Laboratory of Plant Pathology, Department of Agricultural and Environmental Biology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Correspondence
Masashi Suzuki
m-suzuki{at}ynu.ac.jp
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ABSTRACT |
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Present address: Department of Natural Environment and Information, Faculty of Environment and Information Sciences, Yokohama National University, 79-7 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan.
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INTRODUCTION |
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Cucumoviruses have tripartite genomes that consist of three positive-sense, single-stranded RNA molecules, which are designated RNA 1 to 3 in decreasing order of molecular mass. RNA 1 and 2 encode the 1a and 2a proteins, respectively, both of which are necessary for virus replication (Nitta et al., 1988; Hayes & Buck, 1990
). RNA 3 encodes the 3a protein, which is involved in virus movement (Suzuki et al., 1991
; Canto et al., 1997
; Kaplan et al., 1997
). RNA 4, which is transcribed from the negative strand of RNA 3, serves as a messenger RNA for the viral coat protein (CP). CP is involved in symptom determination in several plants (Shintaku et al., 1992
; Takahashi & Ehara, 1993
; Suzuki et al., 1995
; Takeshita et al., 1998
, 2001
). Another subgenomic RNA, RNA 4A, which is transcribed from RNA2, encodes the 2b protein, which is not necessary for replication (Ding et al., 1994
, 1995
). The RNA 4A of subgroup I CMV was reported to accumulate to a very low level compared with that of subgroup II CMV and TAV (Shi et al., 1997
). The 1a protein contains a methyltransferase-like domain in its N-terminal half and a helicase-like domain in its C-terminal half (Mi & Stollar, 1991
; Gorbalenya et al., 1989
). The 2a protein contains a putative RNA-dependent RNA polymerase domain (Argos, 1988
). The 2b protein gene is present all cucumoviruses, but not in the other viruses of the family Bromoviridae, and suppresses gene silencing (Brigneti et al., 1998
).
The interactions between 1a and 2a proteins have been well characterized in Brome mosaic virus (BMV), a member of the genus Bromovirus in the family Bromoviridae that is taxonomically closely related to CMV and PSV. The interacting regions have been mapped on the C-terminal helicase-like domain of the 1a protein and the N-terminal 140 amino acid residues of 2a protein using in vitro immune co-precipitation or in vivo yeast two-hybrid assays (Kao & Ahlquist, 1992; O'Reilly et al., 1995
, 1997
). From results obtained with mutated 1a and 2a proteins, O'Reilly et al. (1995
, 1997
) also postulated that RNA replication was due to the 1a2a interaction. Recently, Kim et al. (2002)
reported that the entire 1a protein and the N-terminal 126 amino acids of the 2a protein of Fny-CMV interacted in an immune co-precipitation assay and yeast two-hybrid assay. They also found that phosphorylation of the N-terminal region of 2a protein prevents the 1a2a interaction.
Generally, genome reassortment is used to determine the components involved in replication, movement and symptom expression in multipartite viruses. Reassortant CMV strains have been well studied (Roossinck & Palukaitis, 1990; Shintaku et al., 1992
; Gal-On et al., 1994
; Zhang et al., 1994
; Suzuki et al., 1995
; Kaplan et al., 1997
; Kim & Palukaitis, 1997
; Karasawa et al., 1997a
; Ryu et al., 1998
; Takeshita et al., 1998
, 2001
; Kobori et al., 2002
) and several reassortants and recombinants between CMV and TAV have also been reported (Fernandez-Cuartero et al., 1994
; Ding et al., 1995
; Fraile et al., 1997
; Salanki et al., 1997
; Masuta et al., 1998
). Similar interspecific reassortants of BMV and Cowpea chlorotic mottle virus (CCMV; genus Bromovirus) have been thoroughly studied. The combination of BMV RNA 1 and CCMV RNA 2 or CCMV RNA 1 and BMV RNA 2 inoculated together with RNA 3 of BMV or CCMV could not replicate in barley protoplasts (Allison et al., 1988
). Using protein expression vectors, however, when the combination of BMV 1a protein and CCMV 2a protein was expressed in Nicotiana benthamiana protoplasts, viral negative-strand RNAs could be synthesized, but positive-strand RNAs and subgenomic RNAs did not accumulate. The converse combination of CCMV 1a and BMV 2a did not support RNA synthesis (Dinant et al., 1993
). Hence, it was believed that heterologous combinations of viral replicase components failed to replicate the viral genome. By contrast, there have been few genetic analyses of interspecific reassortants between CMV and PSV (Hanada & Tochihara, 1980
).
In this study, we prepared interspecific reassortants between CMV and PSV, using infectious RNA transcripts of their genome cDNAs, and analysed viral RNA replication in protoplasts. We also assayed the in vivo compatibility of the replicase components using a yeast two-hybrid system. The results indicated that RNA replication with the interspecific reassortant requires compatibility between the C-terminal half of the 1a protein and the N-terminal region of the 2a protein, and this compatibility is insufficient for the transcription of subgenomic RNA 4.
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METHODS |
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Construction of full-length cDNA clones of PSV RNAs.
The full-length cDNA clones of CMV-Y RNAs were described previously (Suzuki et al., 1991). To establish interspecific reassortants between CMV and PSV, we constructed full-length cDNA clones of PSV-J RNAs 1, 2 and 3 using RT-PCR. Purified PSV-J RNA was first primed with the oligonucleotide 5'-TTTTTTggatccatggTCTCCTATGGAAAC-3', which is complementary to 17 nucleotides (nt) of the 3'-end of all PSV-J RNAs (underlined) (Karasawa et al., 1991
, 1992
) and connected with an NcoI and BamHI linker (lower-case). Its first-strand cDNA was synthesized using SuperScript II reverse transcriptase (Invitrogen), as described by Suzuki et al. (1991)
. The cDNA products were used for PCR with the above primer paired with one of the following three primers: 5'-TTTTTCctgcagGAATTAATACGACGACTCACTATAGTTTTATCACGAGCGTACG-3', which is identical to 19 nt of the 5'-end of PSV-J RNA 1 (underlined) and linked with the T7 RNA promoter (italics) and a PstI recognition site (lower-case); 5'-TTTTTaagcttAATTAATACGACGACTCACTATAGTTTTATCACGAGCGTACG-3', which is identical to 19 nt of the 5' end of PSV-J RNA 2 (underlined) and linked with the T7 RNA promoter (italics) and a HindIII recognition site (lowercase), and 5'-TTTTTaagcttAATTAATACGACGACTCACTATAGTTTTACCAACCAGGAAATC-3', which is identical to 20 nt of the 5' end of PSV-J RNA 3 (underlined) and linked with the T7 RNA promoter sequence (italics) and a HindIII recognition site (lower-case).
The PCR procedure was as described by Suzuki et al. (1995). The resulting PCR products were digested with the corresponding restriction enzymes and cloned into pUC18 (Takara Shuzo, Japan). E. coli strain DH5
was used for the transformation.
Inoculation of plants and protoplasts.
Before in vitro transcription, the three plasmids containing the respective full-length cDNA of PSV RNA 1, 2 and 3 were linearized with NcoI. The linearized plasmids were transcribed with T7 RNA polymerase (Takara), as previously described (Suzuki et al., 2003). The transcripts derived from cDNA of CMV RNA 1, 2 and 3 and PSV RNA 1, 2 and 3 are designated C1, C2, C3, P1, P2, and P3, respectively. Reassortants are designated C1C2P3, P1P2C3, and so on. Reassortants of CMV and PSV transcripts were inoculated onto cowpea and N. benthamiana. Symptoms were examined 1 and 2 weeks post-inoculation on the inoculated leaves and upper leaves, respectively.
Protoplasts prepared from suspension-cultured tobacco BY-2 cells (Nagata & Kumagai, 1999) were inoculated with 2 µg each of the partially purified transcripts by electroporation using a Gene Pulser II (Bio-Rad) (Suzuki et al., 1995
).
Northern blot analysis.
Total RNA was extracted from the infected protoplasts after a 24 h incubation as described by Kroner & Ahlquist (1992), from the inoculated leaves after 1 week, and from the upper non-inoculated leaves after 2 weeks as described by Suzuki et al. (2003)
. The RNA was treated with sample buffer containing formaldehyde and formamide, electrophoresed on agarose gels containing formaldehyde, and transferred to nylon membranes as described (Suzuki et al., 1995
).
Digoxigenin (DIG)-labelled RNA probes were prepared by in vitro transcription of the cloned cDNAs, whose inserts were amplified by PCR and subcloned into pBluescript vectors (Stratagene). The names and locations of the probes are shown in Fig. 1(A). Probe C(1+) is complementary to the sequence from nt 1311 to 1902 of CMV-Y RNA 1, and hybridized the positive-strand of CMV RNA 1. Probe C(2+) is complementary to the sequence from nt 565 to 1016 of CMV-Y RNA 2. Probe C(3+) is complementary to nt 1356 to 1966 of CMV-Y RNA 3. Similarly, probes P(1+), P(2+) and P(3+) are complementary to nt 1433 to 1835 of PSV-J RNA 1, nt 577 to 884 of PSV-J RNA 2 and nt 1330 to 1911 of PSV-J RNA 3, respectively. Probes C(1-), C(2-), C(3-), P(1-), P(2-) and P(3-) are complementary to C(1+), C(2+), C(3+), P(1+), P(2+) and P(3+), respectively. The in vitro transcription procedure followed the manufacturer's instructions (DIG RNA labelling kit; Roche Diagnostics). Prehybridization buffer contains 5x SSC, 50 % deionized formamide, 0·1 % sodium lauroylsarcosine, 7 % SDS, 2 % blocking reagent (Roche Diagnostics) and 200 ng yeast tRNAs ml-1. The viral RNAs on the transferred membrane were hybridized with the probes overnight at 68 °C. After washing the membrane, signals were developed with chemiluminescent substrate CSPD (Roche Diagnostics).
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RESULTS AND DISCUSSION |
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Viral RNA accumulation in the inoculated and upper leaves of cowpea was examined by Northern blot hybridization using mixed probes, each of which specifically hybridized with each CMV and PSV RNA segment (Fig. 1). The RNAs of C1C2C3 and C1C2P3 were not detected in the inoculated or upper leaves (Fig. 1B and C
, lanes 1 to 4). It was reported that very little CMV-Y accumulated in the small necrotic local lesions on inoculated cowpea leaves 7 days post-inoculation (Karasawa et al., 1997a
). By contrast, the accumulated RNAs of P1P2C3 and P1P2P3 in the inoculated leaves were readily detected (Fig. 1B and C
, lanes 5 and 7). No viral RNA was detected from the upper leaves of cowpea infected by P1P2C3 though RNA of P1P2P3 was readily detected (Fig. 1B and C
, lanes 6 and 8). A few bands found between RNA 3 and 4 in Fig. 1
(and also found in Figs 3, 4 and 5
) could be nonspecific signals or degraded viral RNA, which were also detected in the previous papers (Suzuki et al., 1991
, 2003
; Kim & Palukaitis, 1997
).
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Replication of reassortants in protoplasts
To analyse whether the reassortants can replicate in a single cell, we inoculated them into tobacco BY2 protoplasts. Positive- and negative-strand viral RNA accumulation was examined by Northern hybridization using strand-specific probes (Figs 3 and 4). When the reassortants consisting of homologous pairs of RNA 1 and 2 (C1C2 and P1P2) together with either C3 or P3 were analysed, accumulation of all positive-strand RNAs was observed (Fig. 3A and B
; lanes 1, 2, 7 and 8). No positive-strand RNA was detected in C1P2 combined with either C3 or P3 (Fig. 3A and B
, lanes 3 and 4), even after prolonged exposure of the Northern blot (data not shown), suggesting that C1P2 failed to replicate any positive-strand RNA. In contrast, genomic RNA accumulated in P1C2 combined with either C3 or P3 (Fig. 3A and B
; lanes 5 and 6).
Similarly, negative-strand RNA accumulated in protoplasts infected by C1C2, P1P2 or P1C2 combined with either C3 or P3 (Fig. 4A and B, lanes 1, 2, 5 to 8). Negative-strand RNA did not accumulate in the protoplasts inoculated with C1P2 together with either C3 or P3 (Fig. 4A and B
, lanes 3 and 4).
Of note, subgenomic RNA 4 of P1C2 together with either C3 or P3 was not detectable (Fig. 3A, lane 5; Fig. 3B
, lane 6), while positive- and negative-strand RNA 3 was detected (Fig. 3A
, lane 5; Fig. 3B
, lane 6; Fig. 4A
, lane 5; Fig. 4B
, lane 6). Therefore, eight times more RNA of P1C2C3 or P1C2P3 compared with C1C2C3 and P1P2C3, respectively, was blotted and hybridized with probes C(3+) or P(3+), respectively (Fig. 5A and B
). The results indicated that the signal intensities of RNA 3 of P1C2C3 and P1C2P3 were almost equal to those of the homologous combinations C1C2C3 or P1P2P3, respectively (Fig. 5A and B
; lanes 3 and 4). Nevertheless, subgenomic RNA 4 of P1C2C3 and P1C2P3 was not detected (Fig. 5A and B
; lane 4), suggesting decreased subgenome transcription by P1C2 replicase.
It has been reported that reassortants containing heterologous pairs of RNA 1 and 2 of BMV and CCMV could not replicate their genome in barley protoplasts (Allison et al., 1988). Subsequently, DNA-based expression vectors for 1a or 2a proteins of BMV or CCMV were co-transfected in N. benthamiana protoplasts together with RNA 3 transcripts (Dinant et al., 1993
). Interestingly, the heterologous combination of BMV 1a protein (B1a) and CCMV 2a protein (CC2a) could replicate RNA 3, while no RNA 3 was replicated with the combination CC1a and B2a. The accumulation of positive-strand RNA 3 with B1a and CC2a was only 3 % of that with B1a and B2a, and no subgenomic RNA 4 was detected in spite of the 50 % accumulation of negative-strand RNA 3 with B1a and CC2a compared to that with B1a and B2a. Hence, Dinant et al. (1993)
speculated that some aspects of 1a2a interactions in positive-strand and subgenomic RNA synthesis might be distinct from those in negative-strand synthesis.
We observed that the efficiency of positive-strand synthesis with P1C2 combinations was eight times less than that with homologous combinations. Furthermore, our results suggest that the P1C2 combination can synthesize negative- and positive-strand RNA, but fails to transcribe subgenomic RNA 4.
Interaction between 1a and 2a proteins in yeast
We assayed the interaction between 1a and 2a proteins of CMV and PSV in a yeast two-hybrid system to investigate whether the replication of reassortants in protoplasts was correlated with 1a2a interactions. An interaction is detected as the growth of the yeast transformants on (-L/-W/-A/-H) plates. P2(1834) fused with GAL4 AD was designated ADP2(1834), and the plasmid containing the ADP2(1834) DNA clone was designated pADP2(1834) (Fig. 2). pADP2(1834) was co-transformed with pBDP1(63995) or pBDP1(4761005), which contained the DNA sequence of the fusion protein of GAL4 BD and P1(63995) or P1(4761005), respectively (Fig. 2
). The transformed colonies readily grew on (-L/-W/-A/-H) plates (Table 1
, columns 1 and 3). In contrast, the transformants containing pADP2(1834) and pBDP1(63318) or pBDP1(723995), did not grow on (-L/-W/-A/-H) plates (columns 2 and 4). These results indicate that the C-terminal half of 1a protein (aa 476 to 1005) of PSV interacts with 2a protein of PSV and that the N-terminal region (aa 63318) of 1a protein might not be required for the interaction. When pBDP1(63995), which expressed a large fragment of 1a protein, and pADP2(1834) were co-transformed, colonies survived on the (-L/-W/-A/-H) plates, but there were approximately ten times fewer colonies than on the (-L/-W) plates (column 1). It was reported that smaller fusion proteins generally work better in the two-hybrid system, possibly because they can enter the nucleus more efficiently than larger proteins (Fields & Sternglanz, 1994
). We postulated that 1a and 2a proteins of PSV are both so large that fusion proteins might have difficulty entering the nucleus.
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Subsequently, the interspecies 1a2a interactions were analysed. We used C1(481993) and C2(1312), parts of the 1a and 2a proteins of CMV that are homologous to P1(4761005) and P2(13352) of PSV, respectively (Fig. 2). Although the colonies co-transformed with pBDC1(481993) and pADC2(1312) grew well on (-L/-W/-A/-H) plates (Table 1
, column 8), the transformants with pBDC1(481993) and pADP2(13352) did not grow (column 9). Interestingly, colonies co-transformed with pBDP1(4761005) and pADC2(1312) grew on (-L/-W/-A/-H) plates (column 10), indicating that the C-terminal half (aa 476 to 1005) of PSV 1a protein interacted with the N-terminal region (aa 1 to 312) of CMV 2a protein. This positive interaction was not nonspecific because BDP1(4761005), BDC1(481993), ADP2(13352) and ADC2(1312) did not interact with the control fusion protein (columns 11 to 14).
The interaction was also assayed using -galactosidase activity. The results corresponded to those of the growth assay without the combination BDC1(481993) and ADP2(13352) (column 9). The interaction between C1(481993) and P2(13352) might be very weak, as suggested by the combination of BDBMV 1a insertion mutants and ADN-terminal BMV 2a (O'Reilly et al., 1997
).
In conclusion, all these results indicate that an interaction between 1a and 2a proteins is involved in viral RNA replication of interspecific reassortants (Fig. 6). Interestingly, although the synthesis of positive- and negative-strand RNAs by replicase between PSV 1a and CMV 2a proteins did not decrease so drastically compared to that by homologous replicase, the subgenomic transcription ability decreased severely. Very low accumulation of subgenomic RNA 4 in the P1C2-reassortant should be followed by little translation of CP molecules. We hypothesize that the non-pathogenicity of the reassortant of P1C2 on cowpea might be caused by a lack of CP, because CP is required for cell-to-cell movement and systemic spread of CMV (Suzuki et al., 1991
; Canto et al., 1997
).
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ACKNOWLEDGEMENTS |
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Received 31 December 2002;
accepted 10 March 2003.