Institute of Molecular Plant Sciences, Gorlaeus Laboratories, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands1
Author for correspondence: John Bol. Fax +31 71 5274469. e-mail j.bol{at}chem.leidenuniv.nl
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Abstract |
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Previously, we transformed tobacco plants with incomplete DNA copies of AMV RNAs 1 and 2 (Taschner et al., 1991 ). In these P12 plants, the integrated cDNAs lack the 5-terminal 36 nucleotides of RNA 1 and the 3-terminal 10 nucleotides of RNA 2. The P12 plants can be infected with RNA 3 without the requirement for CP in the inoculum. The truncated RNAs 1 and 2 are not replicated by the transgenic RdRp in healthy or RNA 3-infected P12 plants. In the present work, we have engineered transgenic plants that express full-length RNAs 1 and 2 and viral RdRp. An analysis of steps in the replication cycle that occurred in the absence and presence of RNA 3 provided further insight in the role of CP in viral RNA replication at the whole plant level.
The AMV cDNAs 1 and 2, each surrounded by the 35S promoter of Cauliflower mosaic virus and the transcriptional terminator of the nopaline synthase gene (Neeleman et al., 1991 ), were inserted in two steps as KpnIPvuII and SstIPvuII fragments, respectively, in a tandem arrangement into the binary vector pMOG800. The resulting plasmid, pMOG-AMV1+2, was mobilized into Agrobacterium tumefaciens strain LBA4404. Following leaf disc transformation of Nicotiana tabacum cv. Samsun NN (Horsch et al., 1985
), 19 independent plant lines carrying AMV cDNAs 1 and 2 were regenerated on kanamycin-containing MS medium (R12 lines: R12-1, 3 to 8, 10, 11, 14, 15 to 25). To determine the levels of viral RNA in the 19 individual transformants (T0), total RNA was extracted from healthy leaves of the primary transformants and analysed by Northern blot hybridization using 32P-labelled, random primed probes of cDNA 1 and 2 (van der Kuyl et al., 1991
a; Sambrook et al., 1989
). Equal loading of the lanes was checked by ethidium bromide staining of ribosomal RNAs (not shown). In the 19 primary transformed R12 plants, the accumulation of viral plus-strand and possible minus-strand RNAs (Fig. 1
, line numbers on top of the lanes) was generally lower than in P12 plants (Fig. 1
, lane P). The transcription of the RNA 1 and 2 transgenes appeared to be independent as RNAs corresponding to the two transgenes accumulated in different ratios in individual lines. Such variable transgene expression levels and absence of coexpression of cotransferred transgenes have often been reported (Peach & Velten, 1991
, and references therein).
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To investigate whether an active replication complex is assembled in R12 plants, the T1 generation of 13 R12 lines (R12-1, 4 to 8, 11, 17 to 19, 21, 24, 25) was infected with (in)complete mixtures of AMV virions and RNA 3 transcripts in the presence or absence of CP. The inoculum RNA 3 was in vitro transcribed with T7 RNA polymerase from clone pAL3 (Neeleman et al., 1991 ) and used either on its own or supplemented with CP molecules in an RNA 3:CP molecule ratio of 1:40. P12 virus particles (AMV virions containing RNAs 3 and 4) were isolated from RNA 3-infected P12 plants and a complete set of AMV virions was purified from AMV-infected nontransgenic tobacco plants (van Vloten-Doting & Jaspars, 1972
). Three leaf halves of two plants per line were inoculated with one of the different inocula (Neeleman et al., 1993
). Total nucleic acids were extracted from inoculated leaves 5 days post-inoculation (p.i.) and analysed by Northern blot hybridization using a 32P-labelled, random-primed probe mixture containing AMV cDNAs 1 to 3 (Fig. 2
and data not shown for lines R12-1, 7, 11, 18, 19, 21, 24). The replication patterns of viral and transgene-derived RNAs from six R12 lines displayed in Fig. 2
represent an overall picture for the population of R12 plants tested and demonstrate the interclonal variability. The exposure time of the blots did not permit detection of the transgenic viral RNAs in mock-inoculated plants (Fig. 2
, lanes 5). Inoculation with the complete AMV genome revealed that none of the R12 lines showed resistance to AMV infection (Fig. 2
, lanes 4). When R12 plants were inoculated with RNA 3 (Fig. 2
, lanes 1), RNA 3 plus CP (Fig. 2
, lanes 2) or P12 virus particles containing RNAs 3 and 4 (Fig. 2
, lanes 3), the transgene-derived RNAs 1 and 2 accumulated together with RNAs 3 and 4 although the level of replication varied between different transgenic lines.
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To determine whether the transgenically expressed RNAs 1 and 2 were replicating in R12 plants prior to RNA 3 inoculation, the presence of minus-strand RNAs 1 and 2 in the T1 generation of healthy R12 lines was investigated. Total nucleic acids extracted from mock-inoculated and RNA 3-infected plants 5 days p.i. were loaded onto two denaturing gels for detection of plus-strand RNA (Fig. 3A, lane numbers on top of the gel) or minus-strand RNA (Fig. 3B
, line numbers below the gel). For Northern blot hybridization, strand-specific digoxigenin (DIG)-labelled probes (Roche) were transcribed with T7 RNA polymerase from AMV cDNAs 1 and 2 cloned behind the T7 promoter in a sense or antisense direction (Neeleman & Bol, 1999
). The specificity of the probes was verified by hybridization to minus-strand transcripts of cDNAs 1 and 2 (Fig. 3
, lanes 1 and 2) and plus-strand virion RNAs (Fig. 3
, lane 3). For each healthy R12 line, accumulation of plus-strand RNAs 1 and 2 was paralleled by accumulation of roughly similar amounts of minus-strand RNAs 1 and 2 (Fig. 3 A
and B
, lanes 5 to 12). This suggests that in healthy R12 plants the transgenic replicase copied the transcripts of the viral cDNAs into minus-strand RNAs and then subsequent steps in the replication cycle were blocked. When healthy plants of R12 lines 5 and 6 (Fig. 3
, lanes 6 and 7) were inoculated with RNA 3, a massive accumulation of plus-strand RNAs 1 and 2 was observed whereas little or no increase in the accumulation of minus-strand RNAs 1 and 2 occurred (Fig. 3
, lanes 13 and 14). Possibly, the amount of minus-strand RNA synthesized in healthy R12 plants is largely sufficient to act as template for the synthesis of plus-strand RNAs 1 and 2 that is induced by the RNA 3-encoded CP.
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When AMV minus-strand RNA accumulation is successfully initiated in protoplasts from nontransgenic plants or P12 plants, CP expressed from RNA 3 is required for asymmetric plus-strand RNA accumulation in both protoplast systems (van der Kuyl et al., 1991a ,b
; Neeleman & Bol, 1999
). Our results with the R12 plants demonstrate for the first time the requirement of CP for plus-strand RNA accumulation at the whole plant level.
Viruses from the genus Bromovirus neither require CP in the inoculum to initiate infection nor the RNA 3-encoded CP for plus-strand RNA synthesis (Pacha et al., 1990 ). This may explain the observation of Mori et al. (1992)
that in protoplasts from plants transformed with full-length copies of RNAs 1 and 2 of Brome mosaic virus (BMV), replication of the transgenic RNAs 1 and 2 is largely independent on infection of the protoplasts with RNA 3. In contrast to the susceptibility of R12 plants to AMV infection, tobacco plants expressing multiplying RNAs 1 and 2 of Cucumber mosaic virus (CMV) exhibit resistance to challenge inoculation with CMV (Suzuki et al., 1996
). Similarly, protoplasts from plants transformed with replicable BMV RNAs 1 and 2 show resistance to BMV (Kaido et al., 1995
). Expression of replicating RNA of Potato virus X in transgenic plants consistently resulted in the activation of a gene silencing mechanism (Angell & Baulcombe, 1997
). It has been proposed that the inability of BMV P2 transgenic plants to replicate BMV RNA 2 is due to RNA 2-specific gene silencing (Iyer & Hall, 2000
). In R12 plants, the transgene transcription levels and the replication events that occur in the absence of AMV RNA 3 might be insufficient to activate a gene silencing mechanism.
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Acknowledgments |
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Footnotes |
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b Present address: Instituto de Bioquímia y Biología Molecular, Universidad Nacional de la Plata, Calles 47 y 115, (1900) La Plata, Argentina.
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References |
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Bol, J. F., van Vloten-Doting, L. & Jaspars, E. M. J.(1971). A functional equivalence of top component a RNA and coat protein in the initiation of infection by alfalfa mosaic virus. Virology 46, 73-85.[Medline]
de Graaff, M., Man int Veld, M. R. & Jaspars, E. M. J.(1995). In vitro evidence that the coat protein of alfalfa mosaic virus plays a direct role in the regulation of plus and minus-RNA synthesis: implications for the life cycle of alfalfa mosaic virus. Virology 208, 583-589.[Medline]
Dever, T. E.(1999). Translation initiation: adept at adapting. Trends in Biochemical Sciences 24, 398-403.[Medline]
Gallie, D. R.(1991). The cap and poly(A) tail function synergistically to regulate mRNA translational efficiency. Genes & Development 5, 2108-2116.[Abstract]
Horsch, R. B., Fry, J. E., Hoffmann, N. L., Eichholtz, D., Rogers, S. G. & Fraley, R. T.(1985). A simple and general method for transferring genes into plants. Science 227, 1229-1231.
Houser-Scott, F., Ansel-McKinney, P., Cai, J.-M. & Gehrke, L.(1997). In vitro genetic selection analysis of alfalfa mosaic virus coat protein binding to 3-terminal AUGC repeats in viral RNAs. Journal of Virology 71, 2310-2319.[Abstract]
Houwing, C. J. & Jaspars, E. M. J.(1993). Coat protein stimulates replication complexes of alfalfa mosaic virus to produce virion RNAs in vitro. Biochimie 75, 617-622.[Medline]
Imataka, H., Gradi, A. & Sonenberg, N.(1998). A newly identified N-terminal amino acid sequence of human eIF4G binds poly(A) binding protein and functions in poly(A)-dependent translation. EMBO Journal 17, 7480-7489.
Iyer, L. M. & Hall, T. C.(2000). Virus recovery is induced in Brome mosaic virus p2 transgenic plants showing synchronous complementation and RNA-2-specific silencing. Molecular PlantMicrobe Interactions 13, 247-258.
Kaido, M., Mori, M., Mise, K., Okuno, T. & Furusawa, I.(1995). Inhibition of brome mosaic virus (BMV) amplification in protoplasts from transgenic tobacco plants expressing replicable BMV RNAs. Journal of General Virology 76, 2827-2833.[Abstract]
Mori, M., Mise, K., Okuno, T. & Furusawa, I.(1992). Expression of brome-mosaic virus-encoded replicase genes in transgenic tobacco plants. Journal of General Virology 73, 169-172.[Abstract]
Neeleman, L. & Bol, J. F.(1999). Cis-acting functions of alfalfa mosaic virus proteins involved in replication and encapsidation of viral RNA. Virology 254, 324-333.[Medline]
Neeleman, L., van der Kuyl, A. C. & Bol, J. F.(1991). Role of alfalfa mosaic virus coat protein gene in symptom formation. Virology 181, 687-693.[Medline]
Neeleman, L., van der Vossen, E. A. G. & Bol, J. F.(1993). Infection of tobacco with alfalfa mosaic virus cDNAs sheds light on the early function of the coat protein. Virology 196, 883-887.[Medline]
Olsthoorn, R. C. L., Mertens, S., Brederode, F. Th. & Bol, J. F.(1999). A conformational switch at the 3 end of a plant virus RNA regulates viral replication. EMBO Journal 18, 4856-4864.
Pacha, R. F., Allison, R. F. & Ahlquist, P.(1990). Cis-acting sequences required for in vivo amplification of genomic RNA 3 are organized differently in related bromoviruses. Virology 174, 436-443.[Medline]
Peach, C. & Velten, J.(1991). Transgene expression variability (position effect) of CAT and GUS reporter genes driven by linked divergent T-DNA promoters. Plant Molecular Biology 17, 49-60.[Medline]
Quadt, R., Rosdorff, H. J. M., Hunt, T. W. & Jaspars, E. M. J.(1991). Analysis of the protein composition of alfalfa mosaic virus RNA-dependent RNA polymerase. Virology 182, 309-315.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Suzuki, M., Masuata, C., Takanami, Y. & Kuwata, S.(1996). Resistance against cucumber mosaic virus in plants expressing the viral replicon. FEBS Letters 379, 26-30.[Medline]
Taschner, P. E. M., van der Kuyl, A. C., Neeleman, L. & Bol, J. F.(1991). Replication of an incomplete alfalfa mosaic virus genome in plants transformed with viral replicase genes. Virology 181, 445-450.[Medline]
van der Kuyl, A. C., Neeleman, L. & Bol, J. F.(1991a). Complementation and recombination between alfalfa mosaic virus RNA 3 mutants in tobacco plants. Virology 183, 731-738.[Medline]
van der Kuyl, A. C., Neeleman, L. & Bol, J. F.(1991b). Role of alfalfa mosaic virus coat protein in regulation of the balance between viral plus and minus strand RNA synthesis. Virology 185, 496-499.[Medline]
van der Vossen, E. A. G., Neeleman, L. & Bol, J. F.(1994). Early and late functions of alfalfa mosaic virus coat protein can be mutated separately. Virology 202, 891-903.[Medline]
van Vloten-Doting, L. & Jaspars, E. M. J.(1972). The uncoating of alfalfa mosaic virus by its own RNA. Virology 48, 699-708.[Medline]
Vende, P., Piron, M., Castagné, N. & Poncet, D.(2000). Efficient translation of rotavirus mRNA requires simultaneous interaction of NSP3 with the eukaryotic translation initiation factor eIF4G and the mRNA 3) end. Journal of Virology 74, 7064-7071.
Received 2 August 2000;
accepted 16 October 2000.