1 Department of Entomology and Plant Pathology, Oklahoma State University, NRC 127, Stillwater, OK 74078, USA
2 Department of Biochemistry and Molecular Biology, Oklahoma State University, NRC 127, Stillwater, OK 74078, USA
3 Electron Microscopy Facility, Oklahoma State University, NRC 127, Stillwater, OK 74078, USA
4 Department of Statistics, Oklahoma State University, NRC 127, Stillwater, OK 74078, USA
5 Department of Plant and Soil Sciences, Oklahoma State University, NRC 127, Stillwater, OK 74078, USA
Correspondence
Jeanmarie Verchot-Lubicz
Verchot{at}okstate.edu
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ABSTRACT |
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INTRODUCTION |
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The most extensive research on SBWMV has been conducted using Japanese and US isolates of the virus. However, it has not been determined yet whether the US and Japanese strains of SBWMV are related or if they are separate species. Phylogenetic comparisons of US and Japanese strains of SBWMV indicated that the CP of the Japanese strain is related more closely to European wheat mosaic virus (EWMV) than to the US strain of SBWMV (Shirako et al., 2000). Pairwise comparisons indicated that the RNA1-encoded viral replicases of the US and Japanese viruses share 78 % homology and the viral CPs share 82 % sequence homology (Shirako et al., 2000
). Further research comparing virus host range, cytological effects of the virus and serological relationships should to be conducted to determine the relatedness of these two viruses.
An infectious clone of the Japanese strain of SBWMV was prepared and was used in reports exploring viral CP functions (Yamamiya & Shirako, 2000). Research using the US isolate of SBWMV is limited by the lack of a stable infectious cDNA clone of the virus. Full-length cDNA clones used for making infectious transcripts of the US strain of SBWMV have not been obtained due to instability in Escherichia coli cells (unpublished data).
The RNA1 sequences of SBWMV, Oat golden stripe virus, Sorghum chlorotic spot virus, Cereal wheat mosaic virus and EWMV encode a single 37K protein, which has been suggested to be the virus MP. SBWMV 37K has been assigned to the 30K superfamily of virus MPs, based on amino acid sequence alignments (Melcher, 1990, 2000
). The MPs of the genera Furovirus, Dianthovirus, Alfamovirus, Tenuivirus, Cucumovirus and Bromovirus formed a cluster that radiated from a common point (Melcher, 2000
). However, in the previous analysis, only SBWMV 37K was included as a representative of the genus Furovirus. This may have been a reason for the lack of subdefinition of the relationships between the furovirus, dianthovirus, alfamovirus and tenuivirus MPs (Melcher, 2000
).
This study combines three different approaches in an attempt to characterize better the US isolate of SBWMV 37K. First, to test the hypothesis that SBWMV 37K is a virus MP, experiments were conducted to determine if 37K has the ability to move between adjacent cells (Carrington et al., 1996). Plasmids containing the GFP gene fused to the 37K ORF were delivered by biolistic bombardment to wheat leaves and protein movement was monitored. Second, SBWMV-infected and 37K-expressing transgenic wheat plants were used to study protein subcellular targeting to determine if 37K, like other virus MPs, associates with plant cell walls. Third, amino acid sequence comparisons of the furovirus 37K proteins were conducted to identify conserved amino acid sequence motifs and secondary structural elements among members of this genus. More members of each genus were included in a multiple sequence comparison to determine if the relationships between the furovirus, dianthovirus, alfamovirus and tenuivirus MPs may be defined better.
In many studies using viruses that infect dicotyledonous plants, there are reports in which transgenic tobacco plants expressing virus MPs were used to study plasmodesmata gating activity, protein subcellular targeting and complementation of movement-defective viruses. While transgenic tobacco plants have often been used in plant virus studies, transgenic monocotyledonous plants have never been used to study plant viral proteins. Recent successes in preparing transgenic wheat plants have made this avenue of research possible for plant virologists. Herein, we demonstrate that SBWMV 37K can be expressed in transgenic wheat. Subcellular targeting of 37K was studied and the results of these experiments suggest that transgenic monocotyledonous plants may become a tool for studying proteins of viruses that infect monocotyledonous plants.
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METHODS |
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Four plasmids, pAHC25-GFP, pAHC25-GFP:37K, pAHC25-GFP:CP and pAHC25-GFP:RT, were prepared and used to transform E. coli JM109 cells. The plasmid pAHC25 (Christensen & Quail, 1996) contains two expression cassettes: the first contains the bar gene fused to the ubi promoter and nos terminator and the second contains the uidA gene fused to the ubi promoter and nos terminator. The uidA gene was replaced by the fused gene inserts in all pAHC25 constructs. PCR products were amplified using a phosphorylated forward primer and a reverse primer containing a SacI restriction site. For all constructs, pAHC25 was digested with SmaI/SacI, while PCR products were digested with SacI. Linearized plasmid and digested PCR products were gel-purified and ligated.
pGFP was prepared using the EGFP gene (Clontech). EGFP was amplified by PCR using a phosphorylated EGFP forward primer (5'-GCCACCATGGTGAGCAAGGG-3') and an EGFP reverse primer (5'-GATGAGCTCCGCTTTACTTGTACAG-3'), which contains the translation stop codon and additional sequences encoding a SacI site (underlined).
To prepare the GFP37K fusions, the EGFP gene was fused to the SBWMV 37K ORF using overlapping PCR. For GFP37K, EGFP was amplified by PCR using the EGFP forward primer (indicated above) and a reverse primer containing an additional 14 nt corresponding to the 5' end of the 37K-encoding sequence (5'-TCCTGTGAGCCCATCTTGTACAGCTCGTCCATGCC-3'). A 37K forward primer (5'-GGACGAGCTGTACAAGATGGGCTCACAGGATGTC-3') containing an additional 16 nt and corresponding to the 3' end of the EGFP-encoding sequence and a reverse 37K primer (5'-GATGAGCTCCGCTCTACACACTATC-3') containing an additional 13 nt and encoding a SacI restriction site (underlined) were used to amplify the 37K-encoding sequence. The GFP and 37K PCR products were annealed and then amplified by PCR using the GFP forward and 37K reverse primers.
To prepare the GFPCP fusion, a phosphorylated CP forward primer (5'-GCCACCATGGCGGTAAATAAAGG-3') and a CP reverse primer (5'-CCTCGCCCTTGCTCACCATACTCGAACCTTCCCATTTCAAGT-3') containing an additional 19 nt and corresponding to the EGFP gene was used to amplify the CP-encoding sequence. The EGFP-encoding sequence was amplified using a forward primer (5'-GGGAAGGTTCGAGTATGGTGAGCAAGGGCGAGG-3'), which contains 14 nt overlapping the CP-encoding sequence, and the EGFP reverse primer (described above). The CP and EGFP PCR products were annealed and then amplified by PCR using the CP forward primer and the EGFP reverse primer.
To prepare the GFPRT fusion, a phosphorylated EGFP forward primer (described above) and a EGFP reverse primer (5'-GAGACGCCGTCCCGCTTGTACAGCTCGTCCATGCCG-3') containing 14 nt overlapping the RT domain were used to amplify EGFP. The RT domain was amplified using a forward primer (5'-GGACGAGCTGTACAAGCGGGACGGCGTCTCGGGAAAG-3'), which contains 16 nt overlapping the EGFP sequence, and a reverse primer (5'-GATGAGCTCCGCTTTAGGACGCCATAG-3'), which contains sequences 3' of the translation stop codon and which encodes a SacI site (underlined). The EGFP and RT-PCR products were annealed and amplified using the EGFP forward primer and the RT reverse primer.
Since the RT domain contains an internal SacI site located within the SBWMV RNA2, around nt 2154, the initial pAHC25-GFP:RT plasmid lacked the 3' region of the RT domain. Therefore, a fragment of the RT domain was amplified by PCR using a forward primer (5'-GCCCTCGAGCTCTTTGATAGAGCCGG-3'), corresponding to nt 21492175 within the SBWMV RNA2, and the RT reverse primer (described above). The PCR product and pAHC25-GFP:RT plasmid were digested with SacI and ligated.
Plant material and biolistic bombardment.
Leaves of hard red winter wheat (cv. Vona) and tobacco (cv. Petit Havana) were bombarded with 10 µg plasmid mixed with 1 mg of 1 µm gold particles. Of the DNA/gold mixture, 10 µl was loaded onto a carrier disk and bombarded to detached leaves, as described previously (Yang et al., 2000). Leaves were observed at 1 and 3 days post-bombardment (p.b.) using an epifluorescence microscope to detect GFP expression (Yang et al., 2000
).
The 37K-expressing transgenic wheat plants were produced following bombardment of immature wheat embryos. Immature embryos of the wheat variety Bobwhite were cultured for 5 days in the dark on a callus induction medium (CIM) containing 4·3 g MS salts l-1, MS vitamins, 2 % sucrose, 1·5 mg 2,4-dichlorophenoxyacetic acid l-1, 0·150 g L-asparagine l-1 and 2·5 g phytagel l-1 (all chemicals from Sigma) before microprojectile bombardment. Osmotic treatment before and after bombardment was performed on CIM medium with 0·4 M mannitol. The PDS-1000/He apparatus (Bio-Rad) was used for biolistic transformation. Plasmid DNA-coated gold particles were dried on a macrocarrier membrane (Bio-Rad) and bombarded to immature wheat embryos with a 1100 PSI rupture disk. Following bombardment, immature embryos were kept on the same medium overnight and transferred to callus selection medium (CIM medium with 1·5 mg bialophos l-1) for 56 weeks at 20 °C in the dark. Bialophos-resistant callus was transferred to shoot initiation medium (MS medium containing 2 % sucrose, 0·5 mg dicamba l-1, 1·5 mg bialophos l-1 and 2·5 g phytagel l-1) for 34 weeks at 20 °C under a photoperiod of 16 h. Regenerated shoots were transferred to Magenta boxes with root induction medium (containing half-strength CIM medium and lacking 2,4-dichlorophenoxyacetic acid) for 45 weeks at 20 °C under the above conditions of light.
Plantlets were transferred from root induction medium to soil. T0 plants were self-pollinated and grown to maturity in a greenhouse. T1 plants were used for immunoblot analysis and immunogold histochemistry.
Immunoblot analysis of transgenic wheat leaves.
Immunoblot analysis was conducted using leaf extracts from 37K-expressing T1 transformants. For a single transgenic line, 10 T1 plants were grown for 2 weeks in a growth chamber and approximately 100 mg leaf tissue was ground in 100 µl protein extraction buffer (100 mM Tris/HCl, pH 7·5, 10 mM KCl, 5 mM MgCl2, 0·4 M sucrose, 10 % glycerol and 10 mM -mercaptoethanol) and were subjected to immunoblot analyses following electrophoresis using a 12·5 % SDS-polyacrylamide gel. Proteins were transferred to Hybond-P membranes (Amersham) and immunoblot analyses were conducted using rabbit anti-37K serum and the ECL chemiluminescence detection kit (Amersham). Rabbit antiserum raised against bacterially produced 37K was obtained from J. Sherwood (University of Georgia, GA, USA).
Fixation of plant material and immunolabelling.
For electron microscopy studies, segments of transgenic and non-transgenic wheat leaves were collected and embedded in LR-White, as described previously (Littlefield et al., 1998). Leaf samples were fixed for 3 h at room temperature under vacuum in a solution containing 0·8 % glutaraldehyde, 4·0 % paraformaldehyde and 0·1 M phosphate buffer (pH 7·2). Samples were rinsed in 0·1 M phosphate buffer, dehydrated in a graded series of water and ethanol and then infiltrated and embedded in LR-White resin (Ted Pella).
Thin sections (70 nm) of LR-White-embedded leaf segments were cut with a diamond knife on a Sorvall MT 6000 ultramicrotome and mounted onto Formvar-coated, gold-gilded copper grids. Immunogold labelling to detect transgenically expressed 37K was carried out as described previously (Verchot et al., 2001). The dilution of rabbit anti-37K serum tested ranged from 1 : 50 to 1 : 1000; the dilution of 20 nm gold-conjugated secondary antiserum ranged from 1 : 50 to 1 : 320. Grids were incubated at room temperature with primary antiserum for 90 min and secondary antiserum for 2 h. Grids containing sections of non-transgenic or transgenic leaf tissue were incubated with anti-37K serum, anti-TMV serum or with buffer only, and then with secondary antiserum. The anti-37K serum and anti-TMV serum were each cross-adsorbed with wheat leaf extracts prior to immunolabelling.
Microscopy.
GFP fluorescence in wheat and tobacco leaves was studied using a Nikon E600 epifluorescence microscope with a Nikon B2A filter cube (containing a 470490 nm excitation filter, a DM505 dichroic mirror and a BA520 barrier filter). Images were captured using the Optronics MagnaFire camera (Intelligent Imaging Innovations) attached to the Nikon E600 microscope. All images were compiled using Adobe Photoshop, version 4.0 (Adobe Systems).
Statistical analyses.
All data analysis was conducted using procedures from SAS and a significance level of 0·05 was used for all mean comparisons. Data in Table 1, comparing the effects of plasmid and leaves, were assessed using chi-squared tests with PROC FREQ. Each factor's simple effects were analysed by fixing the other factors.
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RESULTS |
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The plasmid pAHC25-GFP:37K contains GFP fused to the 5' end of the SBWMV 37K gene. The fused genes were inserted adjacent to the ubi promoter. Plasmids containing GFP fused to the SBWMV CP- or RT-encoding sequences were also tested to determine if these viral proteins have the ability to move from cell to cell. GFP was fused to the 3' end of the CP ORF, replacing the coding sequence for the RT domain in pAHC25-GFP:CP. Similarly, GFP was fused to the 5' end of the RT-encoding sequence, replacing the CP ORF in the plasmid pAHC25-GFP:RT. A plasmid expressing only GFP was used as a control.
These plasmids were delivered to single epidermal cells of wheat and tobacco leaves by biolistic bombardment and epifluorescence microscopy was used to study protein cell-to-cell movement at 1 and 3 days p.b. Wheat and tobacco leaves were bombarded with plasmids to determine if protein cell-to-cell movement is due to a specific activity of the viral protein.
Fluorescence was detected primarily in single epidermal cells in source leaves bombarded with plasmids expressing GFP alone (Fig. 1). On rare occasions, GFP was detected in two or more adjacent cells in tobacco or wheat leaves (Table 1
). Occasions of GFP fluorescence in adjacent cells were reported in previous studies and might be due to simultaneous delivery of plasmids to neighbouring cells during bombardment (Itaya et al., 1997
; Krishnamurthy et al., 2002
; Mitra et al., 2003
; Morozov et al., 1997
; Yang et al., 2000
).
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When statistical comparisons were made among plant species, GFP, GFPCP and GFPRT performed the same in all plant species (Table 1). There were no significant differences between plant species in the proportions of sites containing fluorescence in cell clusters (Table 1
, P<0·05). However, the proportions of cell clusters in tobacco and wheat leaves containing GFP37K were significantly different (Table 1
, P<0·05). GFP37K accumulated primarily in single cells in tobacco leaves (Table 1
).
These data suggest that GFP37K, but not GFPCP or GFPRT, has the activities needed to move from cell to cell in wheat plants. Lack of GFP37K movement in tobacco leaves might be an indication that 37K movement requires factors present in wheat but not tobacco to move from cell to cell.
Subcellular accumulation of SBWMV 37K
SBWMV-infected wheat leaves and 37K-expressing transgenic wheat leaves were used to study the subcellular pattern of protein accumulation. Immunoblot analyses using anti-37K serum and leaf extracts from infected non-transgenic wheat and 37K-expressing transgenic wheat were conducted (Fig. 2). The anti-37K serum reacted with one polypeptide. The electrophoretic mobilities of the 37K products derived from either SBWMV-infected leaves or transgenic leaves were similar. The antiserum did not react with polypeptides in healthy non-transgenic leaves, indicating that the antiserum is specific for 37K (Fig. 2
).
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SBWMV 37K resembles the 30K superfamily of virus MPs
MPs of viruses belonging to related genera exhibit limited conservation of amino acid identity (Koonin et al., 1991; Melcher, 2000
; Mushegian & Koonin, 1993
). To test whether furoviral 37K proteins showed a degree of conservation characteristic of 30K MPs, we aligned the amino acid sequences of 43 representative viruses of this genera, chosen as described in Methods (complete alignment available through http://opbs.okstate.edu/virevol/index.html). The alignment revealed one position invariant in 42 of the 43 sequences: Trp256 (Fig. 5
). Extensive conservation of the positions of hydrophobic, particularly aliphatic, residues occurred N-terminal of the invariant Trp at positions 252254 within the sequence alignment. Conserved hydrophobic positions alternated with variable positions at three places in the alignment: (1) a region 20 positions N-terminal of the Trp, (2) near residue 143 and (3) near residue 168. The latter two surround a patch of conserved hydrophobic residues. Similar patches occurred near the invariant Trp at positions 152 and 200. Conservation of residue identity within genera and among several genera in the family also occurred. Two motifs, suggested previously to be near invariant and diagnostic of 30K superfamily members, were not invariant in this alignment. The LXD motif (positions 169171, Fig. 5
) tolerated Met, Cys, Ile, Trp and Phe substitutions for the Leu residue and Asn, Gln, Glu and Tyr substitutions for the Asp residue. The ilarvirus alfalfa mosaic virus (AlMV) clade showed the greatest variation at those positions. What appeared previously to be an aligned position consisting exclusively of Gly residues was, in the new alignment, broken up into different positions due to realignment (positions 238, 246, 247 and 250). Nevertheless, the grouping remains a valid one, exhibiting a significance score of 5·4 for the alignment of the ilarvirus AlMV clade with the other proteins.
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DISCUSSION |
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Using electron microscopy, we observed that SBWMV 37K accumulates in plant cell walls (Figs 3 and 4). The pattern of 37K accumulation is similar to that observed in studies of the TMV and CMV MPs (Itaya et al., 1997
, 1998
; Oparka et al., 1997
; Tomenius et al., 1987
). The 37K proteins were also found scattered in the cytoplasm and sometimes there was labelling in electron-dense regions within the cytoplasm. Similar data were obtained using SBWMV-infected and 37K-expressing transgenic wheat leaves, suggesting that other SBWMV proteins did not affect the pattern of protein accumulation in SBWMV-infected leaves.
The clear demonstration by phylogenetic analysis that furoviral 37K proteins are related most closely to the dianthovirus MPs should facilitate analysis of the role of the furoviral 37K proteins in virus movement. The structurefunction relationships of the dianthovirus Red clover necrotic mosaic virus have been well studied (Fujiwara et al., 1993; Geisman-Cookmeyer & Lommel, 1993
). Comparisons should lead us more rapidly to an understanding of the differences in movement characteristics of these two sets of proteins.
Observation that the tips of the branches in the MP tree (Fig. 2) were all approximately the same distance from the root of the tree is intriguing. This suggests that the rate of change of sequence in the MPs might be the same in all lineages. Factors that may have increased or decreased the rate of evolution may have similar influences on the evolution of all MPs of this family. The firmness of this conclusion depends on the accuracy of assignment of the root of the tree. Firm conclusion that the molecular clock has progressed at the same rate in all lineages awaits independent confirmation that of these MPs, those of the tenuiviruses were the first to diverge from the lineage that gave rise to the other MPs in the family.
Although some of the phylogenetic placements of the MPs are inconsistent with current taxonomic practice, it must be emphasized that Fig. 2 describes only the relationships among the amino acid sequences of the MPs and thus describes evolution of the MPs and not of the viruses that encode them. Horizontal transfer of genes among viruses is known to occur, so that any virus may be an amalgam of parts with different evolutionary histories. Nevertheless, the conflicts observed may require those that wish to base taxonomy on sequence to reanalyse some classifications. The most obvious is the classification of AlMV as an alfamovirus, separate from the ilarviruses when its MP appears to have diverged from one of several ilarvirus lineages existing at the time of its divergence.
Analyses of intercellular transport, protein subcellular accumulation patterns and amino acid sequence alignments support the notion that SBWMV 37K is a furovirus MP. In this study, we provide the first experimental evidence to demonstrate that SBWMV 37K has a pattern of subcellular accumulation and the ability to move from cell to cell, reminiscent of the MPs within the 30K superfamily. Transgenically expressed proteins are often used to study viral protein functions and subcellular targeting. This is the first report to use transgenic wheat expressing viral proteins for a similar purpose.
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ACKNOWLEDGEMENTS |
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Received 25 April 2003;
accepted 17 July 2003.