RNAs 1 and 2 of Alfalfa mosaic virus, expressed in transgenic plants, start to replicate only after infection of the plants with RNA 3

Vera Tholea,1, Maria-Laura Garciab,1, Clemens M. A. van Rossum1, Lyda Neeleman1, Frans T. Brederode1, Huub J. M. Linthorst1 and John F. Bol1

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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RNAs 1 and 2 of the tripartite genome of Alfalfa mosaic virus (AMV) encode the two viral replicase subunits. Full-length DNA copies of RNAs 1 and 2 were used to transform tobacco plants (R12 lines). None of the transgenic lines showed resistance to AMV infection. In healthy R12 plants, the transcripts of the viral cDNAs were copied by the transgenic viral replicase into minus-strand RNAs but subsequent steps in replication were blocked. When the R12 plants were inoculated with AMV RNA 3, this block was lifted and the transgenic RNAs 1 and 2 were amplified by the transgenic replicase together with RNA 3. The transgenic expression of RNAs 1 and 2 largely circumvented the role of coat protein (CP) in the inoculum that is required for infection of nontransgenic plants. The results for the first time demonstrate the role of CP in AMV plus-strand RNA synthesis at the whole plant level.


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The genome of Alfalfa mosaic virus (AMV) is divided among three plus-strand RNAs which are separately encapsidated. The RNA 1-encoded P1 protein with putative methyltransferase and helicase activity and the RNA 2-encoded P2 protein containing the GDD motif of viral plus-strand RNA polymerases have been identified as subunits of the purified AMV RNA-dependent RNA polymerase complex (RdRp; Quadt et al., 1991 ). The P3 protein encoded by RNA 3 is involved in virus movement, and the subgenomic RNA 4, which is synthesized from the 3’ half of minus-strand RNA 3, serves as messenger RNA for the multifunctional coat protein (CP). Initiation of infection by the three genomic RNAs requires CP (or RNA 4) in the inoculum (Bol et al., 1971 ). In infected protoplasts, CP in the inoculum appears to be required in a step of the replication cycle prior to viral minus-strand RNA synthesis whereas CP expressed from RNA 3 is required for plus-strand RNA accumulation (Neeleman & Bol, 1999 ). Also in vitro, plus-strand RNA synthesis by the purified AMV RdRp is stimulated by CP (de Graaff et al., 1995 ). Moreover, CP is required for cell-to-cell movement of the virus (van der Kuyl et al., 1991a ; van der Vossen et al., 1994 ).

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 KpnI–PvuII and SstI–PvuII 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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Fig. 1. Accumulation of AMV RNA 1 and 2 transgene transcripts in primary R12 transformants (T0). Northern blot analysis of 10 µg total RNA extracted from 19 R12 lines (designated 1, 3, 4 to 8, 10, 11, 15 and 17 to 25), a P12 plant (lane P) and a nontransgenic tobacco plant (lane N). The blot was probed with random-primed 32P-labelled fragments of AMV cDNA 1 (BglII fragment of pCa17T; Neeleman et al., 1993 ) and cDNA 2 (BglII–BamHI fragment of pCa27T; Neeleman et al., 1993 ), which detect both plus-strand and minus-strand AMV RNAs. The position of AMV RNAs 1 and 2 is indicated on the left of the autoradiogram.

 
In subsequent experiments, kanamycin-resistant T1 plants grown from seeds of self-pollinated transformed plants were used. Accumulation of transgenic RNA in the T1 progeny was similar to that in the corresponding T0 plants. Isolation of poly(A) RNA from total RNA extracts of the T1 plants using the PolyATtract mRNA isolation kit (Pharmacia) revealed that most of the transgenic RNAs 1 and 2 contained a poly(A) tail (result not shown).

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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Fig. 2. Accumulation of transgene-derived and viral AMV RNAs after inoculation of R12 plants (T1) with (in)complete mixtures of AMV virions or RNA 3. Northern blot analysis of six selected R12 lines (R12-4, 5, 6, 8, 17 and 25). For each R12 line, total nucleic acid extracts from plants inoculated with RNA 3 (lane 1), RNA 3 and CP (lane 2), P12 virus particles containing RNAs 3 and 4 (lane 3) and virus particles containing the complete AMV genome (lane 4) were loaded in comparison to samples from mock-inoculated plants (lane 5). For each sample, the amount of total nucleic acids applied corresponded to 5 mg of fresh leaf material. The blots were probed with a mixture of random primed 32P-labelled cDNA 1 (BglII fragment of pCa17T), cDNA 2 (BamHI–BglII fragment of pCa27T) and cDNA 3 (XhoI–ApaI fragment of pAL3), which detect both plus-strand and minus-strand AMV RNAs. The position of AMV RNAs 1 to 4 is indicated on the left of the two panels.

 
In all R12 lines analysed, replication of inoculum RNA 3 and transgenically expressed RNAs 1 and 2 could be initiated in the absence of CP, although for lines R12-8 and R12-25 (Fig. 2, lane 1) a longer exposure of the blot was required to reveal RNA accumulation. Initiation of infection of line R12-5 was fully independent of the presence of CP in the inoculum, but addition of CP to inocula containing RNA 3 stimulated infection of the other lines shown in Fig. 2 (compare lanes 1 and 2). Although the CP dependency of the initiation of infection did not correlate with the level of transgenic RNAs 1 and 2, a correlation with the level of functional replicase activity in different R12 lines cannot be ruled out. Infection with RNA 3 of P12 plants was not stimulated by CP in the inoculum but a possible variation in CP dependency of the infection between different P12 lines was not investigated (Taschner et al., 1991 ).

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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Fig. 3. Accumulation of plus-strand (A) and minus-strand (B) transgene-derived RNAs 1 and 2 in healthy and RNA 3-infected R12 plants. Total nucleic acid extracts from healthy R12 plants (R12 lines 4, 5, 6, 8, 17, 18, 19 and 25; lanes 5 to 12) and RNA 3-infected R12 lines 5 and 6 (lanes 13 and 14) were analysed by Northern blot hybridization in amounts corresponding to 50 µg of fresh leaf material. As controls, the blot was loaded with minus-strand transcripts from cDNA 1 (-1; lane 1), minus-strand transcripts from cDNA 2 (-2; lane 2), plus-strand virion RNAs (V; lane 3) and total RNA from a healthy nontransgenic tobacco plant (H; lane 4). The blots were probed with DIG-labelled minus-strand RNAs 1 and 2 transcribed with T7 RNA polymerase from BamHI-linearized antisense cDNAs 1 or 2 (panel A) or plus-strand RNAs 1 and 2 transcribed from EcoRI/BglII-linearized sense cDNAs 1 and 2 (panel B), respectively. The blots shown in this figure were exposed for the same time. The position of AMV RNAs 1 and 2 is indicated on the left of the autoradiograms. The numbers on top of panel (A) represent lane numbers; numbers at the bottom of panel (B) denote the numbers of transgenic lines.

 
When nontransgenic protoplasts are inoculated with AMV genomic RNAs, minus-strand RNA accumulation is detectable only when CP is added to the inoculum. However, when P12 protoplasts are inoculated with RNA 3, minus-strand RNA 3 accumulation requires neither CP in the inoculum nor expression of CP from RNA 3 (van der Vossen et al., 1994 ; Neeleman & Bol, 1999 ). This demonstrates that CP is not required for minus-strand RNA synthesis per se, and we have proposed that in nontransgenic protoplasts CP is required in a step prior to the initiation of minus-strand RNA synthesis, possibly translation of inoculum RNAs (Neeleman et al., 1993 ; Neeleman & Bol, 1999 ; Olsthoorn et al., 1999 ). Apparently, this step is circumvented in R12 plants as minus-strand RNAs 1 and 2 are synthesized in the absence of the RNA 3-encoded CP. Translation of cellular mRNAs is synergistically enhanced by an interaction between the 3’ poly(A) tail and 5’ capstructure that is mediated by the poly(A)-binding protein (PABP) and translation initiation factors eIF4E and eIF4G (Dever, 1999 ; Gallie, 1991 ; Imataka et al., 1998 ). The rotavirus NSP3 protein has been shown to interact with the 3’ end of the non-polyadenylated viral mRNAs and eIF4G to substitute for PABP in the enhancement of translation (Vende et al., 2000 ). Possibly, the binding of PABP to the poly(A) tail of the transgenic RNA 1 and 2 transcripts in R12 plants may compensate for the putative role of CP in translation of these RNAs in nontransgenic plants. It should be noted that other models for the early function of CP have been proposed (de Graaff et al., 1995 ; Houser-Scott et al., 1997 ; Houwing & Jaspars, 1993 ).

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.


   Acknowledgments
 
V.T. and M.-L.G. were supported by EU fellowships.


   Footnotes
 
a Present address: John Innes Centre, Brassica and Oilseeds Research Department, Norwich Research Park, Norwich NR4 7UH, UK.

b Present address: Instituto de Bioquímia y Biología Molecular, Universidad Nacional de la Plata, Calles 47 y 115, (1900) La Plata, Argentina.


   References
Top
Abstract
Main text
References
 
Angell, S. A. & Baulcombe, D. C.(1997). Consistent gene silencing in transgenic plants expressing a replicating potato virus X RNA. EMBO Journal 16, 3675-3684.[Abstract/Free Full Text]

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 in’t 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.[Abstract/Free Full Text]

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 Plant–Microbe 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.[Abstract/Free Full Text]

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.[Abstract/Free Full Text]

Received 2 August 2000; accepted 16 October 2000.