Abstract
Transgenic tobacco plants expressing hammerhead ribozyme (Rz), which specifically cleaves Chrysanthemum Stunt Viroid (CSVd) RNA, were produced via Agrobacterium-mediated transformation. To analyze the effects of Rz in cells, fire-fly luciferase (Luc) reporter genes, driven by the Cauliflower Mosaic Virus 35S promoter, which have the entire sense or anti-sense cDNA of the CSVd genome in between the Luc ORF and nopalin synthase terminator were constructed. Using particle bombardment, the Luc reporter constructs were delivered into the leaves of the transgenic tobacco plants. Levels of Luc mRNA and Luc enzyme activity were lower in the transgenic plants than in control wild-type plants, suggesting that the transcript of the Luc reporter gene was made unstable in the presence of Rz. On the other hand, mutations at the cleavage sites of the CSVd sequence in the reporter constructs suppressed the reduction of Luc enzyme activity in transgenic plants. A possibility of post-transcriptional gene silencing was ruled out. A direct evidence of the cleavage activity in cells by the experiment of rapid amplification of 5′ cDNA end could not obtained, but the present results suggest that the levels of Luc mRNA and Luc enzyme activity was affected by the ribozymes.
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References
Akama K, Shiraishi H, Ohta S, Nakamura K, Okada K, Shimura Y (1992) Efficient transformation of Arabidopsis thaliana: comparisons of the efficiencies with various organs plant ecotype and Agrobacterium stains. Plant Cell Rep 12:7–11
Akashi H, Kawasaki H, Kim WJ, Akaike T, Taira K, Maruyama A (2002) Enhancement in the cleavage activity of a hammerhead ribozyme by cationic comb-type polymerase and an RNA helicase in vitro. J␣Biochem 131:687–692
Elmayan T, Vaucheret H (1996) Expression of single copies of a strongly expressed 35 S transgene can be silenced post-transcriptionally. Plant J 9:787–797
Gallie DR, Feder JN, Schimke RT, Walbot V (1991) Post-transcriptional regulation in higher eukaryote: The role of the reporter gene in controlling expression. Mol Gen Gent 228:258–264
Guo H-S, Xie Q, Fei J-F, Chua N-H (2005) MicroRNA directs mRNA cleavage of the transcription factor NAC1 to downregulate auxin signals for Arabidopsis lateral root development. Plant Cell 17:1376-1386
Han S, Wu Z, Yang H, Wang R, Yie Y, Xie L, Tien P (2000) Ribozyme-mediated resistance to Rice Dwarf Virus and the transgene silencing in the progeny of transgenic rice plants. Transgenic Res 9:195–203
Haseloff J, Symons RH (1981) Chrysanthemum Stunt Viroid: primary sequence and secondary structure. Nucleic Acid Res 9:2741–2752
Hendry P, McCall MJ, Stewart TS, Lockett TJ (2004) Redesigned and chemically-modified hammerhead ribozymes with improved activity and serum stability. BMC Chemical Biology 4:doi:1-/1186/1472-6769-1-1
Horst RK, Langhans P (1977) Effect of Chrysanthemum Stunt, chlorotic mottle, aspermy and mosaic on flowering and rooting of chrysanthemums. Phytopatholoty 67:9–14
Hosokawa M, Otake A, Ohishi K, Ueda E, Hayashi T, Yasawa S (2004a) Elimination of Chrysanthemum Stunt Viroid from an infected chrysanthemum cultivar by shoot regeneration from a leaf primordium-free shoot apical meristem dome attached to a root tip. Plant Cell Rep 22:859–863
Hosokawa M, Ueda E, Ohishim K, Otake A, Yazawa S (2004b) Chrysanthemum Stunt Viroid disturbs the photoperiodic response for flowering of chrysanthemum plants. Planta 220:64–70
Kawasaki H, Taira K (2002) A functional gene discovery in the Fas-mediated pathway to apoptosis by analysis of transiently expressed randomized hybrid-ribozyme libraries. Nucleic Acid Res 30:3609–3614
Klein D, Denis M, Ricordi C, Pastori RL (2000) Assessement of ribozyme cleavage efficiency using reverse transcriptase real-time PCR. Mol Biotech 14:180– 195
Llave C, Xie Z, Kasschau KD, Carrington JC (2002) Cleavage of Scarecrow-like mRNA targets directed by class of Arabidopsis miRNA. Science 297:2053–2056
McIntyer CL, Manners JM (1996) Strategies for the suppression of peroxidase gene expression in tobacco. I. Designing efficient ribozymes. Transgenic Res 5:257–262
Neese P, Symons RH (1985) Domains in viroids: Evidence of intermolecular RNA rearrangements and their contribution to viroid evolution. Proc Natl Acad Sci USA 82:4582–4586
Perriman R, Delves A, Gerlach WI (1992) Expected target site specificity for hammerhead ribozyme. Gene 113:157–163
Que Q, Wang H-Y, English JJ, Jorgensen RA (1997) The frequency and degree of cosuppression by sense chalcone synthase transgenes are dependent on transgene promoter strength and are reduced by premature nonsense codons in the transgene coding sequence. Plant Cell 9:1357–1368
Qi Y, Ding B (2002) Replication of Potato Spindle Tuber Viroid in cultured cells of tobacco and Nicotiana benthamiana; the role of specific nucleotides in determining replication level for host adaptation. Virology 302:445–456
Sano T, Candresse T, Hmmond RW, Diener O, Owens RA (1992) Identification of multiple structural domains regulating viroid pathogenicity. Proc Natl Acad Sci USA 89:10104–10108
Schubert D, Lechtenberg B, Forsbach A, Gils M, Bahadur S, Schmidt R (2004) Silencing in Arabidopsis T-DNA transformants: The predominant role of gene-specific RNA sensing mechanisms versus position effects. Plant Cell 16:2562–2572
Tsuro M, Kubo T, Shizukawa Y, Takemoto T, Inaba K (2005) Agrobacterium rhizogenes is a useful transporter for introducing T-DNA of the binary plasmid into the chrysanthemum, Dendranthema grandiflorum, Kitamura, genome. Plant Cell Tiss Org Cult 81:175–181
Van Leeuwen W, Hagendoorn MJM, Ruttink T, Voecke RV, Van Der Plas LHW, Van Der Krol A (2000) The use of the luciferase reporter system for in plant gene expression studies. Plant Mol Biol Rep 18:143a-143t
Vanish NK, Kore AR, Eckstein F (1998) Recent developments in the hammerhead ribozyme field. Nucleic Acid Res 26:5237–5242
Wegener D, Steineck P, Herget T, Petereit I, Philipp C, Schreier PH (1994) Expression of a reporter gene is reduced by a ribozyme in transgenic plants. Mol Gen Genet 245:465–470
Yang X, Yie Y, Zhu F, Liu Y, Kang L, Wang X, Tien P (1997) Ribozyme-mediated high resistance against Potato Spindle Tuber Viroid in transgenic potatoes. Proc Natl Acad Sci USA 94:4861–4865
Zhu Y, Qi Y, Xun Y, Owens R, Ding B (2002) Movement of Potato Spindle Tuber Viroid reveals regulatory points of phloeme-mediated RNA traffic. Plant Physiol 130:138–146
Zoumadakis M, Neubert WJ, Tabler M (1994) The influence of imperfectly paired helices I and III on the catalytic activity of hammerhead ribozymes. Nucleic Acid Res 22:5271–5278
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Ando, Y., Oda, K., Iwabuchi, M. et al. Assessment of ribozyme-mediated gene suppression using a luciferase reporter gene containing the sequence of Chrysanthemum Stunt Viroid as a target. Mol Breeding 18, 209–218 (2006). https://doi.org/10.1007/s11032-006-9028-z
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DOI: https://doi.org/10.1007/s11032-006-9028-z