The optimal temperature for RNA replication in cells infected by Soil-borne wheat mosaic virus is 17 °C

Shuichi Ohsato1, Masaki Miyanishi2 and Yukio Shirako2

1 Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
2 Asian Center for Bioresources and Environmental Sciences (ANESC), University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan

Correspondence
Yukio Shirako
shirako{at}ims.u-tokyo.ac.jp


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Systemic infection of wheat plants with Soil-borne wheat mosaic virus (SBWMV) requires temperatures below 20 °C. Here we examine the cause of the temperature sensitivity by inoculating infectious in vitro transcripts of SBWMV RNA1 and RNA2 to barley mesophyll protoplasts. After RNA inoculation, protoplasts were incubated at temperatures between 15 and 25 °C for up to 48 h. Western blot analysis showed that the capsid protein accumulated most abundantly at 17 °C but was not detectable at 25 °C. Northern blot analysis showed that the wild-type RNA1 and RNA2 and their subgenomic RNAs accumulated most abundantly at 17 °C but were barely detectable at 25 °C. An RNA1 mutant in which the p152 and p211 replicase genes were placed between the 5'- and 3'-untranslated regions also replicated most efficiently at 17 °C but not at 25 °C. Thus, the requirement for temperatures lower than 20 °C for SBWMV infection is primarily determined by replication of RNA1, which encodes the viral RNA replicase.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Natural hosts of Soil-borne wheat mosaic virus (SBWMV), the type species in the genus Furovirus (Torrance, 2000), are winter wheat and winter barley (Brakke, 1971). The virus is transmitted by zoospores of the plasmodiophoraceous fungus Polymyxa graminis (Linford & McKinney, 1954), which germinates from resting spores in the old root debris from infected plants and which transmits the virus to roots of young seedlings during the autumn in infested fields (Brakke et al., 1965; Brakke & Estes, 1967). After overwintering, infected plants start showing conspicuous mosaic symptoms on newly developing leaves, usually accompanied by stunting or rosetting of the plants when the temperature increases during the early and middle spring. Those plants that do not develop severe symptoms will recover from the disease and grow as vigorously as uninfected plants in the late spring and early summer (Brunt & Richards, 1989; Shirako & Wilson, 1999).

Early studies on fungal transmission using root washings containing zoospores or root debris containing resting spores showed that systemic infection of SBWMV occurred when the inoculated plants were grown at temperatures below 20 °C (Brakke & Estes, 1967; Brakke & Rao, 1967; Rao & Brakke, 1969). In such experiments, wheat and barley plants inoculated with SBWMV by mechanical inoculation were grown in a growth cabinet below 20 °C to get systemic infection (McKinney, 1948; Rao & Brakke, 1970). Thus, SBWMV appears to be adapted to host plants that grow under a cool climate from autumn to the following spring. The cause of the requirement for low temperatures for SBWMV infection, whether due to RNA replication, cell-to-cell movement or both, has not been resolved.

The genome of SBWMV consists of a 7·2 kb RNA1 and a 3·6 kb RNA2 (Shirako et al., 2000). RNA1 encodes the p152 and p211 replicase proteins, which are equivalent to the p126 and p183 proteins of Tobacco mosaic virus, in the 5'-terminal region and a p37 putative movement protein in the 3'-terminal region (Fig. 1A, pJS1). RNA2 encodes the capsid protein (CP) and its C-terminally extended protein formed by partial readthrough at the CP termination codon in the 5'-terminal region, and a p19 cysteine-rich protein in the 3'-terminal region (Fig. 1B, pJS2).



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 1. (A) RNA1 constructs. pJS1 (WT) contains the full-length cDNA clone of SBWMV RNA1. In pJS1.Rep, the region between the termination codons for the p211 and p37 genes was deleted from pJS1 by PCR-based deletion mutagenesis. (B) RNA2 constructs. pJS2 (WT) contains the full-length cDNA clone of SBWMV RNA2. In pJS2.N-GFP/p19, the CP gene and the downstream readthrough region in pJS2 were replaced with a GFP gene by a PCR-based fragment exchange and deletion method. Rightward arrows above the rectangular boxes indicate the location of a leaky UGA termination codon.

 
Here, we report on studies of SBWMV RNA replication in barley mesophyll protoplasts inoculated with infectious in vitro transcripts from full-length cDNA clones of RNA1 and RNA2 of a Japanese strain of SBWMV (Yamamiya & Shirako, 2000). Our results showed that the optimal temperature of SBWMV RNA1 replication is 17 °C and that the temperature optimum is determined by the p152 and p211 replicase proteins and/or the 5'- and 3'-untranslated region (UTR) in RNA1.


   METHODS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
cDNA constructs for in vitro transcription of infectious RNA.
Full-length cDNA clones for RNA1 and RNA2 of a Japanese strain of SBWMV (Tochigi-82), pJS1 and pJS2, have been described previously (Yamamiya & Shirako, 2000). pJS1.Rep (Fig. 1A), which contained only the p152 and p211 replicase genes between the 116 nucleotide 5'-UTR and the 559 nucleotide 3'-UTR of RNA1, was constructed by deleting nt 5646–6666 in pJS1 as follows. The nt 3612–5645 region was amplified using primers TP48 (plus sense; nt 3612–3628 of RNA1) and TN91 (minus sense; nt 6667–6675 followed by nt 5630–5645 of RNA1) while nt 6667–7226 was amplified using TN90 (plus sense; nt 5637–5645 followed by nt 6667–6682 of RNA1) and TP25 (minus sense; annealing to the 3'-terminal 20 nts of RNA1) by PCR using pJS1 as the template. The resulting 2·0 kb and 0·6 kb PCR products were combined using TP48 and TP25 by PCR over the sequence common between the two DNA fragments, followed by digestion with BglII (nt 4529 in RNA1) and HindIII (nt 6762 in RNA1). The 1·2 kb BglII–HindIII fragment was cloned into BglII/HindIII-digested vector prepared from pJS1. In pJS2.N-GFP/p19 (Fig. 1B), the CP gene and the downstream readthrough region were replaced with a GFP gene derived from pQBI25 (TaKaRa) using pJS2.N-CP:R-GFP (A. Yamamiya & Y. Shirako, unpublished) as the parental construct in which the nt 864–2502 region in RNA2 was replaced with the 720 nucleotide GFP gene. The nt 330–863 region in pJS2.N-CP:R-GFP was deleted by PCR-based deletion mutagenesis using primers TN77 (plus sense; nt 320–329 in RNA2 followed by nt 1–16 of the GFP gene) and TN78 (minus sense; nt 1–12 of the GFP gene followed by nt 318–329 in RNA2). Two NheI sites, one located upstream of the SP6 promoter sequence of pJS2 and another within the GFP gene, were used for cloning of the PCR-derived insert after digestion with NheI. The cloned inserts derived by PCR amplification were verified by nucleotide sequence analysis using an automated DNA sequencer (ABI 377). In vitro transcription was carried out as described previously using SP6 RNA polymerase (TaKaRa) (Yamamiya & Shirako, 2000).

Preparation of barley mesophyll protoplasts.
Barley mesophyll protoplasts were prepared by the methods of Okuno et al. (1977) and Loesch-Fries & Hall (1980) with minor modifications. Seedlings of barley (Hordeum vulgare L. cv Minorimugi) were grown on 1 : 1 peat moss and vermiculite at 25 °C with 16 h of light per 24 h in a growth cabinet (Sanyo, model MLR350) for 6 days. Plants were watered with half-strength Hoagland's solution (Sigma/Aldrich) every day. Two grams of leaves were cut at about 5 cm from the ground surface and the epidermis was pealed off carefully by hand. Exposed mesophyll tissues were immediately placed in an enzyme solution consisting of 0·65 M mannitol, 2 % Cellulase Onozuka R-10 (Yakult), 0·1 % Macerozyme R-10 (Yakult) and 0·1 % BSA (Fraction V; Sigma/Aldrich), pH 5·7, and incubated without stirring at 30 °C for 3 h in the dark. The enzyme solution containing released protoplasts was collected and centrifuged at 100 g for 3 min. The pellet was suspended in 5 ml 0·65 M mannitol, which was layered on 20 % sucrose in a 15 ml tube, followed by centrifugation at 100 g for 8 min. Protoplasts at the interface were collected, suspended and washed twice in 0·65 M mannitol by centrifugation at 100 g for 5 min. The final pellet was suspended in 0·65 M mannitol at a concentration of 3x105 cells ml-1.

Inoculation of protoplasts with in vitro transcripts.
One ml of protoplast suspension (containing 3x105 cells) was centrifuged at 100 g for 5 min and the supernatant was removed. The pelleted cells were suspended in 20 µl 0·65 M mannitol and 10 µl of in vitro transcripts at a concentration of about 200 ng µl-1 were added, followed by immediate mixing with 100 µl PEG solution (40 % polyethylene glycol, average Mr 1450 from Sigma/Aldrich, 30 mM CaCl2, pH 5·5) (Samac et al., 1983). The mixture was placed on ice for 1 min, followed by addition of 1 ml 0·65 M mannitol and incubation on ice for 15 min. Inoculated protoplasts were washed twice in 0·65 M mannitol by centrifugation and suspended in 1 ml of medium consisting of 0·2 mM KH2PO4, 1 mM KNO3, 1 mM MgSO4, 1 µM KI, 0·1 µM CuSO4, 10 mM CaCl2, 0·65 M mannitol, pH 6·5 (Takebe et al., 1968). The cell suspension was transferred to a 24-well plate (Corning) and incubated at 17 °C, or to microfuge tubes and incubated in heat blocks with Peltier elements (ALB 301, IWAKI) at appropriate temperatures for up to 48 h in the dark.

Expression of CP as the GST fusion protein in Escherichia coli cells and preparation of anti-GST–CP antiserum.
The SBWMV CP gene was fused in-frame to the 3' terminus of the glutathione S-transferase (GST) gene in plasmid pGEX-6P-1 (Amersham Pharmacia). The GST–CP fusion protein was expressed in transformed E. coli strain MC1061 cells by induction with 1 mM IPTG. Insoluble GST–CP inclusion body was isolated from E. coli cells lysed with bacterial protein extraction reagent (Pierce) and denatured in 1x SDS-PAGE sample buffer (1x SB), followed by 7·5 % SDS-PAGE. The 66 kDa GST–CP fusion protein was eluted from gel slices by the method of Hager & Burgess (1980) and 2 mg of the purified fusion protein was used to immunize a rabbit at Sawady Technology, Tokyo, Japan.

Western blot analysis.
Protoplasts from one well of a 24-well plate were collected by centrifugation and suspended in 20 µl 1x SB, followed by heating at 95 °C for 3 min. Ten µl of sample containing proteins from 1·5x104 cells was loaded per lane in a 12·5 % SDS-polyacrylamide gel for electrophoresis. After electrophoresis, proteins were transferred to nitrocellulose membrane (S&S) and subjected to detection of SBWMV CP using anti-GST–CP antiserum and goat anti-rabbit IgG conjugated to alkaline phosphatase (Sigma/Aldrich) using 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium as substrate, as described (Shirako & Ehara, 1986).

Northern blot analysis.
Protoplasts from one well of a 24-well plate were collected by centrifugation and suspended in 0·1 M glycine, pH 9·5, 0·1 M NaCl, 10 mM EDTA, 2 % Triton-X 100. After centrifugation at 10 000 g for 3 min to remove large organelles, SDS was added to the supernatant to a final concentration of 2 % and RNA was extracted with phenol/chloroform and precipitated in ethanol. The final pellet was suspended in water. Extracted RNA was denatured in 40 mM MOPS, pH 7·5, 10 mM Na acetate, 1 mM EDTA, 1·1 M formaldehyde at 65 °C for 5 min, followed by immediate chilling on ice. Denatured RNA was run on a 0·8 % agarose gel prepared in MOPS/formaldehyde buffer, blotted on to a nylon membrane (Hybond-N+; Amersham Pharmacia) and fixed on the membrane with 50 mM NaOH for 20 min. The membrane was rinsed in 2x SSC (0·3 M NaCl, 30 mM Na citrate) for 1 min and incubated at 68 °C in 50 % formamide, 0·02 % SDS, 0·1 % lauroylsarcosine, 2 % block stock solution (Roche) in 5x SSC (hybridization buffer) for 1 h. The membrane was further incubated at 68 °C for 6 h in the hybridization buffer containing digoxigenin (DIG; Roche)-labelled RNA probes. RNA probes annealing to nt 3124–3731 and nt 5692–6218 of RNA1 and nt 2658–3182 of RNA2 were prepared by in vitro transcription of pGEM-T plasmids containing cDNA inserts prepared by PCR using pJS1 or pJS2 as the templates in the presence of DIG–UTP according to the manufacturer's protocol. After washing twice for 5 min in 2 x SSC containing 0·1 % SDS and twice for 15 min in 0·1x SSC containing 0·1 % SDS, the membrane was incubated with anti-DIG alkaline phosphatase-conjugated goat Fab fragment (Roche) at a 10-5 dilution for 30 min. Probe-annealing bands on the membrane were detected using CDP-Star (New England Biolabs) as the substrate in a chemiluminescence detector (LAS1000, Fuji Film). In all Northern blots, the amount of RNA sample loaded was adjusted based on ribosomal RNAs stained with ethidium bromide.


   RESULTS AND DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Establishing infection of barley mesophyll protoplasts with infectious in vitro transcripts from pJS1 and pJS2.N-GFP/p19
Prior to this study, there was no report of an SBWMV or SBWMV RNA infectivity assay using barley or wheat protoplasts. Therefore, we examined the efficiency of infection of infectious in vitro transcripts in barley mesophyll protoplasts using GFP fluorescence as a marker. In vitro transcripts from pJS1 and pJS2.N-GFP/p19 (Fig. 1B) were co-transfected into barley protoplasts and the protoplasts were incubated for 24 h at 17 °C, which is the optimal temperature for systemic infection of wheat plants with SBWMV. As shown in Fig. 2, about 50 % of the protoplasts fluoresced strongly 24 h after transfection using approximately 2 µg each of RNA1 and RNA2 in vitro transcripts for 3x105 cells in 1 ml of the cell suspension buffer. Trials to increase transfection efficiency to more than 50 % using increased amounts of in vitro transcripts were not successful. Since both RNA1 and RNA2 are required for expression of the GFP gene, we assumed that more than 70 % of protoplasts received at least one species of RNA.



View larger version (74K):
[in this window]
[in a new window]
 
Fig. 2. Light microscopy of barley mesophyll protoplasts after transfection with in vitro transcripts from pJS1 and pJS2.N-GFP/p19. (A) Under visible light together with UV light. (B) Under UV light (470–490 nm) using a fluorescent light microscope (Olympus IX-70). Fluorescing cells are bright white. Bar, 25 µm.

 
Detection of CP from barley mesophyll protoplasts incubated at different temperatures after transfection with in vitro transcripts from pJS1 and pJS2
Barley mesophyll protoplasts transfected with in vitro transcripts from pJS1 and pJS2 were incubated at 15, 17, 20, 22 and 25 °C for 48 h. Protoplasts were collected by centrifugation and total proteins were subjected to Western blot analysis using anti-GST–CP antiserum. The 19 kDa CP was detected from protoplasts incubated at 17 °C (Fig. 3, lane 3). A weak band was detected at 15 and 20 °C (Fig. 3, lanes 2 and 4, respectively). A faint band was also detected at 22 °C but no band was detected at 25 °C (Fig. 3, lanes 5 and 6, respectively). At 17 °C, the 83 kDa CP-readthrough (CP-RT) protein was also clearly detected, indicating that translational readthrough at the UGA termination codon occurred efficiently at 17 °C within 48 h after transfection. On the contrary, the 24 kDa N-CP protein, which contains a 40 amino acid extension to the N terminus of the CP due to translational initiation at a CUG codon 120 nucleotides upstream from the AUG initiation codon for CP (Shirako, 1998), was not detectable within 48 h after RNA transfection.



View larger version (39K):
[in this window]
[in a new window]
 
Fig. 3. Western blot analysis of the SBWMV CP from barley mesophyll protoplasts transfected with in vitro transcripts from pJS1 and pJS2. Samples were collected 48 h after RNA transfection. Protoplasts transfected with in vitro transcripts were incubated at 15, 17, 20, 22 and 25 °C. The antiserum was prepared against the GST–CP fusion protein expressed in E. coli cells using pGEX-6P-1.

 
SBWMV RNA replication is most efficient at 17 °C
Barley protoplasts transfected with in vitro transcripts from pJS1 and pJS2 were incubated for 24 h at 15, 17, 20, 22 and 25 °C. Intracellular RNA was extracted, denatured with formaldehyde and run on an agarose gel. Fig. 4(A) shows accumulation of RNA1 in barley protoplasts as indicated by an arrow on the right. Using a probe annealing to nt 5692–6218 of the p37 gene, RNA1 was detected most abundantly at 17 °C (lane 2). The intensity of the RNA1 band decreased in the order 20, 15 and 22 °C (Fig. 4A, lanes 3, 1 and 4, respectively). At 25 °C, RNA1 was detected only as a very faint band (Fig. 4A, lane 5). There was another band detected on this blot at the position of ~1·5 kb (Fig. 4A, asterisk), most intensely at 17 °C and faintly at 20 °C. This band was not detected using a probe annealing to nt 3123–3731 in the p152 gene (Fig. 4A, lane 6). Based on the migration rate and specificity to probes in Northern blots, the 1·5 kb RNA is likely to be the subgenomic RNA for the p37 putative movement protein encoded in the 3'-terminal region. Similarly, RNA2 was detected most abundantly at 17 °C (Fig. 4B, lane 2) using a probe annealing to nt 2658–3182 in the p19 gene on RNA2. RNA2 was also detectable, although less abundantly, at 15, 20 and 22 °C, but was barely detected at 25 °C (lanes 1, 3, 4 and 5, respectively). A 0·9 kb band (Fig. 4B, asterisk) was clearly detected at 17, 20 and 22 °C (Fig. 4B, lanes 2, 3 and 4, respectively). This RNA could be the subgenomic RNA for the p19 cysteine-rich protein encoded in the 3'-terminal region.



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 4. Northern blot analysis of RNA1 (A) and RNA2 (B) replicated in barley mesophyll protoplasts after transfection with in vitro transcripts from pJS1 and pJS2. After RNA transfection, protoplasts were incubated at 15, 17, 20, 22 and 25 °C (lanes 1, 2, 3, 4 and 5, respectively) for 24 h. (A) Blotted membrane was probed with DIG-labelled antisense transcripts annealing to nt 5692–6218 in RNA1 to detect both genomic and subgenomic RNAs (lanes 1–5). Lane 6, RNA was extracted from protoplasts incubated at 17 °C for 24 h and the blotted membrane was probed with DIG-labelled probe annealing to nt 3124–3731 to detect the genomic RNA but not the subgenomic RNA. (B) Blotted membrane was probed with DIG-labelled antisense transcripts annealing to nt 2658–3182 in RNA2 to detect both genomic and subgenomic RNAs. Positions of the genomic RNAs are shown by arrows, whereas bands possibly corresponding to subgenomic RNAs are indicated by asterisks on the right. Sizes of marker RNAs are indicated.

 
The p152 and p211 replicase proteins and/or the 5'- and 3'-terminal UTR of RNA1 determine adaptation of SBWMV RNA replication at 17 °C
When barley mesophyll protoplasts were transfected with in vitro transcripts from pJS1 alone, the full-length RNA1 and the possible subgenomic RNA for the p37 protein were detected most abundantly at 17 °C using a probe annealing to nt 5692–6218 (Fig. 5A, lane 2). These two RNAs were detected in reduced amounts at 15, 20 and 22 °C, but were barely detectable at 25 °C (Fig. 5A, lanes 1, 3, 4 and 5, respectively). This result indicates that RNA1 itself can replicate in the absence of RNA2 and its gene products, and that the requirement for low temperatures is determined by RNA1. We further analysed replication of a mutant RNA1 from pJS1.Rep in which only the p152 and p211 genes are present between the 5'- and 3'-UTRs (Fig. 1A). Using a probe annealing to nt 3124–3731, the 6·2 kb RNA1.Rep band was detected at 17 °C but not at 25 °C (Fig. 5B, lanes 5 and 6, respectively).



View larger version (30K):
[in this window]
[in a new window]
 
Fig. 5. Northern blot analysis of RNA1 from pJS1 and from pJS1.Rep replicated in barley mesophyll protoplasts. (A) After RNA transfection with in vitro transcripts from pJS1, protoplasts were incubated at 15, 17, 20, 22 and 25 °C (lanes 1, 2, 3, 4 and 5, respectively) for 24 h. The blotted membrane was probed with DIG-labelled transcripts annealing to nt 5692–6218 of RNA1. Sizes of marker RNAs are shown on the right. Positions of genomic RNA and possible subgenomic RNA are shown by an arrow and an asterisk, respectively, on the left. (B) After transfection with in vitro transcripts from pJS1 and pJS1.Rep, protoplasts were incubated at 17 and 25 °C for 24 h. The blotted membrane was probed with DIG-labelled transcripts annealing to nt 3124–3731 of RNA1. Lane 1, in vitro transcripts from pJS1; lane 2, RNA from mock-infected protoplasts; lanes 3 and 4, RNA extracted from protoplasts transfected with pJS1 transcripts and incubated at 17 and 25 °C, respectively; lanes 5 and 6, RNA extracted from protoplasts transfected with pJS1.Rep transcripts and incubated at 17 and 25 °C, respectively.

 
The above results clearly indicate that the requirement for lower temperatures for SBWMV infection in plants are primarily determined at the level of viral RNA replication. Since RNA1.Rep showed the same characteristics as wild-type RNA1 in the RNA replication profile, involvement of the p37 putative movement protein and the p19 cysteine-rich protein with unknown function is excluded. The determinant for temperature sensitivity may reside not only in the p152 or p211 replicase proteins but also in the cis elements in the 5'- and 3'-UTRs as in the case of Red clover necrotic mosaic virus, in which the 3'-UTR of RNA1 was shown to contain the determinant for temperature sensitivity (Mizumoto et al., 2002). Whether the p37 putative movement protein also requires low temperatures for its activity in planta remains to be determined.


   ACKNOWLEDGEMENTS
 
We are grateful to Myron Brakke for valuable comments on this manuscript. This work was supported by a grant from the Japan Society for the Promotion of Science.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Brakke, M. K. (1971). Soil-borne wheat mosaic virus. CMI/AAB Descriptions of Plant Viruses, no. 77.

Brakke, M. K. & Estes, A. P. (1967). Some factors affecting vector transmission of soil-borne wheat mosaic virus from root washings and soil debris. Phytopathology 57, 905–910.

Brakke, M. K. & Rao, A. S. (1967). Maintenance of soilborne wheat mosaic virus cultures by transfer through root washings. Plant Dis Rep 51, 1005–1008.

Brakke, M. K., Estes, A. P. & Shuster, M. L. (1965). Transmission of soil-borne wheat mosaic virus. Phytopathology 55, 79–86.

Brunt, A. A. & Richards, K. E. (1989). Biology and molecular biology of furoviruses. Adv Virus Res 36, 1–32.[Medline]

Hager, D. A. & Burgess, R. R. (1980). Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Anal Biochem 109, 79–86.

Linford, M. B. & McKinney, H. H. (1954). Occurrence of Polymyxa graminis in roots of small grains in the United States. Plant Dis Rep 38, 711–713.

Loesch-Fries, L. S. & Hall, T. C. (1980). Synthesis, accumulation and encapsidation of individual brome mosaic virus RNA components in barley protoplasts. J Gen Virol 47, 323–332.

McKinney, H. H. (1948). Wheats immune from soil-borne mosaic viruses in the field, susceptible when inoculated manually. Phytopathology 38, 1003–1013.

Mizumoto, H., Hikichi, Y. & Okuno, T. (2002). The 3'-untranslated region of RNA1 as a primary determinant of temperature sensitivity of Red clover necrotic mosaic virus Canadian strain. Virology 293, 320–327.[CrossRef][Medline]

Okuno, T., Furusawa, I. & Hiruki, C. (1977). Infection of barley protoplasts with brome mosaic virus. Phytopathology 67, 610–615.

Rao, A. S. & Brakke, M. K. (1969). Relation of soil-borne wheat mosaic virus and its fungal vector, Polymyxa graminis. Phytopathology 59, 581–587.

Rao, A. S. & Brakke, M. K. (1970). Dark treatment of wheat inoculated with soil-borne wheat mosaic and barley stripe mosaic viruses. Phytopathology 60, 714–716.

Samac, D. A., Nelson, S. E. & Loesch-Fries, L. S. (1983). Virus protein synthesis in alfalfa mosaic virus infected alfalfa protoplasts. Virology 131, 455–462.

Shirako, Y. (1998). Non-AUG translation initiation in a plant RNA virus: a forty-amino-acid extension is added to the N terminus of the soil-borne wheat mosaic virus capsid protein. J Virol 72, 1677–1682.[Abstract/Free Full Text]

Shirako, Y. & Ehara, Y. (1986). Rapid diagnosis of Chinese yam necrotic mosaic virus infection by electro-blot immunoassay. Ann Phytopathol Soc Jap 52, 453–459.

Shirako, Y. & Wilson, T. M. A. (1999). Furoviruses. In Encyclopedia of Virology, 2nd edn, pp. 587–596. Edited by A. Granoff & R. G. Webster. New York: Academic Press.

Shirako, Y., Suzuki, N. & French, R. C. (2000). Similarity and divergence among viruses in the genus Furovirus. Virology 270, 201–207.[CrossRef][Medline]

Takebe, I., Otsuki, Y. & Aoki, S. (1968). Isolation of tobacco mesophyll cells in intact and active state. Plant Cell Physiol 9, 115–124.

Torrance, L. (2000). Genus Furovirus. In Virus Taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses, pp. 904–908. Edited by M. H. V. van Regenmortel, C. M. Fauquet, D H. L. Bishop & 8 others. San Diego: Academic Press.

Yamamiya, A. & Shirako, Y. (2000). Construction of full-length cDNA clones to Soil-borne wheat mosaic virus RNA1 and RNA2, from which infectious RNAs are transcribed in vitro: virion formation and systemic infection without expression of the N-terminal and C-terminal extensions to the capsid protein. Virology 277, 66–75.[CrossRef][Medline]

Received 1 December 2002; accepted 16 December 2002.



This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Ohsato, S.
Articles by Shirako, Y.
Articles citing this Article
PubMed
PubMed Citation
Articles by Ohsato, S.
Articles by Shirako, Y.
Agricola
Articles by Ohsato, S.
Articles by Shirako, Y.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS