Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan1
Laboratory of Plant Pathology and Biotechnology, Faculty of Agriculture, Kochi University, Nankoku, Kochi 783-8502, Japan2
Author for correspondence: Kazuyuki Mise.Fax +81 75 753 6131. e-mail kmise{at}kais.kyoto-u.ac.jp
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Abstract |
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Introduction |
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In recent years, D- and DI-RNAs have been found and well characterized in many plant viruses, including several tombusviruses (Hillman et al., 1987 ; Burgyan et al., 1989
; Rochon, 1991
;Rochon & Johnson, 1991
), carmoviruses (Li et al., 1989
), potexviruses (White et al., 1991
), broad bean mottle bromovirus (BBMV) (Romero et al., 1993
; Pogany et al., 1995
) and cucumber mosaic cucumovirus (CMV) (Graves & Roossinck, 1995
).
Brome mosaic bromovirus (BMV) is a small, spherical plant virus that infects cereals, including barley (Lane, 1981 ). The genome of BMV consists of three species of messenger-sense single-stranded RNA, 1, 2 and 3 (Ahlquist, 1992
). RNA1 (3·2 kb) and RNA2 (2·9 kb) encode the 1a and 2a proteins, respectively, which are required for virus RNA replication (French et al., 1986
; Kibertis et al., 1981
). RNA3 encodes the 3a protein, which is required for cell-to-cell movement of the virus (Schmitz & Rao, 1996
). Subgenomic RNA4, which encodes the coat protein (CP), is synthesized by the virus replicase from a promoter present in the (-)-strand of RNA3 (Miller et al., 1985
).
We have found D-RNAs in purified virions of wild-type BMV and in several CP mutants after prolonged infection (8 weeks) of barley plants. The D-RNAs were derived from RNA3 by single or double deletions in the 3a protein gene. Here we report on the molecular characterization of two D-RNA clones that were derived from a CP mutant of BMV.
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Methods |
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Plasmid clones.
Plasmids pBTF1, pBTF2 and pBTF3W contain the full-length cDNAs of BMV RNA1, RNA2 and RNA3, respectively (Mori et al., 1991 ; Mise et al., 1992
). Progeny virus derived from infected plants inoculated with in vitro transcripts from these plasmids was referred to as KU2 strain (Nagano et al., 1997
).
A plasmid pBTF3WSS5R25 was constructed as follows. pBTF3W was digested with SalI and SacI and the resulting 0·2 kbp fragment was exchanged with the corresponding fragment of pAT3J5, a plasmid that contains the CP gene of BMV strain ATCC PV-47 (accession number X58459), to create pBTF3WSS5. The nucleotide sequences of the SalI/SacI region differed between pBTF3W and pAT3J5 at six sites [A1312G, T1314C, A1323G, C1350A, A1356G and G1374A, where the two letters refer to nucleotides of pBTF3W and pAT3J5 and numbers indicate nucleotide position (Mise et al., 1994 )]. On the basis of the nucleotide differences, single or double point mutations were introduced into pBTF3W or pBTF3WSS5 by site-directed mutagenesis (Kunkel et al., 1987
). Six amino acids contained in the resulting plasmids are listed in Table 1
. Progeny virus derived from inoculation with in vitro transcripts of pBTF3WSS5R25 together with those from cDNA clones of BMV RNA1 and RNA2 was named R25.
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Nucleotide sequences of two full-length and several partially synthesized cDNA clones of D-RNA were analysed with an automated DNA sequencer (Applied Biosystems, model 373A) according to the manufacturer's recommendations.
In vitro transcription, inoculation, purification of virus and RNA extraction.
All plasmids were linearized with EcoRI and used as templates for in vitro transcription. Capped full-length transcripts were synthesized in vitro by using T7 RNA polymerase (Mori et al., 1991 ).
Barley (Hordeum vulgare L. cv. Gose-shikoku) plants were grown under conditions described previously (Fujita et al., 1996 ). Six-day-old seedlings were used for inoculation. Virions and virion RNA were purified from infected plants as described previously (Okuno & Furusawa, 1979
).
Isolation of protoplasts of barley (cv. Hinode-hadaka) and inoculation of in vitro transcripts and virion RNAs were performed as described previously (Okuno & Furusawa, 1978 ; Kroner & Ahlquist, 1992
). Total RNAs were extracted and virion fractions were obtained by PEG precipitation (Kroner & Ahlquist, 1992
) from infected protoplasts at 24 h after inoculation.
Northern blot analysis.
Total or virion RNA was denatured and separated in a 1·5% agarose gel containing formaldehyde and MOPS and transferred to a nylon membrane (Hybond-N+, Amersham). (+)- and (-)-strand RNAs were detected by using 32P-labelled SP6 transcripts from HindIII-linearized pBSPL10 (Kaido et al., 1995 ) and EcoRI-linearized pBSMI10 (Mori et al., 1993
), respectively. The RNA signals were quantified with a digital radioactive imaging analyser (Fujix BAS 2000, Fuji).
Western blot analysis.
Proteins were extracted from barley protoplasts with sample buffer (Laemmli, 1970 ) and separated by electrophoresis on 15% polyacrylamide gels containing 0·1% SDS (Laemmli, 1970
). Proteins were transferred to PVDF membranes (Millipore) by using a transfer-blot SD semi-dry transfer cell (Bio-Rad) according to the manufacturer's instructions. Accumulation of the 3a protein and the CP of BMV was analysed with anti-BMV 3a monoclonal antibody (Fujita et al., 1998
) and anti-BMV antisera (Nagano et al., 1997
), respectively. The proteins were detected with alkaline phosphatase-conjugated anti-Ig secondary antibody, followed by a colour reaction with 5-bromo-4-chloro-3-indolyl phosphate in combination with nitro blue tetrazolium. Protein bands were scanned by an Epson Scan II (Seiko Epson) and protein accumulation was quantified with the Quantity One program (PDI) version 3.0.
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Results |
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Two full-length cDNA clones of the D-RNA were obtained and their nucleotide sequences were determined. These clones had sequences similar to that of R25 RNA3. However, one clone (D1) had a single large deletion (500 bp) in the 3a ORF (Fig. 2). The other clone (D2) had a similar 500 bp deletion and an additional small deletion (66 bp) (Fig. 2
), both in the 3a ORF. There was no deletion in the CP ORF or the non-coding region (sequence data not shown) in these clones.
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Encapsidation of D-RNA was tested in barley protoplasts. The virion fraction was prepared from infected protoplasts and packaged RNA was analysed by Northern blot analysis. The D-RNA was detected together with BMV genomic RNAs (Fig. 3d, lanes 3 and 4), indicating that D-RNA was encapsidated into virions.
Heterogeneity of D-RNA sequence
To analyse any heterogeneity that may be present in the D-RNA population, virion RNA containing D-RNA was extracted at 8 weeks p.i. and full-length cDNAs were amplified by RTPCR as described above. The PCR products of RNA3 and D-RNA were separated and purified after low-melting-point agarose gel electrophoresis and digested with restriction enzymes FokI, ScaI, ClaI, FbaI, Aor51HI or SacI (Fig. 4a). Digested cDNAs were analysed by agarose gel electrophoresis. Full-length cDNA of R25 RNA3 used as control was completely digested by all the enzymes tested, resulting in the expected fragments corresponding to the enzyme cutting sites (Fig. 4a
). The cDNA products of D-RNA were completely digested by FokI, Aor51HI and SacI. After Aor51HI digestion, D-RNA cDNA as well as RNA3 cDNA produced a 1·0 kbp fragment corresponding to the 3' half of RNA3, indicating that there were no deletions in that region, which includes most of the intercistronic region, the CP ORF and the 3' non-coding region. However, the cDNA of the D-RNAs was not digested by ScaI or ClaI (central part of the 3a ORF), indicating that those sites did not exist in the D-RNA. Since the cDNA of the D-RNA was only partially digested by FbaI (3' region of 3a ORF), the D-RNA population was assumed to be heterogeneous. This restriction enzyme mapping also confirms the sequencing result (Fig. 2
) that a deletion occurred around the central region of 3a ORF. However, the cDNA of the D-RNA was completely digested by FokI, which has no recognition site in the D2 sequence, suggesting that D2 RNA could be a minor species.
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Discussion |
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Second, the D-RNAs were generated by either single or double deletions, exclusively in the 3a ORF (Figs 2 and 4 b
). The deleted regions are nt 369868 of D1 RNA and nt 201266 and 366865 of D2 RNA, suggesting that the regions retained are essential for the accumulation of BMV D-RNA in planta. The D-RNA clones with single deletions had different deletion junctions (Fig. 4b
), which resulted in sizes of deletions from 477 to 500 bp. In barley protoplasts inoculated with in vitro transcripts of D-RNA with either one or two deletions, the D-RNAs were replicated and encapsidated into virions when co-inoculated with BMV genomic RNAs. When present together with the genomic RNAs, the D-RNA reduced the accumulation level of protein 3a (Fig. 3c
). This could be the result of competition for ribosomes between D-RNA and wt RNA3 in the synthesis of the truncated and wt 3a proteins.
The third specific feature of the BMV D-RNAs is their generation. As previously reported, repeated passage at high m.o.i. is required for the generation of DI-RNAs in animal viruses (Holland, 1990 ) and D-/DI-RNA in some plant viruses (Morris & Hillman, 1989
; Knorr et al., 1991
; Graves & Roossinck, 1995
). However, the BMV D-RNAs were generated in rather unique circumstances. Generation of D-RNA was demonstrated by inoculation of barley seedlings with virion inocula either containing or lacking D-RNA. In both cases, the D-RNA was not detected at 16 weeks p.i., but was detected after prolonged infection (8 weeks p.i.) (Table 2
).
These features raise interesting questions; why isn't the D-RNA with deletions in the 3a ORF maintained even in the initially inoculated leaves, and why does the 3a ORF become a target for deletions after prolonged infection? Explanations for these observations could be: (i) since D-RNAs replicate efficiently in protoplasts (Fig. 3a) and are encapsidated into virions (Fig. 3d
), the lack of cell-to-cell movement rather than replication may be responsible for the lack of D-RNA maintenance in initially inoculated leaves. Because the 3a gene has a crucial role in virus cell-to-cell movement (Schmitz & Rao, 1996
), RNA3 with a truncated 3a gene may be less advantageous for further cell-to-cell movement than that with an intact 3a gene. Therefore, the D-RNA may fail to accumulate to detectable levels even in initially inoculated leaves. (ii) It is possible that there may be a unique interaction between either BMV strains or BMV CP mutants and 8-week-old barley plants. Physiological changes in old barley may interact with and/or alter the 3a gene. Alternatively, the intact 3a gene might be dispensable in old barley plants. Therefore, the dispensable region in the 3a gene might be deleted and regions that are presumed to be necessary for RNA replication and encapsidation in plants could be retained.
Previously, D- and DI-RNAs have been found to be associated with CMV (Graves & Roossinck, 1995 ) and BBMV (Romero et al., 1993
; Pogany et al., 1995
), respectively. Both viruses, as well as BMV, belong to the family Bromoviridae and their genomic organization is similar. D-RNAs of CMV and BMV originate from parental genomic RNA3, while DI-RNAs of BBMV are from RNA2. On the other hand, the generation processes are quite different. The CMV D-RNAs are produced upon serial passage of the wild-type Fny strain and are maintained by virus even after additional passage, while the BBMV DI-RNAs occur naturally in strains Tu and Mo (Romero et al., 1993
) and are generated de novo by serial passage at high m.o.i. (Pogany et al., 1995
). In contrast, BMV D-RNAs are produced during prolonged infection of BMV and cannot be maintained even in the initially inoculated leaves. This might result from young-barley-mediated inhibition of D-RNA encapsidation or an increased instability of D-RNA-containing virions, as previously reported in the case of BBMV DI-RNAs in pea (Romero et al., 1993
). Further studies to find a favourable host that could support the maintenance of BMV D-RNAs are needed to investigate whether the existence of D-RNAs has any effect on the symptom development induced by the helper virus.
The mechanism of formation of BMV D-RNA is unknown. However, short regions showing sequence similarity and/or complementarity were found at the 5' and 3' junction sites (Figs 4b and 5
). These local complementarities could juxtapose two molecules of (+)-strand RNA3, which might allow the replicase to switch from one template to another (Fig. 5a
), as proposed previously for recombination events among BMV RNAs (Bujarski & Dzianott, 1991
; Bujarski et al., 1994
). Such a template-switching mechanism has also been largely accepted to be responsible for the generation of D-/DI-RNAs in animal viruses (Huang, 1977
; Holland, 1990
) and in plant viruses (Pogany et al., 1995
; Graves & Roossinck, 1995
).
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Acknowledgments |
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Footnotes |
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b Permanent address: Faculty of Agriculture, Department of Plant Pests and Diseases, Bogor Agriculture University, Bogor 16144, Indonesia.
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References |
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Received 11 March 1999;
accepted 17 May 1999.