ORF6 of Tobacco mosaic virus is a determinant of viral pathogenicity in Nicotiana benthamiana

Tomas Canto, Stuart A. MacFarlane and Peter Palukaitis

Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK

Correspondence
Peter Palukaitis
ppaluk{at}scri.sari.ac.uk


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Tobacco mosaic virus (TMV) contains a sixth open reading frame (ORF6) that potentially encodes a 4·8 kDa protein. Elimination of ORF6 from TMV attenuated host responses in Nicotiana benthamiana without alteration in virus accumulation. Furthermore, heterologous expression of TMV ORF6 from either potato virus X (PVX) or tobacco rattle virus (TRV) vectors enhanced the virulence of both viruses in N. benthamiana, also without effects on their accumulation. By contrast, the presence or absence of TMV ORF6 had no effect on host response or virus accumulation in N. tabacum plants infected with TMV or PVX. TMV ORF6 also had no effect on the synergism between TMV and PVX in N. tabacum. However, the presence of the TMV ORF6 did have an effect on the pathogenicity of a TRV vector in N. tabacum. In three different types of assay carried out in N. benthamiana plants, expression of TMV ORF6 failed to suppress gene silencing. Expression in N. benthamiana epidermal cells of the encoded 4·8 kDa protein fused to the green fluorescent protein at either end showed, in addition to widespread cytosolic fluorescence, plasmodesmatal targeting specific to both fusion constructs. The role of the ORF6 in host responses is discussed.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The nucleotide sequence and genome organization of Tobacco mosaic virus (TMV), the type species member of the genus Tobamovirus, were determined over 20 years ago (Goelet et al., 1982). Despite a better understanding of the functions of the various TMV-encoded proteins in the intervening years, there is still controversy concerning the number of proteins encoded by the TMV genome. Two proteins are produced upon direct translation of the genomic RNA (Knowland et al., 1975). The 5' proximal open reading frame (ORF) encodes a 126 kDa protein involved in replication and containing motifs characteristic of putative methyl transferase and helicase domains (Gorbalenya et al., 1988; Hodgman, 1988; Lewandowski & Dawson, 2000; Rozanov et al., 1992; Watanabe et al., 1999; Young et al., 1987). This protein has been shown to suppress gene silencing (Ding et al., 2004). Readthrough of the amber terminator of this ORF yields a 183 kDa protein (Pelham, 1978), considered to be the viral polymerase (Lewandowski & Dawson, 2000; Watanabe et al., 1999; Young et al., 1987). These readthrough sequences contain domains characteristic of other viral polymerases (Poch et al., 1989). Only the 126 and 183 kDa proteins are required for replication of the genomic RNA or various defective RNAs generated in vitro (Lewandowski & Dawson, 1998, 2000). Three subgenomic RNAs (sgRNAs) have also been identified during infection by TMV (Beachy & Zaitlin, 1977; Hunter et al., 1976; Sulzinski et al., 1985), all of which have been found in association with polyribosomes (Palukaitis et al., 1983). The smallest of these sgRNAs, designated LMC (low molecular mass component), encodes the 3' proximal 17·5 kDa capsid protein (CP) (Hunter et al., 1976; Siegel et al., 1973), while the next-largest sgRNA, designated I2 (intermediate-class RNA 2), encodes the 30 kDa viral movement protein (MP) (Beachy & Zaitlin, 1977; Deom et al., 1987). The I1 sgRNA contains an ORF encoding a 54 kDa protein, which coincides with the readthrough portion of the 183 kDa protein (Sulzinski et al., 1985). There is some question about the expression of the I1 sgRNA, as the 54 kDa protein has not been detected in vivo, unlike the other TMV-encoded proteins.

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 38–45 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 4–5 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 {alpha} (EF-1-{alpha}) (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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Viral constructs and plasmids.
All DNA manipulations were done using standard procedures (Sambrook et al., 1989). The plasmid pTMV004, containing a biologically active, full-length cDNA clone of TMV RNA (U1 strain), with a T7 RNA polymerase promoter sequence fused to the 5' end of the TMV RNA sequence and a KpnI restriction site for linearization at the 3' terminus (Lewandowski & Dawson, 1998), was obtained from S. N. Chapman (Scottish Crop Research Institute). Oligonucleotide-directed mutagenesis and overlapping PCR were used to change the potential initiation codons of ORF6 and prevent the putative expression of the 4·8 kDa protein (referred to in the constructs as ‘4K’). Thus, nt 5667 and 5670 were altered from T to C in the plasmid designated pTMV(ORF6–) (=non-translatable ORF6). The plasmid NW291, containing a biologically active cDNA clone of TMV RNA with the gene encoding a monomeric red fluorescent protein (mRFP1) (Campbell et al., 2002) fused to the TMV MP, was obtained from S. N. Chapman. This plasmid will be referred to as pTMV : 30K : : mRFP1.

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 2419–2432 and a flanking BamHI site, and a primer complementary to nt 2739–2751 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 {beta}-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 999–2144 of the TMV genome and the other complementary to 3'-terminal nt 6149–6402. 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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
TMV ORF6 enhances virulence in N. benthamiana
An examination of the position of TMV ORF6 shows that it overlaps the C terminus of the 30 kDa MP and the N terminus of the CP (Fig. 1). In addition, sequences encoding the N-terminal half of the 4·8 kDa protein are also part of the CP gene sgRNA promoter and leader sequences, required for transcription and translation, respectively, of the LMC sgRNA (Grdzelishvili et al., 2000; Guilley et al., 1979). Thus, it was not possible to delete the sequences containing ORF6 without affecting production of the CP, which is needed for encapsidation as well as the long-distance movement of TMV (Dawson et al., 1988; Siegel et al., 1962). Hence, to examine the effect of ORF6 on infection by TMV, the two adjacent methionine codons at the beginning of ORF6 were modified from AUG to ACG, thus eliminating the ORF (Fig. 1). In the overlapping gene encoding the 30 kDa MP, these alterations did not change the encoded amino acids. This modified TMV was designated TMV(ORF6–).



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Fig. 1. Diagram of the location of ORF6 in the TMV genome. ORF6 overlaps the sequences encoding the MP and CP genes. The initiation codons of ORF6 (nt 5666–5668) and the CP gene (nt 5712–5714) and the termination codons of the MP gene (nt 5707–5709) and the ORF6 (nt 5786–5788) are indicated by double- and single-underlined nucleotides, respectively. The positions of the 5' terminus of the LMC sgRNA (nt 5703) and the boundaries of the promoter for the LMC sgRNA (nt 5546–5757) are indicated.

 
In N. benthamiana, infection by wt TMV induced a severe, systemic necrosis, while infection by TMV(ORF6–) induced severe stunting, with epinasty and rugosity, similar to symptoms induced by a number of other viruses in this host (Fig. 2a, first and second plants from the left, respectively). Thus, the presence or absence of the ORF6 had a major effect on the symptoms induced by TMV in N. benthamiana.



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Fig. 2. Effect of TMV ORF6 on virulence in N. benthamiana plants. Plants were inoculated with: (a) RNAs of wt TMV, TMV missing ORF6 [TMV(ORF6–)], PVX or PVX expressing TMV ORF6 [PVX+(ORF6)]; (b) RNAs of TRV vectors either expressing the TMV ORF6 [TRV+(ORF6), upper row of plants] or the ORF6 sequence with the putative initiation codons altered from methionine to threonine [TRV+(ORF6–), lower row of plants].

 
To determine whether this effect of TMV ORF6 was specific to infection by TMV, two viral vectors, PVX and TRV, expressing the TMV ORF6 were inoculated on to N. benthamiana. PVX expressing the TMV ORF6 was designated PVX+(ORF6). N. benthamiana plants infected by PVX+(ORF6) showed more severe symptoms compared with such plants infected by PVX alone (Fig. 2a, third and fourth plants from the left, respectively). The mild symptoms induced by infection with PVX contrasted with the epinasty, rugosity and some veinal necrosis observed after infection by PVX+(ORF6) (Fig. 2a). Thus, expression of TMV ORF6 in N. benthamiana enhanced the virulence of PVX.

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.



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Fig. 3. Effect of TMV ORF6 on accumulation of viral RNAs and CP in systemically infected plants. (a) Northern blot analysis of wt TMV and TMV(ORF6–) RNA accumulation in systemically infected tissues of different hosts: N. tabacum cv. Samsun nn (at 11 days p.i.); N. benthamiana (8 days p.i.); tomato (14 days p.i.); and N. clevelandii (11 days p.i.). Both viruses accumulated to similar levels in all hosts, except in N. clevelandii, where TMV(ORF6–) accumulated to lower levels than wt TMV. (b) Total proteins were extracted from the inoculated leaves of N. benthamiana plants infected with either wt TMV or TMV(ORF6–) and serially diluted (0·01 and 0·1) and undiluted (1) samples were analysed for the level of TMV CP accumulation by SDS-PAGE and immunoblotting using a TMV CP antiserum. The upper panel shows the immunoblot, while the lower panel shows the Coomassie-stained gel of the samples analysed as a loading control. M, Molecular mass markers, with the molecular mass (kDa) indicated on the right. H, Sample from a non-inoculated plant. (c) Northern blot analysis of PVX and PVX+(ORF6) RNA accumulation in N. tabacum and N. benthamiana (at 12 days p.i.). (d) Northern blot analysis of viral RNA accumulation in N. benthamiana (at 11 days p.i.) inoculated with TRV+(ORF6) or TRV+(ORF6–). In all cases, each sample lane represents an individual plant. Viral genomic and sgRNAs are indicated. The ethidium bromide-stained rRNAs are shown below each blot as controls of loading.

 
After infection by the viral vectors, the level of PVX RNA accumulation was not affected in N. benthamiana plants (Fig. 3c) or protoplasts (not shown) by the presence of TMV ORF6. Similarly, analysis of viral RNA accumulation in systemically infected N. benthamiana leaves showed no significant difference between TRV+(ORF6) and TRV+(ORF6–) (Fig. 3d), indicating that TMV ORF6 also did not increase accumulation of the TRV RNAs, despite its effect on virulence.

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.



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Fig. 4. Effect of TMV ORF6 on the PVX/TMV synergy in N. tabacum cv. Samsun nn. Plants were inoculated with RNAs of wt TMV, TMV(ORF6–), PVX or PVX+(ORF6) (upper row). Plants were also inoculated simultaneously with both viruses in all four possible combinations (lower row): wt TMV plus PVX, wt TMV plus PVX+(ORF6), TMV(ORF6–) plus PVX, and TMV(ORF6–) plus PVX+(ORF6). Plants were photographed at 12 days p.i.

 
In addition to N. tabacum, two other hosts were tested for possible differences in TMV pathogenicity mediated by the ORF6: tomato and N. clevelandii. In tomato, no differences in the symptom severity or virus accumulation induced by TMV compared to TMV(ORF6–) were observed (Fig. 3a, and data not shown), while in N. clevelandii the symptoms induced by TMV(ORF6–) were slightly milder than those induced by TMV (not shown) and virus accumulation was also noticeably lower (Fig. 3a).

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).



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Fig. 5. Assessment of silencing suppression by expression of TMV ORF6. (a) N. benthamiana plants of line 16c, transgenic for expression of the GFP, were agroinfiltrated with bacteria harbouring no binary vector (leaf 1), the vector pROK2 with no insert (leaf 2), pROK2/P1HC expressing the PVY P1-HCPro (leaf 3) or pROK2/4K expressing the TMV ORF6 (leaf 4). Leaves were photographed under UV light at 12 days post-infiltration. Agroinfiltration of leaves using bacteria with no binary vector (leaf 1) enhanced GFP expression relative to the non-infiltrated tissues. (b) Non-transgenic N. benthamiana plants were co-infiltrated with a 1 : 1 (v/v) mixture of A. tumefaciens culture harbouring a GUS-expressing T-DNA and one of the following binary vectors: pROK2 (lane 1) or pROK2 expressing the P1-HCPro of PVY (lane 2), the putative 4·8 kDa protein of TMV ORF6 (lane 3) or the 2b protein of CMV (lane 4). The steady-state levels of GUS mRNA and siRNAs to the GUS gene are shown in the first and third panels from the top, respectively. Ethidium bromide-stained rRNAs are shown below each blot as controls of loading.

 
Enhanced transient expression of a reporter gene co-infiltrated with a viral suppressor has been shown using the p19 protein from Tomato bushy stunt virus (Voinnet et al., 2003). Therefore, non-transgenic N. benthamiana plants were co-infiltrated with a 1 : 1 (v/v) mixture of A. tumefaciens culture harbouring a GUS-expressing T-DNA (Canto et al., 2002) and one of the following binary plasmid vectors: pROK2 (without additional genes) or pROK2 expressing the P1-HCPro gene of PVY, the putative 4·8 kDa protein of TMV ORF6 or the 2b gene of CMV (Fig. 5b). The P1-HCPro and 2b suppressors both succeeded in increasing the steady-state levels of GUS mRNA in infiltrated tissue and in decreasing the levels of accumulated siRNAs to GUS (Fig. 5b, upper and lower panels, respectively). However, expression of the 4·8 kDa protein had no effect on the steady-state levels of GUS mRNA or GUS siRNAs (Fig. 5b).

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.



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Fig. 6. Subcellular distribution of TMV ORF6 fused to GFP. (a) N. benthamiana plants were inoculated with PVX expressing TMV ORF6 fused to GFP at either the C terminus of GFP (PVX+GFP : : 4K) (left panel) or the N terminus of GFP (PVX+4K : : GFP) (right panel). The subcellular distribution of the fusion proteins was determined in epidermal cells at 7 days p.i. by detection of fluorescence using a confocal laser-scanning microscope. Arrows indicate the presence of some of the discrete fluorescence spots associated with the cell walls. (b) Subcellular localization to plasmodesmata of TMV ORF6 fused to GFP. N. benthamiana plants were inoculated with TMV expressing the 30 kDa MP fused to a modified red fluorescent protein (TMV 30K : : mRFP1) together with PVX expressing free GFP (PVX+GFP) (upper row), PVX expressing GFP fused to the C terminus of TMV ORF6 (PVX+4K : : GFP) (middle row) or PVX expressing GFP fused to the N terminus of TMV ORF6 (PVX+GFP : : 4K) (lower row). Infected fluorescent cells were viewed by confocal laser-scanning microscopy at 7 days p.i. using different filters for detecting either green (left column) or red (central column) fluorescence. The panels in the right column show an overlay of both green and red images. Arrows point to some of the punctate fluorescence signals observed in plasmodesmata, indicating co-localization of the TMV ORF6 fused to GFP and the TMV 30 kDa MP fused to mRFP1 (middle and lower rows) and the absence of free GFP from the cell wall in the upper row. Bars, 50 µm (a).

 
Interestingly, the gain setting required for detecting GFP in plants infected by PVX expressing the two 4K/GFP fusion proteins was much higher than was needed to detect the signal for free GFP expressed from PVX. This suggested that the fusion proteins may be turned over more rapidly and do not accumulate to the same extent as free GFP.

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
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Here we have shown that the TMV ORF6 increases the virulence of viruses that carry this gene, without causing an increase in the replication or spread of the virus. This indicates that the TMV ORF6 must act directly on the host rather than by stimulating virus infection directly. The TMV ORF6 also showed some differences in its effects, depending upon both the virus species containing this gene and the host species infected. When ORF6 was present in either the TMV genome or a PVX vector, no changes in virulence were observed after infection in tobacco, but the virulence of the virus was increased greatly after infection in N. benthamiana. By contrast, when TMV ORF6 was present in the TRV vector, enhanced virulence was observed after infection of both tobacco and N. benthamiana. These data suggest that ORF6 is expressed from the virus vectors and probably from TMV in both plant species, but that the effects of the encoded 4·8 kDa protein may depend on other factors associated with infection by the different viruses. For example, both PVX and TMV vectors are suggested to encode relatively weak suppressors of gene silencing, leading to patchy infection of the host (Voinnet et al., 1999), whereas TRV can suppress gene silencing more effectively leading to a more uniform infection (Ratcliff et al., 2001). Moreover, gene silencing is not as efficient in tobacco as in N. benthamiana (Peart et al., 2002). Our results showed that the TMV 4·8 kDa protein did not suppress gene silencing in three different assays and also did not affect the synergistic interaction between TMV and PVX. In the latter interactions, the accumulation of PVX increased, but the accumulation of TMV did not (Palukaitis & Kaplan, 1997).

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-{alpha} (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 1–9 at the N terminus of the ToMV 3·9 kDa protein have been shown not to be essential for binding to EF-1-{alpha}, while certain mutations at aa 10–14 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-{alpha} 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
 
The authors thank Dr S. N. Chapman and Dr N. Wood (Scottish Crop Research Institute) and Dr T. M. A. Wilson (Warwick University, Warwick, UK) for making various materials available. This work was supported by a grant in aid from the Scottish Executive Environment and Rural Affairs Department.


   REFERENCES
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Received 12 May 2004; accepted 17 June 2004.