Abstract
In order to optimise expression of a foreign protein in transgenic plants we investigated the potential benefits of including a viral untranslated leader sequence within a plant transformation vector. A variety of 5 leaders, including the tobacco mosaic virus (TMV) leader sequence and 31 nucleotides of the cauliflower mosaic virus (CaMV) 35S RNA leader, were compared. Viral leader constructs employing the 35S promoter and the reporter β-glucuronidase (GUS) were tested by electroporation into tobacco mesophyll protoplasts and against a cointroduced chloramphenicol acetyl transferase (CAT) gene in transgenic tobacco leaves. In the transient assay system, GUS activities from the viral leaders were compared with those from either a short, random leader or a translational fusion of the CaMV 19S RNA ORF VI to GUS. A two-to-three-fold enhanced level of expression resulted when these leaders were substituted with either the 35S RNA or the TMV leader sequences. This enhancement was further increased, to four-to five-fold, by inclusion of four or seven of the bases from the 35S transcription initiation site adjacent to the TMV leader. In transgenic tobacco the improved GUS levels were maintained from constructs including either the TMV leader (eight-fold) or this sequence with the addition of the 35S transcription initiation site bases (ten-fold). A comparison of GUS enzyme amounts with GUS mRNA amounts, using the CAT gene as an internal standard, revealed that TMV leader-bearing mRNA was translated from four-to six-fold more efficiently than the random leader control.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Altmann M, Blum S, Wilson TMA, Trachsel H: The 5′-leader sequence of tobacco mosaic virus RNA mediates initiation factor-4E-independent, but still initiation factor-4A-dependent translation in yeast extracts. Gene 91: 127–129 (1990).
Baughman G, Howell SH: Cauliflower mosaic virus 35S RNA leader region inhibits translation of downstream genes. Virology 167: 125–135 (1988).
Berry JO, Breiding DE, Klessig DF: Light-mediated control of translational initiation of ribulose-1.5-biphosphate carboxylase in amaranth cotyledons. Plant Cell 2: 795–803 (1990).
Bevan M: Binary Agrobacterium vectors for plant transformation. Nucl Acids Res 12: 8711–8721 (1984).
Browning KS, Lax SR, Humphreys J, Ravel JM, Jobling SA, Gehrke L: Evidence that the 5′-untranslated leader of mRNA affects the requirement for wheat germ initiation factors 4A, 4F, and 4G. J Biol Chem 263: 9630–9634 (1988).
Chandler PM, Huiet L: Primer extension studies on α-amylase mRNAs in barley aleurone 1. Characterization and quantification of the transcripts. Plant Mol Biol 16: 627–635 (1991).
Dawe PK, Lachmansingh AR, Freeling M: Transposonmediated mutations in the untranslated leader of maize adh1 that increase and decrease pollen-specific gene expression. Plant Cell 5: 311–319 (1993).
Devereaux J, Haeberli P, Smithies O: A comprehensive set of sequence analysis programs for the VAX. Nucl Acids Res 12: 387–395 (1984).
Dunsmuir P, Bond D, Lee K, Gidoni D, Townsend J: Stability of introduced genes and stability in expression. In: Gelvin SB, Schilperoort RA (eds) Plant Molecular Biology Manual, pp. Cl: 1–17, Kluwer Academic Publishers, Dordrecht (1988).
Franck A, Guilley H, Jonard G, Richards K, Hirth L: Nucleotide sequence of cauliflower mosaic virus DNA. Cell 21: 285–294 (1980).
Fütterer J, Gordon K, Bonneville JM, Sanfaçon H, Pisan B, Penswick J, Hohn T: The leading sequence of caulimovirus large RNA can be folded into a large stem loop structure. Nucl Acids Res 16: 8377–8390 (1988).
Fütterer J, Gordon K, Feiffer P, Sanfaçon H, Pisan B, 0 Bonneville JM, Hohn T: Differential inhibition of downstream gene expression by the cauliflower mosaic virus 35S RNA leader. Virus Genes 3: 45–55 (1989).
Fütterer J, Gordon K, Sanfaçon H, Bonneville JM, Hohn T: Positive and negative control of translation by the leader sequence of cauliflower mosaic virus pregenomic 35S RNA. EMBO J 9: 1697–1707 (1990).
Gallie DR, Sleat DE, Watts JW, Turner PC, Wilson TMA: The 5′ leader of tobacco mosaic virus RNA enhances the expression of foreign gene transcripts in vitro and in vivo. Nucl Acids Res 15: 3257–3273 (1987).
Gallie DR, Sleat DE, Watts JW, Turner PC, Wilson TMA: A comparison of eucaryotic viral 5-leader sequences as enhancers of mRNA expression in vivo. Nucl Acids Res 15: 8693–8710 (1987).
Gallie DR, Sleat DE, Watts JW, Turner PC, Wilson TMA: Mutational analysis of the tobacco mosaic virus 5′-leader for altered ability to enhance translation. Nucl Acids Res 16: 883–893 (1988).
Gallie DR, Lucas WJ, Walbot V: Visualizing mRNA expression in plant protoplasts: factors influencing efficient mRNA uptake and translation. Plant Cell 1: 301–311 (1989).
Gallie DR, Walbot V: Identification of motifs within the tobacco mosaic virus 5′-leader responsible for enhancing translation. Nucl Acids Res 20: 4631–4638 (1992).
Guerineau F, Lucy A, Mullineaux P: Effect of two consensus sequences preceding the translation initiator codon on gene expression in plant protoplasts. Plant Mol Biol 18: 815–818 (1992).
Guilley H, Dudley RK, Jonard G, Balàzs E, Richards KE: Transcription of cauliflower mosaic virus DNA: detection of promoter sequences and characterization of transcripts. Cell 30: 763–773 (1982).
Harpster MH, Townsend JA, Jones JDG, Bedbrook J, Dunsmuir P: Relative strengths of the 35S cauliflower mosaic virus, 1, 2 and nopaline synthase promoters in transformed tobacco, sugarbeet and oilseed rape callus tissue. Mol Gen Genet 212: 182–190 (1988).
Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT: A simple and general method for transferring genes into plants. Science 227: 1229–1231 (1985).
Jefferson RA: Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5: 387–405 (1987).
Jobling SA, Gehrke L: Enhanced translation of chimaeric messenger RNAs containing a plant viral untranslated leader sequence. Nature 325: 622–625 (1987).
Kozak M: Influence of mRNA secondary structure on initiation by eukaryotic ribosomes. Proc Natl Acad Sci USA 83: 2850–2854 (1986).
Kozak M: An analysis of 5-noncoding sequences from 699 vertebrate messenger RNAs. Nucl Acids Res 15: 8125–8148 (1987).
Kozak M: Leader length and secondary structure moderate mRNA function under conditions of stress. Mol Cell Biol 8: 2737–2744 (1988).
Kozak M: The scanning model for translation: an update. J Cell Biol 108: 229–241 (1989).
Kozak M: Circumstances and mechanisms of inhibition of translation by secondary structure in eukaryotic mRNAs. Mol Cell Biol 9: 5134–5142 (1989).
Kozak M: Effects of long 5′ leader sequences on initiation by eukaryotic ribosomes in vitro. Gene Expression 1: 117–125 (1991).
Kozak M: Structural features in eukaryotic mRNAs that moderate the initiation of translation. J Biol Chem 266: 19867–19870 (1991).
Kuhlemeier C: Transcriptional and post-transcriptional regulation of gene expression in plants. Plant Mol Biol 19: 1–14 (1992).
Lawton MA, Tierney MA, Nakamura I, Anderson E, Komeda Y, Dubé P, Hoffman N, Fraley RT, Beachy RN: Expression of a soybean β-conglycinin gene under the control of the cauliflower mosaic virus 35S and 19S promoters in transformed petunia tissues. Plant Mol Biol 9: 315–324 (1987).
Lücke HA, Chow KC, Mickel FS, Moss KA, Kern HF, Scheele GA: Selection of AUG initiation codons differs in plants and animals. EMBO J 6: 43–48 (1987).
Nielsen DA, Chang T-C, Shapiro DJ: A highly sensitive, mixed-phase assay for chloramphenicol acetyltransferase activity in transfected cells. Anal Biochem 179: 19–23 (1989).
Pooggin MM, Skryabin KG: The 5′-untranslated leader sequence of potato virus X RNA enhances the expression of a heterologous gene in vivo. Mol Gen Genet 234: 329–331 (1992).
Prescott A, Martin C: A rapid method for the quantitative assessment of levels of specific mRNAs in plants. Plant Mol Biol Rep 4: 219–224 (1987).
Shatkin AJ: mRNA caps-old and newer hats. BioEssays 7: 275–277 (1987).
Sleat DE, Hull R, Turner PC, Wilson TMA: Studies on the mechanism of translational enhancement by the 5′-leader sequence of tobacco mosaic virus RNA. Eur J Biochem 175: 75–86 (1988).
Smirnyagina EV, Morozov SY, Radionova NP, Miroschnichenko NA, Solovyev AG, Fedorkin ON, Atabekov JG: Translational efficiency and competitive ability of mRNAs with 5′-untranslated αβ-leader of potato virus X RNA. Biochimie 73: 587–598 (1991).
Wilson TMA, Saunders K, Dowson Day MJ, Sleat DE, Trachsel H, Mundry KW: Effects of the 5′ leader sequence of tobacco mosaic virus RNA, or derivatives thereof, on foreign mRNA and native viral gene expression. In: McCarthy JEG, Tuite MF (eds) Post-transcriptional Control of Gene Expression, pp. 261–275, NATO-ASI Series, Vol H49 (1990).
Watts JW, Dawson JRO, King JM: Infection of plant protoplasts with two or more viruses. In: Ingram DS and Helgeson JP (eds) Tissue Culture Methods for Plant Pathologists, pp. 97–101 Blackwell Scientific Publications (1980).
Yamaya J, Yoshioka M, Meshi T, Okada Y, Ohno T: Expression of tobacco mosaic virus RNA in transgenic plants. Mol Gen Genet 211: 520–525 (1988).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Dowson Day, M.J., Ashurst, J.A., Mathias, S.F. et al. Plant viral leaders influence expression of a reporter gene in tobacco. Plant Mol Biol 23, 97–109 (1993). https://doi.org/10.1007/BF00021423
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00021423