1 Plant Virus GenBank, Division of Environmental and Life Sciences, Seoul Women's University, Seoul 139-774, Korea
2 Scottish Crop Research Institute, Invergowrie DD2 5DA, UK
3 Division of Biological Environment, Kangwon National University, Chuncheon 200-701, Korea
Correspondence
Ki Hyun Ryu
ryu{at}swu.ac.kr
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ABSTRACT |
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Present address: Department of Biochemistry and Biophysics, Texas A & M University, TX 77843, USA.
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INTRODUCTION |
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Various CMV-encoded proteins or combinations of proteins have been implicated in systemic infection in cucurbits (Roossinck, 1991; Saitoh et al., 1999
; Takeshita et al., 2001
) besides those already mentioned. Moreover, a variant of Sny-CMV with two amino acid alterations in the 3a movement protein (MP) increased the accumulation of MP up to 50-fold in tobacco (Gal-On et al., 1996
), but prevented systemic infection in several cucurbit species (Kaplan et al., 1997
). Thus, the factors limiting infection and rates of movement can be subtle and complex, even in the same species or the same cultivar. Most strains of CMV used in previous studies (Owen & Palukaitis, 1988
; Zaitlin et al., 1994
) showed similar symptoms and rates of systemic symptom development to that of Sny-CMV in zucchini squash cv. Black Beauty, i.e. 57 days post-inoculation (p.i.). However, a pepper strain of CMV isolated in Florida (Pf-CMV; Simon, 1957
; Owen & Palukaitis, 1988
) developed much milder symptoms in zucchini squash, starting at 9 days p.i. Unlike M-CMV, systemic infection of zucchini squash by Pf-CMV was not restricted, but there was an apparent delay in symptom development, systemic movement or both. Therefore, to gain further insight into the various interactions between viruses and hosts leading to a delay in symptom development, it was of interest to characterize the nature and genetic basis of the differences in pathogenicity associated with Pf-CMV. This was done by comparison with the better-characterized and faster-moving Fny-CMV used in similar previous studies (Gal-On et al., 1994
; Roossinck & Palukaitis, 1990
; Wong et al., 1999
).
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METHODS |
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Construction of full-length cDNA clones of Pf-CMV RNAs 1, 2 and 3.
Pf-CMV (Owen & Palukaitis, 1988) viral RNAs were extracted from purified virus particles using buffered SDS/phenol extraction (Choi et al., 1999
; Gal-On et al., 1994
). The purified viral RNAs were used as templates for reverse transcription (RT) and PCR. PCR was carried out in a 50 µl reaction volume contained 5 µl RT solution, 5 µl 10x Expand Long Template PCR buffer (Roche Diagnostics) containing 22·5 mM MgCl2, 10 ng forward primers (Rizzo & Palukaitis, 1990
) for RNAs 13 of Pf-CMV, 10 ng reverse primer (Rizzo & Palukaitis, 1990
), 1 mM dNTPs and 2·5 U Expand Long Template Enzyme mixture (Roche) in a programmable thermal cycler (iCycler; Bio-Rad Laboratories), as previously described by Yoon et al. (2002)
. The synthesized, full-length RT-PCR products were directly digested by BamHI and PstI and the digested fragments were purified from an agarose gel using the QIAquick Gel Extraction kit (Qiagen), following the manufacturer's instructions. The purified cDNAs of Pf-CMV were ligated into linearized pUC18, previously digested with PstI and BamHI, to generate constructs pPf-CMV1, pPf-CMV2 and pPf-CMV3 for Pf-CMV RNAs 1, 2 and 3, respectively.
Construction and characterization of pseudorecombinants of Pf- and Fny-CMV.
Fny-CMV cDNA clones pFny109, pFny209 and pFny309 (Rizzo & Palukaitis, 1990) and Pf-CMV clones pPf-CMV1, pPf-CMV2 and pPf-CMV3 were linearized at the 3' end of the inserted sequence using PstI and blunt-ended as described by Zhang et al. (1994)
. Infectious RNAs were generated by in vitro transcription with T7 RNA polymerase (Promega) in the presence of the cap analogue m7GpppG (New England Biolabs) following a procedure described previously (Canto & Palukaitis, 1998
). RNA transcripts from full-length cDNA clones of Pf- and Fny-CMV RNAs 1, 2 and 3 were combined to form the parental RNAs P1P2P3 and F1F2F3, as well as six different pseudorecombinants, F1F2P3, F1P2F3, F1P2P3, P1F2F3, P1F2P3 and P1P2F3. Each parental and pseudorecombinant RNA, without further purification, was inoculated (100250 µg ml1) on to tobacco plants. Subsequently, zucchini squash plants were inoculated mechanically on both cotyledons with sap from the parental and pseudorecombinant CMV-infected tobacco plants. All progeny viruses of the pseudorecombinants were characterized by RT-PCR analysis or by direct sequencing.
Generation of chimeric cDNA constructs between Pf- and Fny-CMVs.
Four sets of reciprocal RNA 3 chimeras were constructed between Pf- and Fny-CMV using common restriction endonuclease sites. The first set of RNA 3 chimeras was constructed using the common BamHI site located upstream of the T7 promoter and the SalI site, which is in the CP gene. A second set of RNA 3 chimeras was constructed by exchanging a fragment using the BamHI and NheI sites, the latter in the 3a gene. A third set of RNA 3 chimeras was constructed using the BamHI and NaeI sites, the latter also in the 3a gene. The fourth set of RNA 3 chimeras was constructed using the NaeI and NheI sites, both within the 3a gene. In vitro transcripts were generated from these RNA 3 cDNA constructs: FP3Sal and PF3Sal (the SalI reciprocals), FP3Nhe and PF3Nhe (the NheI reciprocals), FP3Nae and PF3Nae (the NaeI reciprocals), and FP3Nae/Nhe and PF3Nae/Nhe (the NaeINheI reciprocals). Each of the synthesized chimeric RNA 3s was mixed with transcripts of either Fny-CMV RNAs 1 and 2 or Pf-CMV RNAs 1 and 2 and was inoculated on to tobacco plants. Zucchini squash plants were inoculated with sap derived from tobacco plants infected with the various RNA 3 chimeric viruses.
Three reciprocal sets of RNA 2 chimeras were constructed between Pf- and Fny-CMV. The first set of reciprocal RNA 2 chimeras was constructed using the common PstI site located at the 3' end of the viral RNA sequence and the XbaI site, which was in the central region of the 2a gene. The second set of RNA 2 chimeras was constructed by exchanging a fragment obtained by digestion at the two KpnI sites, one located in the pUC18 vector and the other in the 2a gene. The third set of RNA 2 chimeras was generated using both reciprocal KpnI-digested fragments and KpnIXbaI fragments derived from pFny209 and pPf-CMV2 using three-piece ligations into pFny209 or pPf-CMV2 previously digested with KpnI and XbaI. After introduction of the plasmids into Escherichia coli, the orientations of the resulting inserts in the cDNA clones were identified by digestion with various restriction enzymes. All chimeric constructs were analysed by partial sequencing or restriction enzyme digestions. Infectious in vitro transcripts generated from the chimeric cDNA clones of RNA 2 were mixed with transcripts of either Fny-CMV RNAs 1 and 3 or Pf-CMV RNAs 1 and 3 and were inoculated on to tobacco plants. Zucchini squash plants were inoculated with sap derived from tobacco plants infected with the various RNA 2 chimeric viruses.
Site-directed mutagenesis.
Site-directed mutagenesis was performed to alter either nt 886 of Fny- and Pf-CMV RNA 2 or nt 622 of Fny- and Pf-CMV RNA 3 by the two-step PCR procedure described by Higuchi et al. (1988) using the QuikChange Site-directed Mutagenesis kit (Stratagene). The complementary paired primers covering and flanking the mutation sites were 5'-CCAACGCCAACCCTCGCGACTCCTCCGGATTTAAACCGTGC-3' (plus strand) and 5'-GCACGGTTTAAATCCGGAGGAGTCGCGATGGTTGGCGTTGG-3' (minus strand) for nt 886 of Fny-RNA 2 (mutated nucleotide shown in bold and underlined); 5'-GTTATCGAAAGACATGGTTACACTGGGTATACCGGTACCACAGC-3' (plus strand) and 5'-GCTGTGGTACCGGTATACCCAGTGTAACCATGTCTTTCGATAAC-3' (minus strand) for nt 622 of Fny-RNA 3; 5'-GTCCAACGCCAACCATCGCGATTCCTCCGGATTTAAACCGTG-3' (plus strand) and 5'-CACGGTTTAAATCCGGAGGAATCGCGATGGTTGGCGTTGGAC-3' (minus strand) for nt 886 of Pf-CMV RNA 2; and 5'-GTTATCGAAAGACATGGTTACATTGGGTATACCGGCACCACAG-3' (plus strand) and 5'-CTGTGGTGCCGGTATACCCAATGTAACCATGTCTTTCGATAAC-3' (minus strand) for nt 622 of Pf-CMV RNA 3. These primers were used sequentially to generate the mutants in RNAs 2 and 3, respectively. The reciprocal mutant viruses designated Fny-T/C-CMV and Pf-C/T-CMV were then generated from infectious transcripts of the corresponding plasmids, containing point mutations in RNAs 2 and 3, in combination with wild-type Fny-CMV RNA 1 (Higuchi et al., 1988
). The point mutations in Fny-T/C- and Pf-C/T-CMV were confirmed by sequencing the respective RT-PCR products obtained after mutagenesis, as well as from infected tobacco plants, prior to inoculation of the squash cotyledons.
Preparation and electroporation of zucchini squash protoplasts.
Mesophyll protoplasts were prepared from the fully expanded cotyledons of zucchini squash by modification of previously described protocols (Gal-On et al., 1994; Lee et al., 2001
). Specifically, the detached cotyledons were surface sterilized for 5 min in 0·5 % hypochlorite and rinsed extensively with distilled water. The lower epidermis of the cotyledons was peeled away and the remaining tissue was incubated for 4 h with 20 ml 0·35 M mannitol containing 1·2 % (w/v) Cellulase Onozuka R10 and 0·4 % (w/v) Macerozyme (both from Yakult Pharmaceutical). The residual leaf skeletons were removed and the protoplasts from several cotyledons were pooled, filtered through pre-wetted Miracloth and collected by centrifugation at 37 g for 5 min. The protoplasts were resuspended in 0·35 M mannitol (pH 5·6) and centrifuged as above, twice more. The protoplasts were then diluted to 0·5x106 protoplasts ml1 in 0·35 M mannitol. Electroporation was carried out using 5 µg each RNA transcript at 260 V for 5 ms in an electroporation apparatus (Bio-Rad Laboratories). The protoplasts were then incubated for 16 h in the light in a temperature-controlled growth chamber.
Extraction and analysis of CMV RNAs.
Northern blot hybridization analysis was done to detect CMV RNA accumulation in leaves and protoplasts of zucchini squash. About 200 mg leaf tissue or 1 ml protoplast solution (0·5x106 cells ml1) was extracted with 400 µl 50 mM Tris/HCl (pH 8·0) containing 10 mM EDTA and 2 % SDS. The samples were then extracted with phenol and the RNAs precipitated using standard protocols (Sambrook et al., 1989). Total RNAs were fractioned by electrophoresis on a 1·6 % agarose/formaldehyde denaturing gel and transferred to positively charged nylon membranes (Roche Diagnostics) by the capillary method. The membrane was cross-linked by UV and hybridized to DIG-labelled RNA probes complementary to the 3' non-coding region of all Fny-CMV RNAs (Gal-On et al., 1994
). Labelled RNA probes were detected using the DIG Luminescent Detection kit with CSPD (Roche Diagnostics). All experiments were performed at least three times.
Leaf detachment and leaf-print assays.
Inoculated zucchini squash cotyledons were detached at 12 h intervals from 12 to 96 h p.i. and symptoms were assessed at 10 days p.i., as described previously (Gal-On et al., 1994). Inoculated zucchini squash cotyledons were subjected to leaf-press blot analysis at 3, 5 and 9 days p.i. using antiserum to CMV CP, as described previously (Gal-On et al., 1994
).
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RESULTS |
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Mapping of sequences in Pf-CMV RNAs 2 and 3 involved in delayed infection
Chimeric plasmids were constructed between those containing biologically active, full-length cDNAs of RNA 3 from Pf- and Fny-CMV to delimit further the sequences in RNA 3 responsible, in part, for the differences in phenotype. These were prepared using common restriction endonuclease cleavage sites to generate three pairs of reciprocal, single-exchange chimeras and one pair of reciprocal, double-exchange chimeras (Fig. 3). RNA transcripts of these chimeric RNA 3s were mixed with either Pf-CMV RNAs 1 and 2 or Fny-CMV RNAs 1 and 2, and the types of symptom induced on zucchini squash were assessed. Those RNA 3 chimeras in which the central portion of the MP was derived from Pf-CMV RNA 3 induced the same delayed, chlorotic spot phenotype with Pf-CMV RNAs 1 and 2 as did Pf-CMV RNAs 1, 2 and 3. By contrast, the reciprocal RNA 3 chimeras, in combination with Fny-CMV RNAs 1 and 2, induced the same rapid, systemic mosaic phenotype as Fny-CMV RNAs 1, 2 and 3 (Fig. 3
). The differences in virulence mapped to a region of the encoded 3a MP between the NaeI and NheI restriction sites (nt 247 and 643). This region of the MP contained six, silent, nucleotide differences (not shown), but only one nucleotide (nt 622) encoding an amino acid difference between Fny- and Pf-CMV: Thr-168 in the Pf-CMV 3a MP compared with Ile-168 in the Fny-CMV 3a MP (Fig. 3
).
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Effect of sequence differences on virus replication and movement
To determine whether the differences in sequence between RNA 2 and/or RNA 3 of Pf-CMV compared with Fny-CMV had a significant effect on the replication of the CMV RNAs, the accumulation of various viral RNAs was assessed in zucchini squash protoplasts. The inoculated protoplasts were assessed for virus accumulation at 17 h p.i., since this time after inoculation has previously been shown to reflect the linear phase of CMV RNA replication in zucchini squash protoplasts (Gal-On et al., 1994). Levels of CMV RNA accumulation were virtually identical at this time point for the parental CMV RNAs (Fig. 5
, lanes 1 and 11), as well as the RNAs of the six pseudorecombinant viruses (Fig. 5
, lanes 27), the two double chimeric viruses (Fig. 5
, lanes 8 and 9) and the double mutant designated Fny-T/C-CMV (Fig. 5
, lane 10). Thus, it seemed unlikely that the sequence alterations in either Pf-CMV RNA 2 or 3 significantly affected virus replication.
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To determine whether the differences between Pf- and Fny-CMV that affected the rates of systemic movement as well as the time of appearance and severity of systemic symptoms also affected the rate of local movement in the inoculated cotyledons, leaf-press blots were made from infected zucchini squash cotyledons (Fig. 6). At 3 days p.i., Fny-CMV and the double-chimeric virus F1FP2Kpn/Xba-FP3Nae/Nhe had both invaded a large number of cells in the inoculated cotyledon, while Pf-CMV and the reciprocal double-chimeric virus P1PF2Kpn/Xba-PF3Nae/Nhe showed far fewer cells with virus accumulation (Fig. 6
). At 5 days p.i., the latter two viruses showed larger foci of infection, but were still limited to a few defined zones, while the former two viruses showed more extensive invasion and accumulation throughout most of the cotyledons (Fig. 6
). At 9 days p.i., more cells were infected by Pf-CMV and P1FP2Kpn/Xba-FP3Nae/Nhe than at 5 days p.i., but this was still less than seen for Fny-CMV and F1PF2Kpn/Xba-PF3Nae/Nhe at 3 days p.i. (Fig. 6
). Thus, the sequence alterations that affected the rates of systemic invasion also affected local movement of the corresponding viruses.
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DISCUSSION |
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Several studies with other viruses have implicated the polymerase protein as being involved in virus movement (Chen et al., 1996; Deom et al., 1997
; Nelson & van Bel, 1998
; Hirashima & Watanabe, 2001
, 2003
). Previous work with CMV has shown that two changes in the polymerase protein affected the elicitation of a hypersensitive response and restriction of CMV to the local lesion in cowpea (Kim & Palukaitis, 1997
). Two other studies have implicated both RNA 2 and 3 as being involved in host-specific infection in Lactuca saligna (Edwards et al., 1983
) and radish (Takeshita et al., 1998
). However, in those cases, resistance breakage required changes in both RNA 2 and 3.
Although the CMV 2b protein has been shown to affect virus movement (reviewed by Palukaitis & García-Arenal, 2003), the phenotype described here did not involve sequence differences in the 2b protein. Nevertheless, it is conceivable that the role of the 2a protein in affecting virus movement might relate to differential expression of the subgenomic RNA encoding the 2b protein. Although this seems unlikely, it cannot be ruled out at this time (Wang et al., 2004
).
Neither aa 267 of the 2a protein nor aa 168 of the 3a protein has been implicated previously in any phenotype. Aa 267 of the 2a protein is between two domains (aa 1126 and 290335) in the N-terminal half of the 2a protein shown to be phosphorylated in vitro (Kim et al., 2002). However, it is not known whether the presence of yet another Thr residue in this region of the 2a protein might affect phosphorylation of either domain. Similarly, the presence of another Thr residue in the 3a protein potentially could affect phosphorylation of the 3a protein, leading to some change in specificity of the respective protein. Aa 168 of the 3a protein is located in a putative zinc finger domain (aa 157194) and at the end of a putative nucleic acid binding domain (aa 164168) (Li et al., 2001
). Thus, it is conceivable that the nucleotide sequence change leading to a Thr residue at aa 168 may have affected the function of the MP with regard to RNA binding or entry to the vasculature for systemic infection. Although we do not know why such changes lead to host-specific alterations in movement, such observations are not without precedence. Mutation of aa 20 and 21 of the 3a MP of CMV has been shown to affect movement between epidermal cells and affect local lesion production in cowpea and Chenopodium spp. (Canto & Palukaitis, 1999a
, b
; Li et al., 2001
), while mutation of aa 51 affects movement in bottle gourd (Takeshita et al., 2001
) and aa 51 plus aa 240 affect systemic movement in several other cucurbit species (Kaplan et al., 1997
). Such effects are not limited to CMV in cucurbits. Mutation at one or more of four positions within the 3a gene of Brome mosaic virus (BMV) resulted in a variable frequency of systemic infection in cowpea plants, depending on both the nature of the mutation(s) and the cultivar of cowpea (De Jong et al., 1995
). Moreover, a single codon change in a conserved motif of the BMV 3a gene conferred compatibility with a new host (Fujita et al., 1996
).
It is not known whether the 3a MP interacts directly with either of the components of the CMV replicase, the 1a or 2a proteins (Hayes & Buck, 1990). It also remains to be determined whether phosphorylation of the MP affects its ability to interact with other viral components. Establishing these criteria should provide a better framework within which to consider models for the basis of how the viral polymerase might be affecting viral movement and why in some situations the effects of sequence alterations in the 2a and 3a genes are functionally independent, while in other cases they appear to be co-dependent.
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ACKNOWLEDGEMENTS |
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Received 29 October 2004;
accepted 14 December 2004.