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Suppression of a key gene involved in chlorophyll biosynthesis by means of virus-inducing gene silencing

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Abstract

The ChlH gene coding the H subunit of magnesium chelatase, an enzyme involved in chlorophyll biosynthesis, was silenced in Nicotiana benthamiana plants by infection with tobacco mosaic virus vectors (pTMV-30b) containing 67, 214 or 549 nt long ChlH inserts. Silencing of the nuclear ChlH gene induced a chimeric phenotype with green and yellow/white leaves associated with alterations of chloroplast shape and ultrastructure. The symptoms became first evident around veins of young leaves, and only later in the mesophyll tissues. The efficiency of gene silencing was not dependent on the insert orientation, but was strongly correlated with the size of the ChlH insert, providing a flexible method to modulate the level of gene suppression. Silencing efficiency seemed to be strongly dependent on endogenous ChlH mRNA level of the target tissue. Silencing of the ChlH gene with the longest fragment of 549 nt also lowered the accumulation of ChlD and chlorophyll synthetase mRNAs, i.e. other genes involved in chlorophyll biosynthesis.

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References

  • Baulcombe, D.C. 1996. Mechanisms of pathogen-derived resistance to viruses in transgenic plants. Plant Cell 8: 1833–1844.

    Google Scholar 

  • Baulcombe, D.C. 1999. Fast forward genetics based on virus-induced gene silencing. Curr. Opin Plant Biol. 2: 109–113.

    Google Scholar 

  • Burton, R.A., Gibeaut, D.M., Bacic, A., Findlay, K., Roberts, K., Hamilton, A., Baulcombe, D.C. and Fincher, G.B. 2000. Virus-induced silencing of a plant cellulose synthetase gene. Plant Cell 12: 691–705.

    Google Scholar 

  • Carrington, J.C. 2000. Moving targets. Nature 408: 150–151.

    Google Scholar 

  • Cogoni, C. and Macino, G. 1999. Homology-dependent gene silencing in plants and fungi: a number of variations on the same theme. Curr. Opin. Microbiol. 2: 657–662.

    Google Scholar 

  • Covey, S.N., Al-Kaff, N.S., Làngara, A. and Turner, D.S. 1997. Plants combat infection by gene silencing. Nature 385: 781–782.

    Google Scholar 

  • Falbel, T.G., Meehl, J.B. and Staehelin, L.A. 1996. Severity of mutant phenotype in a series of chlorophyll-deficient wheat mutants depends on light intensity and the severity of the block in the chlorophyll synthesis. Plant Physiol. 112: 821–832.

    Google Scholar 

  • Gibson, L.C.D., Marrison, J.L., Leech, R.M., Jensen, P.E., Bassham, D.C., Gibson, M. and Hunter, C.N. 1996. A putative Mg chelatase subunit from Arabidopsis thaliana cv. C24. Plant Physiol. 111: 61–71.

    Google Scholar 

  • Gräfe, S., Saluz, H.-P., Grimm, B. and Hänel, F. 1999. Mg-chelatase of tobacco: the role of the subunit ChlD in the chelation step of protoporphyrin IX. Proc. Natl. Acad. Sci. USA 96: 1941–1946.

    Google Scholar 

  • Grimm, B. 1998. Novel insights in the control of tetrapyrrole metabolism of higher plants. Curr. Opin. Plant Biol. 1: 245–250.

    Google Scholar 

  • Hammond, S.M., Bernstein, E., Beach, D. and Hannon, G.J. 2000. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404: 293–296.

    Google Scholar 

  • Hansson, A., Kannangara, C.G., von Wettstein, D. and Hansson, M. 1999. Molecular basis for semidominance of missence mutations in the XANTHA-H (42-kDa) subunit of magnesium chelatase. Proc. Natl. Acad. Sci. USA 96: 1744–1749.

    Google Scholar 

  • Härtel, H., Kruse, E. and Grimm, B. 1997. Restriction of chlorophyll synthesis due to expression of glutamate 1-semialdehyde aminotransferase antisense RNA does not reduce the light-harvesting antenna size in tobacco. Plant Physiol. 113: 1113–1124.

    Google Scholar 

  • Hudson, A., Carpenter, R., Doyle, S. and Coen, E.S. 1993. Olive: a key gene required for chlorophyll biosynthesis in Antirrhinum majus. EMBO J. 12: 3711–3719.

    Google Scholar 

  • Inkeep, W.P. and Blomm, P.R. 1985. Extinction coefficients of chlorophyll a and b in N,N-dimethylformamide and 80% acetone. Plant Physiol. 77: 483–485.

    Google Scholar 

  • Jones, L., Hamilton, A.J., Voinnet, O., Thomas, C.L., Maule, A.J. and Baulcombe, D.C. 1999. RNA-DNA interactions and DNA methylation in post-transcriptional gene silencing. Plant Cell 11: 2291–2301.

    Google Scholar 

  • Kennerdell, J.R. and Carthew, R.W. 1998. Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway. Cell 95: 1017–1026.

    Google Scholar 

  • Kjemtrup, S., Sampson, K.S., Peele, C.G., Nguyen, L.V., Conkling, M.A., Thompson, W.F. and Robertson, D. 1998. Gene silencing from plant DNA carried by a Geminivirus. Plant J. 14: 91–100.

    Google Scholar 

  • Konat, G.W., Laszkiewicz, I., Grubinska, B. and Wiggins, C. 1994. PCR Technology: Current Innovations, pp. 37–39. CRC Press, Boca Raton, FL.

    Google Scholar 

  • Kumagai, M.H., Donson, J., Della-Cioppa, G., Harvey, D., Hanley, K. and Grill, L.K. 1995. Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA. Proc. Natl. Acad. Sci. USA 92: 1679–1683.

    Google Scholar 

  • Lindbo, J.A., Silva-Rosales, L., Proebsting, W.M. and Dougherty, W.G. 1993. Induction of a highly specific antiviral state in transgenic plants: implications for regulations for regulation of gene expression and virus resistance. Plant Cell 5: 1749–1759.

    Google Scholar 

  • Montgomery, M.K, Xu, S. and Fire, A. 1998. RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 95: 15502–15507.

    Google Scholar 

  • Nakayama, M., Masuda, T., Bando, T., Yamagata, H., Ohta, H. and Takamiya, K.-I. 1998. Cloning and expression of the soybean ChlH gene encoding a subunit of Mg-chelatase and localization of the Mg2+ concentration-dependent ChlH protein within the chloroplast. Plant Cell Physiol. 39: 275–284.

    Google Scholar 

  • Ngô, H., Tschudi, C., Gull, K. and Ullu, E. 1998. Double-stranded RNA induces mRNA degradation in Trypanosoma brucei. Proc. Natl. Acad. Sci. USA 95: 14687–14692.

    Google Scholar 

  • Palauqui, J.-C. and Vaucheret, H. 1998. Transgenes are dispensable for the RNA degradation step of cosuppression. Proc. Natl. Acad. Sci. USA 95: 9675–9680.

    Google Scholar 

  • Palauqui, J.-C. and Balzergue, S. 1999. Activation of systemic acquired silencing by localised introduction of DNA. Curr. Biol. 9: 59–66.

    Google Scholar 

  • Papenbrock, J., Mock, H.-P., Tanaka, R., Kruse, E. and Grimm, B. 2000a. Role of magnesium chelatase activity in the early steps of the tetrapyrrole biosynthetic pathway. Plant Physiology 122: 1161–1169.

    Google Scholar 

  • Papenbrock, J., Pfündel, E., Mock, H.-P. and Grimm, B. 2000b. Decreased and increased expression of the subunit CHL I diminishes Mg chelatase activity and reduces chlorophyll synthesis in transgenic tobacco plants. Plant J. 22: 155–164.

    Google Scholar 

  • Ratcliff, F.G., Harrison, B.D. and Baulcombe, D.C. 1997. A similarity between viral defense and gene silencing in plants. Science 276: 1558–1560.

    Google Scholar 

  • Ratcliff, F.G., MacFarlane, S.A. and Baulcombe, D.C. 1999. Gene silencing without DNA: RNA-mediated cross-protection between viruses. Plant Cell 11: 1207–1215.

    Google Scholar 

  • Reinbothe, S. and Reinbothe, C. 1996. The regulation of enzymes involved in chlorophyll biosynthesis. Eur. J. Biochem. 237: 323–343.

    Google Scholar 

  • Ruiz, M.T., Voinnet, O. and Baulcombe, D.C. 1998. Initiation and maintenance of virus-induced gene silencing. Plant Cell 10: 937–946.

    Google Scholar 

  • Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd edn, Cold Spring Harbor Laboratory Press, Plainview, NY.

    Google Scholar 

  • Sànchez-Alvarado, A. and Newmark, P.A. 1999. Double-stranded RNA specifically disrupts gene expression during planarian regeneration. Proc. Natl. Acad. Sci. USA 96: 5049–5054.

    Google Scholar 

  • Shivprasad, S., Pogue, G.P., Lewandowski, D.J., Hidalgo, J., Donson, J., Grill, L.K. and Dawson, W.O. 1999. Heterologous sequences greatly affect foreign gene expression in tobacco mosaic virus-based vectors. Virology 255: 312–323.

    Google Scholar 

  • Sijen, T., Wellink, J., Hiriart, J.-B. and van Kammen. 1996. RNA-mediated virus resistance: role of repeated transgenes and delineation of targeted regions. Plant Cell 8: 2277–2294.

    Google Scholar 

  • Thomas, C.L., Jones, L., Baulcombe, D.C. and Maule, A.J. 2001. Size constraints for targeting post-transcriptional gene silencing and for RNA-directed methylation in Nicotiana benthamiana using a potato virus X vector. Plant J. 25: 417–425.

    Google Scholar 

  • Tuschl, T., Zamore, P.D., Lehmann, R., Bartel, D.P. and Sharp, P.A. 1999. Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev. 13: 3191–3197.

    Google Scholar 

  • van der Krol, A.R., Mur, L.A., Mol, J.N. and Stuitje, A.R. 1990. Flavonoid gene in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell 2: 291–299.

    Google Scholar 

  • Vaucheret, H., Béclin, C., Elmayan, T., Feuerbach, F., Godon, C., Morel, J.-B., Mourrain, P., Palauqui, J.-C. and Vernhettes, S. 1998. Transgene-induced gene silencing in plants. Plant J. 16: 651–659.

    Google Scholar 

  • Voinnet, O., Lederer, C. and Baulcombe, D.C. 2000. A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana. Cell 103: 157–167.

    Google Scholar 

  • Walker, C.J. and Willows, R.D. 1997. Mechanism and regulation of Mg-chelatase. Biochem. J. 327: 321–333.

    Google Scholar 

  • Waterhouse, P.M., Graham, M.W. and Wang, M.-B. 1998. Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc. Natl. Acad. Sci. USA. 95: 13959–13964.

    Google Scholar 

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Authors and Affiliations

  1. Department of Biology, Laboratory of Plant Physiology and Molecular Biology, University of Turku, Tykistokatu 6a, 20540, Turku, Finland

    Jean-Baptiste Hiriart, Kirsi Lehto, Esa Tyystjärvi, Teemu Junttila & Eva-Mari Aro

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  1. Jean-Baptiste Hiriart
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  2. Kirsi Lehto
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  3. Esa Tyystjärvi
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  4. Teemu Junttila
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  5. Eva-Mari Aro
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Hiriart, JB., Lehto, K., Tyystjärvi, E. et al. Suppression of a key gene involved in chlorophyll biosynthesis by means of virus-inducing gene silencing. Plant Mol Biol 50, 213–224 (2002). https://doi.org/10.1023/A:1016000627231

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