Summary
Expression of the beet necrotic yellow vein virus (BNYVV) coat protein (CP) gene in transgenic sugar beet hairy roots was accomplished as a step towards CP-mediated virus resistance. A cDNA for the CP gene and its 5′ terminal untranslated leader sequence was prepared from BNYVV RNA, using two oligodeoxynucleotides to prime the synthesis of both strands. Second-strand synthesis and amplification of the cDNA were done by Taq DNA polymerase chain reactions. Run-off transcripts of the cloned cDNA sequence were obtained and translated in vitro, yielding immunoreactive CP. A binary vector construction containing the CP gene under the control of the 35S promoter of cauliflower mosaic virus was prepared and used for Agrobacterium rhizogenes-mediated transformation of sugar beet tissue. Stable integration and expression of the CP gene in sugar beet hairy roots was demonstrated by Southern, Northern, and Western blot analysis, respectively.
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Bouzoubaa S, Ziegler V, Beck D, Guilley H, Richards K, Jonard G (1986) Nucleotide sequence of beet necrotic yellow vein virus RNA-2. J Gen Virol 67:1689–1700
Bouzoubaa S, Quillet L, Guilley H, Jonard G, Richards K (1987) Nucleotide sequence of beet necrotic yellow vein virus RNA-1. J Gen Virol 68:615–626
Burgermeister W, Koenig R (1984) Electro-blot immunoassay — a means for studying serological relationships among plant viruses? Phytopathol Z 111:15–25
Burgermeister W, Koenig R, Weich H, Sebald W, Lesemann D-E (1986) Diversity of the RNAs in 13 isolates of beet necrotic yellow vein virus in Chenopodium quinoa detected by means of cloned cDNAs. J Phytopathol 115:229–242
Cuozzo M, O'Connell KM, Kaniewski W, Fang RX, Chua NH, Tumer NE (1988) Viral protection in transgenic tobacco plants expressing the cucumber mosaic virus coat protein or its antisense RNA. Biotechnology 6:549–557
David C, Chilton MD, Tempé J (1984) Conservation of T-DNA in plants regenerated from hairy root cultures. Biotechnology 2:73–76
Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: Version II. Plant Mol Biol Rep 1 [4]: 19–21
Gallie DR, Sleat DE, Watts JW, Turner PC, Wilson TMA (1987) A comparison of eukaryotic viral 5′-leader sequences as enhancers of mRNA expression in vivo. Nucleic Acids Res 15:8693–8711
Hamill JD, Parr AJ, Robins RJ, Rhodes MJC (1986) Secondary product formation by cultures of Beta vulgaris and Nicotiana rustica transformed with Agrobacterium rhizogenes. Plant Cell Rep 5:111–114
Hoekema A, Huisman MJ, Molendijk L, Elzen PJM van der, Cornelissen BJC (1989) The genetic engineering of two commercial potato cultivars for resistance to potato virus X. Biotechnology 7:273–278
Kallerhoff J, Perez P, Bouzoubaa S, Ben Tahar S, Perret J (1990) Beet necrotic yellow vein virus coat protein-mediated protection in sugar beet (Beta vulgaris L.) protoplasts. Plant Cell Rep 9:224–228
Koenig R, Ehlers U (1989) Influence of RNA composition of beet necrotic yellow vein furovirus isolates and of the sugar beet cultivar on the translocation of the virus in mechanically inoculated sugar beet roots. EPPO Bull 19:527–530
Krieg PA, Melton DA (1984) Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Res 12:7057–7070
Landsmann J, Llewellyn D, Dennis ES, Peacock WJ (1988) Organ-regulated expression of the Parasponia andersonii haemoglobin gene in transgenic tobacco plants. Mol Gen Genet 214:68–73
Lindsey K, Gallois P (1990) Transformation of sugar beet (Beta Vulgaris) by Agrobacterium tumefaciens. J Exp Bot 41:529–536
Loesch-Fries LS, Merlo D, Zinnen T, Burhop L, Hill K, Krahn K, Jarvis N, Nelson S, Halk E (1987) Expression of alfalfa mosaic virus RNA 4 in transgenic plants confers virus resistance. EMBO J 6:1845–1851
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor/NY
Melton DA, Krieg PA, Rebagliati MR, Maniatis T, Zinn K, Green MR (1984) Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res 12:7035–7056
Petit A, David C, Dahl GA, Ellis JG, Guyon P, Casse Delbart F, Tempé J (1983) Further extension of the opine concept: plasmids in Agrobacterium rhizogenes cooperate for opine degradation. Mol Gen Genet 190:204–214
Stark DM, Beachy RN (1989) Protection against potyvirus infection in transgenic plants: evidence for broad spectrum resistance. Biotechnology 7:1257–1262
Stegemann H, Francksen H, Macko V (1973) Potato proteins: genetic and physiological changes, evaluated by one- and two-dimensional PAA-gel-techniques. Z Naturforsch Teil C 28:722–723
Tamada T (1975) Beet necrotic yellow vein virus. Association of Applied Biologists Descriptions of Plant Viruses. Commonwealth Mycological Institute, Kew, England, No. 144
Thomas PS (1980) Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci USA 77:5201–5205
Töpfer R, Matzeit V, Gronenborn B, Schell J, Steinbiss H (1987) A set of plant expression vectors for transcriptional and translational fusions. Nucleic Acids Res 15:5890
Verwoerd TC, Dekker BMM, Hoekema A (1989) A small-scale procedure for the rapid isolation of plant RNAs. Nucleic Acids Res 17:2362
Wilson TMA (1989) Plant viruses. A tool box for genetic engineering and crop protection. Bio Essays 10:179–186
Ziegler V, Richards K, Guilley H, Jonard G, Putz C (1985) Cell-free translation of beet necrotic yellow vein virus: read-through of the coat protein cistron. J Gen Virol 66:2079–2087
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Communicated by G. Wenzel
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Ehlers, U., Commandeur, U., Frank, R. et al. Cloning of the coat protein gene from beet necrotic yellow vein virus and its expression in sugar beet hairy roots. Theoret. Appl. Genetics 81, 777–782 (1991). https://doi.org/10.1007/BF00224989
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DOI: https://doi.org/10.1007/BF00224989