1 Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences (SLU), Box 7080, SE-750 07 Uppsala, Sweden
2 A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russian Federation
3 Department of Applied Biology, University of Helsinki, Finland
Correspondence
E. I. Savenkov
eugene.savenkov{at}vbsg.slu.se
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ABSTRACT |
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These authors contributed equally to this paper.
Present address: The Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK.
Present address: Amersham Biosciences, SE-751 25, Uppsala, Sweden.
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INTRODUCTION |
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Another CRP class is found in viruses of genera Carlavirus and Allexivirus. These proteins have a sequence conservation pattern distinct from that found in the HBFT class (Kanyuka et al., 1992). A carlavirus CRP has been shown to contain a zinc finger-like sequence motif and to bind RNA in vitro (Gramstat et al., 1990
). However, little is known about the functions of carla- and allexiviral CRPs. The CRP encoded by Potato mop-top virus (PMTV; the type member of genus Pomovirus) shows no noticeable amino acid sequence similarity to proteins of the two aforementioned CRP classes (Scott et al., 1994
; Savenkov et al., 2003
).
The tripartite PMTV genome consists of single-stranded RNAs of positive-polarity and contains eight open reading frames (ORFs). RNA1 and RNA2 encode virus replication and encapsidation/transmission functions, respectively (Kashiwazaki et al., 1995; Reavy et al., 1998
; Savenkov et al., 1999
; Sandgren et al., 2001
). RNA3, the smallest RNA in the PMTV genome, encodes a triple gene block (TGB) and an 8 kDa CRP (ORF4) (Scott et al., 1994
; Savenkov, 2002
). Three TGB-encoded proteins are involved in viral cell-to-cell movement (Morozov & Solovyev, 2003
; Zamyatnin et al., 2004
), whereas CRP function(s) is yet unknown. Our previous analysis demonstrated that the CRP gene is dispensable for replication, accumulation, movement or virulence of PMTV in Nicotiana benthamiana plants (Savenkov et al., 2003
). Furthermore, it is not known whether this protein is expressed in infected plants. Other pomoviruses do not encode a CRP similar to the PMTV 8 kDa protein (Koenig et al., 1996
, 1998
). Analysis of the PMTV sequences from several virus isolates revealed that CRP is highly variable (Pe
enková et al., 2004
). Additionally, unlike Swedish and Scottish isolates (Todd), in four Danish isolates the AUG start codon of the CRP ORF is replaced by a GUG codon (Pe
enková et al., 2004
).
This study was carried out to determine whether the PMTV CRP is expressed in virus-infected plants and has any functions in vivo. We report immunological detection of PMTV CRP in infected plants, mapping of the subgenomic RNAs (sgRNAs) associated with RNA3, characterization of PMTV CRP-associated effects on the infection phenotypes of heterologous viruses, analysis of the potential of CRP to suppress RNA silencing and localization of GFP-fused CRP in plant cells.
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METHODS |
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Primer extension.
To map the 5' ends of sgRNAs, primer extension was performed using 5 µg total RNA extract isolated from PMTV-infected N. benthamiana as templates. Template RNAs were annealed with 50 pM primer EXT-p2 (complementary to PMTV RNA3 nt 17911813), EXT-p3 (complementary to PMTV RNA3 nt 20322055) or EXT-CIS (complementary to PMTV RNA3 nt 25102532). [-32P]dATP-labelled reverse transcription was performed using Moloney murine leukaemia virus (Mo-MuLV) reverse transcriptase (MBI Fermentas) according to manufacturer's protocol.
Plant inoculations and analyses.
Inoculation of N. benthamiana plants with recombinant viruses, Western and Northern blot analyses were carried out as described by Yelina et al. (2002) and Savenkov et al. (2003)
, respectively. Agroinfiltration assay and fluorescent microscopy were done according to Yelina et al. (2005)
.
Sequence analysis.
For prediction of transmembrane protein segments, programs TopPred II (Claros & von Heijne, 1994; http://bioweb.pasteur.fr/seqanal/interfaces/toppred.html), TMpred (Hofmann & Stoffel, 1993
; http://www.ch.embnet.org/software/TMPRED_form.html), MEMSAT (Jones et al., 1994
; http://saier-144-37.ucsd.edu/memsat.html) and PRED-TMR (Pasquier et al., 1999
; http://biophysics.biol.uoa.gr/PRED-TMR/) were used.
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RESULTS |
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Run-off reverse transcription reactions were performed with total RNA extracted from PMTV-infected N. benthamiana plants with primers EXT-p2, EXT-p3 and EXT-CIS annealing to positive-sense RNA3-specific RNAs downstream of start codons of TGBp2, TGBp3 and CRP genes, respectively. The primer extension reaction with EXT-p2 was terminated at positions corresponding to residues 1705 and 1706 of the genomic RNA3 (Fig. 2b). Presumably, the 5' terminus of the sgRNA-p2/p3 corresponds to an adenine residue at position 1706, whereas the 1 base longer run-off product may arise from incorporation of a residue complementary to the cap structure, as suggested by White & Mackie (1990)
. Similarly, two major products corresponding to positions 2372 and 2373 were identified in the extension reaction with the primer EXT-CIS (Fig. 2c
). These data indicate that sequences of sgRNA-p2/p3 and sgRNA-CRP start at 42 and 89 bases upstream of the translation initiation codons of TGBp2 and CRP, respectively. As expected, the primer extension reaction with primer EXT-p3 did not result in any extension product (data not shown), confirming the hypothesis that PMTV TGBp3 lacks its own sgRNA.
PMTV CRP influence on BSMV, TMV and PVX infection
BSMV (genus Hordeivirus) encodes a CRP previously known as a virulence determinant for viral infection (Donald & Jackson, 1994) and which was recently demonstrated to be a suppressor of RNA silencing (Yelina et al., 2002
; Bragg & Jackson, 2004
). To determine whether BSMV CRP could be functionally replaced by PMTV CRP, a BSMV (the ND18 strain) chimera was constructed with the PMTV CRP gene replacing that of BSMV (BSMV[PMTV-CRP]; Fig. 3
a) under the control of the
b subgenomic promoter. As reported previously, control inoculations with BSMV resulted in mild mosaic symptoms in the systemically infected N. benthamiana leaves at 1014 days p.i. and local lesions on inoculated Chenopodium amaranticolor leaves at 46 days p.i. (Petty et al., 1994
; Solovyev et al., 1999
). In contrast to BSMV, BSMV[PMTV-CRP] produced no visible symptoms in N. benthamiana and C. amaranticolor (data not shown). In these plants, the BSMV coat protein (CP) was undetectable by Western blotting up to 30 days p.i. (the latest time point observed) (Fig. 4
a and data not shown). By contrast, BSMV from which the CRP gene was deleted remained infectious (Yelina et al., 2002
).
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Protoplasts of Nicotiana tabacum were inoculated to distinguish between the two possible mechanisms, PMTV CRP-mediated suppression of virus accumulation in initially infected cells and an inhibited BSMV cell-to-cell movement. BSMV CP could be detected in BSMV-, BSMV[PMTV-CRP-stop]-and BSMV[del-CRP]-inoculated protoplasts, in contrast to those inoculated with BSMV[PMTV-CRP], at 48 h p.i. (Fig. 4b). These results indicated that PMTV CRP could suppress BSMV accumulation at the single cell level.
The CRP gene of PMTV was inserted into engineered cDNAs of TMV and PVX to determine whether it has any inhibitory effects on infection with viruses from genera other than Hordeivirus. A TMV-based expression vector TMV30B (Shivprasad et al., 1999) was modified to carry the PMTV CRP gene under the control of an sgRNA promoter (TMV[PMTV-CRP]; Fig. 3b
). A control construct TMV[PMTV-CRP-stop] contained the CRP gene with a stop codon introduced 6 nt downstream of the PMTV CRP start codon (Fig. 3b
). The recombinants were inoculated onto N. benthamiana and N. tabacum cv. Samsun (nn) plants. In both hosts, the parental construct TMV30B caused no visible symptoms on the inoculated leaves and displayed mild mosaic symptoms in the systemically infected leaves, as described previously (Li et al., 1999
; Shivprasad et al., 1999
). Similar symptoms were induced by TMV[PMTV-CRP-stop] (data not shown), whereas TMV[PMTV-CRP] showed a different phenotype. In N. benthamiana, localized necrotic lesions about 2 mm in diameter first appeared on TMV[PMTV-CRP]-inoculated leaves at 4 days p.i. and expanded later to cover a substantial part of the leaf (Fig. 5
a), which resulted in a fast death of the leaf. In N. tabacum, leaves inoculated with TMV[PMTV-CRP] displayed small necrotic lesions about 12 mm in diameter at 3 days p.i. (Fig. 5b
). Thus, TMV[PMTV-CRP] induced a necrotic reaction in inoculated leaves in both N. benthamiana and N. tabacum. In systemically infected leaves of N. benthamiana TMV[PMTV-CRP] caused mild mosaic symptoms closely resembling those caused by TMV30B at 79 days p.i. (data not shown). The upper non-inoculated leaves in the TMV[PMTV-CRP]-inoculated N. tabacum plants remained symptomless up to 30 days p.i.
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The PMTV CRP gene was inserted into an engineered PVX cDNA to yield a PVX[PMTV-CRP] chimera (Fig. 3c). This construct was inoculated onto N. benthamiana plants, along with wt PVX used as a control. Systemic infection with wt PVX caused mild mosaic symptoms in the upper non-inoculated leaves at 7 days p.i., while the inoculated leaves remained symptomless (Yelina et al., 2002
; data not shown). In contrast, PVX-[PMTV-CRP] caused localized necrosis on the inoculated N. benthamiana leaves at 5 days p.i., followed by the development of severe necrosis and wilting of the whole upper non-inoculated leaves 910 days p.i. (Fig. 5e
). These plants died 23 days later. To confirm further that the necrotic phenotype induced by PVX[PMTV-CRP] is attributable to the PMTV CRP expression from the PVX vector, we engineered a PVX[PMTV-CRP-FLAG] chimera and inoculated it onto N. benthamiana. PVX[PMTV-CRP-FLAG] induced disease phenotype similar to that of PVX[PMTV-CRP] chimera (necrosis of the upper non-inoculated leaves). Expression of the PMTV CRP-FLAG was assayed by Western blotting with anti-FLAG antibodies (Fig. 5g
) as before. Overall, this finding suggests that PMTV CRP expressed in the PVX genome dramatically enhances symptom severity of PVX. Moreover, in all experiments, Danish type of PMTV CRP (Fig. 7b
), when expressed from three viral vectors, induced disease phenotypes and had an effect on viral vector expression similar to that observed for the original Swedish type of PMTV CRP (data not shown).
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The ability of PMTV CRP to induce necrotic reaction in both N. benthamiana and N. tabacum when expressed from a heterologous virus prompted us to test CRP for anti-silencing properties. To this end, we employed a previously described assay that involves Agrobacterium-mediated co-expression of a reporter gene GFPC3 with dsGF (a double-stranded inducer of GFPC3-targeted silencing) and a candidate silencing suppressor (Yelina et al., 2005). Infiltration of N. benthamiana leaves with a GFPC3-containing culture resulted in bright GFP fluorescence visible under long-wave UV at 3 days p.i., whereas a co-infiltration with two Agrobacterium cultures carrying GFPC3 and dsGF gave no visible fluorescence under the same conditions due to efficient silencing of the GFP gene, as demonstrated by Yelina et al. (2005)
. When A. tumefaciens carrying PMTV CRP gene was mixed with cultures carrying GFPC3 and dsGF prior to inoculation, the infiltrated area showed no visible fluorescence, whereas a culture carrying the CRP gene of Poa semilatent virus (PSLV; genus Hordeivirus), a known silencing suppressor (Yelina et al., 2005
), resulted in restored GFP fluorescence in the infiltrated area (data not shown). To confirm visual observations, the infiltrated areas were examined by Western blotting with a GFP-specific antiserum and Northern blotting with a GFP-specific probe. Three repeated experiments gave similar results. The representative data of blot analyses are shown in Fig. 6
(a). The levels of GFP and its mRNA were considerably reduced in the presence of dsGF, whereas co-expression with PSLV CRP resulted in slightly elevated levels of GFP and mRNA accumulation as compared with areas expressing GFP only (Fig. 6a
). The levels of GFP and its mRNA accumulation observed in areas co-infiltrated with PMTV CRP were consistently similar to those expressing GFP and dsGF (Fig. 6a
). Moreover, both PMTV CRP and PSLV CRP (used as a control) had no effect on the levels of GFP-specific small interfering RNA (siRNA) accumulation in leaf areas expressing GFP and dsGF (Fig. 6a
). Thus, PMTV CRP failed to suppress RNA silencing in the agroinfiltration assay. In parallel experiments carried out in N. tabacum plants, PMTV CRP also failed to exhibit silencing suppression activity (data not shown).
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To analyse whether PMTV CRP could influence virus-induced RNA silencing in the course of infection, we used a previously described cross-protection assay (Yelina et al., 2002). Co-inoculation of N. benthamiana plants with TMVGFP (GFP-tagged TMV; Fig. 3b
) and PVX-GF, a PVX derivative carrying a portion of the GFP gene (Fig. 3c
), resulted in blocked systemic movement of TMVGFP due to RNA silencing triggered by two replicating chimeras carrying the homologous GFP sequence (Yelina et al., 2002
). When a derivative of PVX-GF expressing PSLV CRP (PVX-PS
b-GF; Yelina et al., 2002
) was inoculated first, followed by inoculation of the same leaves with TMVGFP 3 days later, extensive apical necrosis induced by CRP expression from PVX-GF-CRP was observed in the systemically infected leaves. This was preceded by appearance of GFP fluorescence in systemically infected leaves, which indicated systemic infection with TMVGFP allowed by the suppression of RNA silencing by PSLV CRP in the co-inoculated leaves (Yelina et al., 2002
; data not shown). In contrast, when PVX-GF was modified to express PMTV CRP (PVX[PMTV-CRP]-GF; Fig. 3c
) and used for inoculation followed by inoculation of the same leaves with TMVGFP, no GFP fluorescence was observed in the upper leaves before their necrosis and death induced by PTMV CRP (data not shown). Thus, PMTV CRP was unable to suppress virus-induced RNA silencing in the cross-protection assay.
Subcellular localization of GFP-fused PMTV CRP
Analysis of PMTV CRP amino acid sequence revealed that the central region of the protein is highly hydrophobic (Fig. 7a), suggesting that PMTV CRP could interact with cell membranes. CRP sequences of six PMTV isolates were analysed using four different prediction algorithms (see Methods). The central CRP region was always predicted, with a high probability, to represent a transmembrane segment (Fig. 7b
). As a result of amino acid substitutions, the length of the predicted CRP transmembrane segment in four Danish isolates was 5 aa longer than in the Sw and Todd isolates (Fig. 7b
). However, its hydrophobic properties were preserved (Fig. 7b
). Taken together, these data suggest that PMTV CRP is an integral membrane protein.
To analyse the subcellular localization of PMTV CRP, we fused the CRP gene with the GFP gene and cloned the resulting construct under the control of the 35S promoter (plasmid pRT-CRPGFP). Microprojectile bombardment was used to introduce the construct to leaves of N. benthamiana. Confocal laser scanning microscopy of the leaves bombarded with pRT-CRPGFP revealed that the GFP-fused CRP accumulated in amorphous structures distributed throughout the cell (Fig. 7c). As PMTV CRP was predicted to interact with cell membranes, CRPGFP was co-expressed with ERYFP, a marker of endoplasmic reticulum (ER) (Zamyatnin et al., 2004
). Surprisingly, co-expression with CRPGFP drastically changed the localization of ERYFP, which was found in CRPGFP-containing amorphous bodies (Fig. 7c
). Similarly, ERYFP-containing bodies were observed in cells in which ERYFP was co-expressed with non-fused PMTV CRP (data not shown). Therefore, these structures originated from the ER membranes. Moreover, CRPGFP-induced rearrangements of the native ER structure, showing a destructive effect on the cell endomembrane system.
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DISCUSSION |
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As a starting point, CRP was immunologically detected in PMTV-infected leaves, and a CRP-specific sgRNA was mapped, showing that CRP is a functional gene.
In a previous study, we demonstrated that PMTV CRP is not required for systemic movement of the virus and symptom induction in N. benthamiana (Savenkov et al., 2003). Here, we report that PMTV CRP expression from PVX or TMV vectors enhances the virulence of both viruses and results in systemic necrosis in PVX[PMTV-CRP]-infected N. benthamiana and localized necrotic reaction in N. benthamiana and N. tabacum leaves inoculated with TMV[PMTV-CRP] (Fig. 5a and b
). Many viral suppressors of RNA silencing have similar effects when expressed in the PVX or TMV genome background (e.g. Pruss et al., 1997
; Brigneti et al., 1998
; Li et al., 1999
; Yelina et al., 2002
). However, PMTV CRP failed to suppress gene silencing in three different assays carried out in N. benthamiana plants. Thus, PMTV CRP resembles the 4·8 kDa protein of TMV shown to enhance virulence of PVX and TMV, even though it is not an RNA silencing suppressor (Canto et al., 2004
). Conversely, it has been shown that some of the mutations in the Cucumber mosaic virus 2b protein, which prevented elicitation of necrosis by the protein, did not prevent its silencing suppression activity (Lucy et al., 2000
). Our data show that the PMTV CRP functions as virulence determinant are manifested depending on the viral genetic background from which the CRP is expressed. Indeed, in N. benthamiana, the host plant in which the PMTV infection phenotype does not depend on the presence of the CRP gene (Savenkov et al., 2003
), expression of CRP induces necrotic plant responses to PVX and TMV infections. Similarly, N. tabacum cv. Samsun plants infected with Tomato aspermy virus (TAV; genus Cucumovirus) exhibit mild systemic symptoms, whereas expression of TAV 2b protein from the TMV or PVX genomes triggered the hypersensitive response in this host (Li et al., 1999
). It is plausible to assume that PMTV CRP, when expressed in the native viral genetic background, may form complexes with other PMTV-encoded products, thereby preventing strong response in N. benthamiana plants. Alternatively, TMV and PVX might provide functions which, when combined with PMTV CRP action, result in induction of a strong plant response. We cannot exclude the possibility that PMTV CRP acts in a dose-dependent manner: the observed strong plant response to CRP accumulation could be attributed to its overexpression from PVX and TMV vectors (Shivprasad et al., 1999
).
When PMTV CRP was expressed from a BSMV genome in which the BSMV CRP ORF was replaced with that of PMTV, no detectable accumulation of BSMV was observed. This finding was unexpected, since the BSMV CRP is dispensable for virus replication and cell-to-cell movement in N. benthamiana, C. amaranticolor and barley (Hordeum vulgare). A mutant virus in which the gene encoding the BSMV CRP is deleted is able to infect barley plants systemically, but is restricted to the inoculated leaves of N. benthamiana (Petty et al., 1990; Yelina et al., 2002
). The presence of translatable PMTV CRP gene in BSMV genome led to suppression of virus accumulation at a single cell level that represents an example of extreme virus resistance (Valkonen, 2002
). This might imply that the PMTV CRP-induced response in N. benthamiana is greatly enhanced by BSMV infection. Interestingly, a similar extreme resistance to PMTV CRP-expressing BSMV was also found in C. amaranticolor, the local lesion indicator host of the virus. These data suggest that the PMTV CRP-induced plant response may be common for different host species. PMTV CRP was expressed from BSMV under control of
b subgemomic promoter.
b subgenomic promoter is the strongest in BSMV genome, and sgRNA
is the most abundant sgRNA throughout replication (Johnson et al., 2003
). Therefore, inhibitory effect of the PMTV CRP on BSMV infection might be a consequence of PMTV-CRP overexpression and/or, perhaps, a result of the mis-regulation or the absence of regulation of the BSMV gene expression, as was reported before for PSLV
b protein expressed in the BSMV genome background (Yelina et al., 2002
).
In this study, we have shown that PMTV CRP is an integral membrane protein associated with ER-derived membranes in plant cells. Importantly, expression of GFP-fused CRP in epidermal cells by microprojectile bombardment induced dramatic rearrangements of the ER structure (Fig. 7c). Replication of many plant RNA viruses is known to occur in association with ER membranes (Restrepo-Hartwig & Ahlquist, 1999
; Carette et al., 2002
; Ritzenthaler et al., 2002
; Han & Sanfacon, 2003
). Our data suggest that the ER membrane rearrangements induced by the PMTV CRP can be favourable (or neutral) for PMTV, TMV and PVX replication, but deleterious for BSMV replication.
This study, while reporting the ability of PMTV CRP to dramatically enhance viral symptoms and disease severity of heterologous viruses, does not resolve the putative CRP function(s) in PMTV infection. The putative TMV ORF6-encoded protein shows properties similar to those of PMTV CRP and behaves as the virulence determinant in N. benthamiana but not in other hosts such as N. tabacum and tomato (Lycopersicon esculentum) (Canto et al., 2004). Therefore, assuming the possibility of a similar host-specific PMTV CRP action, further studies involving a variety of other PMTV host species are required to determine the role of this protein in the virus life cycle.
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ACKNOWLEDGEMENTS |
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Received 11 April 2005;
accepted 16 June 2005.
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