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1 Department of Crop Sciences, Section Plant Virology, University of Göttingen, Grisebachstrasse 6, D-37077 Göttingen, Germany
2 Institute of Sugar Beet Research, Department of Phytopathology, Holtenser Landstrasse 77, D-37079 Göttingen, Germany
Correspondence
Mark Varrelmann
varrelmann{at}ifz-goettingen.de
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ABSTRACT |
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These authors contributed equally to this work. 
Two supplementary figures showing CLUSTAL_X alignments are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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; Roossinck, 1997), and combine high population densities with small genomes and template-replicating enzymes. Viral replicases possess a high mutation rate, due to missing proofreading abilities of the RNA-dependent RNA polymerases (RdRps) (Domingo et al., 1999, 2002). The mechanism of RNA recombination, which joins two molecules of different parental/replicational origin, has two main functions. In addition to representing a source of variation, recombination can rescue viral templates by reversing deleterious mutations (Aaziz & Tepfer, 1999a; Fernández-Cuartero et al., 1994; White & Morris, 1994).
Despite its important function for variability and evolution of RNA viruses, knowledge about the underlying mechanism of recombination, including the identification and functionality of the RdRp domains involved, is still uncertain. Knowledge of RNA primary (sequence composition and similarity) and secondary structure parameters, which can modify recombination, is available for only a few species. Evidence for the occurrence of the RNA recombination phenomenon either by means of in silico sequence comparison (reviewed by Chare & Holmes, 2006) or by its occurrence in mixed infections (Aaziz & Tepfer, 1999a; de Wispelaere et al., 2005; Meier & Truve, 2006), or under experimental conditions by applying high selection pressure, has been given for numerous plant virus species (Aaziz & Tepfer, 1999b; Simon & Bujarski, 1994). However, knowledge concerning RNA recombination mechanisms and parameters affecting these processes has been obtained mainly from a limited number of positive-strand RNA model virus species, namely brome mosaic virus (BMV) and cowpea chlorotic mottle virus, both belonging to the genus Bromovirus (Alejska et al., 2005; Allison et al., 1996; Bruyere et al., 2000; Figlerowicz & Bujarski, 1998) in supergroup III (van der Heijden & Bol, 2002). Supergroup II RNA recombination model viruses are tomato bushy stunt virus (TBSV) (genus Tombusvirus) (Panavas & Nagy, 2003), turnip crinkle virus (TCV) (genus Carmovirus) (Cascone et al., 1993; Zhang et al., 1991) and the RNA bacteriophage Qβ (Brown & Gold, 1996). Animal-infecting recombination model viruses include poliovirus (Jarvis & Kirkegaard, 1992) and mouse hepatitis virus (Banner et al., 1990; Fu & Baric, 1992).
It is generally accepted that replicative RNA recombination is driven by the ability of the viral replicase to switch templates during nascent-strand synthesis from a donor to an acceptor RNA molecule and by the use of nascent strands as primers to complete synthesis on the acceptor strand. Nevertheless, the involvement of host factors cannot be excluded. Several variants of nascent-strand transfer are conceivable (Gmyl & Agol, 2005; Nagy & Simon, 1997), but experimental evidence is still lacking. Important structural factors of the template or nascent strand that are used for classification have been identified in these model viruses and can be subdivided into primary factors, such as sequence similarity, AU-rich motifs, sequence complementarity and replicative cis elements, and secondary structures, such as stem–loops (Bruyere et al., 2000; Nagy & Bujarski, 1993, 1995; Nagy et al., 1998; Olsthoorn et al., 2002; Shapka & Nagy, 2004). So far, non-replicative RNA recombination, possibly based on RNA breakage and ligation, has only been detected in bacteriophage Qβ and polioviruses (Chetverin et al., 1997; Chetverina et al., 1999; Gallei et al., 2004; Gmyl et al., 1999, 2003), but not yet in plant viruses.
Recombination model viruses that belong to the genera Bromovirus and Tombusvirus possess icosahedral particle morphology. Tombusviruses encode one molecule of replication-associated protein and a readthrough polymerase, both necessary for replication. In contrast, bromoviruses possess a replicase molecule with polymerase and helicase functions plus methyltransferase on two separate proteins (reviewed by van der Heijden & Bol, 2002). We have recently established a recombination assay for potato virus X (PVX) (Draghici & Varrelmann, 2009), another plus-strand RNA virus with flexuous, rod-shaped particle morphology, representing a replication model virus with a single replicase molecule (Batten et al., 2003; Verchot-Lubicz et al., 2007). This in vivo assay is based on Agrobacterium tumefaciens-mediated expression of a green fluorescent protein (GFP)-labelled PVX isolate with a partial coat protein (CP) open reading frame (ORF) deletion in parallel with an intact CP transcript plus 3' non-translated region (ntr). When we applied PVX replicase insertion-scanning mutants (Draghici et al., 2009), we were able to identify the involvement of a separate viral replicase domain in replicational RNA recombination, thereby supporting previous mutational studies of BMV (Dzianott et al., 2001; Figlerowicz et al., 1997, 1998; Nagy et al., 1995) and cucumber necrosis virus (Panaviene & Nagy, 2003) replicase.
Applying this in vivo recombination assay, which exerts a high selection pressure on the generation of recombinants, we aimed to study efficiently the parameters affecting PVX RNA recombination. PVX CP transcripts from isolates harbouring several mutations were used to determine the number and location of recombination-junction sites. Several PVX mutants containing CP deletions with different lengths of overlap of the intact CP gene were tested for their influence on the observed time point and frequency of reconstitution. Another CP gene derived from a different PVX type with only limited sequence similarity was tested for possible occurrence of non-homologous recombination events, frequency of reconstitution and composition of movement-competent recombination products. By using CP and 3'-ntr secondary-structure predictions, we aimed to find evidence for the influence of RNA secondary structure on the location of recombination-junction sites.
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CP transcripts were co-expressed with CP–3'-ntr transcripts in Nicotinana benthamiana leaf patches, the agroinfiltration of 65 plants (each inoculated four times) resulted in 60 systemically PVX–GFP-infected plants at 14 days post-infection (p.i.) (Draghici & Varrelmann, 2009). Such a frequent occurrence of recombination, leading to the reconstitution of a complete CP (711 nt in length), was possible because two continuous homologous stretches of CP, nt 1–514 and 646–711, plus the entire 3'-ntr of 72 nt in length, were available for template switching. The intact CP+3'-ntr transcript, however, did not contain any nucleotide mismatch compared with the deletion mutant and therefore did not allow determination of the RdRp template switch for subsequent reconstitution. We intended to analyse the relationship between frequency of CP gene reconstitution by means of recombination and the length of the overlapping sequence. This was achieved by the combined use of PVX CP mutants carrying deletions of variable length plus intact CP-UK3 transcripts. We expected a proportional relationship and a possible minimal length of sequence overlap to be necessary for successful CP gene reconstitution. Moreover, we aimed to find evidence for whether PVX recombines mainly via replicative RdRp template switching between homologous sequences or whether RNA-ligation activity might represent an alternative recombination mechanism. Several 35S-PVX-GFP-CP deletion mutants were constructed (35S-PVX-GFP-CP-0, -10, -20, -50, -101, -200, -300, -401, -500 and -600 in Fig. 1) and each was co-expressed with 35S-CP-UK3 in 20 infiltrated leaf patches in two independent experiments. Virally expressed GFP fluorescence was monitored over time by using UV illumination, from 3 until 17 days p.i. (Table 1).
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). In addition to this observation, the total number of agroinfiltrated patches displaying virus movement observed at 17 days p.i. correlated positively with the length of sequence overlap between deletion mutant and intact CP transcript.
Use of intact PVX CP genes with sequence variability enables determination of the composition of the recombinant CP sequence and the number of RdRp template switches
The recombination assay did not allow determination of the number of template switches or preferred recombination sites up- and downstream (3' and 5') of the CP gene (nt 514–646) deletion because the overlapping parental sequences were identical. To apply intact genes possessing nucleotide exchanges that can serve as markers for this purpose, four different PVX type X isolates were used for cloning and sequencing of the CP gene+3'-ntr region (PVX-14, -17, -18 and -847). CLUSTAL_X (Thompson et al., 1997) alignment revealed 100 % sequence identity of the 3'-ntr between all isolates (data not shown) and 20–31 nt exchanges in the entire 711 nt CP ORF (95.6–97.2 % sequence similarity) compared with CP-UK3 (Fig. 2). Most of these mutations were silent and located at the 5' end of the deletion in CP-UK3, leading to only three amino acid exchanges (data not shown). Therefore, we assumed functionality of chimeric CPs, and no selection of recombinants with specific compositions was expected. All 35S-driven CP+3'-ntr expression clones (35S-CP-PVX-14, -17, -18 and -847) plus the 35S-PVX-GFP-CP control were applied in the assay. The treatment was tested in five N. benthamiana plants, each being inoculated at four positions. Surprisingly, in both independent repetitions, the first recombinants occurred at least 1 day earlier (at 7 instead of 8–9 days p.i.) when the divergent CP genes were applied instead of the CP-UK3 transcript (Table 2). However, the reconstitution frequency within the time period remained comparable.
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Recombinant sequence alignment of the different treatments and repetitions revealed several interesting features. Independently of the four CP sequences, many recombinants from independent plants displayed variable properties [number of template switches (recombination events), mutations], which in some cases resulted in recombinants with variable amino acid composition (see Supplementary Fig. S1, available in JGV Online; summarized in Table 2). Although the deletion in the CP-UK3 gene could have been repaired by an RdRp template switch 5'-proximal to the PvuII deletion from the 35S-CP transcript to the intact PVX-UK3 template strand and completion of viral minus-strand synthesis, this did not occur frequently. Thirteen of 20 recombinants were a product of more than one template switch. In contrast, three recombinants with seven template switches (PVX-14 Rec4 and PVX-847 Rec1 and 2) were observed. Additionally, sequence analysis revealed that most of the recombinant sequences (14 of 20) were unique. No evidence for the selection of a specific region in the variable amino acids was found (Table 2). All amino acid differences between PVX-UK3 and PVX-14, -17, -18 and -847 were located in the N-terminal 60 aa (data not shown). The CP-UK3 deletion (aa 17–215) did not overlap with these mutations. Theoretically, this would allow the reconstitution of a recombinant CP with wild-type composition. However, several recombinants encoding chimeric or even the entire intact divergent CP sequence were detected (Table 2). Comparison between recombinants derived from one treatment, as well as between different treatments, did not reveal preferred regions for RdRp template switches or indications of recombinational hot spots. Even the recombination events leading to CP gene reconstitution by only a single template switch, which was found in seven recombinants, seemed to be distributed homogeneously across the CP ORF (Supplementary Fig. S1).
A CP transcript plus 3'-ntr from a phylogenetically distant PVX type Bi isolate reconstitutes a PVX-UK3 CP deletion mutant and enables a more precise determination of recombination sites
The long distance between nucleotide exchanges when different intact, type X CP genes (PVX-14, -17, -18 and -847) were applied did not allow the determination of preferred regions or stretches for recombination. Secondly, sequence identity was too high to enable us to study the influence of limited sequence similarity on the frequency of CP reconstitution. To achieve this, a phylogenetically more heterologous isolate of PVX was required. Santa Cruz & Baulcombe (1995) compared PVX CP genes from different PVX type X isolates displaying 97–99 % similarity and from type B (Bi and Bii subtypes) isolates showing lower similarity (77 % identity at the nucleic acid level). In order to apply a CP gene containing a higher number of mutations in the recombination assay, an intact Bi type CP gene was selected. This experiment should supply information as to whether recombination occurs at all and, if so, at what relative frequency, the preferred sites and whether a specific chimeric CP is selected. A PVX type B isolate (PVX-4) was used for cloning and sequencing of the CP gene+3'-ntr. This revealed a PVX Bi type strain CP gene and 3'-ntr displaying only 75.1 % CP gene and 76.8 % CP gene+3'-ntr sequence similarity to CP-UK3. As described by Santa Cruz & Baulcombe (1995), the CP-PVX-4 Bi type isolate contained an insertion of 33 nt in the 3' end (see Supplementary Fig. S2, available in JGV Online). For precise determination of the recombination-junction sites, the relatively uniform spread of 187 nucleotide exchanges in the CP ORF and five nucleotide exchanges in the 3'-ntr appeared promising. Compared with recombination with type X CP genes, the relative recombination frequency observed with CP-PVX-4 transcripts was reduced severely. Only 21 of 208 double-infiltrated patches displayed virus movement at 14 days p.i., compared with 72 of 104 patches of CP-UK3 control that were tested in parallel.
To determine the sequence composition of different individual recombinants, 15 plants showing systemic GFP fluorescence (named PVX-4-Rec1 to -Rec15) were used for cloning and sequencing of the recombinant CP gene+3'-ntr. A CLUSTAL_X alignment of the CP gene plus 3'-ntr of PVX-UK3, PVX-4 and 15 recombinants revealed that all recombinants were unique at the nucleotide level (Supplementary Fig. S2). Again, a single template switch would have been sufficient to reconstitute a complete, functional CP gene. However, this was only detected in PVX-4-Rec1, 5, 7, 12 and 14. The remaining 10 recombinants exhibited between two and seven template switches. The 3'-ntr composition of the different recombinants showed that the most 3' nucleotide exchange, at position 750 in the UK3 sequence (corresponding to position 783 in PVX-4; Santa Cruz & Baulcombe, 1995), T (PVX-UK3) 
C (PVX-4), was shown to be PVX-4 in 10 of 15 recombinants (Supplementary Fig. S2). This demonstrated that, in the majority of the recombinations observed, the initiation of minus-strand synthesis had occurred on the intact PVX-4 transcript. In 15 recombinant CP genes analysed, 45 recombination-junction sites were observed, of which 27 were unique (Supplementary Fig. S2, sequences between shaded sections). CLUSTAL_X alignment of the amino acid composition revealed that only PVX-4-Rec2, 4, 10, 15 and 1, 12 (respectively) possessed identical composition, providing no evidence for selection of a chimeric CP with specific composition (data not shown). Remarkably, none of the recombinants had acquired the extreme N-terminal part of PVX-4 and only PVX-4-Rec3 had acquired the entire PVX-4 CP specific 11 aa insertion. The recombination sites detected in the recombinants were not distributed randomly over the CP gene and 3'-ntr, but clustered to five sites in proximity to the deletion in CP-UK3 (designated I–V in Supplementary Fig. S2), where 21 of 45 template switches were observed. The remaining sites appeared to be spread uniformly over the possible sequence range available for recombination.
Precise homologous RNA recombination of PVX occurs preferentially at predicted stem–loop structures
Assuming that recombination occurs during minus-strand synthesis, all parental sequences containing regions of possible RdRp template switch (Supplementary Fig. S2) were used for RNA secondary-structure prediction with the program Mfold 3.2 (Zuker, 2003). Analysis of regions I–V (Fig. 3) revealed a folding that leads to stem–loop structures. Remarkably, four of five recombination sites overlapped with the predicted loop and one template switch could be located within the loop. Analysis of the remaining 22 junction sites with more uniform distribution resulted in the prediction of recombination sites overlapping with a predicted loop structure in 17 cases (data not shown).
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DISCUSSION |
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) provides an important detection system for the identification of homologous recombinants with high probability and frequency within the functional PVX genome. It has been demonstrated here that this recombination system presents a powerful tool to study factors influencing the parameters of virus RNA recombination. PVX is a member of supergroup III (alpha-like virus) with rod-shaped particle morphology, and possesses a replicase protein differing in structure and functional domains from those encoded by the recombination model plant viruses described so far (Goldbach & de Haan, 1994; Habili & Symons, 1989; Koonin & Dolja, 1993; reviewed by van der Heijden & Bol, 2002).
According to the current classification by Nagy & Simon (1997), replicative RNA recombination of model viruses characterized to date can be categorized into the following systems: similarity-essential (class 1, with either precise or aberrant end products), similarity non-essential (class 2) and similarity-assisted (class 3). In class 1, substantial sequence similarity is essential for recombination; RNA secondary stem–loop structures are required for class 2 recombination to occur; class 3 recombination combines features of classes 1 and 2. Our results obtained and described above enable the assignment of the observed recombination parameters to an RNA recombination type defined by Nagy & Simon (1997).
Recombination rate and selection pressure
The application of intact PVX CP genes with variable sequence composition enabled determination of the number and positions of template switches leading to the formation of the intact CP gene by means of homologous recombination. As explained above, we have no evidence to specify whether the recombinant sequence was a product of virus plus- or minus-strand synthesis. When recombinants that represent a product of a single RdRp template switch were detected, these could be generated following minus-strand transcription initiation on the 3'-ntr of the intact transcript and completion of the viral genome synthesis after one template switch upstream of the CP deletion. Alternatively, recombination could have occurred during plus-strand synthesis following 5'-ntr replication initiation, as the virus deletion mutant itself was replication-competent (Draghici & Varrelmann, 2009). When an intact CP gene containing several mutations was offered for reconstitution of the defective viral gene, several recombinants were shown to be a product of at least two to seven template switches, indicating that, in this assay, PVX recombines at a rate higher than required to reconstitute the deletion. Most of the type X CP (PVX-14, -17, -18 and -847)-derived recombinants were unique, demonstrating that no strong selection pressure was exerted on the selection of a recombinant with a specific amino acid composition. This might be explained by the fact that these relatively few coding differences do not affect the functionality of the recombinant CP. To our knowledge, this high rate of template switching is a unique observation, compared with previous studies where only the number of template switches necessary for functional reconstitution of the gene was reported (Allison et al., 1996; Borja et al., 1999; Gal-On et al., 1998; Varrelmann et al., 2000).
As the assay selects for precise homologous recombinants, all other recombination events, including those that lead to non-viable virus or debilitated mutants, remained undetected. It must be stressed that the recombination assay with high selection pressure does not allow deduction of the ability of PVX to recombine under natural conditions. Moreover, the viral genes and genomes were co-expressed in numerous cells via agroinfiltration. These artificial conditions might explain the frequent observation of recombinants, although evidence for this is currently lacking. The ability of the virus to recombine can only be depicted when minimal selection, i.e. in mixed infection, is applied (de Wispelaere et al., 2005), but recombinants do not occur frequently enough to study recombination parameters efficiently. However, in addition to the possible analysis of the recombination mechanism and parameters, the system might be useful to test virus-derived sequences for their ability to support recombination in virus-resistant transgenic plants. This will possibly help to evaluate biosafety issues before starting tedious plant-transformation experiments.
Recombination-mediated frequency of CP gene reconstitution is dependent on the length and similarity of homologous sequence overlap
The observation that the frequency of CP gene reconstitution was proportional to the length of the homologous sequence available for template switching is consistent with the findings of Nagy & Bujarski (1995). The relative recombination frequency observed in our assay obviously decreased linearly when the homologous sequence overlap was reduced from 600 to 50 nt. The conclusion may be drawn that the hypothetical heteroduplex formation (hybridization of parental strands), bringing homologous regions into proximity (Romanova et al., 1986), is dependent on the length of the homologous sequence, or is simply formed more frequently if the overlapping homologous region is longer. This confirmed the hypothesis that similarity is essential for precise reconstitution of the functional CP gene. This is strengthened by the observation that the sequence-divergent CP-4, although similar in length to CP-UK3, strongly reduced the frequency of CP gene reconstitution. More evidence is supplied by the observation that no recombination was detected on the scale of this assay when the overlap was reduced from 50 to 20 nt or below. On the other hand, the distance between mutations in CP-4–UK3 recombinants (Supplementary Fig. S2) varied between 2 and 21 nt, indicating that mismatches were tolerated at least to some extent in the homologous sequence stretch. Nagy & Bujarski (1995) provided evidence that nucleotide identity as short as 15 nt between parental BMV sequences supported efficient homologous recombination. In order to discern limits more precisely, deletion mutants with a more detailed resolution and higher number of independent repetitions are required. Taken together, these results prompted us to conclude that PVX homologous recombination does not occur frequently by a mechanism based on breakage and ligation, as described previously (see Introduction). To study replication-independent breakage and ligation recombination by host cell-encoded RNA ligase, two PVX deletion constructs would be required, separating the RdRp gene into two non-overlapping fragments. Although a critical length of homologous sequence overlap was determined with the system applied, non-replicative recombination by breakage and ligation cannot be excluded entirely.
Junction sites of homologous recombination are influenced by RNA secondary structure: evidence for similarity-assisted recombination
Intermolecular RNA secondary structures such as stem–loops have been shown to be important in directing non-homologous or similarity non-essential recombination sites in TCV, TBSV and cucumoviruses (Carpenter et al., 1995; Cascone et al., 1993; Chare & Holmes, 2006; de Wispelaere et al., 2005; Nagy & Simon, 1998; Nagy et al., 1998; Suzuki et al., 2003; White & Morris, 1994), whereas Nagy & Bujarski (1995) did not find a clear correlation between RNA secondary structure and homologous recombination sites. However, a high proportion (21 of 27) of independent recombination-junction sites between mutations were found in recombinants formed between X and Bi type CP genes and co-localized to predicted stem–loop structures. This finding provides strong evidence for an involvement of RNA secondary structure in precise homologous PVX recombination. These double-stranded structures might lead to RdRp stalling or template dissociation (Jager & Pata, 1999; Nagy & Simon, 1997) and possibly direct the template switch to the non-structured loop. This finding, however, is in contrast to the homologous recombination results obtained with BMV (Olsthoorn et al., 2002), where a reduction in recombinant accumulation and no evidence for enhanced RdRp pausing and detachment were detected when stem–loop structures were introduced into the parental sequences. On the other hand, it has been demonstrated for BMV that intercistronic subgenomic CP promoter (sgPr) sequences, consisting of an AU-rich region and an RdRp-binding stem–loop structure, increase homologous recombination activity (Bruyere et al., 2000; Nagy et al., 1999; Wierzchoslawski et al., 2004). However, as the PVX CP ORF represents the most 3' ORF in the genome, there is no evidence for the presence of an internal sgPr in the CP ORF. Considering that the experimental system selected amplification of only movement-competent CP genes and that only products of precise recombination events (no mismatch mutations in proximity to the junction sites) were detected, the results obtained allow classification of the precise recombination into the similarity-assisted group as defined by Nagy & Simon (1997). Another example for similarity-assisted recombination has been given (Cascone et al., 1993; Nagy et al., 1998) for TCV satellite RNA; however, recombination hot spots were observed to cluster at the base of a hairpin motif. By using this recombination system, it should be possible to reduce successively the sequence similarity of intact genes offered for recombination, in order to analyse the similarity-dependent limits of precise homologous recombination. Thereby, the functional interchangeability of CP genes from more distant potexviruses could be investigated and the occurrence of aberrant homologous or even non-homologous recombination (if possible at all in PVX) could be forced, as described for other viruses (Carpenter & Simon, 1996; Figlerowicz, 2000; Figlerowicz et al., 1998; Hajjou et al., 1996). In addition, this experimental system can be adapted for the easy and straightforward characterization of recombination parameters of other viruses, where full-length cDNA clones for agroinfection or particle bombardment are available. The detection of recombinants between phylogenetic distant X and Bi type CP genes demonstrates the suitability of RNA recombination under high selection pressure to design new viral RNA genomes with different biological properties. In future, PVX mutants or mutant libraries of the viral RdRp can be produced easily and assayed in vivo for their recombination abilities. Furthermore, the influence of RNA structural elements [i.e. internal enhancer elements, such as cis-acting elements; summarized by Hu et al. (2007)] and crossover sites displaying varying sequence similarity on recombination frequency and accuracy can be investigated.
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; Kirkegaard & Baltimore, 1986), PVX type X isolates (Santa Cruz & Baulcombe, 1995) from different geographical origins [isolates PVX-14, PVX-17 (both Germany), PVX-18 (Afghanistan) and PVX-847 (Iran), kindly supplied by Stefan Winter (Plant Virus Department, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany)] were obtained and propagated on N. benthamiana plants. A PVX subtype Bi isolate, PVX-4 (group 4) (Santa Cruz & Baulcombe, 1995), displaying higher sequence divergence from UK3, was kindly provided by Rene van der Vlugt (Plant Research International, Wageningen, the Netherlands).
Sequence analysis.
Sequence alignment was carried out by using the neighbour-joining algorithm in the CLUSTAL_X version 1.83 program (Thompson et al., 1997). The RNA secondary structures of parental (+)-strand CP gene+3'-ntr sequences were predicted for PVX-UK3 and PVX-4 at 37 °C with 150 bp maximum distance between paired bases by using Mfold version 3.2 (http://frontend.bioinfo.rpi.edu/applications/mfold/cgi-bin/rna-form1.cgi) based on free-energy minimization (Mathews et al., 1999; Zuker, 2003).
Clones and infectious transcripts.
The cauliflower mosaic virus (CaMV) 35S promoter-driven PVX plant expression plasmid cloned in a binary vector for agroinfection (pgR106; GenBank accession no. AY297843
[GenBank]
) (Jones et al., 1999; Lu et al., 2003) used in this study was supplied by David Baulcombe (Sainsbury Laboratory, Norwich, UK). The construction of a PVX (isolate UK3) plant expression vector expressing GFP, named 35S-PVX-GFP, was described previously (Draghici & Varrelmann, 2009).
35S-PVX-GFP-derived mutants.
The identity of clones was confirmed via DNA sequencing by a commercial company (Eurofins MWG Operon, Martinsried, Germany). All recombinant plasmid manipulations were carried out as described by Sambrook et al. (1989) unless otherwise specified. The construction of a movement-defective PVX CP mutant with a 514–646 nt PvuII deletion, named 35S-PVX-GFP-CP, was described previously (Draghici & Varrelmann, 2009). To generate CP deletion mutants with different lengths in 35S-PVX-GFP, the following strategy was applied. The 3' end of the GFP gene, the sgPr and the 5' end of the CP gene with variable lengths were PCR-amplified with specific primers using 35S-PVX-GFP as template. The upper primer CP-del-up (5'-AATCTGCCCTTTCGAAAGAT-3') (in all primers, restriction sites are underlined and virus-specific sequences are in italics), homologous to a part of the GFP ORF, contained a Bsp119I restriction site for subsequent exchange in 35S-PVX-GFP and was used for the generation of all CP deletion mutants. Different lower CP primers (supplied with an XhoI restriction site) were used. Together with primer CP-del-up, this approach produced CP gene fragments of different lengths. The different PCR fragments were Bsp119I/XhoI-cloned into 35S-PVX-GFP, replacing most of the CP ORF and resulting in the generation of CP mutants with a 3' end of fixed length (nt 653–711) and a 5' end of variable length (0, nt 0–10, –20, –50, –101, –200, –300, –401, –500 and –600, respectively). The resulting clones were named 35S-PVX-GFP-CP-0, -10, -20, -50, -101, -200, -300, -401, -500 and -600, respectively (Fig. 1) and were subsequently applied in the recombination assay.
Construction of binary vectors expressing complete CP gene+3'-ntr in vivo transcripts derived from different PVX isolates.
The exact CP ORF and complete 3'-ntr regions of PVX-UK3, PVX-14, PVX-17, PVX-18 and PVX-847 were RT-PCR-amplified from total RNA extracts (RNeasy; Qiagen) of virus-infected N. benthamiana leaf tissue, introducing flanking SacI and BamHI restriction sites. The following primers were used: CP-up (5'-TTAGAGCTCACCATGTCAGCACCAGCTAGCAC-3') and poly-T-low (Draghici & Varrelmann, 2009). In order to amplify the respective sequence from a PVX Bi type isolate, PVX-4, a derivative upper primer, CP4-up (5'-TTAGAGCTCACCATGACTACACCAGCTAGCAC-3'), was applied. The PCR fragments were SacI/BamHI-cloned into a binary vector, pBIN61S, under control of the enhanced CaMV 35S promotor and poly(A) terminator (Silhavy et al., 2002). Plasmid inserts were sequenced and named 35S-CP-UK3, -PVX-14, -PVX-17, -PVX-18, -PVX-847 and -PVX-4.
Cloning and sequencing of CP gene and 3'-ntr from recombinant PVX isolates.
Upper leaves of N. benthamiana plants were selected for the recombination assay. Leaves displaying systemic infection, as monitored by GFP fluorescence, were used for total RNA extraction and RT-PCR amplification of the PVX CP gene and 3'-ntr, as described previously (Draghici & Varrelmann, 2009).
Recombination assay.
In general, the recombination assay was performed as described previously (Draghici & Varrelmann, 2009). To produce PVX-GFP-UK3 with a recombinant CP gene, 35S-PVX-GFP-CP was co-infiltrated with 35S-CP-UK3, -PVX-14, -PVX-17, -PVX-18, -PVX-847 or -PVX-4. Agroinfiltrated leaf patches were examined for GFP fluorescence with a hand-held long-wave UV lamp (Black Ray model B100 AP, 100 W, UV products) to visualize occurrence and movement of recombinant PVX–GFP. Photographs were taken using a digital camera (Nikon D50).
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ACKNOWLEDGEMENTS |
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, Hungary) is acknowledged for providing the pBIN61S plasmid. We thank M. Stevens for critically reading the manuscript. This research was financed by a fellowship (grant no. VA202/3-2) of the Deutsche Forschungsgemeinschaft. |
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Received 30 June 2009;
accepted 23 October 2009.
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