Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK
Correspondence
Peter Palukaitis
ppaluk{at}scri.sari.ac.uk
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In general, during the derivation of viral genome organizations from the sequences of their genomes, separate ORFs of less than 10 kDa have tended to be ignored, unless they occurred in several strains of the same virus or in different viral species in the same genus. In one such example, an ORF (which we refer to as ORF6) encoding a protein of 3845 aa was described as being present in the genomes of TMV and the tobamoviruses Tomato mosaic virus (ToMV) and Tobacco mild green mosaic virus (Morozov et al., 1993). Transcription of RNA corresponding to the ToMV ORF6 from a cDNA clone and translation in vitro yielded a protein of approximately 45 kDa (Morozov et al., 1993
). The ORF6-encoded protein of ToMV has a predicted molecular mass of 3·9 kDa, while that of TMV has a predicted molecular mass of 4·8 kDa. The in vitro-translated protein of ToMV also bound very strongly to a translation factor of 50 kDa, later shown to be eukaryotic elongation factor 1
(EF-1-
) (Fedorkin et al., 1995
). However, ORF6 was not present in all tobamoviruses and no specific function could be ascribed to the encoded protein, although it was suggested to be a regulator of mRNA translation or gene expression (Morozov et al., 1993
). Thus, this study was undertaken to determine whether the TMV ORF6 actually has functionality in vivo and to analyse the possible role(s) of the putatively expressed protein. We have characterized the effects observed in planta when ORF6 was prevented from being expressed from the TMV genome, as well as when ORF6 was expressed from two heterologous viral vectors, Potato virus X (PVX) and Tobacco rattle virus (TRV). We have also determined the subcellular distribution of the TMV-encoded 4·8 kDa protein and examined whether this protein might function as a silencing suppressor.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The plasmid pTX.P3C2 402, a plant expression vector containing a biologically active cDNA clone of PVX RNA expressed using a T7 promoter (Baulcombe et al., 1995), was obtained from S. N. Chapman and was used to generate additional vectors for gene expression. The TMV ORF6 was amplified by PCR and inserted into the EagI/NsiI sites of the vector polylinker in pTX.P3C2 402 to generate the plasmid pPVX+(ORF6). The same PVX vector and restriction sites were used for the generation of plasmids pPVX+4K : : GFP and pPVX+GFP : : 4K, which contained genes encoding the green fluorescent protein (GFP) fused to either the C or N terminus of the TMV 4·8 kDa protein, respectively. The plasmid, pTXS.GFP (Boevink et al., 1996
) was used to generate RNA transcripts of PVX expressing free GFP (PVX+GFP).
The plasmid pTRV-GFPc contained a full-length cDNA clone of TRV RNA 2 (strain PpK20) fused to a T7 RNA promoter and the gene encoding the GFP, but lacking the TRV 2b and 2c genes (MacFarlane & Popovich, 2000). pTRV-GFPc was modified to express the TMV ORF6 or the same sequence lacking initiation codons, to generate pTRV+(ORF6) or pTRV+(ORF6), respectively. For the former, the sequences of TMV ORF6 were amplified by PCR from plasmid pPVX+(ORF6) with primers containing an RcaI at the initiation codon and a KpnI site downstream of the termination codon. The digested fragment was inserted into NcoI/KpnI linearized pTRV-GFPc. For the latter, the corresponding sequence was amplified by PCR from plasmid pTMV(ORF6) with primers containing an HpaI upstream of the initiation codon and a KpnI site downstream of the termination codon. The digested fragment was inserted into HpaI/KpnI-linearized pTRV-GFPc.
The gene encoding the TMV 4·8 kDa protein was amplified by PCR and inserted into the BamHI/SacI sites of the plasmid pROK2 to generate the plasmid pROK2/4K. The same gene was also amplified as a 3' fusion to the sequences encoding the GFP from plasmid pPVX+4K : : GFP, using primers containing BamHI and KpnI sites flanking the initiation and termination codons, and inserted into the BamHI/KpnI sites of pROK2, to generate plasmid pROK2/4K : : GFP. Both plasmids were introduced into cells of Agrobacterium tumefaciens strain LBA4404, as described previously (Canto & Palukaitis, 1998).
The 2b gene of the Fny strain of Cucumber mosaic virus (CMV) was amplified by PCR from the full-length cDNA clone of the viral RNA 2, pFny209 (Rizzo & Palukaitis, 1990), using a primer homologous to nt 24192432 and a flanking BamHI site, and a primer complementary to nt 27392751 with a flanking SacI site. After digestion with both restriction enzymes, the fragment was inserted into the binary vector pROK2 linearized with the same enzymes to generate pROK2/2b.
Plants, viruses and inoculation.
Tobacco (Nicotiana tabacum cv. Samsun nn or cv. Samsun NN), Nicotiana benthamiana, tomato (Lycopersicon esculentum cv. Moneymaker) and Nicotiana clevelandii plants were propagated and maintained at 25 °C (Canto & Palukaitis, 1998, 2002
). Plants were inoculated with T7 RNA transcripts of TMV, TMV(ORF6), TMV30K : : mRFP1, PVX, PVX+(ORF6), PVX+4K : : GFP, PVX+GFP : : 4K, PVX+GFP, TRV+(ORF6) or TRV+(ORF6) (the latter two in combination with TRV RNA 1), using the plasmids described above after linearization with the appropriate restriction enzymes (Canto & Palukaitis, 2002
; Mueller et al., 1997
).
Three sets of experiments were performed to assess whether the 4·8 kDa protein acted as a viral suppressor of gene silencing: (i) N. benthamiana line 16c plants were agroinfiltrated for a silencing suppression assay with A. tumefaciens cultures harbouring one of the following binary plasmid vectors: (a) pROK2, (b) pROK2/4K or (c) pROK2/P1HC (Canto et al., 2002), as described previously (Canto et al., 2002
). (ii) Silencing of GFP expression was achieved first in the GFP-transgenic, N. benthamiana line 16c plants (Ruiz et al., 1998
) agroinfiltrated with the bacteria harbouring binary plasmid vectors containing pROK2-GFP. Then the plants were inoculated with PVX, PVX+(ORF6) or Potato virus Y (PVY) to detect suppression of pre-existing gene silencing. (iii) Non-transgenic N. benthamiana plants were co-infiltrated with a 1 : 1 (v/v) mixture of A. tumefaciens culture harbouring the
-glucuronidase (GUS)-expressing binary vector pGPTV(+35P,+NosT) (Canto et al., 2002
) and one of the following binary plasmid vectors: (a) pROK2, (b) pROK2/4K, (c) pROK2/P1HC or (d) pROK2/2b, the last expressing the 2b protein of CMV. Plants were infiltrated as described previously (Canto et al., 2002
). Silencing and silencing suppression were monitored using a Black Ray long-wavelength UV lamp (UV Products), a fluorescence stereomicroscope (Leica) (Canto et al., 2002
) or Northern blot analysis.
Protoplasts were prepared from either N. tabacum or N. benthamiana, transfected with RNA transcripts and incubated, all as described previously (Canto & Palukaitis, 1999, 2002
; Gal-On et al., 1994
).
Detection of viral RNAs and viral-encoded proteins.
Nucleic acids and proteins were extracted from plants and protoplasts and analysed by Northern blot hybridization and protein immunoblotting, respectively, as described previously (Canto & Palukaitis, 2001, 2002
). Two TMV RNA probes were used in this work: one complementary to nt 9992144 of the TMV genome and the other complementary to 3'-terminal nt 61496402. RNA probes complementary to the 3'-terminal 157 nt of the PVX genome, to the MP gene of TRV RNA 1 and the 5' NTR and CP gene of TRV RNA 2, and to the entire GUS gene were also used. Polyclonal antisera to TMV CP (from T. M. A. Wilson via S. N. Chapman), GFP (Molecular Probes) or a mixture of synthetic peptides corresponding to the N-terminal 15 aa and the C-terminal 15 aa of the TMV 4·8 kDa protein (Sigma Genosys) were used as primary antibodies.
Fluorescence imaging.
GFP- and mRFP1-derived fluorescence in individual cells was detected and recorded using a Leica TCS SP spectral confocal laser-scanning microscope (Gillespie et al., 2002). Fluorescence of whole leaves was imaged and recorded as reported previously (Canto & Palukaitis, 2002
; Canto et al., 2002
).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
TRV expressing TMV ORF6 [designated TRV+(ORF6)] induced systemic necrosis on N. benthamiana (Fig. 2b, upper row of plants). By contrast, a TRV vector expressing the TMV ORF6 sequence in which the two methionine codons at the beginning of ORF6 were modified from AUG (methionine) to ACG (threonine) [designated TRV+(ORF6)] showed only leaf curling and a systemic yellow mottle (Fig. 2b
, lower row of plants), similar to symptoms induced by TRV expressing GFP (not shown). Thus, TMV ORF6 also enhanced TRV virulence in N. benthamiana.
TMV ORF6 does not enhance virus accumulation in N. benthamiana
Despite the difference in symptom severity, analysis of viral RNAs in whole N. benthamiana plants showed no substantial alteration in accumulation of either the genomic RNA or the LMC sgRNAs of TMV(ORF6) compared to wt TMV (Fig. 3a). Moreover, levels of CP accumulation in N. benthamiana leaves inoculated with either wt TMV or TMV(ORF6) were similar (Fig. 3b
), indicating that the mutations in the CP sgRNA promoter of TMV(ORF6) did not affect expression of the CP gene. Thus, the presence or absence of ORF6 did not significantly affect accumulation of either the viral RNAs or the CP in whole plants. Similarly, the accumulation of TMV(ORF6) was comparable to that of wt TMV in N. benthamiana protoplasts (not shown). Thus, the enhanced virulence of TMV by ORF6 apparently was not due to either higher levels of virus replication or increased cell-to-cell movement of virus.
|
TMV ORF6 is not a pathogenicity determinant in N. tabacum
Infection of N. tabacum cv. Samsun nn plants by TMV(ORF6) resulted in stunting of the plants and the production of mosaic symptoms identical to those induced by wt TMV (Fig. 4, upper row, first and second plants from the left, respectively). Infection of N. tabacum cv. Samsun NN plants, expressing resistance to TMV via the N gene, also gave similar results for wt TMV and TMV(ORF6), i.e. the number, size and time of appearance of the necrotic local lesions was not altered (data not shown). No difference in the level of RNA accumulation was found in infected N. tabacum cv. Samsun nn plants (Fig. 3a
) or protoplasts (not shown). Thus, in N. tabacum, the presence or absence of TMV ORF6 encoding the 4·8 kDa protein had no apparent effect on TMV replication, movement or virulence.
|
Infection of N. tabacum by PVX or PVX+(ORF6) induced only very mild symptoms (Fig. 4, upper row, third and fourth plants from the left, respectively) and there was also no significant difference in viral RNA accumulation (Fig. 3c
). Thus, the TMV 4·8 kDa protein also did not appear to enhance the virulence, replication or spread of PVX in N. tabacum.
Several other viral genes expressed from the PVX vector have been shown to enhance virulence in either N. tabacum or N. benthamiana (Brigneti et al., 1998; Burgyán et al., 2000
; Li et al., 1999
; Pruss et al., 1997
; Qiu et al., 2002
; Scholthof et al., 1995
; Thomas et al., 2003
). Some of these genes have been shown to be determinants of viral synergy between the corresponding virus pairs and some have also been shown to be suppressors of RNA silencing (Anandalakshmi et al., 1998
; Brigneti et al., 1998
; Pruss et al., 1997
; Qiu et al., 2002
; Qu & Morris, 2002
; Qu et al., 2003
; Thomas et al., 2003
). To determine whether the TMV ORF6 affected synergistic interactions between TMV and PVX, both viruses, with or without ORF6, were co-inoculated on to N. tabacum plants. In N. benthamiana, TMV alone induced systemic necrosis and collapse of the plants (Fig. 2a
, first plant from the left). Thus, it was not possible to determine whether TMV plus PVX induced any synergism. In N. tabacum cv. Samsun nn plants infected by TMV plus PVX, the plants showed severe stunting and limited leaf necrosis, more severe than that induced by either virus alone in this host (Fig. 4
, lower row, first plant from left). Elimination of ORF6 from TMV and/or expression of TMV ORF6 from PVX did not affect the synergy induced by the combination of these viruses in N. tabacum (Fig. 4
, lower row, second, third and fourth plants from the left). Analysis of the accumulation levels of both TMV and PVX RNAs showed that the presence or absence of ORF6 did not influence the accumulation levels of either viral RNA (data not presented), similar to the effects observed in plants infected by either virus alone, with or without ORF6 (Fig. 3a and c
). Thus, TMV ORF6 itself did not influence synergy in tobacco between TMV and PVX.
Unlike expression of ORF6 from either TMV or PVX (Fig. 4), a TRV vector expressing TMV ORF6 [TRV+(ORF6)] induced large necrotic lesions in N. tabacum cv. Samsun nn that coalesced, together with extensive necrosis on the midribs and petiole, resulting in collapse of the leaf by 4 days post-inoculation (p.i.). Non-inoculated leaves remained asymptomatic and did not contain detectable viral RNAs (not shown). By contrast, infection by TRV+(ORF6) induced few or no symptoms on the inoculated leaf and produced only minor stunting of plant growth. Examination of the accumulation of TRV RNAs in the inoculated leaves prior to complete necrosis of those leaves showed that the TMV ORF6 did not increase the accumulation of the TRV RNAs (not shown).
Expression of the putative TMV 4·8 kDa protein does not suppress gene silencing
Three assays were performed to determine whether the product of TMV ORF6 could suppress silencing in N. benthamiana, all with negative results. In the first assay, N. benthamiana plants transgenic for the GFP gene were agroinfiltrated with bacteria harbouring T-DNAs expressing the PVY P1-HCPro gene, the TMV ORF6 or no additional genes. All three T-DNAs contained the nopaline synthase transcription terminator (NosT) signal sequence in the expression cassette. This sequence contained sufficient sequence identity with the 3' non-translated region of the transgene transcript to induce local silencing of the transgenically expressed GFP gene (Fig. 5a, leaf 2), as reported previously (Canto et al., 2002
). Expression of P1-HCPro from T-DNA in the agroinfiltrated leaves could suppress this silencing (Fig. 5a
, leaf 3). By contrast, expression of TMV ORF6 from T-DNA in the agroinfiltrated leaves did not suppress silencing mediated by the NosT sequences (Fig. 5a
, leaf 4). Similarly, GFP expression in transgenic N. benthamiana plants (line 16c) previously silenced by agroinfiltration, could not be restored by infection with PVX or PVX+(ORF6), whereas infection with PVY suppressed silencing and led to GFP expression in the upper leaves (data not presented). Comparable results for PVX and PVY have been reported previously by Brigneti et al. (1998)
and Voinnet et al. (1999)
.
|
TMV 4·8 kDa protein fused to GFP is distributed within the cytoplasm and to plasmodesmata
To determine the subcellular distribution of the 4·8 kDa protein, TMV ORF6 was fused to the GFP gene and the fusion proteins were localized in planta by confocal microscopy. The TMV 4·8 kDa protein was fused to either the N or C terminus of GFP, generating 4K : : GFP or GFP : : 4K, respectively. The genes containing the sequences encoding these fusion proteins were expressed from PVX vectors PVX+4K : : GFP and PVX+GFP : : 4K, after inoculation on to N. benthamiana (Fig. 6). 4K : : GFP was also expressed transiently from pROK2 agroinfiltrated into the same host (not shown). The fluorescence produced by both 4K : : GFP and GFP : : 4K in infected epidermal cells was distributed in the cytoplasm, cytoplasmic inclusions and the nucleus, as occurred for free (non-fused) expressed GFP (Baulcombe et al., 1995
; Boevink et al., 1996
). However, both 4K : : GFP and GFP : : 4K proteins also partitioned uniquely to discrete spots in the cell wall, which suggested association with plasmodesmata (arrows in Fig. 6a
). Localization of the fusion proteins to plasmodesmata was confirmed by co-inoculation of TMV expressing the 30 kDa MP fused to the modified red fluorescent protein, mRFP1 (30K : : mRFP1) and PXV expressing 4K : : GFP, GFP : : 4K or free GFP. TMV MP is known to localize to plasmodesmata in newly infected epidermal cells (Ding et al., 1992
; Gillespie et al., 2002
; Tomenius et al., 1987
) and in these cells the TMV 30K : : mRFP1 signal was detectable only in plasmodesmata (Fig. 6b
, middle column). The 30K : : mRFP1 signal was not visible at the wavelength setting used to detect free GFP expressed from PVX (Fig. 6b
, upper left panel); the latter was localized to the cytoplasm and nucleus, but was absent from the cell wall. However, fluorescence from TMV 30K : : mRFP1 and either 4K : : GFP or GFP : : 4K both co-localized to plasmodesmata connecting adjacent epidermal cells (Fig. 6b
, middle and lower-right panels). Thus, in epidermal cells, the distribution of the TMV 4·8 kDa protein fused to GFP was partitioned between the nucleus, the cytoplasm and plasmodesmata.
|
Antisera prepared separately to peptides corresponding in sequence to the N-terminal 15 aa and the C-terminal 15 aa of the TMV 4·8 kDa protein did not detect the 4·8 kDa protein in Western blots of proteins extracted from either TMV-infected N. tabacum or N. benthamiana. Moreover, Western blots of proteins extracted from N. benthamiana infected by either PVX+4K : : GFP or PVX+GFP : : 4K did not show any reaction with either the 4·8 kDa-specific antisera or the antiserum to GFP, although the GFP antiserum could detect GFP present in a 100-fold dilution of the extract of a plant infected with PVX expressing free GFP (data not presented). Thus, it was not possible biochemically to establish unequivocally the presence of the TMV 4·8 kDa protein in the various infected plants.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The in vitro-expressed 3·9 kDa protein of ToMV and the 4·8 kDa protein of TMV have been shown to bind strongly to EF-1- (Fedorkin et al., 1995
; Morozov et al., 1993
). This translation factor is a cytoplasmic protein. The presence of most of the GFP-tagged TMV 4·8 kDa protein in the cytoplasm is consistent with its interaction with a cytoplasmic protein. Amino acids at positions 19 at the N terminus of the ToMV 3·9 kDa protein have been shown not to be essential for binding to EF-1-
, while certain mutations at aa 1014 were shown to affect this binding (Morozov et al., 1993
). Whether these mutants also affected both the binding of the TMV 4·8 kDa protein to EF-1-
and the virulence associated with the TMV 4·8 kDa protein remains unknown.
Morozov et al. (1993) suggested that the ToMV 4·8 kDa protein may have some effect on translation regulation or cellular gene expression. On the other hand, from earlier studies, it is clear that infection by TMV does not in general affect translation of host proteins per se (Siegel et al., 1978
; Van Loon & Van Kammen, 1970
; Van Telgen et al., 1985
; Zaitlin & Hariharasubramanian, 1972
), although there are some effects on the steady-state levels of accumulation of specific plant mRNAs (Itaya et al., 2002
). Our results on the poor accumulation of GFP fused to the TMV 4·8 kDa protein suggest that the 4·8 kDa protein may have affected protein turnover. However, whether this plays a role in pathogenicity remains to be determined.
The association of some of the TMV 4·8 kDa protein with plasmodesmata was unexpected, since this gene product has no essential role in virus movement (Figs 2, 3 and 4, and data not shown). This localization of the 4·8 kDa protein may suggest some role in regulating the intercellular transport of signal molecules as an explanation for the effects of the 4·8 kDa protein on increasing the virulence of viruses.
How is the ORF6 expressed from the TMV genome? There is no evidence for an sgRNA specific for expression of ORF6. However, Morozov et al. (1993) showed that a low level of the ORF6 was expressed in vitro from the I2 sgRNA of ToMV and suggested that the same occurred from TMV. Thus, it is conceivable that ORF6 is expressed at a low level from an sgRNA associated with expression of the 30 kDa MP, possibly through an internal ribosome entry site (IRES). The CP of TMV was not expressed from the I2 sgRNA of TMV, but was expressed from the I2 sgRNA of a crucifer-infecting tobamovirus (Cr-TMV) (Ivanov et al., 1997
). This CP gene expression was shown to be via an IRES upstream of the CP gene of Cr-TMV. Perhaps the location of ORF6, which is not present in this corresponding region of Cr-TMV between the MP and CP genes, prevented the use of an IRES-based strategy for expression of the TMV CP (Ivanov et al., 1997
), since the initiation codon of ORF6 would precede that of the CP ORF and be the preferential site of translation initiation. Thus, this same IRES might facilitate the translation of ORF6.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Baulcombe, D. C., Chapman, S. & Santa Cruz, S. (1995). Jellyfish green fluorescent protein as a reporter for virus infection. Plant J 7, 10451053.[CrossRef][Medline]
Beachy, R. N. & Zaitlin, M. (1977). Characterization and in vitro translation of the RNAs from less-than-full-length, virus-related, nucleoprotein rods present in tobacco mosaic virus preparations. Virology 81, 160169.[CrossRef][Medline]
Boevink, P., Santa Cruz, S., Hawes, C., Harris, N. & Oparka, K. J. (1996). Virus-mediated delivery of the green fluorescent protein to the endoplasmic reticulum of plants cells. Plant J 10, 935941.[CrossRef]
Brigneti, G., Voinnet, O., Li, W.-X., Ji, L.-H., Ding, S.-W. & Baulcombe, D. C. (1998). Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana. EMBO J 17, 67396746.
Burgyán, J., Hornyik, C., Szittya, G., Silhavy, D. & Bisztray, G. (2000). The ORF1 products of tombusviruses play a crucial role in lethal necrosis in virus-infected plants. J Virol 74, 1087310881.
Campbell, R. E., Tour, O., Palmer, A. E., Steinbach, P. A., Baird, G. S., Zacharias, D. A. & Tsien, R. Y. (2002). A monomeric red fluorescent protein. Proc Natl Acad Sci U S A 99, 78777882.
Canto, T. & Palukaitis, P. (1998). Transgenically expressed cucumber mosaic virus RNA 1 simultaneously complements replication of cucumber mosaic virus RNAs 2 and 3 and confers resistance to systemic infection. Virology 250, 325336.[CrossRef][Medline]
Canto, T. & Palukaitis, P. (1999). Are tubules generated by the 3a protein necessary for cucumber mosaic virus movement? Mol Plant Microbe Interact 12, 985993.
Canto, T. & Palukaitis, P. (2001). A cucumber mosaic virus (CMV) RNA 1 transgene mediates suppression of the homologous viral RNA 1 constitutively and prevents CMV entry into the phloem. J Virol 75, 91149120.
Canto, T. & Palukaitis, P. (2002). A novel N gene-associated, temperature-independent resistance to the movement of Tobacco mosaic virus vectors, neutralized by a Cucumber mosaic virus RNA1 transgene. J Virol 76, 1290812916.
Canto, T., Cillo, F. & Palukaitis, P. (2002). Generation of siRNAs by T-DNA sequences does not require active transcription or homology to sequences in the plant. Mol Plant Microbe Interact 15, 11371146.[Medline]
Dawson, W. O., Bubrick, P. & Grantham, G. L. (1988). Modifications of the tobacco mosaic virus coat protein gene affecting replication, movement and symptomatology. Phytopathology 78, 783789.
Deom, C. M., Oliver, M. J. & Beachy, R. N. (1987). The 30-kilodalton gene product of tobacco mosaic virus potentiates virus movement. Science 237, 389394.
Ding, B., Haudenshield, J. S., Hull, R. J., Wolf, S., Beachy, R. N. & Lucas, W. J. (1992). Secondary plasmodesmata are specific sites of localization of the tobacco mosaic virus movement protein in transgenic tobacco plants. Plant Cell 4, 915928.
Ding, X.-S., Liu, J., Cheng, N.-H., Folimonov, A., Hou, Y.-M., Bao, Y., Katagi, C., Carter, S. A. & Nelson, R. S. (2004). The Tobacco mosaic virus 126-kDa protein associated with virus replication and movement suppresses RNA silencing. Mol Plant Microbe Interact 17, 583592.[Medline]
Fedorkin, O. N., Denisenko, O. N., Sitkov, A. S., Zelenina, D. A., Lukashova, L. I., Morozov, S. Y. & Atabekov, J. G. (1995). The tomato mosaic virus small gene-product forms stable complex with translation elongation factor EF-1-. Dokl Acad Nauk 343, 703704.
Gal-On, A., Kaplan, I., Roossinck, M. J. & Palukaitis, P. (1994). The kinetics of infection of zucchini squash by cucumber mosaic virus indicate a function for RNA 1 in virus movement. Virology 205, 280289.[CrossRef][Medline]
Gillespie, T., Boevink, P., Haupt, S., Roberts, A. G., Toth, R., Valentine, T., Chapman, S. & Oparka, K. J. (2002). Functional analysis of a DNA-shuffled movement protein reveals that microtubules are dispensable for the cell-to-cell movement of Tobacco mosaic virus. Plant Cell 14, 12071222.
Goelet, P., Lomonossoff, G. P., Butler, P. J. G., Akam, M. E., Gait, M. J. & Karn, J. (1982). Nucleotide sequence of tobacco mosaic virus RNA. Proc Natl Acad Sci U S A 79, 58185822.[Abstract]
Gorbalenya, A. E., Koonin, E. V., Donchenko, A. P. & Blinov, V. M. (1988). A conserved NTP-motif in putative helicases. Nature 333, 22.[Medline]
Grdzelishvili, V. Z., Chapman, S. N., Dawson, W. O. & Lewandowski, D. J. (2000). Mapping of the Tobacco mosaic virus movement protein and coat protein subgenomic RNA promoters in vivo. Virology 275, 177192.[CrossRef][Medline]
Guilley, H., Jonard, G., Kukla, B. & Richards, K. E. (1979). Sequence of 1000 nucleotides at the 3' end of tobacco mosaic virus RNA. Nucleic Acids Res 6, 12871307.[Abstract]
Hodgman, T. C. (1988). A new superfamily of replicative proteins. Nature 333, 2223.[Medline]
Hunter, T. R., Hunt, T., Knowland, J. & Zimmern, D. (1976). Messenger RNA for the coat protein of tobacco mosaic virus. Nature 260, 759764.[Medline]
Itaya, A., Matsuda, Y., Gonzales, R. A., Nelson, R. S. & Ding, B. (2002). Potato spindle tuber viroid strains of different pathogenicity induces and suppresses expression of common and unique genes in infected tomato. Mol Plant Microbe Interact 15, 990999.[Medline]
Ivanov, P. A., Karpova, O. V., Skulachev, M. V., Tomashevskaya, O. L., Rodionova, N. P., Dorokhov, Y. L. & Atabekov, J. G. (1997). A tobamovirus genome that contains an internal ribosome entry site functional in vitro. Virology 232, 3243.[CrossRef][Medline]
Knowland, J., Hunter, T., Hunt, T. & Zimmern, D. (1975). Translation of tobacco mosaic virus RNA and isolation for the messenger for TMV coat protein. INSERM Colloq (Inst Nat Sant Rech Méd) 47, 211216.
Lewandowski, D. J. & Dawson, W. O. (1998). Deletion of internal sequences results in tobacco mosaic virus defective RNAs that accumulate to high levels without interfering with replication of the helper virus. Virology 251, 427437.[CrossRef][Medline]
Lewandowski, D. J. & Dawson, W. O. (2000). Functions of the 126- and 183-kDa proteins of tobacco mosaic virus. Virology 271, 9098.[CrossRef][Medline]
Li, H.-W., Lucy, A. P., Guo, H.-S., Li, W.-X., Ji, L.-H., Wong, S.-M. & Ding, S.-W. (1999). Strong host resistance targeted against a viral suppressor of the plant gene silencing defence mechanism. EMBO J 18, 26832691.
MacFarlane, S. A. & Popovich, A.-H. (2000). Expression of foreign proteins in roots from tobravirus vectors. Virology 267, 2935.[CrossRef][Medline]
Morozov, S. Y., Denisenko, O. N., Zelenina, D. A., Fedorkin, O. N., Solovyev, A. G., Maiss, E., Casper, R. & Atabekov, J. G. (1993). A novel open reading frame in tobacco mosaic virus genome coding for a putative small, positively charged protein. Biochimie 75, 659665.[CrossRef][Medline]
Mueller, A.-M., Mooney, A. L. & MacFarlane, S. A. (1997). Replication of in vitro tobravirus recombinants shows that the specificity of template recognition is determined by 5' non-coding but not 3' non-coding sequences. J Gen Virol 78, 20852088.[Abstract]
Palukaitis, P. & Kaplan, I. B. (1997). Synergy of virus accumulation and pathology in transgenic plants expressing viral sequences. In Virus-resistant Transgenic Plants: Potential Ecological Impact, pp. 7784. Edited by M. Tepfer & E. Balázs. Berlin: Springer.
Palukaitis, P., García-Arenal, F., Sulzinski, M. A. & Zaitlin, M. (1983). Replication of tobacco mosaic virus. VII. Further characterization of single- and double-stranded virus-related RNAs from TMV-infected plants. Virology 131, 533545.[CrossRef]
Peart, J. R., Cook, G., Feys, B. J., Parker, J. E. & Baulcombe, D. C. (2002). An EDS1 orthologue is required for N-mediated resistance against tobacco mosaic virus. Plant J 29, 569579.[CrossRef][Medline]
Pelham, H. R. B. (1978). Leaky UAG termination codon in tobacco mosaic virus RNA. Nature 272, 469471.[Medline]
Poch, O., Sauvaget, I., Delarue, M. & Tordo, N. (1989). Identification of four conserved motifs among RNA-dependent polymerase encoding elements. EMBO J 8, 38673874.[Abstract]
Pruss, G., Ge, X., Shi, X. M., Carrington, J. C. & Vance, V. B. (1997). Plant viral synergism: the potyviral genome encodes a broad-range pathogenicity enhancer that transactivates replication of heterologous viruses. Plant Cell 9, 859868.
Qiu, W., Park, J.-W. & Scholthof, H. B. (2002). Tombusvirus P19-mediated suppression of virus-induced gene silencing is controlled by genetic and dosage features that influence pathogenicity. Mol Plant Microbe Interact 15, 269280.[Medline]
Qu, F. & Morris, T. J. (2002). Efficient infection of Nicotiana benthamiana by Tomato bushy stunt virus is facilitated by the coat protein and maintained by p19 through suppression of gene silencing. Mol Plant Microbe Interact 15, 193202.[Medline]
Qu, F., Ren, T. & Morris, T. J. (2003). The coat protein of turnip crinkle virus suppresses posttranscriptional gene silencing at an early initiation step. J Virol 77, 511522.[CrossRef][Medline]
Ratcliff, F., Martín-Hernández, A.-M. & Baulcombe, D. C. (2001). Tobacco rattle virus as a vector for analysis of gene function by silencing. Plant J 25, 237245.[CrossRef][Medline]
Rizzo, T. M. & Palukaitis, P. (1990). Construction of full-length cDNA clones of cucumber mosaic virus RNA1, RNA2 and RNA3: generation of infectious RNA transcripts. Mol Gen Genet 222, 249256.[Medline]
Rozanov, M. N., Koonin, E. V. & Gorbalenya, A. E. (1992). Conservation of the putative methyltransferase domain: a hallmark of the Sindbis-like supergroup of positive-strand RNA viruses. J Gen Virol 73, 21292134.[Abstract]
Ruiz, M. T., Voinnet, O. & Baulcombe, D. C. (1998). Initiation and maintenance of virus-induced gene silencing. Plant Cell 10, 937946.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual. 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Scholthof, H. B., Scholthof, K.-B. G. & Jackson, A. O. (1995). Identification of tomato bushy stunt virus host-specific symptom determinants by expression of individual genes from a potato virus X vector. Plant Cell 7, 11571172.
Siegel, A., Zaitlin, M. & Sehgal, O. P. (1962). The isolation of defective tobacco mosaic virus strains. Proc Natl Acad Sci U S A 48, 18451851.[Medline]
Siegel, A., Zaitlin, M. & Duda, C. T. (1973). Replication of tobacco mosaic virus. IV. Further characterization of viral related RNAs. Virology 53, 7583.[CrossRef]
Siegel, A., Hari, V. & Kolacz, K. (1978). The effect of tobacco mosaic virus infection on host and virus-specific protein synthesis in protoplasts. Virology 85, 494503.[CrossRef][Medline]
Sulzinski, M. A., Gabard, K. A., Palukaitis, P. & Zaitlin, M. (1985). Replication of tobacco mosaic virus. VIII. Characterization of a third subgenomic TMV RNA. Virology 145, 132140.[CrossRef]
Thomas, C. L., Leh, V., Lederer, C. & Maule, A. J. (2003). Turnip crinkle virus coat protein mediates suppression of RNA silencing in Nicotiana benthamiana. Virology 306, 3341.[CrossRef][Medline]
Tomenius, K., Clapham, D. & Meshi, T. (1987). Localization by immunogold cytochemistry of the virus-coded 30K protein in plasmodesmata of leaves infected with tobacco mosaic virus. Virology 160, 363371.
Van Loon, L. C. & Van Kammen, A. (1970). Polyacrylamide disc electrophoresis of the soluble leaf proteins from Nicotiana tabacum var "Samsun" and "Samsun NN". II. Changes in protein constitution after infection with tobacco mosaic virus. Virology 40, 199211.
Van Telgen, H. J., Van Der Zaal, E. J. & Van Loon, L. C. (1985). Evidence for an association between viral coat protein and host chromatin in mosaic-diseased tobacco leaves. Physiol Plant Pathol 26, 8398.
Voinnet, O., Pinto, Y. M. & Baulcombe, D. C. (1999). Suppression of gene silencing: a general strategy used by diverse DNA and RNA viruses of plants. Proc Natl Acad Sci U S A 96, 1414714152.
Voinnet, O., Rivas, S., Mestre, P. & Baulcombe, D. (2003). An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J 33, 949956.[CrossRef][Medline]
Watanabe, T., Honda, A., Iwata, A., Ueda, S., Hibi, T. & Ishihama, A. (1999). Isolation from tobacco mosaic virus-infected tobacco of a solubilized template-specific RNA-dependent RNA polymerase containing a 126K/183K protein heterodimer. J Virol 73, 26332640.
Young, N. D., Forney, J. & Zaitlin, M. (1987). Tobacco mosaic virus replicase and replicative structures. J Cell Sci 7, S277S285.
Zaitlin, M. & Hariharasubramanian, V. (1972). A gel electrophoretic analysis of proteins from plants infected with tobacco mosaic and potato spindle tuber viruses. Virology 47, 296305.[CrossRef][Medline]
Received 12 May 2004;
accepted 17 June 2004.