1 Department of Plant Pathology, Cornell University, Ithaca, NY 14853, USA
2 Department of Biological Sciences, The National University of Singapore, Kent Ridge, Singapore 117543, Singapore
3 Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK
Correspondence
Peter Palukaitis
ppaluk{at}scri.sari.ac.uk
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ABSTRACT |
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INTRODUCTION |
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D RNAs and defective interfering (DI) RNAs have been described for a number of plant viruses (see reviews by Graves et al., 1996; Simon & Nagy, 1996
; White, 1996
; White & Morris, 1999
). DI and D RNAs usually contain in-frame deletions within one or more genes and in some cases may be associated with changes in pathology (Graves et al., 1996
). In the case of the cucumovirus Cucumber mosaic virus (CMV), a D RNA derived from RNA 3 of CMV has been described (Graves & Roossinck, 1995
). This D RNA, designated D RNA 3
, was found associated with a local lesion isolate of the Fny strain of CMV (Fny-CMV) and contained a 156 nt in-frame deletion within the 3a gene encoding the movement protein. In addition, a second D RNA 3 molecule (D RNA 3
), which contained an in-frame deletion of 309 nt within the Fny-CMV 3a gene, was also described, although it was not characterized further (Graves & Roossinck, 1995
). By definition, such D RNAs could be maintained by the associated wild-type (WT) virus, as well as some other strains of CMV. However, it is not clear whether these D RNA 3s were originally present at subliminal levels in the population and subsequently were amplified to high levels, perhaps following further mutation, or whether they truly arose de novo.
During the passage of CMV derived from cDNA clones, two D RNA 3s were generated. These D RNA 3s were characterized and compared with those described previously, with regard to both the nature of the deletion and their biological properties. Host-specific maintenance of one D RNA 3 was observed and shown to be due to host-specific encapsidation.
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METHODS |
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Sap from a tobacco plant inoculated with transcripts representing Fny-CMV RNAs 1, 2 and 3 (Rizzo & Palukaitis, 1990) was inoculated into two tobacco plants and passaged at 2-weekly intervals for a total of 20 passages. Leaves from passages 220 were dried and stored at 4 °C. Dried leaves from passages 2, 3 and 20 were homogenized in 50 mM sodium phosphate, pH 7, and the slurry was used to inoculate two to four tobacco plants. Virus was purified from these infected plants 2 weeks post-inoculation and viral RNAs were extracted and stored at 20 °C (Palukaitis et al., 1992
).
Various sources of CMV RNA 3 were used in different inoculations. In some experiments, RNA transcripts were derived from full-length cDNA clones of Fny-CMV RNA 3 (pFny309) or strain M CMV (M-CMV) RNA 3 (pMCMV3; Shintaku et al., 1992). In other experiments, RNA 3 transcripts generated from a cDNA clone of D RNA 3-1 (see below) were used.
Construction of D RNA 3-1.
All standard manipulations were done as described by Sambrook et al. (1989). cDNA was synthesized from gel-purified Fny-CMV D RNA 3, using avian myeloblastosis virus reverse transcriptase and a primer (5'-TGGTCTCCTTTTGGAG-3') complementary to the 16 nt at the 3'-end of Fny-CMV RNA 3. PCR was done using a primer corresponding to the 10 nt at the 5'-end of Fny-CMV RNA 3, preceded by a T7 promoter sequence and a 5'-terminal BamHI site (Owen et al., 1990
) and a primer complementary to nt 12821299 (containing a SalI site) of Fny-CMV RNA 3. The PCR product was digested with BamHI and SalI and incubated in a three-piece ligation reaction. The other components of the ligation included the larger fragment of pUC18 cleaved with BamHI and PstI and the smaller fragment of pFny309 digested with SalI and PstI (containing the capsid protein gene and 3' non-coding region). Escherichia coli DH5
cells were transformed by the ligation reaction products. One such transformant contained a plasmid with an insert designated D RNA 3-1. The 3a gene of D RNA 3-1 was sequenced. Other cDNA clones from the same transformation event showed the same sequence. In addition, the non-coding regions as well as the genes encoding the capsid protein and 3a protein were sequenced directly from the gel-purified D RNA 3, to confirm that D RNA 3-1 contained a deletion typical of the D RNA 3 population and that only the 3a gene contained a deletion in the Fny-CMV D RNA 3.
Characterization of progeny viral RNAs.
Viral RNAs were extracted from virions and total plant RNAs were extracted from plants, both as described by Kaplan et al. (1995). The various RNAs were analysed by agarose gel electrophoresis and Northern blot hybridization as described previously (Kaplan et al., 1995
). Blots were probed with 32P-labelled transcripts complementary to the 3'-terminal 200 nt of Fny-CMV RNA 3 (Gal-On et al., 1994
). WT RNA 3 and D RNA 3-1 were subjected to RT-PCR, using a primer complementary to the intergenic region (nt 11351148) for reverse transcription and the same primer plus a primer corresponding to the sequence to the first 10 nt at the 5' end of Fny-CMV RNA 3 (see above) for the PCR step. PCR products were analysed by agarose gel electrophoresis.
The D RNA designated [M] D RNA 3-2, detected after inoculation of tobacco plants with transcripts of Fny-CMV RNAs 1 and 2 plus M-CMV RNA 3 (Canto & Palukaitis, 1998), was amplified from total viral RNA containing this D RNA by RT-PCR. The primers used corresponded to the first 13 nt at the 5' end of Fny-CMV RNA 3 and sequences complementary to the 15 nt at the 3' end of Fny-CMV RNA 3, respectively. The nature of the deletion in [M] D RNA 3-2 was established by sequence analysis of the PCR product.
Preparation, infection and analysis of virus accumulation and encapsidation in zucchini squash protoplasts.
Protoplasts were prepared from leaves of zucchini squash plants and infected by electroporation using viral RNAs, all as described previously (Lee et al., 2001). Protoplasts were incubated for 24 or 48 h and then processed. This experiment was done four times. Total RNAs were extracted and purified from the infected protoplasts as described by Gal-On et al. (1994)
. Virions were isolated from infected protoplasts and RNAs were extracted from the isolated virions, both as described by Osman et al. (1998)
. Total plant RNAs and viral RNAs were analysed by Northern blot hybridization or by RT-PCR, both as indicated above, except that the CMV probe was labelled with digoxigenin and the blots were processed following the manufacturer's instructions (Roche Diagnostics).
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RESULTS |
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The lineage of the generation of D RNA 3-1 was analysed. The first virus preparation containing the progenitor of D RNA 3-1 was derived after passage in tobacco of a virus preparation that did not contain visible D RNA 3. This parental virus preparation was derived from passage 3a (Fig. 2). This was a separate lineage of a 20-passage experiment starting from passage 2, i.e. the virus from passage 3a was obtained from plants inoculated with an extract of dried leaves containing passage 2 of Fny-CMV derived from an inoculation involving RNA transcripts of the biologically active Fny-CMV cDNA clones. When dried leaves from the original passages 3 and 20 were used as inocula (Fig. 2
), no D RNA 3 was detectable in virions of the successive passages (data not presented). Viral RNAs obtained from these virions (extracted from passages 4a and 21) were each inoculated into five tobacco plants at a concentration of 1 mg ml1 to determine whether the appearance of D RNA 3 was stimulated by a high concentration of inoculum, but no D RNAs were visible in the progeny virions (data not shown). Thus, the D RNA 3 was most probably generated in subsequent passages of virus derived from the second-passage dried leaves (i.e. after passage 3a), rather than during the initial 20 passages in tobacco.
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These experiments demonstrated that D RNAs derived from CMV RNA 3 could appear spontaneously in CMV RNAs derived from cDNA clones, although the generation of D RNAs was rare. In addition, although the virions formed by the capsid proteins of Fny-CMV and M-CMV are known to differ in their stability (Ng et al., 2000), this did not affect the ability of these capsid proteins to encapsidate the respective D RNA 3s.
Effects of host on maintenance and encapsidation of D RNA 3-1
To assess potential host effects on the accumulation of the D RNA 3s, transcripts of the cDNA clone of D RNA 3-1 were mixed with Fny-CMV RNAs 1, 2 and 3, also derived from transcripts, and were passaged serially in tobacco plants. D RNA 3-1 was supported by the Fny-CMV RNAs in tobacco (Fig. 3a) and had no effect on the yield of virus or the symptoms induced by Fny-CMV (data not presented). By contrast, when sap or purified virus obtained from tobacco infected by Fny-CMV containing D RNA 3-1 was passaged through squash plants, D RNA 3-1 was not readily detectable (Fig. 3a and b
). Analysis of the gel containing virion RNAs shown in Fig. 3(a)
could not unambiguously establish whether D RNA 3-1 was absent from such virions or was simply maintained at a reduced level, due to the presence of a background of degraded genomic RNAs. Therefore, small cotyledons of squash plants were inoculated with Fny-CMV RNA, with and without D RNA 3-1, and virion RNAs were analysed at 2 weeks post-inoculation (Fig. 3b
). In these preparations, which were made before the cotyledons began to deteriorate due to abscission, breakdown of the larger RNAs did not obscure the presence of stainable levels of D RNA 3-1. Nevertheless, no D RNA 3-1 could be detected in virions extracted from either the cotyledons or true leaves.
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DISCUSSION |
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The D RNA 3s described here did not affect the yield of virus or the symptoms induced by the corresponding viruses (Fny-CMV induces a light green/dark green mosaic on tobacco, while the capsid protein of M-CMV induces a yellow, systemic chlorosis). This was also reported for the two D RNA 3s described previously, D RNA 3 and D RNA 3
, both found associated with WT Fny-CMV (Graves & Roossinck, 1995
). Those D RNA 3s, differing in the nature of the deletion within the 3a gene from the D RNA 3s reported here (Fig. 6
), were derived from a local lesion isolate and not from cDNA clones (Graves & Roossinck, 1995
). Nevertheless all four natural D RNA 3s of CMV have been derived and maintained by the Fny-CMV replicase. Which factors lead to the spontaneous generation of D RNA 3s with Fny-CMV remains unknown.
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The differential accumulation of D RNA 3-1 in tobacco vs squash (Figs 1, 3 and 4) was due to an inability of the D RNA 3-1 to become encapsidated in squash cotyledons and to accumulate in upper leaves of squash plants (Figs 4b and 5
). The failure to accumulate in upper leaves is probably due to an inability of D RNA 3-1 to move systemically in the absence of encapsidation rather than an inhibition of cell-to-cell movement, since the latter process does not require either encapsidation (Kaplan et al., 1998
) or interaction between CMV RNAs and the capsid protein (Kim et al., 2004
). It seems likely that virion formation is essential for systemic movement in squash, since it was shown that CMV capsid protein mutants that did not form virions still supported systemic movement in some Nicotiana species but not in squash (Kaplan et al., 1998
). Moreover, the capsid protein of M-CMV did not promote systemic infection in squash (Wong et al., 1999
). This might suggest that either there is a fundamental difference in how CMV moves in squash compared with tobacco or that movement in squash is more restrictive than in tobacco. The data presented here do not distinguish between these alternatives.
Why is there host-specific encapsidation of D RNA 3-1? Such host-specific encapsidation was also observed for a DI RNA 2 of the bromovirus Broad bean mottle virus (Romero et al., 1993). In that case, it was suggested that the lack of encapsidated DI RNA 2 may have been due to a lower stability of such virions in pea compared with broad bean, although why only virions containing the DI RNA would be less stable in one host than another was not explained (Romero et al., 1993
). Moreover, CMV strains with considerable differences in virion stability (Ng et al., 2000
) did not show strain-specific effects on encapsidation of D RNAs (Figs 1 and 3
; Canto & Palukaitis, 1998
). The assembly of most plant viruses is not well characterized, although generally assembly of RNA viruses with only a single type of capsid protein has been considered to be a process that does not require scaffolding proteins, but only interactions between the viral RNAs and the capsid proteins. Thus, in the absence of known host-specific effects on assembly or stability of virions, it may be that differences in the interactions of genomic, subgenomic and D/DI RNAs with host components in various plants may determine whether particular RNAs are available for encapsidation or can be mobilized to the site(s) of assembly. This in turn may then determine whether the RNAs are encapsidated or not. Studying the host-specific encapsidation of D RNAs may provide some enlightenment of this selection process and ultimately contribute to a better understanding of the CMV encapsidation process as such.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 4 June 2004;
accepted 12 August 2004.
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