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Assessment of CaMV-mediated gene silencing and integration of CaMV into GM plants with a 35S RNA promoter

Published online by Cambridge University Press:  20 December 2007

Julie Squires
Affiliation:
Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK
Jennifer Stephens
Affiliation:
Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK
James E. Shoelz
Affiliation:
Div. of Plant Sciences, University of Missouri, Columbia, MO 65211, USA
Peter Palukaitis
Affiliation:
Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK

Abstract

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Four GM plant species (Arabidopsis thaliana, Brassica napus, Nicotiana benthamiana and N. tabacum), each expressing the gene encoding the jellyfish green fluorescent protein (GFP) regulated by the cauliflower mosaic virus (CaMV) 35S RNA promoter, were assessed for the extent of transgene silencing and viral genome integration following infection by CaMV. The first three species are systemic hosts of CaMV, while N. tabacum is only a local host for a few strains of CaMV. A generalized systemic silencing of the GFP transgene was not observed in a total of 100 plants of each species infected with CaMV, although some localized loss of GFP was observed in CaMV-infected N. benthamiana leaves, and some loss of fluorescence was observed in older leaves of uninfected as well as infected plants. Progeny seedlings obtained from the above infected plants also did not exhibit transgene silencing showing that virus infection did not affect the stability of the transgene. These progeny plants also did not show signs of virus infection, indicating that the presence of the CaMV 35S RNA promoter sequences in the plant genome did not potentiate seed transmission of the virus. Integration of infective CaMV into the CaMV 35S RNA promoter could not be detected in 944 samples taken from leaves of the above infected plant species or in 2912 samples taken from progeny seedlings. Based on a detection limit of one copy per 4000 haploid genomes, we conclude that if integration of virus does occur into the CaMV 35S RNA promoter, then it occurs at such a low frequency as to be insignificant.

Type
Research Article
Copyright
© ISBR, EDP Sciences, 2007

References

Al-Kaff, NS, Covey, SN, Kreike, MM, Page, AM, Pinder, R, Dale, PJ (1998) Transcriptional and posttranscriptional gene silencing in response to a pathogen. Science 279: 21132115 CrossRef
Al-Kaff NS, Kreike MM, Covey SN, Pitcher R, Page AM, Dale PJ (2000) Plants rendered herbicide-susceptible by cauliflower mosaic virus-elicited suppression of a 35S promoter-regulated transgene. Nature Biotechnol. 18: 995–999
Assaad, FF, Signer, ER (1990) Cauliflower mosaic virus P35S promoter activity in Escherichia coli. Mol. Gen. Genet. 223: 517520 CrossRef
Ballas, N, Broido, S, Soreq, H, Loyter, A (1989) Efficient functioning of plant promoters and poly(A) sites in Xenopus oocytes. Nucleic Acids Res. 17: 78917903 CrossRef
Baulcombe, DC, Saunders, GR, Bevan, MW, Mayo, MA, Harrison, BD (1986) Expression of biologically active viral satellite RNA from the nuclear genome of transformed plants. Nature 321: 446449 CrossRef
Bevan, MW, Mason, SE, Goelet, P (1985) Expression of tobacco mosaic virus coat protein by the cauliflower mosaic virus promoter in plants transformed by Agrobacterium. EMBO J. 4: 19211926
Cullen, DW, Lees, AK, Toth, IK, Duncan, JM (2001) Conventional PCR and real-time quantitative PCR detection of Helminthosporium solani in soil and on potato tubers. Eur. J. Plant Pathol. 107: 387398 CrossRef
Franck, A, Guilley, H, Jonard, G, Richards, K, Hirth, L (1980) Nucleotide sequence of cauliflower mosaic virus. Cell 21: 285294 CrossRef
Gal, S, Pisan, B, Hohn, T, Grimsley, N, Hohn, B (1992) Agroinfection of transgenic plants leads to viable cauliflower mosaic virus by intermolecular recombination. Virology 187: 525533 CrossRef
Goldberg, K-B, Kiernan, J, Shepherd, RJ (1991) A disease syndrome associated with expression of gene VI of caulimoviruses may be a nonhost reaction. Mol. Plant-Microbe Interact. 4: 182189 CrossRef
Gracia, O, Shepherd, RJ (1985) Cauliflower mosaic virus in the nucleus of Nicotiana. Virology 146: 141145 CrossRef
Guilley, H, Dudley, RK, Jonard, G, Balázs, E, Richards, KE (1982) Transcription of cauliflower mosaic virus DNA: Detection of promoter sequences, and characterization of transcripts. Cell 30: 763773 CrossRef
Halfhill, MD, Richards, HA, Mabon, SA, Stewart, CN Jr (2001) Expression of GFP and Bt genes in Brassica napus and hybridization with Brassica rapa. Theor. Appl. Genet. 103: 659667 CrossRef
Harper, BK, Stewart, CN Jr (2000) Patterns of green fluorescent protein expression in transgenic plants. Plant Mol. Biol. Rep. 18: 121a141i
Haseloff J, Siemering KR, Prasher DC, Hodge S (1997) Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc. Natl. Acad. Sci USA 94: 2122–2127
Ho, MW, Ryan, A, Cummins, J (1999) Cauliflower mosaic viral promoter - a recipe for disaster? Microb. Ecol. Health Dis. 11: 194197 CrossRef
Ho, MW, Ryan, A, Cummins, J (2000) Hazards of transgenic plants containing the cauliflower mosaic virus viral promoter. Microb. Ecol. Health Dis. 12: 611 CrossRef
Hull R (1984) Caulimovirus group. In CMI/AAB Description of Plant Viruses No. 295. Unwin Bros. Ltd., Old Woking, Surrey, UK
Hull R, Covey SN, Dale P (2000) Genetically modified plants and the 35S promoter: assessing the risks and enhancing the debate. Microb. Ecol. Health Dis. 12: 1–5
Jefferson, RA, Kavanagh, TA, Bevan, MW (1987) GUS fusions: $\beta$ -glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6: 39013907
Love AJ, Laval V, Geri C, Laird J, Tomos AD, Hooks MA, Milner JJ (2007) Components of Arabidopsis defense- and ethylene-signaling pathways regulate susceptibility to Cauliflower mosaic virus by restricting long-distance movement. Mol. Plant-Microbe Interact. 20: 659–670
Mitsuhara, I, Ugaki, M, Hirochika, H, Ohshima, M, Murakami, T, Gotoh, Y, Katayose, Y, Nakamura, S, Honkura, R, Nishimiya, S, Ueno, K, Mochizuki, A, Tanimoto, H, Tsugawa, H, Otsuki, Y, Ohashi, Y (1996) Efficient promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledonous plants. Plant Cell Physiol. 37: 4959 CrossRef
Morel, JB, Tepfer, M (2000) Pour une évaluation scientifique des risques : le cas du promoteur 35S. Biofutur 201: 3235 CrossRef
Odell, JT, Nagy, F, Chua, NH (1985) Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313: 810812 CrossRef
Ow, DW, Jacobs, JD, Howell, SH (1987) Functional regions of the cauliflower mosaic virus 35S RNA promoter determined by the use of the firefly luciferase gene as a reporter of promoter activity. Proc. Natl. Acad. Sci. USA 84: 48704874 CrossRef
Pobjecky, N, Rosenberg, GH, Dinter-Gottlieb, G, Kaufer, D (1990) Expression of the beta-glucuronidase gene under the control of the CaMV 35S promoter in Schizosacharomyces pombe. Mol. Gen. Genet. 220: 314316 CrossRef
Ruiz, MT, Voinnet, O, Baulcombe, DC (1998) Initiation and maintenance of virus-induced gene silencing. Plant Cell 10: 937946 CrossRef
Rüth, J, Hirt, H, Schweyen, RJ (1992) The cauliflower mosaic virus 35S promoter is regulated by cAMP in Saccharomyces cerevisiae. Mol. Gen. Genet. 235: 365372 CrossRef
Rüth, J, Schweyen, RJ, Hirt, H (1994) The plant transcription factor TGA1 stimulates expression of the CaMV 35S promoter in Saccharomyces cerevisiae. Plant Mol. Biol. 25: 323328 CrossRef
Schoelz JE (2006) Viral determinants of resistance versus susceptibility. In Loebenstein G, Carr JP, eds, Natural Resistance Mechanisms of Plants to Viruses, Springer, The Netherlands, pp 13–33
Schoelz, JE, Wintermantel, WM (1993) Expansion of viral host range through complementation and recombination in transgenic plants. Plant Cell 5: 16691679 CrossRef
Schoelz, JE, Shepherd, RJ, Daubert, SD (1986a) Region VI of cauliflower mosaic virus encodes a host range determinant. J. Mol. Cell Biol. 6: 26322637 CrossRef
Schoelz, JE, Shepherd, RJ, Richins, DR (1986b) Properties of an unusual strain of cauliflower mosaic virus. Phytopathology 76: 451454 CrossRef
Sun, L, Cai, H, Xu, W, Hu, Y, Lin, Z (2002) CaMV 35S promoter directs beta-glucuronidase expression in Gonoderma lucidum and Pleurotus citrinopileatus. Mol. Biotechnol. 20: 239244 CrossRef
Tepfer, M, Gaubert, S, Leroux-Coyau, M, Prince, S, Houdebine, LM (2004) Transient expression in mammalian cells of transgenes transcribed from the Cauliflower mosaic virus 35S promoter. Environ. Biosafety Res. 3: 9197 CrossRef
Vlasák, J, Šmahel, M, Pavlik, A, Pavingerová, D, Břiza, J (2003) Comparison of hCMV intermediate early and CaMV 35S promoters in both plant and human cells. J. Biotechnol. 103: 197202 CrossRef
Yu, W, Murfett, J, Schoelz, JE (2003) Differential induction of symptoms in Arabidopsis by P6 of Cauliflower mosaic virus. Mol. Plant-Microbe Interact. 16: 3542 CrossRef