Cucumber mosaic virus 2a polymerase and 3a movement proteins independently affect both virus movement and the timing of symptom development in zucchini squash

Seung Kook Choi1,{dagger}, Peter Palukaitis2, Byoung Eun Min1, Mi Yeon Lee1, Jang Kyung Choi3 and Ki Hyun Ryu1

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The basis for differences in the timing of systemic symptom elicitation in zucchini squash between a pepper strain of Cucumber mosaic virus (Pf-CMV) and a cucurbit strain (Fny-CMV) was analysed. The difference in timing of appearance of systemic symptoms was shown to map to both RNA 2 and RNA 3 of Pf-CMV, with pseudorecombinant viruses containing either RNA 2 or RNA 3 from Pf-CMV showing an intermediate rate of systemic symptom development compared with those containing both or neither Pf-CMV RNAs. Symptom phenotype was shown to map to two single-nucleotide changes, both in codons for Ile at aa 267 and 168 (in Fny-CMV RNAs 2 and 3, respectively) to Thr (in Pf-CMV RNAs 2 and 3). The differential rate of symptom development was shown to be due to differences in the rates of cell-to-cell movement in the inoculated cotyledons, as well as differences in the rate of egress of the virus from the inoculated leaves. These data indicate that both the CMV 3a movement protein and the CMV 2a polymerase protein affect the rate of movement of CMV in zucchini squash and that these two proteins function independently of each other in their interactions with the host, facilitating virus movement.

{dagger}Present address: Department of Biochemistry and Biophysics, Texas A & M University, TX 77843, USA.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains of a plant virus are usually defined by differences in biological properties (Hull, 2002). Such differences may be host specific. For example, the Fny, Pf, A9 and LS strains of Cucumber mosaic virus (CMV) all infect tobacco and cucumber, but show differences in infection and pathogenicity in Solanum spp. (Valkonen et al., 1995). Similarly, the Fny and Sny strains were differentiated initially on the basis of the timing and intensity of symptom development in zucchini squash (Cucurbita pepo), although these strains did not show such differences in tobacco or cucumber. In the case of Fny- and Sny-CMV, these host-specific differences were due to changes in the 1a protein, affecting the rates of both cell-to-cell and systemic movement of virus, but not replication (Gal-On et al., 1994; Roossinck & Palukaitis, 1990), which in turn resulted in different rates of systemic symptom development. By contrast, in the case of the M strain of CMV, very slow movement in inoculated cotyledons of zucchini squash correlated with a lack of systemic infection, associated with two specific amino acid alterations in the capsid protein (CP) (Wong et al., 1999). In that instance, the inability to infect plants systemically was due to a host-specific response restricting movement, since M-CMV infected tobacco and cucumber systemically with the same timing as other strains of CMV (Rao & Francki, 1982). Co-infection of M-CMV with a potyvirus was able to neutralize the restriction of systemic movement by M-CMV (Choi et al., 2002).

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. 5–7 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).


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Plants, virus source and plant maintenance.
Tobacco plants (Nicotiana tabacum cv. Xanthi-nc) at the four-leaf stage were used for propagation of virus as described previously (Canto et al., 2001). Both Fny- and Pf-CMV were propagated in tobacco plants and purified essentially by the method of Peden & Symons (1973). Inocula were sap from CMV-infected plants, purified virus or in vitro transcripts generated from cDNA clones (Zhang et al., 1994). Cotyledons of zucchini squash (C. pepo cv. Black Beauty) were inoculated with sap from infected tobacco plants or various transcripts of pseudorecombinants and chimeras as described below. Inoculated plants were grown in controlled greenhouse conditions with the temperature at 26 °C (day) and 20 °C (night).

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 1–3 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 (100–250 µg ml–1) 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 NaeI–NheI 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 KpnI–XbaI 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 ml–1 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 ml–1) 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).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Association of RNAs 2 and 3 with delayed infection by Pf-CMV
The genetic basis for the delayed viral symptom response to infection by Pf-CMV was first delimited by the use of mixed inocula consisting of the Pf-CMV RNAs combined with Fny-CMV RNA 1, 2 or 3, generated from RNA transcripts of full-length cDNA clones. This approach has been validated previously as a preliminary screen (Ryu et al., 1998). The mixed inocula were established first on tobacco plants, where they reached similar virus titres (not shown), and were then used to inoculate zucchini squash cotyledons. The timing of symptom development as well as the nature of the symptom response by 14 days p.i. was determined. Plants infected with Pf-CMV alone as well as those infected with Pf-CMV RNAs and Fny-CMV RNA 1 (abbreviated to P1P2P3+F1) both developed mild symptoms consisting of chlorotic spots on systemic leaves starting at 9 days p.i. (Table 1). These symptoms were much milder than the severe stunting and mosaic symptoms developed after infection by Fny-CMV alone, beginning at 4 days p.i. (Table 1 and Fig. 1). Addition of Fny-CMV RNA 2 or 3 to Pf-CMV in the inoculum resulted in intermediate symptoms developing in the zucchini squash plants, beginning at 6 or 7 days p.i. (Table 1). Thus, while Pf-CMV RNA 1 did not appear to affect the delayed symptom response, Pf-CMV RNAs 2 and 3 both showed some effect that could be compensated for by the addition of the corresponding Fny-CMV RNAs. However, addition of either Fny-CMV RNA 2 or 3 alone did not completely reverse the delay and mild symptom response associated with infection by Pf-CMV.


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Table 1. Severity and timing of symptoms in zucchini squash after inoculation with Pf-CMV, Fny-CMV, pseudorecombinants and mutated Fny-CMV

 


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Fig. 1. Systemic symptoms at 14 days p.i. on zucchini squash plants inoculated with CMV strains, pseudorecombinant RNAs, chimeras and the mutant viruses Fny-T/C- and Pf-C/T-CMV. Fny, Fny-CMV; Pf, Pf-CMV; P1F2F3 and F1P2P3, pseudorecombinant viruses contained the indicated RNAs from Fny- and Pf-CMV; P1FP2Kpn/Xba-FP3Nae/Nhe, a pseudorecombinant virus containing RNA 1 of Pf-CMV and chimeric recombinants of RNAs 2 and 3, as indicated in the text; F1PF2Kpn/Xba-PF3Nae/Nhe, a pseudorecombinant virus containing RNA 1 from Fny-CMV and chimeric recombinants of RNAs 2 and 3, as indicated in the text; Pf-C/T and Fny-T/C, Pf- and Fny-CMV containing point mutations from C to T and from T to C, respectively, at nt 886 of RNA 2 and nt 622 of RNA 3 in the respective cDNA clones; Mock, mock-inoculated leaves.

 
Accumulation of viral RNAs in the non-inoculated leaves of the above infected plants was ascertained at 7 days p.i. (Fig. 2), a time at which plants infected with Pf-CMV or Pf-CMV+Fny-1 had not yet shown the development of systemic symptoms but plants infected with the other inocula combinations had (Table 1). This analysis showed that, although the plants infected with either Pf-CMV alone or Pf-CMV+Fny-1 did not display symptoms, they already contained low levels of accumulated viral RNAs, while those plants infected with Pf-CMV+Fny-2, Pf-CMV+Fny-3 or Fny-CMV alone, all of which displayed symptoms before or at 7 days p.i., accumulated higher levels of viral RNAs (Fig. 2). The levels of accumulation of the Fny-CMV RNAs were consistently higher than the other two mixed inocula, probably because zucchini squash plants infected by Fny-CMV already showed systemic symptoms by 4 days p.i. (Table 1) and the Fny-CMV RNAs were detectable in the systemic leaves at 3 days p.i. (Gal-On et al., 1994). These differences were not due to differential ability of the Fny-CMV probe to hybridize to the Fny-CMV RNAs versus the Pf-CMV RNAs (Owen & Palukaitis, 1988).



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Fig. 2. Accumulation of CMV RNAs on zucchini squash plants inoculated with either wild-type or mixed CMV RNAs. The RNA mixtures indicated below were inoculated on to zucchini squash cotyledons and total RNAs extracted at 7 days p.i. were analysed by Northern blot hybridization. Arrows indicate the positions of CMV RNAs in the upper panel. Pf, Pf-CMV RNAs; Pf+1, Pf-CMV RNAs plus RNA 1 of Fny-CMV; Pf+2, Pf-CMV RNAs plus RNA 2 of Fny-CMV; Pf+3, Pf-CMV RNAs plus RNA 3 of Fny-CMV; Fny, Fny-CMV RNAs; Healthy, mock-inoculated leaves. The lower panel shows stained rRNA loading controls from the various samples.

 
The above data suggested that both Pf-CMV RNAs 2 and 3 were involved in the differential infection phenotypes of Pf-CMV compared with Fny-CMV. To verify this, both parental and all six reassorted viruses were generated from biologically active cDNA clones and tested on zucchini squash. Again, both the timing of systemic symptom development and the intensity of symptoms produced were assessed (Table 1). Plants infected by all combinations of viral RNAs that contained either Fny-CMV RNA 2 or 3 showed an intermediate timing of symptom development and an intermediate symptom response compared with those plants infected by the Fny- and the Pf-CMV RNAs. However, plants infected by the viruses designated P1F2F3, which contained Fny-CMV RNAs 2 and 3, showed the same timing and severe symptoms as plants infected by the reconstituted Fny-CMV (F1F2F3) (Table 1 and Fig. 1). Likewise, plants infected by the reciprocal combination, F1P2P3, showed the same timing and severity of symptoms as the reconstituted Pf-CMV (P1P2P3) (Table 1 and Fig. 1). Thus, both RNAs 2 and 3 of Pf-CMV contain determinants limiting infection of Pf-CMV relative to Fny-CMV.

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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Fig. 3. Localization of sequences in CMV RNA 3 specifying the phenotypes of the chimeric CMVs on zucchini squash. Pf-CMV RNAs 1 and 2 (P1+P2) or Fny-CMV RNAs 1 and 2 (F1+F2) were combined with transcripts of cDNA clones containing various RNA 3 chimeras and inoculated on to tobacco plants. Virus from these infected tobacco plants was then inoculated on to zucchini squash cotyledons. Systemic symptoms were assessed at 7 days p.i. as either chlorotic spots (CS) or severe mosaic (SM). RNA 3 transcripts were derived from the following plasmids to generate the corresponding viruses (given in parentheses): pPf-CMV3 (Pf-CMV); pFny309 (Fny-CMV); pPF3Sal (PF3Sal-CMV); pFP3Sal (FP3Sal-CMV); pPF3Nhe (PF3Nhe-CMV); pFP3Nhe (FP3Nhe-CMV); pPF3Nae (PF3Nae-CMV); pFP3Nae (FP3Nae-CMV); pPF3Nae/Nhe (PF3Nae/Nhe-CMV); pFP3Nae/Nhe (FP3Nae/Nhe-CMV). The transcripts of RNAs 1 and 2 used for inoculation with RNA 3 transcripts of chimeric or wild-type clones are indicated at the top of the figure. Pf-CMV RNA 3 sequences are indicated by open rectangles and Fny-CMV RNA 3 sequences by filled rectangles. The encoded amino acids at positions 7 and 168 in the 3a gene that differ between the two strains are indicated at the top, with the amino acid difference correlating to the change in phenotype circled. The locations of restriction endonuclease sites used for making the chimeras are shown at the bottom.

 
To identify those sequences in Pf-CMV RNA 2 that were associated with the difference in phenotype between Pf- and Fny-CMV, two pairs of reciprocal, single-exchange chimeras and one pair of reciprocal, double-exchange chimeras were constructed (Fig. 4). RNA transcripts of these chimeras were combined with either Fny-CMV RNAs 1 and 3 or Pf-CMV RNAs 1 and 3, and the symptoms induced on zucchini squash were assessed. The differences in virulence mapped to a region of the 2a polymerase protein between the KpnI and XbaI restriction sites (nt 840 and 1209). Those chimeras in which this portion of the encoded 2a polymerase protein was derived from Pf-CMV RNA 2 induced the same delayed, chlorotic spot phenotype with Pf-CMV RNAs 1 and 3 as Pf-CMV RNAs 1, 2 and 3. On the other hand, the reciprocal chimeras in combination with Fny-CMV RNAs 1 and 3 induced the same rapid, systemic, mosaic phenotype as Fny-CMV RNAs 1, 2 and 3 (Fig. 4). Although the 2a protein encoded by Fny-CMV RNA 2 differed from the 2a protein encoded by Pf-CMV RNA 2 by three amino acids, only one amino acid difference, Ile-267 in the Fny-CMV 2a protein compared with Thr-267 in the Pf-CMV 2a protein, was present in the delimited region (Fig. 4), corresponding to differences at nt 886. There were also four silent nucleotide changes in this region (not shown).



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Fig. 4. Localization of sequences in CMV RNA 2 specifying the phenotypes of CMV strains on zucchini squash. Either Pf-CMV RNAs 1 and 3 (P1+P3) or Fny-CMV RNAs 1 and 3 (F1+F3) were combined with transcripts of cDNA clones containing various RNA 3 chimeras and inoculated on to tobacco plants. Virus from infected tobacco was inoculated on to zucchini squash cotyledons. The systemic symptoms in zucchini squash were assessed at 7 days p.i. as either CS (chlorotic spots) or SM (severe mosaic). RNA 2 transcripts were derived from the following plasmids to generate the corresponding viruses (in parentheses): pPf-CMV2 (Pf-CMV); pFny209 (Fny-CMV); pPF2Xba (PF2Xba-CMV); pFP2Xba (FP2Xba-CMV); pPF2Kpn (PF2Kpn-CMV); pFP2Kpn (FP2Kpn-CMV); pPF2Kpn/Xba (PF2Kpn/Xba-CMV); pFP2Kpn/Xba (FP2Kpn/Xba-CMV). Pf-CMV RNA 2 sequences are indicated by open rectangles and Fny-CMV RNA 2 sequences are indicated by filled rectangles. The transcripts of RNAs 1 and 3 used for inoculation with RNA 2 transcripts of the chimeric or wild-type clones are indicated at the top of the figure. The amino acids at 2a protein positions 60, 76 and 267 that are different between the two strains are indicated at the top, with the amino acid difference correlating with the change in phenotype circled. The locations of restriction endonuclease sites used for making the chimeras are shown at the bottom.

 
To confirm that both Thr-168 in the 3a protein and Thr-267 in the 2a protein of Pf-CMV were involved in the differences in phenotype induced by infection with Pf-CMV compared with Fny-CMV, the DNA sequences of the corresponding codons in both the 2a and 3a ORFs of Fny-CMV were mutated from ATT (Ile) to ACT (Thr). Zucchini squash plants inoculated with RNA transcripts of Fny-CMV RNA 1 plus transcripts of RNAs 2 and 3 from the above T-to-C-mutated Fny-CMV cDNAs showed mild chlorotic spots (Fny-T/C in Fig. 1), which first appeared 9 days p.i. The phenotype was identical to that observed after inoculation of Pf-CMV RNA 1 plus the double-exchange chimeras in which most of the sequence derived from Fny-CMV RNAs 2 and 3, except for those sequences between the specified restriction sites (i.e. the double chimeric virus P1FP2Kpn/Xba-FP3Nae/Nhe in Fig. 1). By contrast, infection by the reciprocal, double chimeric virus containing mostly Pf-CMV RNA sequences in RNAs 2 and 3, except in the above-identified regions, showed symptoms typical of infection by Fny-CMV (i.e. F1PF2Kpn/Xba-PF3Nae/Nhe in Fig. 1). To confirm these results, the reciprocal point mutants were generated in RNAs 2 and 3 of Pf-CMV and co-inoculated on to zucchini squash plants with RNA transcripts of Pf-CMV RNA 1. This virus (designated Pf-C/T in Fig. 1) showed systemic mosaic symptoms, which first appeared 5 days p.i. This demonstrated that only the changes in RNA 2 at nt 886 and in RNA 3 at nt 622 were required for the differences in phenotype, and that the silent nucleotide changes in RNAs 2 and 3 did not contribute to the differences in timing or severity of symptoms.

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 2–7), 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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Fig. 5. Analysis of CMV RNA accumulation in protoplasts of zucchini squash. RNA accumulation of CMV was detected by Northern blot hybridization using DIG-labelled RNA probes complementary to the 3' non-translated region common to each CMV RNA. Zucchini squash protoplasts were infected with RNAs of all the pseudorecombinant viruses, Fny-T/C-CMV and the viruses containing the various RNA 2/RNA 3 chimeras. Lane 1, Pf-CMV; lane 2, F1P2P3; lane 3, P1F2F3; lane 4, P1F2P3; lane 5, F1P2F3; lane 6, P1P2F3; lane 7, F1F2P3; lane 8, P1PF2Kpn/Xba-PF3Nae/Nhe; lane 9, F1FP2Kpn/Xba-FP3Nae/Nhe; lane 10, Fny-T/C-CMV; lane 11, Fny-CMV. Arrows indicate the positions of the CMV RNAs. The lower panel shows stained rRNA loading controls from the samples.

 
To determine whether the differences in phenotype between Fny- and Pf-CMV were due to different rates of systemic infection, the inoculated zucchini squash cotyledons were detached at different times p.i. and the plants assessed for systemic infection. Plants inoculated with reconstituted Fny-CMV (designated F1F2F3, generated from infectious transcripts) showed systemic invasion from the inoculated cotyledons between 24 and 36 h p.i. (Table 2). By contrast, plants inoculated with reconstituted Pf-CMV (designated P1P2P3) showed systemic invasion by 60 h p.i. or even later (Table 2). Thus, there was a distinct difference in the rate of systemic movement of Pf-CMV compared with Fny-CMV.


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Table 2. Time course of systemic movement of Pf-CMV, Fny-CMV and pseudorecombinant viruses in zucchini squash

Results are shown as number of plants showing symptoms at 10 days p.i./number of plants inoculated. F1P2F3 was a pseudorecombinant virus formed from transcripts of Fny-CMV RNAs 1 and 3 and Pf-CMV RNA 2. The other pseudorecombinant viruses were formed from different combinations of the RNAs of Fny- and Pf-CMV. Control, plants in which the cotyledons were not detached.

 
Additional time-course studies on the rate of systemic movement of the six pseudorecombinant viruses formed between the RNAs of Fny- and Pf-CMV were done to confirm that these differences in rates of systemic invasion related to the nature of RNAs 2 and 3. The pseudorecombinant viruses that contained RNAs 2 and 3 from the same strain (P1F2F3 and F1P2P3) showed the same rate of systemic invasion as the corresponding parental viruses (F1F2F3 and P1P2P3, respectively), i.e. the source of the RNA 1 did not appear to affect the rate of systemic movement, at least in these combinations (Table 2). Moreover, the pseudorecombinant viruses containing RNAs 2 and 3 from different strains (P1F2P3, F1F2P3 and P1P2F3) generally showed intermediate rates of systemic movement between the extremes of the parental viruses, although some plants infected by the pseudorecombinant virus F1P2F3 showed a rate of systemic invasion similar to that of F1F2F3 or P1F2F3 (Table 2). Nevertheless, the overall pattern of the variation in the rate of systemic infection (Table 2) was consistent with differences in the timing of appearance and severity of symptoms (Table 1). This suggested that the sequence alterations in both the 2a polymerase protein and the 3a MP were affecting the rate of systemic movement.

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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Fig. 6. Leaf-press analysis of cell-to-cell movement of CMV in zucchini squash cotyledons. Cotyledons of zucchini squash were inoculated with Fny-CMV, P1PF2Kpn/Xba-PF3Nae/Nhe, F1FP2Kpn/Xba-FP3Nae/Nhe, Pf-CMV or buffer (Mock). As the times indicated, inoculated cotyledons were detached and blotted on to nitrocellulose membranes. The membranes were probed with antiserum against CP of CMV and anti-CP antibodies detected using a secondary antibody conjugated to alkaline phosphatase. The purple colour foci show the presence of CMV CP.

 

   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The molecular basis of the differences in timing and severity of symptoms in squash by Pf-CMV compared with Fny-CMV were shown to be due to differences in virus movement, both cell-to-cell in the inoculated tissue and via the vasculature to upper leaves. There appeared to be no major effect on virus replication. These phenotypic differences mapped to nucleotides encoding single amino acid changes in both the 2a polymerase and the 3a MP, in both cases involving changes in sequence from Ile (in Fny-CMV) to Thr (in Pf-CMV). Viruses containing either RNA 2 or 3 of Pf-CMV gave an intermediate phenotype with regard to the rate of systemic movement or timing of symptom appearance compared with viruses containing both RNAs from the same strain. Thus, each protein (2a or 3a) appeared to act independently of the other in modulating virus movement, and the presence of both sequence alterations had an additive effect on virus movement. Moreover, these effects were not due to generalized alterations in the functions of the 2a and 3a proteins, since they were specific to zucchini squash and were not seen in tobacco (data not shown). Thus, they most likely affected specific host interactions involved in facilitating virus movement.

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 1–126 and 290–335) 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 157–194) and at the end of a putative nucleic acid binding domain (aa 164–168) (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.


   ACKNOWLEDGEMENTS
 
This research was supported by a grant from the BioGreen 21 Program of the Rural Development Administration and in part by the Post-doctoral Fellow Program of the Korea Science & Engineering Foundation (KOSEF), in part by a grant from the SIGNET (SRC, R11-2003-008-02002-0) and in part by a grant from Plant Virus GenBank (R21-2004-000-10014-0) from the KOSEF, Republic of Korea. P. P. was supported by a grant-in-aid from the Scottish Executive Environment and Rural Affairs Department.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 29 October 2004; accepted 14 December 2004.