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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Abstract |
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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 pKYLX7135S2 (Maiti et al., 1993
; CPR, LMVCP) and all were used to transform tobacco (Horsch et al., 1985
).
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Fig. 2(A1) shows that in two of the four CCP5.2 plants infected with TAV-P, a 550 bp fragment was amplified by RTPCR. 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 RTPCR 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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In both experimental systems described here, detection of (-)-strand transcripts synthesized from mRNAs of viral transgenes was difficult, due to very poor RTPCR 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
).
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Acknowledgments |
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P.-Y. Teycheney and R. Aaziz contributed equally to this work.
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References |
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Received 4 October 1999;
accepted 14 December 1999.