1 Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
2 Graduate School of Biotechnology, Korea University, Seoul 139-774, Korea
3 Plant Virus GenBank, Division of Environmental and Life Sciences, Seoul Women's University, Seoul, 139-774, Korea
4 Division of Biological Environment, Kangwon National University, Chunchon 200-701, Korea
Correspondence
Chikara Masuta
masuta{at}res.agr.hokudai.ac.jp
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the nucleotide sequence of the genomic RNA1 of HL-CMV is AB186043.
Supplementary figures showing representative results of Y-sat detection and Northern blot analysis of the CMV mutant constructs are available in JGV Online.
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INTRODUCTION |
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CMV has a broad host range and a large number of CMV isolates have been reported (Palukaitis & García-Arenal, 2003). However, CMV isolates from lily are capable of infecting only a small number of host plant species, due to their specific adaptation to lily plants (Masuta et al., 2002
; Ryu et al., 2002
) and typical CMV strains are incapable of systemic spread in lily. CMV strains are divided into three groups (subgroups IA, IB and II) and these categories are based upon serological relationships and nucleic acid similarities (Roossinck, 2002
; Roossinck et al., 1999
). Although CMV was isolated independently from different lily species in separate countries (Japan and Korea), RNA3 from these isolates showed a very high sequence similarity (Masuta et al., 2002
; Ryu et al., 2002
). Chen et al. (2001)
also reported that nucleotide sequences of CMV isolated from lily in the Netherlands, France and Taiwan had high conservation of the CP gene sequence. These reports indicate that sequences of lily strains of CMV are similar, regardless of their geographical origins. Furthermore, these data suggest that lily strains of CMV became adapted to lily in their evolutionary history.
Several studies have mapped host-range determinants to RNA3 (Carrère et al., 1999; Ryu et al., 1998
). However, some reports suggest that RNAs1 and 2 are responsible for further restriction in host range. For example, a local, hypersensitive response to CMV infection was mapped to the 2a gene in RNA2 (Kim & Palukaitis, 1997
). Studies on a distantly related virus (Brome mosaic virus), the type member of the family Bromoviridae, also showed that host-specificity determinants existed on RNAs1 and 2, as well as on RNA3 (Allison et al., 1988
; Mise et al., 1993
). The determinants of the systemic infection in edible lily were mapped to the 5' untranslated region (UTR) and the region between the FbaI and SpeI sites in HL-CMV RNA1 (Yamaguchi et al., 2005
).
Satellite RNAs (satRNAs) depend on helper viruses for replication and encapsidation and usually attenuate the diseases induced by the helper virus (Roossinck et al., 1992). Some satRNAs of CMV exacerbate the pathogenicity of CMV in specific hosts. For example, Y-satellite RNA (Y-sat) was found to induce bright yellow symptoms on tobacco plants (Takanami, 1981
). In tomato, lethal necrosis is induced by CMV containing particular satRNAs (Kaper & Waterworth, 1977
). Specific satRNA sequences involved in inducing such symptoms have been identified (Devic et al., 1989
; Hidaka & Hanada, 1994
; Jaegle et al., 1990
; Kuwata et al., 1991
; Masuta & Takanami, 1989
; Sleat & Palukaitis, 1990
).
There are some reports on the CMV strains that cannot support satRNAs (Gal-On et al., 1995; McGarvey et al., 1995
; Roossinck & Palukaitis, 1991
). For example, Sny-CMV can support replication of WL1-satRNA in tobacco, but not in zucchini (Gal-On et al., 1995
; Roossinck & Palukaitis, 1991
). Roossinck et al. (1997)
found that the amino acid residue at position 978 of the 1a protein in Sny-CMV was important for WL1-satRNA replication in zucchini. It was apparent that lily CMV isolates were unable to support the replication of satRNAs in any of the host plants tested. In this study, we identified CMV sequences responsible for the inability of HL-CMV to support Y-sat.
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METHODS |
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Virus inoculation.
The virus-free lily cultivar Hakugin, which had been grown from tissue-cultured bulbs, was supplied by the Hokkaido Central Agricultural Experiment Station (Naganuma-Cho, Hokkaido, Japan). The plants were dusted with carborundum, rub-inoculated and maintained in a greenhouse under natural light conditions (1016 h) at 2028 °C. Viral infection and satRNA replication were confirmed by several methods, including ELISA, RT-PCR, immunocapture RT-PCR (IC-RT-PCR) and Northern blot hybridization.
IC-RT-PCR.
Samples from infected plants were homogenized in 10 mM sodium phosphate (pH 7·5). Homogenates were incubated for 1 h at 37 °C in a 96-well plate that was previously coated with anti-CMV antibody. After two subsequent washing steps with PBS/Tween buffer (1x PBS, 0·5 g Tween 20 l1), RT-PCR was performed as described previously (Ryu et al., 2002) by using the Core RT-PCR system (Perkin Elmer). In order to detect CMV and Y-sat, primer pairs consisting of CMCP5 (GAGTCATGGACAAATCTGAATC) and CMCP3 (GGAACACGGAATCAGACTGG), and Y-sat5 (GATGGAGAATTGCGTAGAGG) and Y-sat3 (GGAGAATGTATAGACATTCAC), respectively, were used for the IC-PCR.
Cloning of the full-length cDNAs of HL-CMV and production of pseudorecombinant viruses.
Pseudorecombinant viruses were created between HL-CMV and Y-CMV. The three genomic RNAs of HL-CMV and Y-CMV were designated H1H3 and Y1Y3, respectively. Cloning of the full-length cDNA for H1, H2 and H3 was described previously (Masuta et al., 2002; Yamaguchi et al., 2005
). The transcripts were synthesized from the cDNA clones by T7 RNA polymerase (TaKaRa) in the presence of the m7G(5')ppp(5')G cap analogue (Invitrogen). Transcripts for each genomic RNA were mixed in various combinations and inoculated onto N. benthamiana.
Frameshift construct.
For the construction of Yfs1d, the biologically active clone of Y-CMV RNA1 was digested with XbaI at nucleotide position 383 and the termini were blunt-ended by using a Blunting High kit (Toyobo). The 3' ends following nt 31383361 of Y1d and Yfs1d were deleted to restrict Y1 replication.
Viral transfection of protoplasts.
Protoplasts were prepared from leaves of N. benthamiana and were used as the source of material for viral transfection. Specifically, the abaxial epidermis of the leaves was removed by peeling with forceps and the peeled leaves were immersed in an enzyme solution containing 2 % cellulase Onozuka R-10 (Yakult Honsha), 0·5 % Macerozyme R-10 (Yakult Honsha) and 0·5 M mannitol (pH 5·8). After incubation at 25 °C for 3 h, the protoplasts were collected by centrifugation at 400 r.p.m. for 3 min and were washed twice with 0·5 M mannitol solution. Approximately 1x106 protoplasts were inoculated with in vitro RNA transcripts in the presence of polyethylene glycol. After the mixtures were maintained for 15 min at 0 °C, the inoculated protoplasts were collected and washed with 0·5 M mannitol. Samples were subsequently incubated for 12, 20 or 24 h in the dark at 25 °C in incubation buffer (0·2 mM KH2PO4, 1 mM KNO3, 1 mM MgSO4, 1 µM KI, 0·1 µM CuSO4, 10 mM CaCl2 and 10 % mannitol) and the accumulation of Y-sat was detected by Northern blot hybridization analysis.
Northern blot anlysis.
Detection of viral RNAs in the protoplasts was accomplished by Northern blot hybridization essentially as described previously (Masuta et al., 1998). Total RNAs were extracted from the infected protoplasts by using TRIzol reagent (Invitrogen) according to the supplier's instructions. In the satRNA detection, digoxigenin (DIG)-labelled RNA probes (in sense and antisense orientations) were in vitro-transcribed from the PCR-amplified Y-sat sequence with T7 polymerase. In the detection of CMV RNAs in protoplasts, a DIG-labelled antisense RNA was in vitro-transcribed from the PCR-amplified RNA4 sequence with T7 polymerase. For detection of CMV genomic RNAs in the upper leaves, Northern blot hybridization was performed to detect the genomic RNAs by using a DIG-labelled DNA probe that corresponded to the 3'-end sequence of Y-CMV RNA3 (352 bp).
RNA1 chimeric constructs.
The chimeric structures of the RNA1 recombinants were created by using four unique restriction enzymes (XbaI, FbaI, SpeI and StuI). The in vitro transcripts from the RNA1 chimeric constructs were mixed with Y2 and Y3 and were inoculated onto N. benthamiana.
Point-mutation constructs.
Point mutations at positions 862, 876 and 891 were introduced by recombinant PCRs using the following primer pairs: 5'-terminal primer (CCCTTCCGAAAGGAACCCATTC) and 3'-terminal primer (GAATGGGTTCCTTTCGGAAGGG), 5'-terminal primer (GTTTCTCAATCTAAAGTGAGAAG) and 3'-terminal primer (CTTCTCACTTTAGATTGAGAAAC), and 5'-terminal primer (CGCTAGTGTGACATTGGTTGACTTG) and 3'-terminal primer (CAAGTCAACCAATGTCACACTAGCG), respectively. Underlined bases in the aforementioned primers reflect the nucleotides that were mutated. PCR products were subcloned into the pGEM-T Easy vector (Promega) and were sequenced fully in order to confirm their sequence integrity. The 200 nt fragment between the SpeI and StuI sites containing the mutation was substituted for the corresponding region in pCHL1.
PVX vector construct.
The PVX vector (Baulcombe et al., 1995) was obtained from D. C. Baulcombe, The Sainsbury Laboratory, Norwich, UK, and Y-sat was amplified by RT-PCR. Plus- and minus-sense sequences of Y-sat were cloned into the PVX vector in order to create PVX-Y-sat(+) and PVX-Y-sat(), respectively. The in vitro transcripts were then inoculated onto N. benthamiana leaves.
RT-PCR.
Total nucleic acids were extracted from the infected leaves by the conventional phenol/chloroform method and reverse transcription was accomplished by using an RNA LA PCR kit (TaKaRa) according to the supplier's instructions. In order to amplify CMV RNA1, we used the primer pair 5'-terminal primer (GACTTGTAAGTCTCTCTACTGGCG) and 3'-terminal primer (TGGTCTCCTTGTGGAGCC), and we used the primer pair 5'-terminal primer (T7 promoter-GTTTTGTTTGATGGA) and 3'-terminal primer (GGGTCCTGTGTAGGAATGATAGACATTC) for amplification of Y-sat.
Determination of Y-sat(HL) sequence.
Total nucleic acids were extracted from the plants in which Y-sat was detected. Single- and double-stranded RNAs of Y-sat(HL) were isolated from the agarose gels (1·5 % LowMelt agarose gel; BioExpress). The RNAs were boiled for 5 min and were then added to the reaction solution [40 mM Tris/HCl (pH 8·0), 8 mM MgCl2, 5 mM dithiothreitol, 2 mM spermidine, 100 mM ATP and 10 U poly(A) polymerase (TaKaRa)]. After incubation at 37 °C for 30 min, the poly(A)-tailed RNAs were primed with an oligo(dT)20-M4 adaptor primer (TaKaRa) at 40 °C for 1 h. A series of PCR fragments was then amplified by using pairs of several internal specific primers and the M4 primer (GTTTTCCCAGTCACGAC). The PCR products were cloned into the pGEM-T Easy vector (Promega) and sequenced for verification.
Dot-blot hybridization.
DIG-labelled DNA probes were prepared from the Y-sat clones by PCR using the primer pair described above. Total nucleic acid extracts were mixed with an equal volume of the sample buffer containing 1x MOPS, 50 % formamide and 17·5 % formaldehyde. After denaturing at 65 °C, the samples were spotted onto Biodyne nylon membrane (Pall) and the blots were cross-linked with UV illumination for 5 min. The pre-hybridization buffer contained 5x SSC, 50 % formamide, 7 % SDS, 2 % blocking reagent (Roche Diagnostics) and 0·1 mg herring sperm ml1 (Invitrogen). The blots were then hybridized with the probes overnight at 65 °C and positive signals were detected by using CDP-Star reagent according to the manufacturer's instructions (Tropix).
Computer analysis.
Analysis of the 1a protein was performed by the PredictProtein program (http://www.predictprotein.org/), which is operated by the Columbia University Bioinformatics Center. Program SEG, which was developed by Wootton & Federhen (1996), was also utilized for data analysis.
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RESULTS |
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Accelerated generation of Y-sat supported by HL-CMV
As shown by the schematic illustrations of various constructs in Fig. 5(a), a PVX vector was used to express Y-sat. Plus- and minus-sense sequences of Y-sat were cloned into the PVX vector in order to create PVX-Y-sat(+) and PVX-Y-sat(), respectively. The synthesized transcripts generated from the PVX vector constructs were inoculated onto N. benthamiana and thereby enabled Y-sat sequences to be supplied continuously in the PVX-infected plants. Seven days after PVX inoculation, HL-CMV was superinoculated onto the same plants that had been pre-infected with the PVX vector constructs. satRNA replication by CMV could be discriminated by the detection of the strand complementary to that expressed from the PVX vector, either by Northern hybridization or by dot-blot hybridization. The plants infected with Y-CMV and the PVX vector constructs induced bright yellow symptoms within 1 month of Y-CMV inoculation. On the other hand, the plants infected with HL-CMV and the PVX vector constructs did not show such bright yellow symptoms within 1 month, indicating no Y-sat accumulation. However, when observed for 2 months after HL-CMV inoculation, three out of the four plants infected with HL-CMV plus PVX-Y-sat() induced bright yellow symptoms, indicating that Y-sat accumulation occurred. Total RNAs were extracted from the plants as a method to confirm Y-sat accumulation. Sequencing of the amplified Y-sat revealed three nucleotide differences at the 3' end, compared with the authentic Y-sat (Fig. 5b
). We designated this mutant as Y-sat(HL). Y-sat(HL) was isolated from the plants infected with HL-CMV plus PVX-Y-sat(), but it was not generated with HL-CMV plus PVX-Y-sat(+). We then confirmed that HL-CMV could support the replication of the transcript from a cDNA clone of Y-sat(HL). The 3' end of Y-sat(HL) actually reverted in the progeny Y-sat when a transcript of Y-sat(HL) was inoculated with Y-CMV. We also tested whether Y-sat(HL) amplified by HL-CMV was maintained in lily by inoculating it onto the surface of lily leaves. Although Y-sat(HL) was created in N. benthamiana, we detected Y-sat accumulation in the systemically infected lily leaves (data not shown).
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DISCUSSION |
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In the next line of our investigation, we demonstrated that a frameshift mutation in the 1a protein of CMV-Y abolished the ability to support Y-sat (Fig. 2). This result revealed that the protein translated from Y1, rather than the Y1 RNA itself, was essential for Y-sat replication. Roossinck & Palukaitis (1991)
showed previously that the inability of Sny-CMV to support WL1-satRNA in zucchini also mapped to RNA1. McGarvey et al. (1995)
mapped the inability of Ix-CMV to support the replication of a necrogenic satRNA to RNA1. In addition, the inability of a strain of Peanut stunt virus (PSV-W) to support the replication of PSV-satRNA in tobacco was also mapped to RNA1 (Hu et al., 1998
). As PSV is another cucumovirus, it is therefore concluded that the 1a proteins of cucumoviruses are important for satRNA replication. Beyond the data presented in the previously mentioned studies, we successfully identified two amino acid residues (at positions 876 and 891) in the 1a protein responsible for the inability of HL-CMV to support Y-sat in the present study. It is apparent that Thr at position 876 seems to be more important than Met at position 891. This conclusion was reached due to the observation that a single amino acid transition at position 876 allowed Y-sat to replicate with HL-CMV, although the ability was still poor (Fig. 4
). Although Roossinck et al. (1997)
suggested previously that the amino acid residue at position 978 in Sny-CMV was important for satRNA replication in squash, it is not important in the combination of HL-CMV and Y-sat in N. benthamiana.
To determine whether these mutations affect the structure of the 1a protein, we analysed the 1a protein of HL-CMV by using an online program for secondary-structure prediction (PredictProtein). Among the predicted secondary structures, we found one unique difference in the locations of the low-complexity region (LCR) (Wootton & Federhen, 1996). Because LCR can signify interdomain regions of protein structures, LCR is usually found to reside between two domains. It is important to note that the 1a protein of HL-CMV and Ly2-CMV has an LCR between motifs IV and V in the helicase domain, separating the two motifs spatially (Fig. 6a
). On the other hand, there is no LCR in the helicase domains of the other CMV strains (Fig. 6b
). The structural changes in the helicase domain of lily CMVs caused by inserting an LCR may have coincidentally affected the ability to support satRNAs; motif IV was thought to be important for eIF-4A helicase activity (Buck, 1996
). Although the amino acid residue at position 891 of Ly2-CMV was Leu instead of Met, Thr at position 876, which induces an LCR between motifs IV and V, was also found in Ly2-CMV (Fig. 6b
).
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In this study, we demonstrated that transient expression of Y-sat sequences through a PVX vector accelerated generation of Y-sat(HL) that was supported by HL-CMV (Fig. 5). Although all of the plants inoculated with HL-CMV plus PVX-Y-sat(+) induced simple mosaic symptoms without Y-sat, three out of the four plants inoculated with HL-CMV plus PVX-Y-sat() showed bright yellow symptoms with Y-sat (Fig. 5a
). Continuous supply of the minus strand of Y-sat resulted in the generation of the replicative Y-sat [Y-sat(HL)] in the presence of HL-CMV. Sequencing of Y-sat(HL) revealed that there were three nucleotide changes at the 3' end (Fig. 5b
). Considering that the synthesis of the () strand of Y-sat was hardly detected in the protoplast experiment, it is likely that the HL replicase may not recognize the 3' end of Y-sat efficiently. It is conceivable that the replicase could bind to the 3' end of the () RNA and this binding would allow it to copy the satRNAs. The 3' end of (+) satRNA will eventually be selected to be fit for replication by accumulation of random mutations. In fact, Burgyán & García-Arenal (1998)
reported previously that the 3' end of satRNA lacking the last three Cs was repaired in planta in the presence of CMV, indicating that CMV has an ability to restore the 3' end of a satRNA. In addition, Moriones et al. (1991)
reported that minor sequence changes might have a major effect on the fitness of sequence variants of satRNAs in different hosts.
However, unless a satRNA and its helper virus at least coexist, no satRNA variants will be generated. It is important to consider here the rather peculiar lily propagation through bulbs; lily CMV isolates are transmitted vertically to progeny plants, although they are occasionally carried by aphids. Once a CMV strain has adapted to lily plants by changing some amino acid residues in the 1a protein and, accordingly, lost the ability to support satRNAs, any challenge inoculation by foreign CMV may be defeated through cross-protection. This can be concluded because most ordinary strains of CMV cannot infect lily plants efficiently. There is thus little chance that a satRNA can be incorporated successfully into lily CMV from the outside.
We believe that there must be a specific host factor(s) in lily that should be somehow different from the corresponding factor in many other plants, which associates with the 1a protein and affects the ability of CMV to support satRNAs. After all, without a certain level of initial replication by using lily factor(s), a satRNA cannot accumulate random mutations on its RNA sequence to increase fitness to lily CMV isolates.
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ACKNOWLEDGEMENTS |
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Received 24 March 2005;
accepted 30 April 2005.
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