Skip to main content

Advertisement

SpringerLink
Log in

A ribozyme gene and an antisense gene are equally effective in conferring resistance to tobacco mosaic virus on transgenic tobacco

  • Original Paper
  • Published:
Molecular and General Genetics MGG Aims and scope Submit manuscript

Abstract

Ribozymes of the hammerhead class can be designed to cleave a target RNA in a sequence-specific manner and can potentially be used to specifically modulate gene activity. We have targeted the tobacco mosaic virus (TMV) genome with a ribozyme containing three catalytic hammerhead domains embedded within a 1 kb antisense RNA. The ribozyme was able to cleave TMV RNA at all three target sites in vitro at 25°C. Transgenic tobacco plants were generated which expressed the ribozyme or the corresponding antisense constructs directed at the TMV genome. Six of 38 independent transgenic plant lines expressing the ribozyme and 6 of 39 plant lines expressing the antisense gene showed some level of protection against TMV infection. Homozygous progeny of some lines were highly resistant to TMV; at least 50% of the plants remained asymptomatic even when challenged with high levels of TMV. These plants also displayed resistance to infection with TMV RNA or the related tomato mosaic virus (ToMV). In contrast, hemizygous plants of the same lines displayed only very weak resistance when inoculated with low amounts of TMV and no resistance against high inoculation levels. Resistance in homozygous plants was not overcome by a TMV strain which was altered at the three target sites to abolish ribozyme-mediated cleavage, suggesting that the ribozyme conferred resistance primarily by an antisense mechanism.

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

Similar content being viewed by others

References

  • An G, Watson BD, Stachel S, Gordon MP, Nester EW (1985) New cloning vehicles for transformation of higher plants. EMBO J 4:277–284

    Google Scholar 

  • Bariana HS, Shannon AL, Chu PWG, Waterhouse PM (1994) Detection of five seed-borne legume viruses in one sensitive multiplex polymerase chain reaction test. Phytopathology 84:1201–1205

    Google Scholar 

  • Barnes WM (1990) Variable patterns of expression of luciferase in transgenic tobacco leaves. Proc Natl Acad Sci USA 87:9183–9187

    Google Scholar 

  • Benfey PN, Chua NH (1989) Regulated genes in transgenic plants. Science 244:174–181

    Google Scholar 

  • Bruening G (1989) Compilation of self-cleaving sequences from plant virus satellite RNAs and other sources. Methods Enzymol 180:546–558

    Google Scholar 

  • Cameron FH, Jennings PA (1989) Specific gene suppression by engineered ribozymes in monkey cells. Proc Natl Acad Sci USA 86:9139–9143

    Google Scholar 

  • Cameron FH, Jennings PA (1994) Multiple domains in a ribozyme construct confer increased suppressive activity in monkey cells. Antisense Res Develop 4:87–94

    Google Scholar 

  • Church GM, Gilbert W (1984) Genomic cloning. Proc Natl Acad Sci USA 81:1991–1995

    Google Scholar 

  • Cotten M, Birnstiel ML (1989) Ribozyme-mediated destruction of RNA in vivo. EMBO J 8:3861–3866

    Google Scholar 

  • Cuozzo M, O'Connell KM, Kaniewski W, Fang RX, Chua NH, Tumer NE (1988) Viral protection in transgenic tobacco plants expressing the cucumber mosaic coat protein or its antisense RNA. Bio/Technology 6:549–557

    Google Scholar 

  • Dawson WO, Beck DL, Knorr DA, Grantham GL (1986) cDNA cloning of the complete genome of tobacco mosaic virus and production of infectious transcripts. Proc Natl Acad Sci USA 83:1832–1836

    Google Scholar 

  • Day AG, Bejarano ER, Buc KW, Burrell M, Lichtenstein CP (1991) Expression of an antisense viral gene in transgenic tobacco confers resistance to the DNA virus tomato golden mosaic virus. Proc Natl Acad Sci USA 88:6721–6725

    Google Scholar 

  • De Carvalho F, Gheysen G, Kushnir S, Van Montagu M, Inze D, Castresana C (1992) Suppression of β-1,3-glucanase transgene expression in homozygous plants. EMBO J 7:2595–2602

    Google Scholar 

  • De Carvalho Niebel F, Frendo P, Van Montagu M, Cornelissen M (1995) Post-transcriptional cosuppression of β-1,3-glucanase genes does not affect accumulation of transgene nuclear mRNA. Plant Cell 7:347–358

    Google Scholar 

  • De Feyter RC, Yang Y, Gabriel DW (1993) Gene-for-genes interactions between cottonR genes andXanthomonas campestris pv.malvacearum avr genes. Mol Plant Microbe Interacts 6:225–237

    Google Scholar 

  • De Haan 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

    Google Scholar 

  • Fitchen JH, Beachy RN (1993) Genetically engineered protection against viruses in transgenic plants. Annu Rev Microbiol 47:739–763

    Google Scholar 

  • Goelet P, Lomonosoff GP, Butler PJG, Akam ME, Gait MJ, Karn J (1982) Nucleotide sequence of tobacco mosaic virus RNA. Proc Natl Acad Sci USA 79:5818–5822

    Google Scholar 

  • Hart CM, Fischer B, Neuhaus JM, Meins F Jr (1992) Regulated inactivation of homologous gene expression in transgenicNicotiana sylvestris plants containing a defense-related tobacco chitinase gene. Mol Gen Genet 235:179–188

    Google Scholar 

  • Haseloff J, Gerlach WL (1988) Simple RNA enzymes with new and highly specific endoribonuclease activity. Nature 334:585–591

    Google Scholar 

  • Hemenway C, Fang RX, Kaniewski WK, Chua NH, Tumer NE (1988) Analysis of the mechanism of protection in transgenic plants expressing the potato virus X coat protein or its antisense RNA. EMBO J 7:1273–1280

    Google Scholar 

  • Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general method of transferring genes into plants. Science 227:1229–1231

    Google Scholar 

  • Kaniewski W, Lawson C, Sammons B, Haley L, Hart J, Delannay X, Tumer NE (1990) Field resistance of transgenic Russet Burbank potato to effects of infection by potato virus X and potato virus Y. Bio/Technology 8:750–754

    Google Scholar 

  • Kawchuk LM, Martin RR, McPherson J (1991) Sense and antisense RNA-mediated resistance to potato leafroll virus in Russet Burbank potato plants. Mol Plant Microbe Interact 4:247–253

    Google Scholar 

  • Kunkel TA, Roberts JD, Zakour RA (1987) Rapid and efficient specific mutagenesis without phenotypic selection. Methods Enzymol 154:367–382

    Google Scholar 

  • Lawson C, Kaniewski W, Haley L, Rozman R, Newell C, Sanders P, Tumer NE (1990) Engineering 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

    Google Scholar 

  • Lindbo JA, Dougherty WG (1992) Pathogen-derived resistance to a potyvirus: Immune and resistant phenotypes in transgenic tobacco expressing altered forms of a potyvirus coat protein nucleotide sequence. Mol Plant Microbe Interact 5:144–153

    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 5:1749–1769

    Google Scholar 

  • McDonnell RE, Clark RD, Smith WA, Hinchee MA (1987) A simplified method for the detection of neomycin phosphotransferase II activity in transformed plant tissues. Plant Mol Biol Rept 5:380–386

    Google Scholar 

  • Mueller E, Gilbert J, Davenport G, Brignet G, Baulcombe DC (1995) Homology-dependent resistance: transgenic virus resistance in plants related to homology-dependent gene silencing. Plant J 7:1001–1013

    Google Scholar 

  • Namba S, Ling K, Gonsalves C, Gonsalves D, Slightom JL (1991) Expression of the gene encoding the coat protein of cucumber mosaic virus (CMV) strain WL appears to provide protection to tobacco plants against infection by several different CMV strains. Gene 107:181–188

    Google Scholar 

  • Nelson RS, Powell Abel P, Beachy RN (1987) Lesions and virus accumulation in inoculated transgenic tobacco plants expressing the coat protein gene of tobacco mosaic virus. Virology 158:126–132

    Google Scholar 

  • Nelson A, Roth DA, Johnson JD (1993) Tobacco mosaic virus infection of transgenicNicotiana tabacum plants is inhibited by antisense constructs directed at the 5′ region of viral RNA. Gene 127:227–232

    Google Scholar 

  • Pang S-Z, Slightom JL, Gonsalves D (1993) Different mechanisms protect transgenic tobacco against tomato spotted wilt and impatients necrotic spotTospoviruses. Bio/Technology 11:819–824

    Google Scholar 

  • Perriman R, Delves A, Gerlach WL (1992) Extended target-site specificity for a hammerhead ribozyme. Gene 113:157–163

    Google Scholar 

  • Perriman R, Graf L, Gerlach WL (1993) A ribozyme that enhances gene suppression in tobacco protoplasts. Antisense Res Develop 3:253–263

    Google Scholar 

  • Perriman R, Bruening G, Dennis ES, Peacock WJ (1995) Effective ribozyme delivery in plant cells. Proc Natl Acad Sci USA 92:6175–6179

    Google Scholar 

  • Powell PA, Stark DM, Sanders PR, Beachy RN (1989) Protection against tobacco mosaic virus in transgenic plants that express tobacco mosaic virus antisense RNA. Proc Natl Acad Sci USA 86:6949–6952

    Google Scholar 

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

    Google Scholar 

  • Rezaian MA, Skene KGM, Ellis JG (1988) Anti-sense RNAs of cucumber mosaic virus in transgenic plants assessed for control of the virus. Plant Mol Biol 11:463–471

    Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  • Sarver N, Cantin EM, Chang PS, Zaia JA, Ladne PA, Stephens DA, Rossi JJ (1990) Ribozymes as potential anti-HIV-1 therapeutic agents. Science 247:1222–1225

    Google Scholar 

  • Saxena SK, Ackerman EJ (1990) Ribozymes correctly cleave a model substrate and endogenous RNA in vivo. J Biol Chem 265:17106–17109

    Google Scholar 

  • Shaw JG, Plaskitt KA, Wilson TMA (1986) Evidence that tobacco mosaic virus particles disassemble cotranslationally in vivo. Virology 148:326–336

    Google Scholar 

  • Shimayama T, Nishikawa S, Taira K (1995) Generality of the NUX rule: kinetic analysis of the results of systematic mutations in the trinucleotide at the cleavage site of hammerhead ribozymes. Biochemistry 34:3649–3654

    Google Scholar 

  • Steinecke P, Herget T, Schreier PH (1992) Expression of a chimeric ribozyme gene results in endonucleolytic cleavage of target messenger RNA and a concomitant reduction of gene expression in vivo. EMBO J 11:1525–1530

    Google Scholar 

  • Symons RH (1989) Self-cleavage of RNA in the replication of small pathogens of plants and animals. TIBS 14:445–450

    Google Scholar 

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

    Google Scholar 

  • Walker JC, Howard EA, Dennis ES, Peacock WJ (1987) DNA sequences required for anaerobic expression of the maize alcohol dehydrogenase 1 gene. Proc Natl Acad Sci USA 84:6624–6628

    Google Scholar 

  • Wegener D, Steinecke P, Herget T, Petereit I, Philipp C, Schreier P (1994) Expression of a reporter gene is reduced by a ribozyme in transgenic plants. Mol Gen Genet 245:465–470

    Google Scholar 

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

    Google Scholar 

  • Zhao JJ, Pick L (1993) Generating loss-of-function phenotypes of thefushi tarazu gene with a targeted ribozyme inDrosophila. Nature 365:448–451

    Google Scholar 

Download references

Author information

Author notes

  1. M. Young

    Present address: Department of Plant Pathology, Montana State University, 59717, Bozeman, MT, USA

  2. W. Gerlach

    Present address: R. W. Johnson Pharmaceutical Research Institute, Johnson and Johnson Research GPO, Box 3331, 2001, Sydney, NSW, Australia

Authors and Affiliations

  1. CSIRO Division of Plant Industry, GPO Box 1600, 2601, Canberra, ACT, Australia

    R. de Feyter, M. Young, K. Schroeder, E. S. Dennis & W. Gerlach

Authors
  1. R. de Feyter
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. Young
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. K. Schroeder
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. E. S. Dennis
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. W. Gerlach
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

de Feyter, R., Young, M., Schroeder, K. et al. A ribozyme gene and an antisense gene are equally effective in conferring resistance to tobacco mosaic virus on transgenic tobacco. Molec. Gen. Genet. 250, 329–338 (1996). https://doi.org/10.1007/BF02174391

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02174391

Key words

Search

Navigation