro, EM (2002) Suppression of a key gene involved in chlorophyll biosynthesis by means of virus-inducing gene silencing. Plant Mol. Biol. 50: 213224 CrossRef
Hong, Y, Stanley, J, van Wezel R (2003) Novel system for the simultaneous analysis of geminivirus DNA replication and plant interactions in Nicotiana benthamiana. J. Virol. 77: 1331513322 CrossRef
Horsch, RB, Fry, JE, Hoffmann, NL, Eicholtz, D, Rogers, SG, Fraley, RT (1985) A simple and general method of transferring genes into plants. Science 277: 12291231
Jefferson, RA, Kavanagh, TA, Bevan, MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6: 39013907
Joshi, RL, Joshi, V (1991) Strategies for expression of foreign genes in plants. Potential use of engineered viruses. FEBS Lett. 281: 18 CrossRef
Kobayashi, K, Sarrobert, C, Ares, X, Rivero, MM, Maldonado, S, Robaglia, C, Mentaberry, A (2004) Over-expression of potato virus X TGBp1 movement protein in transgenic tobacco plants causes developmental and metabolic alterations. Plant Physiol. Biochem. 42: 731738 CrossRef
Kopertekh L, Jüttner G, Schiemann J (2004a) Site-specific recombination induced in transgenic plants by PVX virus vector expressing bacteriophage P1 recombinase. Plant Sci. 166: 485–492
Kopertekh L, Jüttner G, Schiemann J (2004b) PVX-Cre-mediated marker gene elimination from transgenic plants. Plant Mol. Biol. 55: 491–500
Kumagai, MH, Donson, J, della-Cioppa, G, Harvey, D, Hanley, K, Grill, LK (1995) Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA. Proc. Natl. Acad. Sci. USA 92: 16791683 CrossRef
Kumagai, MH, Keller, Y, Bouvier, F, Clary, D, Camara, B (1998) Functional integration of non-native carotenoids into chloroplasts by viral-derived expression of capsanthin-capsorubin synthase in Nicotiana benthamiana. Plant J. 14: 305315 CrossRef
Li, Y, Geng, Y, Song, H, Zheng, G, Huan, L, Qiu, B (2004) Expression of a human lactoferrin N-lobe in Nicotiana benthmiana with potato virus X-based agroinfection. Biotechnol. Lett. 26: 953957 CrossRef
McCormick, AA, Reinl, SJ, Cameron, TI, Vojdani, F, Fronefield, M, Levy, R, Tuse, D (2003) Individualized human scFv vaccines produced in plants: humoral anti-idiotype responses in vaccinated mice confirm relevance to the tumor Ig. J. Immunol. Methods 278: 95104 CrossRef
MacFarlane, SA, Popovich, AH (2000) Efficient expression of foreign proteins in roots from tobravirus vectors. Virology 267: 2935 CrossRef
Mor, TS, Moon, YS, Palmer, KE, Mason, HS (2003) Geminivirus vectors for high-level expression of foreign proteins in plant cells. Biotechnol. Bioeng. 81: 430437 CrossRef
Mori, M, Fujihara, N, Mise, K, Furusawa, I (2001) Inducible high-level mRNA amplification system by viral replicase in transgenic plants. Plant J. 27: 7986 CrossRef
Morozov, SYu, Fedorkin, ON, Juttner, G, Schiemann, J, Baulcombe, DC, Atabekov, JG (1997) Complementation of a potato virus X mutant mediated by bombardment of plant tissues with cloned viral movement protein genes. J. Gen. Virol. 78: 20772083 CrossRef
Nolasco, G, de Blas, C, Torres, V, Ponz, F (1993) A method combining immunocapture and PCR amplification in a microtiter plate for the detection of plant viruses and subviral pathogens. J. Virol. Methods 45: 201218 CrossRef
Orman, BE, Celnik, RM, Mandel, AM, Torres, HN, Mentaberry, AN (1990) Complete cDNA sequence of a South American isolate of potato virus X. Virus Res. 16: 293305 CrossRef
Page, JE, Hause, G, Raschke, M, Gao, W, Schmidt, J, Zenk, MH, Kutchan, TM (2004) Functional analysis of the final steps of the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway to isoprenoids in plants using virus-induced gene silencing. Plant Physiol. 134: 14011413 CrossRef
Perez Filgueira, DM, Zamorano, PI, Dominguez, MG, Taboga, O, Del Medico Zajac, MP, Puntel, M, Romera, SA, Morris, TJ, Borca, MV, Sadir, AM (2003) Bovine herpes virus gD protein produced in plants using a recombinant tobacco mosaic virus (TMV) vector possesses authentic antigenicity. Vaccine 21: 42014209
Pogue, GP, Lindbo, JA, Garger, SJ, Fitzmaurice, WP (2002) Making an ally from an enemy: plant virology and the new agriculture. Annu. Rev. Phytopathol. 40: 4574 CrossRef
Rabindran, S, Dawson, WO (2001) Assessment of recombinants that arise from the use of a TMV-based transient expression vector. Virology 284: 182189 CrossRef
Ratcliff, F, Martin-Hernandez, AM, Baulcombe, DC (2001) Technical Advance. Tobacco rattle virus as a vector for analysis of gene function by silencing. Plant J. 25: 23745 CrossRef
Sablowski RW, Baulcombe DC, Bevan M (1995) Expression of a flower-specific Myb protein in leaf cells using a viral vector causes ectopic activation of a target promoter. Proc. Natl. Acad. Sci. USA 92: 6901–6905
Saitoh, H, Kiba, A, Nishihara, M, Yamamura, S, Suzuki, K, Terauchi, R (2001) Production of antimicrobial defensin in Nicotiana benthamiana with a potato virus X vector. Mol. Plant Microbe Interact. 14: 111115 CrossRef
Santa Cruz, S, Chapman, S, Roberts, AG, Roberts, IM, Prior, DAM, Oparka, KJ (1996) Assembly and movement of a plant virus carrying a green fluorescent protein overcoat. Proc. Natl. Acad. Sci. USA 93: 62866290 CrossRef
Santa Cruz, S, Roberts, AG, Prior, DA, Chapman, S, Oparka, KJ (1998) Cell-to-cell and phloem-mediated transport of potato virus X. The role of virions. Plant Cell 10: 495510 CrossRef
Scholthof, KB, Mirkov, TE, Scholthof, HB (2002) Plant virus gene vectors: biotechnology applications in agriculture and medicine. Genet. Eng. (NY) 24: 6785
Schubert, J, Matousek, J, Mattern, D (2004) Pathogen-derived resistance in potato to Potato virus Y-aspects of stability and biosafety under field conditions. Virus Res. 100: 4150 CrossRef
Selth, LA, Randles, JW, Rezaian, MA (2004) Host responses to transient expression of individual genes encoded by Tomato leaf curl virus. Mol. Plant Microbe Interact. 17: 2733 CrossRef
Shivprasad, S, Pogue, GP, Lewandowski, DJ, Hidalgo, J, Donson, J, Grill, LK, Dawson, WO (1999) Heterologous sequences greatly affect foreign gene expression in tobacco mosaic virus-based vectors. Virology 255: 312323 CrossRef
Tepfer, M (2002) Risk assessment of virus-resistant transgenic plants. Annu. Rev. Phytopathol. 40: 467491 CrossRef
Töpfer, R, Maas, C, Höricke-Grandpierre, C, Schell, J, Steinbiss, HH (1993) Expression vectors for high-level gene expression in dicotyledonous and monocotyledonous plants. Methods in Enzymology 217: 6678 CrossRef
Turpen TH September 22, 1998. Viral amplification of recombinant messenger RNA in transgenic plants. United States Patent No. 5 811 653
Valentine, T, Shaw, J, Blok, VC, Phillips, MS, Oparka, KJ, Lacomme, C (2004) Efficient virus-induced gene silencing in roots using a modified tobacco rattle virus vector. Plant Physiol. 136: 39994009 CrossRef
Verch, T, Hooper, DC, Kiyatkin, A, Steplewski, Z, Koprowski, H (2004) Immunization with a plant-produced colorectal cancer antigen. Cancer Immunol. Immunother. 53: 9299 CrossRef
Verchot, J, Angell, SM, Baulcombe, DC (1998) In vivo translation of the triple gene block of potato virus X requires two subgenomic mRNAs. J. Virol. 72: 83168320
Vigne, E, Komar, V, Fuchs, M (2004) Field safety assessment of recombination in transgenic grapevines expressing the coat protein gene of Grapevine fanleaf virus. Transgenic Res. 13: 165179 CrossRef
Wagner, B, Hufnagl, K, Radauer, C, Wagner, S, Baier, K, Scheiner, O, Wiedermann, U, Breiteneder, H (2004) Expression of the B subunit of the heat-labile enterotoxin of Escherichia coli in tobacco mosaic virus-infected Nicotiana benthamiana plants and its characterization as mucosal immunogen and adjuvant. J. Immunol. Methods 287: 203215 CrossRef
Wright, KM, Duncan, GH, Pradel, KS, Carr, F, Wood, S, Oparka, KJ, Cruz, SS (2000) Analysis of the N gene hypersensitive response induced by a fluorescently tagged tobacco mosaic virus. Plant Physiol. 123: 13751386 CrossRef
Yusibov, V, Hooper, DC, Spitsin, SV, Fleysh, N, Kean, RB, Mikheeva, T, Deka, D, Karasev, A, Cox, S, Randall, J, Koprowski, H (2002) Expression in plants and immunogenicity of plant virus-based experimental rabies vaccine. Vaccine 20: 31553164 CrossRefPubMed
Zhang, G, Leung, C, Murdin, L, Rovinski, B, White, KA (2000) In planta expression of HIV-1 p24 protein using an RNA plant virus-based expression vector. Mol. Biotechnol. 14: 99107 CrossRef
Zhao, Y, Hammond, J, Tousignant, ME, Hammond, RW (2000) Development and evaluation of a complementation-dependent gene delivery system based on cucumber mosaic virus. Arch. Virol. 145: 22852295 CrossRef
Ziegler, A, Cowan, GH, Torrance, L, Ross, HA, Davies, HV (2000) Facile assessment of cDNA constructs for expression of functional antibodies in plants using the potato virus X vector. Molecular Breeding 6: 327335 CrossRef
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