Department of Virus Research, John Innes Centre, Colney, Norwich Research Park, Norwich NR4 7UH, UK1
Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK2
Author for correspondence: Jeffrey Davies. Fax +44 1603 450045. e-mail jeffrey.davies{at}bbsrc.ac.uk
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Abstract |
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Introduction |
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The mechanism of movement of the bipartite geminiviruses of the genus Begomovirus has been investigated intensively. Two proteins encoded by the B DNA component are involved in movement, BV1 (BR1) and BC1 (BL1) (Lazarowitz, 1992 ). The BC1 protein of Bean dwarf mosaic virus (BDMV) increases the size exclusion limit (SEL) of plasmodesmata of cells into which it is injected, and the protein mediates viral DNA transport from cell to cell (Noueiry et al., 1994
; Rojas et al., 1998
) and is thus regarded as the viral movement protein. In contrast, the BC 1 protein of Squash leaf curl virus (SqLCV) does not bind DNA but is present in virus-induced tubules that cross the walls of meristematic phloem tissues (Ward et al., 1997
). The BV1 protein is nuclear localized, binds to viral DNA (Noueiry et al., 1994
; Pascal et al., 1994
) and acts as a nuclear shuttle protein. Interaction of the two proteins regulates the directionality of intracellular viral DNA transport (Sanderfoot et al., 1996
). The CP of the bipartite viruses is dispensable for systemic infection in an adapted host (Gardiner et al., 1988
; Pooma et al., 1996
), although it can aid the movement or pathogenicity of such viruses (Qin et al., 1998
). In contrast, the CP of mastreviruses is required for plant infection (Boulton et al., 1989b
, 1993
; Liu et al., 1998
; Woolston et al., 1989
). The E. coli-expressed CPs of MSV (Liu et al., 1997
) and the monopartite begomovirus Tomato yellow leaf curl virus (TYLCV) interact with viral DNA and localize in the nucleus in insect and plant cells (Kunik et al., 1998
; Liu et al., 1997
; 1999; Palanichelvam et al., 1998
). Furthermore, microinjection of MSV CP with fluorescently labelled viral DNA into plant cells has demonstrated that the MSV CP can facilitate transport of viral DNA into the nucleus, suggesting that the CPs of the monopartite viruses may perform at least some of the functions of BV1 (Liu et al., 1999
). Although the CP of SqLCV has also been shown to bind ssDNA and localize to the nucleus, these functions are also provided by the BV1 protein (Qin et al., 1998
), and thus CP-mediated nuclear targeting may be required only in initially infected cells following insect transmission of begomovirus particles.
The MPs of mastreviruses are required for cell-to-cell movement (Boulton et al., 1993 ; Liu et al., 1998
); all have a stretch of hydrophobic amino acids that are predicted to form a transmembrane structure (Boulton et al., 1993
). The MSV MP is associated with plasmodesmata in infected tissue (Dickinson et al., 1996
), and an MSV MPGFP fusion translocates from cell to cell when expressed in epidermal cells of maize leaves (Kotlitzky et al., 2000
). However, the precise role of MSV MP in virus movement is still unknown. Likely, there is functional analogy to the BC1 of the begomoviruses. Towards understanding the MSV MP functions and MSV movement mechanism, the DNA- and CP-binding capacity of E. coli-expressed MP was analysed using south-western, gel overlay and immunoprecipitation assays. The effect of MSV MP on CP-mediated viral DNA transport was investigated by microinjection. The results showed that MP, unlike CP, was unable to bind viral DNA, but can interact with CP. Furthermore, the MP prevented, or diminished, nuclear accumulation of an MSV CPDNA complex in microinjected plant cells. A model for the intracellular transport of MSV DNA is suggested.
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Methods |
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Expression and purification of MSV CP were carried out as described (Liu et al., 1999 ). Where appropriate, the E. coli-expressed CP and MP were compared with native protein (in the form of total protein extracts of MSV-infected maize) by SDSPAGE and immunoblotting.
Preparation of anti-MSV MP serum.
Anti-MSV MP serum was raised by intramuscular injection into a rabbit as described (Pinner & Markham, 1990 ) except that 100 µg of purified 6xHis-MP was used for each immunization. Serum was recovered from blood collected from the rabbit 10 days after the third boost and tested against 6xHis-MP and infected plant extracts by immunoblotting. This antiserum was used for south-western and gel overlay experiments.
South-western analysis of MPDNA binding.
To assess MSV MPDNA binding, both purified 6xHis-MP and total E. coli cell extracts were used for south-western analysis as described by Sukegawa & Blobel (1993) . All samples and controls were prepared, fractionated by SDSPAGE, and transferred to membrane as described previously for CP extracts (Liu et al., 1997
). The [
-32P]dCTP-labelled MSV and pUC probes were prepared as before (Liu et al., 1997
) and added to reaction buffer containing 100 or 250 mM KCl for binding assays.
Gel overlay assays.
To investigate MSV MPCP interaction in vitro, the gel overlay assay was used following the method described by Schwank et al. (1995) except that MP or CP was visualized using immunochemical staining. Cells expressing CP (collected from 100 ml of culture) were sonicated in 20 ml of lysis buffer (10 mM TrisHCl pH 8·0, 100 mM NaCl, 1 mM EDTA, 10% glycerol, 1 mM DTT, 1 mM PMSF). For cells expressing MP, 0·1% Triton X-100 was added to solubilize the MP. A total of 20 µl of each cell extract was fractionated by SDSPAGE on a 15% separating gel and electrotransferred onto a nitrocellulose membrane. After renaturation of the proteins, the membrane on which CP extracts were bound was overlaid with 20 ml of total extract of cells expressing 6xHis-MP from pETMP, whereas the membrane onto which MP extracts were bound was overlaid with the extract containing CP. After washing the membranes, immunoblotting with anti-MP or anti-CP serum, respectively, was used to determine whether the proteins used in the overlay buffer were bound to immobilized proteins and therefore retained on the membrane.
Immunoprecipitation assay.
To determine whether the MP and CP could interact in infected plants, immunoprecipitation assays were used. About 2 g of MSV-infected maize leaf was ground to a fine powder in liquid nitrogen and the total proteins were extracted by grinding for another 2 min in 2 ml HEGKMND buffer pH 7·5 (Blanar & Rutter, 1992 ) lacking MgCl2 but containing 5 mM EDTA. The supernatant was collected by centrifugation at 13000 r.p.m. for 30 min at 4 °C. The MSV MP or CP present in the supernatant was immunoprecipitated using anti-MSV MP or CP serum as described by Blanar & Rutter (1992)
except that the CP or MP was subsequently visualized using immunochemical staining. Immunoblotting with anti-MSV CP serum was used to identify CP in the sample precipitated with anti-MP serum, while anti-MSV MP serum was used to determine whether MP co-precipitated with the CP. Anti-NADP-dependent malic enzyme (NADP-ME) serum (a gift from Jane Langdale, Dept of Plant Sciences, University of Oxford, UK) was used to determine whether proteins abundant in maize tissue were present in the precipitate.
Microinjection.
Microinjection of maize and tobacco leaf cells and labelling of MSV DNA with TOTO-1 dye (Molecular Probes) was carried out as described previously (Liu et al., 1999 ). 6xHis-MP was co-injected into the cells with MSV TOTO-1-ss or -dsDNA to assess its ability to mediate cell-to-cell transport of DNA. The effect of the MSV 6xHis-MP on the localization of CP DNA in the cells was investigated by injecting TOTO-1-DNA, CP and MP together in a ratio of 1:1:1 into the cells. The CP (0·5 mg/ml) was first mixed with the TOTO-1-DNA for 15 min, and then the MP (0·5 mg/ml) was added. The mixture was injected into the cells with an equal volume of Texas Red dextran. The effect of 6xHis-MP at 1 mg/ml was also tested. An MP-nonexpressing extract of pET3a-transformed E. coli cells was used, in place of the 6xHis-MP, for the control injections.
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Results |
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MSV MP affects CP-mediated nuclear transport of DNA in tobacco and maize cells
Although interaction between the MP and CP have been shown in vitro (Fig. 3) and in plant extracts (Fig. 4
), the data do not show that an MPCP complex can be produced in living cells, nor do they suggest a function for the complex. We have previously suggested (Liu et al., 1999
; Kotlitzky et al., 2000
) that the MPCP interaction could be important for regulating the directionality of DNA (or a CPDNA complex) transport, for example by directing it from the nucleus to the cell periphery. Microinjection studies were used, therefore, to determine whether the 6xHis-MP could affect nuclear accumulation of MSV DNA in the presence of MSV CP. We have previously shown (Liu et al., 1999
) that the MSV CP can facilitate nuclear transport of TOTO-1-labelled MSV DNA in both maize and tobacco cells, with nuclear fluorescence being clearly visible within 5 min and being maintained for at least 15 min after injection. However, in the current study when MP was present in the injection mixture (at both concentrations tested), negligible nuclear fluorescence was seen in the cells injected with TOTO-1-labelled ds (Fig. 5A
D
) or ssDNA (Fig. 5I
L
), even at 15 min after injection. This inhibition of nuclear accumulation of MSV DNA was not caused by the presence of contaminating E. coli proteins because when purified extracts of E. coli transformed with pET3a were used in place of the 6xHis-MP, the nuclei in the injected cell showed clear fluorescence (Fig. 5E
H
, M
P
). Microinjection of the MP and TOTO-1-DNA alone did not result in nuclear accumulation of fluorescence (not shown). The relatively high level of autofluorescence in the injected cells made it impossible to determine the site to which the labelled DNA was redirected when the MP was present in the injection mixture. The results of the microinjections are summarized in Table 1
. Fluorescence was not observed in other neighbouring cells after injection of higher levels of DNA or longer incubation times. Indeed, fluorescence usually began to fade about 30 min after injection (not shown).
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Discussion |
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Many plant virus MPs have been shown to bind viral nucleic acid and interact with plasmodesmata (reviewed in Carrington et al., 1996 ). However, the His-tagged MSV MP did not bind viral DNA (Fig. 2
). This result suggests that MSV MP might be functionally different from the majority of plant virus MPs, although an association MSV MP with plasmodesmata of infected plants has been observed (Dickinson et al., 1996
). As MSV CP interacts with DNA and mediates the nuclear transport of viral DNA (Liu et al., 1997
, 1999
), it is possible that MSV MP co-operates with the CP to move the viral DNA from the infected cell nucleus to the periphery of the cell, thereby enabling cell-to-cell movement through the plasmodesmata. Such interaction of the MP with the CP is indicated by the gel overlay assays (Fig. 3
) and the MPCP complex immunoprecipitated from MSV-infected plant cells (Fig. 4
). It is thus proposed that the MSV MP moves a CPDNA complex from cell to cell. Since no MSV MP has been detected associated with purified MSV particles (Mullineaux et al., 1988
), it is likely that the movement is accomplished in the form of a nucleoprotein complex. However, the structure of the MPCPDNA complex and the regulation of its formation is unknown.
The CP-mediated nuclear transport of MSV DNA in maize and tobacco cells was disrupted in the presence of the 6xHis-MP (Fig. 5). We therefore propose that the MP requires the assistance of the CP, which binds to viral DNA (Liu et al., 1997
), to facilitate MSV movement towards the cell periphery and we cannot rule out the possibility that MP facilitates nuclear export of the CP or CPDNA. Clearly, there is some analogy with begomovirus nuclear shuttling and cell-to-cell movement functions. The limited protein-coding capacity of geminivirus monopartite genomes requires proteins to have multiple functions compared to those encoded by the bipartite begomoviruses. Indeed, it has been shown that the begomovirus BV1 and CP proteins exhibit functional redundancy; the SqLCV CP localizes to the nucleus and masks BV1 mutants (Qin et al., 1998
). The evolutionary relationship of these proteins is underlined by their amino acid similarity (Kikuno et al., 1984
). The close relationship between mastrevirus and begomovirus CPs has also been reported (Mullineaux et al., 1985
).
The MSV MP has many of the functions of the begomovirus BC1 protein, but the means by which the MSV complex (MPCPDNA) moves from cell to cell is not yet clear. For example, it is not certain whether the MP binds at plasmodesmata to enable CPDNA to move through and if it moves with the complex or remains at the binding site. Dickinson et al. (1996) reported that MSV MP is associated with the secondary plasmodesmata in MSV-infected tissue, but no stable association was detected by Kotlitzky et al. (2000)
using an MSV MPGFP fusion, although in this case no other viral proteins were present in the cell. It is also not clear how the directionality of the movement is regulated. Nuclear targeting of the MSV CP is mediated by a nuclear localization signal located at the N terminus of the protein (Liu et al., 1997
, 1999
). It is possible that this signal is blocked by the presence of the MP, although other modifications of the CP (such as a change in phosphorylation state) cannot be ruled out. We propose a model in which CP moves DNA into the nucleus, but CPMP interaction redirects MSV DNA to the cell periphery for movement to a neighbouring cell.
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Acknowledgments |
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Footnotes |
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References |
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Boulton, M. I., Buchholz, W. B., Marks, M. S., Markham, P. G. & Davies, J. W.(1989a). Specificity of Agrobacterium-mediated delivery of maize streak virus DNA to members of the Gramineae. Plant Molecular Biology 12, 31-40.
Boulton, M. I., Steinkellner, H., Donson, J., Markham, P. G., King, D. I. & Davies, J. W.(1989b). Mutational analysis of the virion-sense genes of maize streak virus. Journal of General Virology 70, 2309-2323.[Abstract]
Boulton, M. I., King, D. I., Markham, P. G., Pinner, M. S. & Davies, J. W.(1991). Host range and symptoms are determined by specific domains of the maize streak virus genome. Virology 181, 312-318.[Medline]
Boulton, M. I., Pallaghy, C. K., Chatani, M., MacFarlane, S. & Davies, J. W.(1993). Replication of maize streak virus mutants in maize protoplasts: evidence for a movement protein. Virology 192, 85-93.[Medline]
Briddon, R. W. & Markham, P. G.(1995). Geminiviridae. In Virus Taxonomy. Sixth Report of the International Committee on Taxonomy of Viruses , pp. 158-165. Edited by F. A. Murphy, C. M. Fauquet, D. H. L. Bishop, S. A. Ghabrial, A. W. Jarvis, G. P. Martelli, M. A. Mayo& M. D. Summers. Vienna & New York:Springer-Verlag.
Carrington, J. C., Kasschau, K. D., Mahajan, S. K. & Schaad, M. C.(1996). Cell-to-cell and long distance transport of viruses in plants. Plant Cell 8, 1669-1681.
Davies, J. W., Stanley, J., Donson, J., Mullineaux, P. M. & Boulton, M. I.(1987). Structure and replication of geminivirus genomes. Journal of Cell Science Supplement 7, 95-107.[Medline]
Deom, C. M., Lapidot, M. & Beachy, R. N.(1992). Plant virus movement proteins. Cell 69, 221-224.[Medline]
Dickinson, V. J., Halder, J. & Woolston, C. J.(1996). The product of maize streak virus ORF V1 is associated with secondary plasmodesmata and is first detected with the onset of viral lesions. Virology 220, 51-59.[Medline]
Gamer, J., Bujard, H. & Bukau, B.(1992). Physical interaction between heat shock proteins DnaK, DnaJ, and GrpE and the bacterial heat shock transcription factor sigma 32. Cell 69, 833-842.[Medline]
Garcia, J. A., Riechmann, J. L. & Lain, S.(1989a). Proteolytic activity of the plum pox potyvirus NIa-like protein in Escherichia coli. Virology 170, 362-369.[Medline]
Garcia, J. A., Riechmann, J. L., Martin, M. T. & Lain, S.(1989b). Proteolytic activity of the plum pox potyvirus NIa-protein on excess of natural and artificial substrates in Escherichia coli. FEBS Letters 257, 269-273.[Medline]
Gardiner, W., Sunter, G., Brand, L., Elmer, J. S., Rogers, S. & Bisaro, D.(1988). Genetic analysis of tomato golden mosaic virus: the coat protein is not required for systemic spread or symptom development. EMBO Journal 7, 899-904.
Gilbertson, R. L. & Lucas, W. J.(1996). How do viruses traffic on the vascular highway? Trends in Plant Science 1, 260-268.
Goodman, R. M.(1981). Geminiviruses. In Handbook of Plant Virus Infection & Comparative Diagnosis , pp. 879-910. Edited by E. Kustak. New York:Elsevier/North Holland Biomedical Press.
Grimsley, N., Hohn, T., Davies, J. W. & Hohn, B.(1987). Agrobacterium-mediated delivery of infectious maize streak virus into maize plants. Nature 325, 177-179.
Howell, S. H.(1984). Physical structure and genetic organisation of the genome of maize streak virus (Kenyan isolate). Nucleic Acids Research 12, 7359-7375.[Abstract]
Kikuno, R., Toh, H., Hayashida, H. & Miyata, T.(1984). Sequence similarity between putative gene products of geminiviral DNAs. Nature 308, 562.[Medline]
Kotlitzky, G., Boulton, M. I., Pitaksutheepong, C., Davies, J. W. & Epel, B.(2000). Intracellular and intercellular movement of maize streak geminivirus V1 and V2 proteins transiently expressed as green fluorescent protein fusions. Virology 274, 32-38.[Medline]
Kunik, T., Palanichelvam, K., Czosnek, H., Citovsky, V. & Gafni, Y.(1998). Nuclear import of the capsid protein of tomato yellow leaf curl virus (TYLCV) in plant and insect cells. Plant Journal 13, 393-399.[Medline]
Lazarowitz, S. G.(1988). Infectivity and complete nucleotide sequence of the genome of a South African isolate of maize streak virus. Nucleic Acids Research 16, 229-249.[Abstract]
Lazarowitz, S. G.(1992). Geminiviruses: genome structure and genome function. Critical Reviews in Plant Sciences 11, 327-349.
Lazarowitz, S. G., Pinder, A. J., Damsteegt, V. D. & Rogers, S. G.(1989). Maize streak virus genes essential for systemic spread and symptom development. EMBO Journal 8, 1023-1032.
Liu, H., Boulton, M. I. & Davies, J. W.(1997). Maize streak virus coat protein binds single- and double-stranded DNA in vitro. Journal of General Virology 78, 1265-1270.[Abstract]
Liu, L., Davies, J. W. & Stanley, J.(1998). Mutational analysis of bean yellow dwarf virus, a geminivirus of the genus Mastrevirus that is adapted to dicotyledonous plants. Journal of General Virology 79, 2265-2274.[Abstract]
Liu, H., Boulton, M. I., Thomas, C. L., Prior, D. A., Oparka, K. J. & Davies, J. W.(1999). Maize streak virus coat protein is karyophyllic and facilitates nuclear transport of viral DNA. Molecular PlantMicrobe Interactions 12, 894-900.
McLean, B. G., Waigmann, E., Citovsky, V. & Zambryski, P.(1993). Cell-to-cell movement of plant viruses. Trends in Microbiology 1, 105-109.[Medline]
Maia, I. G. & Bernardi, F.(1996). Nucleic acid-binding properties of a bacterially expressed potato virus Y helper component-proteinase. Journal of General Virology 77, 869-877.[Abstract]
Morris-Krsinich, B. A. M., Mullineaux, P. M., Donson, J., Boulton, M. I., Markham, P. G., Short, M. N. & Davies, J. W.(1985). Bidirectional transcription of maize streak virus DNA and identification of the coat protein gene. Nucleic Acids Research 13, 7237-7256.[Abstract]
Mullineaux, P. M., Donson, J., Morris-Krsinich, B. A. M., Boulton, M. I. & Davies, J. W.(1984). The nucleotide sequence of maize streak virus DNA. EMBO Journal 3, 3063-3068.[Abstract]
Mullineaux, P. M., Donson, J., Stanley, J., Boulton, M. I., Morris-Krsinich, B. A. M., Markham, P. G. & Davies, J. W.(1985). Computer analysis identifies sequence homologies between potential gene products of maize streak virus and those of cassava latent virus and tomato golden mosaic virus. Plant Molecular Biology 5, 125-131.
Mullineaux, P. M., Boulton, M. I., Bowyer, P., van der Vlugt, R., Marks, M., Donson, J. & Davies, J. W.(1988). Detection of a non-structural protein of MW 11000 encoded by the virion DNA of maize streak virus. Plant Molecular Biology 11, 57-66.
Nagar, S., Pedersen, T. J., Carrick, K. M., Hanley-Bowdoin, L. & Robertson, D.(1995). A geminivirus induces expression of a host DNA synthesis protein in terminally differentiated plant cells. Plant Cell 7, 705-719.
Noueiry, A. O., Lucas, W. J. & Gilbertson, R. L.(1994). Two proteins of a plant DNA virus coordinate nuclear and plasmodesmal transport. Cell 76, 925-932.[Medline]
Palanichelvam, K., Kunik, T., Citovsky, V. & Gafni, Y.(1998). The capsid protein of tomato yellow leaf curl virus binds cooperatively to single-stranded DNA. Journal of General Virology 79, 2829-2833.[Abstract]
Pascal, E., Sanderfoot, A. A., Ward, B. M., Medville, R., Turgeon, R. & Lazarowitz, S. G.(1994). The geminivirus BR1 movement protein binds single-stranded DNA and localizes to the cell nucleus. Plant Cell 6, 995-1006.
Pinner, M. S. & Markham, P. G.(1990). Serotyping and strain identification of maize streak virus isolates. Journal of General Virology 71, 1635-1640.[Abstract]
Pooma, W., Gillette, W. K., Jeffrey, J. L. & Petty, I. T.(1996). Host and viral factors determine the dispensability of coat protein for bipartite geminivirus systemic movement. Virology 218, 264-268.[Medline]
Qin, S., Ward, B. M. & Lazarowitz, S. G.(1998). The bipartite geminivirus coat protein aids BR1 function in viral movement by affecting the accumulation of viral single-stranded DNA. Journal of Virology 72, 9247-9256.
Rojas, M. R., Noueiry, A. O., Lucas, W. J. & Gilbertson, R. L.(1998). Bean dwarf mosaic geminivirus movement proteins recognize DNA in a form- and size-specific manner. Cell 95, 105-113.[Medline]
Sanderfoot, A. A. & Lazarowitz, S. G.(1995). Co-operation in viral movement. The geminivirus BL1 movement protein interacts with BR1 and redirects it from the nucleus to the cell periphery. Plant Cell 7, 1185-1194.
Sanderfoot, A. A., Ingham, D. J. & Lazarowitz, S. G.(1996). A viral movement protein as a nuclear shuttle. The geminivirus BR1 movement protein contains domains essential for interaction with BL1 and nuclear localization. Plant Physiology 110, 23-33.
Schwank, S., Ebbert, R., Rautenstrauss, K., Schweizer, E. & Schuller, H. J.(1995). Yeast transcriptional activator INO2 interacts as an Ino2p/Ino4p basic helix-loop-helix heteromeric complex with the inositol/choline-responsive element necessary for expression of phospholipid biosynthetic genes in Saccharomyces cerevisiae. Nucleic Acids Research 23, 230-237.[Abstract]
Stanley, J., Boulton, M. I. & Davies, J. W. (1999). Geminiviridae. In Embryonic Encyclopedia of Life Sciences CD-ROM. http://www.els.net.
Studier, F. W. & Moffatt, B. A.(1986). Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. Journal of Molecular Biology 189, 113-130.[Medline]
Sukegawa, J. & Blobel, G.(1993). A nuclear pore complex protein that contains zinc finger motifs, binds DNA, and faces the nucleoplasm. Cell 72, 29-38.[Medline]
Towbin, H., Staehelin, T. & Gordon, J.(1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences, USA 76, 4350-4354.[Abstract]
Ward, B. M., Medville, R., Lazarowitz, S. G. & Turgeon, R.(1997). The geminivirus BL1 movement protein is associated with endoplasmic reticulum-derived tubules in developing phloem cells. Journal of Virology 71, 3726-3733.[Abstract]
Woolston, C. J., Reynolds, H. V., Stacey, N. J. & Mullineaux, P. M.(1989). Replication of wheat dwarf virus DNA in protoplasts and analysis of coat protein mutants in protoplasts and plants. Nucleic Acids Research 17, 6029-6041.[Abstract]
Wright, E. A., Heckel, T., Groenendijk, J., Davies, J. W. & Boulton, M. I.(1997). Splicing features in maize streak virus virion- and complementary-sense gene expression. Plant Journal 12, 1285-1297.[Medline]
Received 9 August 2000;
accepted 6 October 2000.