Synthesis of (-)-strand RNA from the 3' untranslated region of plant viral genomes expressed in transgenic plants upon infection with related viruses

Pierre-Yves Teycheney1, Rachid Aaziz1, Sylvie Dinant1, Katalin Salánki2, Colette Tourneur1, Ervin Balázs2, Mireille Jacquemond3 and Mark Tepfer1

INRA, Laboratoire de biologie cellulaire, F-78026 Versailles cedex, France1
Agricultural Biotechnology Center, POB 411, H-2100 Gödöllö, Hungary2
INRA, Domaine Saint Maurice, Station de pathologie végétale, F-84143 Montfavet cedex, France3

Author for correspondence: Pierre-Yves Teycheney. Fax +33 1 30 83 30 99. e-mail teychene{at}versailles.inra.fr


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When expressed in transgenic tobacco plants, transgene mRNA that includes the 3' untranslated region (3' UTR) of Lettuce mosaic virus served as template for synthesis of complementary (-)-strand RNA following an infection by Tobacco etch virus, Tobacco vein mottle virus or Pepper mottle virus, but not when infected with Cucumber mosaic virus. Deletion of the 3' UTR from the transgene abolished the synthesis of (-)-strand transcripts. Similar results were obtained in transgenic tobacco plants expressing mRNA that includes the RNA3 3' UTR of Cucumber mosaic virus when infected with Tomato aspermy virus. These results show that the viral RNA-dependent RNA polymerase of several potyviruses and Tomato aspermy virus have the ability to recognize heterologous 3' UTRs when included in transgene mRNAs, and to use them as transcription promoters.


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Potyviruses and cucumoviruses are agronomically important groups of (+)-stranded plant RNA viruses, but they are strikingly different both structurally and in their modes of expression and replication. The potyviruses (Potyviridae) are members of the Picorna-associated supergroup, and have a monopartite, polyadenylated RNA genome, which is covalently linked to a small peptide (VPg) at its 5' end. The viral RNA is encapsidated in flexuous rod-shaped particles, and encodes a single polyprotein which is co- and post-translationally processed by three virus-encoded proteases (Dougherty & Semler, 1993 ). Cucumoviruses (Bromoviridae) are members of the Alpha-associated supergroup, and their genome is composed of one monocistronic and two bicistronic RNAs, which are encapsidated in icosahedral particles. The 5' ends of cucumoviral RNAs are capped, and their 3' ends can adopt a tRNA-like structure that can be aminoacylated with tyrosine (for review see Palukaitis et al., 1992 ).

Despite extensive functional studies that have succeeded in characterizing the roles of most of the proteins encoded by potyviral genomes (Schaad et al., 1997 ), little is known about the viral genome replication process itself. However, recent studies showed that 3'-terminal sequences with specific secondary structures, located both within the coat protein (CP) coding region and the 3' untranslated region (UTR), are essential for Tobacco etch virus (TEV) replication (Mahajan et al., 1996 ; Haldeman-Cahill et al., 1998 ). It has also been shown that interaction between the TEV VPg-proteinase (Nia) and RNA polymerase (Nib) plays an important role during viral RNA replication (Daros et al., 1999 ). In the case of plant members of the Alpha-associated supergroup, including Brome mosaic virus (BMV; Chapman & Kao, 1999 ) and Turnip yellow mosaic virus (TYMV; Deiman et al., 1998 ), several studies have shown that the 3'-terminal tRNA-like region contains the essential elements of the promoter for (-)-strand synthesis (for review see Pogue et al., 1994 ). By analogy, it is generally assumed that this is also the case for Cucumber mosaic virus (CMV). Deletion analysis of CMV RNA3 confirmed that the tRNA-like region is essential for initiation of replication (Boccard & Baulcombe, 1993 ).

The 3'-terminal promoters of (-)- or (+)-sense RNA synthesis are thought to be essential determinants of the template specificity of viral RNA-dependent RNA polymerases (RdRps). However, at present, there is no evidence concerning the determinants of template specificity for potyvirus replication. In contrast, for CMV, there is evidence that the promoter of (-)-strand RNA synthesis can be recognized by certain heterologous viral replicases. For instance, pseudo-recombinants and recombinants created with CMV and Tomato aspermy virus (TAV) can replicate normally both in protoplasts and in entire plants (Perry & Francki, 1992 ; Moriones et al., 1994 ; Salánki et al., 1997 ). In addition, a BMV recombinant in which the 3' terminus of RNA3 was replaced by that of CMV could be replicated by BMV replicase (Rao & Grantham, 1994 ), suggesting that the RdRp of the more distantly related BMV can also recognize the CMV 3'-terminal promoter. Similarly, a Tobacco mosaic virus (TMV) recombinant in which the 3' terminus was replaced by that of BMV could be replicated by TMV replicase (Ishikawa et al., 1991 ).

Since many of the viral sequences expressed in transgenic plants that confer virus resistance include 3' UTRs, it was of interest to determine if this region could promote the synthesis of complementary (-)-strand RNAs from cellular transcripts of such transgenes upon infection of transgenic plants. Homologous recognition was first shown to take place in plants expressing the TYMV 3' UTR when infected by TYMV (Zaccomer et al., 1993 ). This could favour recombination between transcripts of a viral transgene and the genome of an infecting virus (Greene & Allison, 1994 ), and thus it is now generally suggested to delete the 3' UTR from the viral transgene in order to reduce the frequency of recombination (Greene & Allison, 1996 ).

The CP genes of either Lettuce mosaic virus (LMV) or CMV with or without their respective 3' UTRs were expressed in transgenic tobacco plants (Fig. 1). Construct CCP contains the CMV-TrK7 CP gene and its entire 3' UTR (Szász et al., 1998 ), and CPR contains the CMV-R CP gene and only the first 51 residues of its 3' UTR (M. Jacquemond and others, unpublished). CMV strains TrK7 and R are very closely related strains of subgroup II, since their RNA3 share an overall similarity of 98·3%, and their respective 3' UTR are 99% identical (Carrère et al., 1999 ). Construct LMVCP contains the LMV-O CP coding sequence into which a start codon has been engineered and the entire 3' UTR of LMV-O genome (Dinant et al., 1997 ). Construct LMVCPBIO (C. Kusiak and others, unpublished) is a modified version of LMVCP in which the amino terminus of LMVCP has been changed from MVDAKLDAG to MVTG and the last 195 residues of its 3' UTR deleted. Constructs were cloned into binary vectors pGA482 (Hall et al., 1993 ; CCP), pZp Hygro (generously provided by J. Arrieta; LMVCPBIO) or pKYLX71–35S2 (Maiti et al., 1993 ; CPR, LMVCP) and all were used to transform tobacco (Horsch et al., 1985 ).



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Fig. 1. Schematic representation of the constructs used to express the CP genes of CMV and LMV in transgenic tobacco plants. Construct CCP (Szász et al., 1998 ) includes the last 141 nucleotides of the RNA3 intergenic region of CMV-TrK7 (Salánki et al., 1994 ), its entire CP gene coding region and 3' UTR. Construct CPR (M. Jacquemond and others, unpublished) contains the last 17 nucleotides of the RNA3 intergenic region of CMV-R, its entire CP gene coding region and the first 51 nucleotides of its 3' UTR. Construct LMVCP (Dinant et al., 1997 ) contains the TMV {Omega}' leader sequence, fused to the coding sequence of LMV-O in which a start codon has been engineered, and the entire 3' UTR of the LMV-O genome. Construct LMVCPBIO (C. Kusiak and others, unpublished) is a modified version of LMVCP in which the encoded amino terminus of LMVCP has been modified and the last 195 nucleotides of its 3' UTR have been deleted. The CMV RNA3 intergenic region is shown by black boxes. Hatched boxes represent the CMV or LMV 3' UTR and grey boxes represent the {Omega}' leader sequence of TMV subgenomic RNA. Numbers refer to nucleotide positions on CMV-R RNA3 or LMV-O genomic RNA (EMBL accession nos X97704 and Y18138 respectively). Arrows indicate the position of oligonucleotides used for RT–PCR experiments. 35S, Cauliflower mosaic virus (CaMV) 35S promoter; 35S2, CaMV 35S promoter with duplicated enhancer; rbcs 3', 3' non-coding region of the gene encoding pea ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit. Approximately 235 nucleotides of this fragment will be present in the transgene mRNA; nos 3', 3' non-coding region of the Agrobacterium tumefaciens nopaline synthase gene. Approximately 190 nucleotides of this fragment will be present in the transgene mRNA.

 
Homozygous R2 transgenic plants expressing CMV constructs were infected with the related cucumovirus TAV-P, whereas homozygous R2 transgenic plants expressing LMV constructs were infected by CMV-R and various potyviruses: TEV, Tobacco vein mottling virus (TVMV), Turnip mosaic virus (TuMV), Potato virus Y (PVY), Pepper mottle virus (PepMoV) and Plum pox virus (PPV). Total RNA was extracted from young leaves showing systemic symptoms, treated twice with RNase-free DNase I, and used in RT–PCR experiments as described by Zaccomer et al. (1993) to detect RNA complementary to the transgene mRNAs. Primers R(+) (position 1348–1365 on CMV-R RNA3) and O(+) (position 9072–9091 on LMV-O genomic RNA) used in the reverse transcription step were specific to (-)-strand transcripts from CMV-R and LMV-O transgenes respectively. For the detection of CMV CP (-)-strand transcripts, 35 PCR cycles were performed following the reverse transcription step, using primers R(+) and R(-) (the latter at position 1898–1879 on CMV-R RNA3), each cycle consisting of denaturation (94 °C, 30 s), annealing (52 °C, 30 s) and extension (72 °C, 30 s). For the detection of LMV CP (-)-strand transcripts, PCR conditions using primers O(+) and O(-) (the latter at position 9868–9849 on LMV-O genomic RNA) were as described by Zaccomer et al. (1993) , except that the annealing temperature was 51 °C. (-)- and (+)-strand in vitro T7 transcripts of LMV CP were used as reverse transcription positive and negative controls, respectively.

Fig. 2(A1) shows that in two of the four CCP5.2 plants infected with TAV-P, a 550 bp fragment was amplified by RT–PCR. Given the specificity of primer R(+), the 550 bp fragment resulted from the synthesis of a (-)-strand RNA from the CMV transcript, following infection by TAV-P. PCR amplification without reverse transcription showed that the PCR products resulted from RT–PCR and not from PCR amplification of the transgene (Fig. 2A2). Digestion with restriction enzymes confirmed that the amplified DNA fragment was specific of the transgene (data not shown). A similar amplified band was never observed either in CPR6.2 plants infected by TAV-P, or in mock-inoculated plants of either line. These results, which were reproduced several times, show that the TAV-P RdRp can synthesize complementary RNA from cellular transcripts of the CCP transgene, despite the low level of accumulation of CCP5.2 mRNA (Fig. 3), and that the presence of the last 269 nucleotides of the CMV-R 3' UTR is required for this to occur.



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Fig. 2. Detection by RT–PCR of RNA complementary to the initial transcripts of CMV-R (A) or LMV-O (B) CP genes in transgenic plants upon infection by heterologous viruses. A1, Detection of complementary transcripts of CMV transgenes in CCP5.2 transgenic plants infected by TAV-P. RT–PCR products were separated on 1% agarose gels. White arrows indicate the position of RT–PCR products of the expected size. L, 1 kb DNA ladder; 0*, PCR negative control; 0, RT–PCR negative control; CCP5.2, transgenic line expressing the CCP construct; CPR6.2, transgenic line expressing the CPR construct. m1, m2, mock-inoculated plants; 1–4, plants infected by TAV-P. A2, Reverse transcription control experiments: reverse transcriptase was added (+RTase) or omitted (-RTase) prior to PCR. 1–3, CCP5.2 plants mock-inoculated (1) or infected by TAV-P (2, 3). B1, Detection of complementary transcripts of LMV-O transgenes in 11C5 transgenic plants infected by TVMV, TEV or PepMoV. RT–PCR products were separated on 1% agarose gels. L, 1 kb DNA ladder; 60, 62, transgenic lines expressing the CPLMVBIO construct. 11C5, 5D10, transgenic lines expressing the CPLMV construct; NT, non-transformed control plant; CpLMV RNA (-), RT–PCR product synthesized from in vitro synthesized (-)-strand transcript of the CPLMV construct. B2, Reverse transcription control experiments. Reverse transcriptase was added (+RTase) or omitted (-RTase) prior to PCR. 11C5, 5D10, 60, 62, transgenic plants infected by TVMV; CpLMV RNA (-), in vitro synthesized (-)-strand transcript of the CpLMV construct; CpLMV RNA (+), in vitro synthesized (+)-strand transcript of the CpLMV construct.

 


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Fig. 3. Northern blot analysis of transgenic plants expressing viral transgenes. Ten µg of total RNA from homozygous transgenic plants was separated on 1% agarose gel under denaturing conditions and blotted on a nylon membrane. Membranes were hybridized with a radioactive probe specific to the CMV-R CP gene (A) or LMV-O CP gene (B). NT, Non-transformed control plant; CMV, 1 ng of CMV-R total genomic RNAs was loaded on the gel as a positive control.

 
Fig. 2(B1) shows that in transgenic tobacco line 11C5, which expresses the LMVCP construct, infection by three distinct potyviruses, TVMV, TEV and PepMoV, made it possible to amplify an LMV-specific 796 bp fragment by RT–PCR. Amplification of an equivalent band was not observed in plants infected by other potyviruses, including PVY, TuMV and PPV (data not shown), although PVY and PepMoV are very closely related, or by CMV-R. An equivalent band was never amplified from mock-inoculated plants expressing the LMVCP construct, nor from virus- or mock-inoculated plants expressing the LMVCPBIO construct, which lacks most of the LMV-O 3' UTR (Fig. 2B1). PCR amplification without reverse transcription showed that PCR products resulted from RT–PCR and not from PCR amplification of the transgene (Fig. 2B2). Digestion with restriction enzymes confirmed that the amplified DNA fragment was specific of the transgene (data not shown). The presence of the last 185 nucleotides of the LMV-O 3' UTR is necessary to promote the synthesis of a complementary RNA from transcripts of the transgene. Thus, the TEV, TVMV and PepMoV RdRps have the ability to initiate the transcription of (-)-strand RNA from the LMV-O 3' UTR contained in cellular mRNAs of the CPLMV transgene, despite the low level of accumulation of CPLMV transcripts (Fig. 3). To the best of our knowledge, this is the first observation that potyviral RdRps can act in trans on a viral promoter situated internally on a cellular mRNA. The lack of specificity of the TEV, TVMV and PepMoV RdRps observed here suggests that sequences located elsewhere on the viral genome are more important determinants of substrate specificity, as has been shown for tobraviruses (Mueller et al., 1997 ). Lack of template specificity may be more widespread than previously thought, since it was shown that initiation of replication in Poliovirus-1, the type-member of the Picornaviridae, is not strictly template specific (Todd et al., 1997 ).

In both experimental systems described here, detection of (-)-strand transcripts synthesized from mRNAs of viral transgenes was difficult, due to very poor RT–PCR yields. One element that may contribute to this is that the levels of accumulation of transgene mRNA from the constructs including the 3' UTR was lower than those from constructs lacking them (Fig. 3). Another interesting point is that (-)-strand RNA was not observed in all plants transformed with the same transgene, whether they belonged to the same homozygous lines (Fig. 2A1) or to different ones (Fig. 2B1). If the phase of active synthesis of viral (-)-strand RNA is short, then there may also be only a brief phase during which RNA complementary to the cellular mRNA can be synthesized. Thus we cannot exclude that the other potyviral RdRps tested could also have recognized the LMV CP transgene-derived RNAs if a larger number of plants had been tested.

In order to better characterize the sequence requirements for initiation of either TAV-P, TEV, TVMV or PepMoV RdRps, the 3' UTR sequences of the poty- and cucumoviruses used in this work were compared. No significant homology nor conserved secondary structures were found between the 3' UTR of LMV-O and that of TEV, TVMV or PepMoV (data not shown). Nevertheless, our results suggest that the genomic RNAs of these viruses must share essential features with LMV-O genomic RNA that are recognized by their RdRps. In contrast, the CMV-R and TAV-P 3' UTRs are 66% identical, with several blocks of up to 25 identical nucleotides. Current available data do not permit pinpointing specific motifs or secondary structures involved in interactions between CMV and TAV viral RNA 3' UTRs and their RdRps, as could be done for CMV and BMV (Rao & Grantham, 1994 ), but here again, our results indicate that such features must exist. Furthermore, the presence of extra-viral sequences at the 3' end of CCP or LMVCP transgene mRNAs, due to the cloning steps in the binary vectors, does not prevent the recognition of their 3' UTR by viral RdRps.

The work presented here fully supports the suggestion that one should avoid including the 3' UTR in CP-encoding transgenes (Greene & Allison, 1996 ). Experimental data currently favour a template switching mechanism to explain viral recombination (for review see Aaziz & Tepfer, 1999a ). Therefore, the use of viral transgenes including 3' UTRs would be expected to favour recombination between transgene-derived and viral RNAs, particularly in the case of related viruses that are prone to recombination under natural conditions when co-infecting common hosts (Aaziz & Tepfer, 1999b ).


   Acknowledgments
 
We are grateful to P. Gognalons (INRA Montfavet, France) for providing TEV and TVMV strains used in this work. We thank I. Touton for technical assistance. Plasmid pZpHygro was a gift from J. Arrieta (CGEB, Havana, Cuba). P.-Y.T. is supported by a fellowship from the Association pour la Recherche sur les Nicotianées (ARN). R.A. was supported by a fellowship from INRA.

P.-Y. Teycheney and R. Aaziz contributed equally to this work.


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Received 4 October 1999; accepted 14 December 1999.