- Email: [email protected]
Studying the movement of plant viruses using green fluorescent protein
reviews
Studying the m o v e m e n t of plant viruses using green fluorescent protein opo,,o,o,,o BoevinkandSimonSantaCruz Recent studies with green fluorescent protein (GFP) are providing n e w insights into the mechanisms of virus movement in plants. The gfp gene has been inserted into the genomes of a number of plant viruses, enabling production of either the free protein or a fusion with a virus-encoded protein. Fusion of GFP to the coat protein of potato virus X (PVX) has produced a functional virus with a GFP 'overcoat' that can be traced as it moves. Many plant viruses are known to use 2novement proteins' to assist in their transit through plasmodesmata. Movement protein-GFP fusions can be expressed from the parental virus or, in the absence of a full-length infectious clone, using an alternative expression vector. Such fusions retain their capacity to target plasmodesmata and are providing n e w information on the factors responsible for trafficking viral RNA to, and through, the plasmodesmal pore. ost plant viruses are able to move both locally (between cells) and systemically (over long distances). Local movement occurs through plasmodesmata, the small channels that interconnect higher plant cells ~-3. Systemic movement is dependent on the entry of the virus (or an infectious component of the virus) into the long-distance transport system of the phloem 2,4. However, although some viruses have the capacity to move systemically
(e.g. if introduced directly into the phloem by a feeding insect), they may not possess the ability to escape from the phloem in sink tissues. Such phloem-limited viruses 1 appear to lack the correct 'key(s)' to allow local movement out of the phloem. Other viruses have proteins that potentiate both local and systemic virus movement. For example, in tobacco mosaic virus (TMV), local movement is facilitated by a 30 kDa movement protein ~that targets plasmodesmata during virus infection~,7, binds to singlestranded nucleic acids s and 'gates' plasmodesmata to a much higher than normal size exclusion limit s. AlthoUgh TMV does not require its coat protein for local movement, this protein is apparently essential for efficient long-distance movement 10. In contrast to this, some viruses require their coat protein in addition to movement protein(s) for local movementH-l~; other viruses can dispense with the coat protein for both local and systemic movement~4,15. Fig. 1. Leaf trichome cells of Nicotiana clevelandii systemically infected with potato virus X To date, unravelling the (PVX) constructs allowing expression of the green fluorescent protein (GFP). In (a), the GFP is roles of different virusbeing released as a free protein in virus-infected cells and remains restricted to the cytosol and encoded genes in local and nucleus. In (b), the PVX vector is carrying a GFP fusion to the 3a movement protein of cucumsystemic movement has ber mosaic virus. The GFP-movement protein fusion has been targeted to plasmodesmata on been hindered by the lack of the end walls of the subapical (neck) cell during intercellular virus movement. Note the absence a suitable in vivo marker for of labelling on the lateral walls - all trichome lateral walls lack plasmodesmata. In (c), the GFP is fused to the viral coat protein of PVX to produce a functional virus with a GFP 'overcoat'4~. tracking the key elements Note that the virus adopts different morphologies in different cells. In the apical (tip) cell the of movement with adequate virus forms flexuous, spindle-shaped aggregates, while in the neck cell the aggregated virus cellular resolution. Hence forms a continuous doughnut-shaped ring. This image was reconstructed from a series of conthe introduction of GFP focal sections taken through the trichome. All the images were obtained with a confocal laser as a noninvasive reporter scanning microscope. Scale bars represent 10 ~m. in a range of systems 1~ has opened up a unique
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reviews opportunity to study viral trafficking in plants (a recent review 2 highlighted some of the current problems in studying this phenomenon). Here, the ways in which GFP may aid in the noninvasive detection of virus movement are reviewed.
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Green fluorescent protein as a reporter of virus infection
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The marker protein 13-glucuronidase (GUS) has previously been used as a reporter of virus infection and to study ," the roles of different viral genes in both local and systemic movement 14,17,1s. ",-" Histochemical staining of tissue infected with viruses expressing GUS . f 'll allows the virus to be localized to specific cells and tissues. However, the staining procedure is destructive and does not allow for continuous monitorFig. 2. Association of the movement protein of tobacco mosaic virus (TMV)with the ing of the viral infection. In contrast to cytoskeleton of infected Nicotiana benthamiana plants. The GFP was fused to the this procedure, detection of GFP movement protein of TMV and released as a fusion protein from the TMV vector24. depends only on excitation of the protein The cells shown are close to the infection front and depict different stages of the using light of the appropriate waveassociation of the movement protein-GFP fusion with the cytoskeleton. In some length and does not require the addition cells the fusion protein is present as discrete aggregates, but other cells lack such structures. The image was obtained using a confocal laser scanning microscope. of exogenous substrates or cofactors19,20. Scale bar represents 20 ~m. Courtesy of Roger Beachy and Denton Prior. Baulcombe et al. 21 were the first to introduce the gfp gene into a plant virus, using the potexvirus PVX. Transcription of the gfp mRNA was directed by a viral promoter It was subsequently shown that the local and systemic sequence. Following infection of plants with the modified movement of this coat protein-lacking mutant could be virus, GFP accumulated in the cytoplasm and nucleus of restored when it was inoculated onto transgenic tobacco infected cells2~,22(Fig. la). Green fluorescent protein has also expressing the PVX coat protein• Further observations claribeen expressed as the free protein using as vectors both TMV fied the role of the protein. First, microinjection studies using (Ref. 23) and cowpea mosaic virus (J. Wellink and J. van Lent, fluorescent dextran probes showed that transgenic exprespers. commun.). Detection of fluorescence in virus-infected sion of the coat protein did not alter the plasmodesmal size cells is facilitated by the high expression level of GFP when directed by viruses such as PVX and TMV. The fluorescent signal from tissue infected with either TMV or PVX is sufficiently strong to allow macroscopic imaging under long,:. "~ wavelength ultraviolet light, thereby providing a means of studying systemic :~': virus movement as the infection ~. ~" p r o g r e s s e s 21,22,24. ~" =,
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Replacing viral proteins with green fluorescent protein The use of GFP also provides novel opportunities for studying mutant viral genomes, by the replacement of virusencoded proteins with GFP. This approach has already yielded information on the requirement of viral genes for local movement. For example, when the coat protein gene of PVX was replaced with the GFP gene 21, it was found that the resulting virus was capable of replication and GFP production, but was restricted to single cells on the leaf epidermis; this demonstrates that the coat protein of PVX is essential for local movement.
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Fig. 3. Transverse section of a leaf ofNicotiana benthamiana infected with potato virus X expressing a fusion of green fluorescent protein (GFP) with the 3a movement protein of cucumber mosaic virus. The movement protein-GFP fusion is exclusively localized to the plasmodesma pit fields of the palisade mesophyll. The image was obtained using a confocal laser scanning microscope. Scale bar represents 20 ~m.
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reviews exclusion limit. Second, in uninfected coat protein-containing transgenic tobacco, the protein was shown by immunolocalization to be in the cytoplasm only; however, infection of these transgenic plants with the PVX coat protein deletion mutant resulted in targeting of the coat protein to plasmodesmata (in wild-type PVX infections, the coat protein is also localized at plasmodesmata). These observations suggest that, for PVX, the coat protein is important for trafficking the viral RNA to the plasmodesmal pore, but is not directly involved in gating 25.
Fusion of green fluorescent protein to viral proteins Although the release of free GFP in cells is a valuable marker for the presence of virus and the roles of different viral genes in determining local and systemic movement, an alternative approach is to create fusions between GFP and viral proteins. This exploits one of the principal advantages of GFP as a reporter- its ability to fluoresce in fusion proteins 2~ - and opens up the possibility for noninvasive imaging of viral proteins during the infection process. Green fluorescent protein-movement protein fusions The role of the movement protein from TMV in plasmodesmal gating was initially demonstrated using transgenic plants constitutively expressing the movement protein 9. Microinjection of the mesophyll cells of these plants with fluorescently labelled dextrans and peptides demonstrated that the size exclusion limit was considerably in excess (>10kDa) of their 'basal' limit (
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on the cell wall 23,24(Fig. 2); this suggests that the interaction of the movement protein with microtubules occurs during virus infection of tissues as well as in protoplasts. The cytoskeleton (particularly microtubules) has also been shown to play a role in viral trafficking in animal systems. For example, the directional movement of herpes simplex virus in cells may be mediated by a minus-end directed motor such as cytoplasmic dynein 34. The recent studies on plant viruses, using GFP fusion approaches, have begun to open up the search for the plant host factors that interact with viral movement protein-RNA complexes and allow them to reach plasmodesmata from their sites of synthesis in the cytoplasm. The challenge now is to determine precisely how the movement protein of TMV uses the cytoskeleton to traffick the viral RNA to the plasmodesmata (if this is indeed the transport pathway) and to dissect the stages involved in the association of the movement protein-viral RNA complex with the cytoskeleton. Not all viral movement proteins necessarily associate with the plant cytoskeleton in order to locate plasmodesmata. In the case of cells infected with cowpea mosaic virus, protein tubules are assembled that span the plasmodesmal pore; these may form in situ at the plasma membrane of isolated protoplasts 3~. The formation of tubules requires a 48 kDa virus-encoded protein, and may also require hostencoded protein(s) 11(although it is not yet known if these proteins associate in any way with the cytoskeleton during viral movement). Recently, J. Wellink and J. van Lent made a GFP fusion to the C-terminus of the 48 kDa movement protein by replacing the coat protein-coding region of the virus (unpublished). The substitution prevented the virus from moving systemically (as this virus requires its coat protein for local movement3~), but GFP-tagged tubules formed on the plasma membrane of infected protoplasts. In the case of the potyviruses, pinwheel structures, composed of a virus-encoded protein, are frequently observed at or near plasmodesmata 37,3s.Little is known of the function of these structures, but they are certainly candidates for GFP fusion, which would allow their precise distribution in an advancing infection front to be studied. Noninvasive location of pinwheels would aid in the selection of infected cells for electron microscopy and for injection studies with fluorescent dextrans to determine if they have a role in plasmodesmal gating. As pointed out by Gilbertson and Lucas 2, the formation of assembly sites for movement proteins at or close to the plasmodesmal pore may obviate the need for an association of the viral movement protein with the cytoskeleton. Different viruses are likely to locate plasmodesmata in different ways, and in this respect movement protein-GFP fusions may play an important role in dissecting the pathway leading to the plasmodesmal pore.
Alternative vectors for studying movement protein targeting and function A functional analysis of the movement proteins from some viruses may be hindered either by lack of availability of a full-length infectious clone, or because of the inability to modify the viral genome without losing infectivity. Thus, although the specific protein(s) implicated in viral movement may have been identified and sequenced, it could be impossible to express these as GFP fusions from the parental virus. One approach to overcome this is to fuse the protein to GFP and direct its expression in the plant independently of the parental virus.
reviews We have been exploring the potential of PVX as an episomal vector for the expression of GFP fusion proteins; PVX has proven to be a versatile vector for the expression of foreign genes in plants and, because of its high multiplication rate, the levels of foreign gene expression can exceed those typically obtained in plants transformed with other vectors 17. Foreign proteins expressed in this manner are under the transcriptional control of a viral promoter. We have used this strategy to study the behaviour of the movement protein of TMV following its release as a movement protein-GFP fusion from the PVX vector22.Under PVX-directed expression, the TMV movement protein-GFP fusion was targeted to plasmodesmata within cells at the infection front. In addition, the cytoskeleton became labelled in many of the infected cells, confirming the observations ofHeinlein et al.24 that the movement protein of TMV associates with the cytoskeleton. These results, which are based on the use of an alternative vector, indicate that the association of the TMV movement protein with the cytoskeleton is an intrinsic property of the movement protein itself, and is not attributable to an interaction with other TMV-encoded proteins. The intercellular movement function of cucumber mosaic virus has been attributed to the 3a protein, a 30 kDa protein that shows weak homology to the TMV-encoded movement protein 39.This protein has been shown to gate plasmodesmata to allow the passage of fluorescent dextrans 32,4°and can itself traffick between cells 32. In addition, transgenic plants expressing this viral protein have been shown to potentiate the movement of viral RNA between adjacent mesophyll cells% To date, however, direct evidence that the 3a protein targets to plasmodesmata during viral movement has not been presented. Indeed, there is some evidence, from cell fractionation studies of virus-infected plants and transgenic plants expressing the 3a protein, that the 3a protein is distributed between a number of subcellular fractions 4°,42,43. Attempts to introduce a 3a-GFP fusion into cells using cucumber mosaic virus as the vector have not yet been successful (P. Palukaitis, pers. commun.). However, expression of a 3a-GFP fusion protein from the PVX vector has allowed the targeting of the fusion protein to be studied in plants (P. Boevink, K. Oparka and S. Santa Cruz, unpublished). Within developing infection sites, the fusion protein was exclusively targeted to plasmodesmata of epidermal and mesophyll cells. Palisade parenchyma cells showed striking plasmodesmal localization with the 3a-GFP fusion (Fig.3). Using this approach, the targeting of viral movement proteins to plasmodesmata can now be studied in vivo, opening up new opportunities for studying the interaction between the movement protein and plasmodesma proteins. The ability to assess movement protein targeting noninvasively raises some interesting new questions concerning movement protein function: • Do the movement proteins of different viruses target the same types of plasmodesmata when released from a common vector? • Do movement proteins discriminate between the plasmodesmata of different cell types (or the plasmodesmata within a single cell)? • Do movement proteins target the plasmodesmata associated with the cells of the phloem, particularly those 'specialized' plasmodesmata 44,45 that connect the sieve element with its companion cell? Hopefully the answers to these questions will come from studies in which movement protein-GFP targeting is mapped in
relation to the cellular anatomy of the leaf, and in particular the nature and distribution of plasmodesmata connecting different cell types. The PVX-based vector is clearly very useful as a delivery system for studying the targeting capabilities of putative movement proteins from different viruses. However, a drawback of the approach is that it is difficult to separate the movement functions of the foreign movement protein from those of the vector. An alternative approach would be to insert movement protein-GFP fusions into a PVX vector whose own movement functions have been deleted. The behaviour of the foreign movement protein could then be studied independently. In addition, the capacity of the foreign movement protein(s) to allow the local and systemic movement of the vector could be studied. The selective deletion of specific sequences from the movement protein, when fused to GFP, may yield important new information as to which domains are essential for different functions, such as association of the movement protein with host factors or targeting to plasmodesmata.
Isolation of plasmodesmal proteins involved in movement protein recognition An area of significant current interest is the development of techniques for the isolation and characterization of plasmodesma-associated proteins (PAPs). To date, a number of candidate PAPs have been isolated from cell wall fractions 464s, but the role of such proteins in plasmodesmal function has not yet been elucidated. It is possible that movement protein-GFP fusions will aid in the isolation of plasmodesmal proteins with a specific role in movement protein recognition. The movement protein of TMV is known to occur at the plasmodesmal pore (from immunolocalization) ~,7, and recent work with movement protein-GFP fusions confirms that the movement protein is targeted to plasmodesmata during virus infection 23. Also, a TMV movement protein-GFP fusion (expressed from the TMV vector) remained bound to the cell wall and retained its fluorescence following extraction of the cell contents and extensive washing (B. Epel et al., pers. commun.). In addition to immunocytochemical studies using antibodies raised against putative PAPs, PAP genes could be reinserted into an appropriate vector, such as PVX, allowing the production of PAP-GFP fusions in cells; it would then be possible to determine whether such a protein could be relocated to plasmodesmata. The use of transgenic plants expressing deletions or modified PAPs involved in movement protein recognition remains a future goal, but would provide a powerful tool in the study of movement proteinplasmodesma interactions. By expressing movement proteinGFP fusions of a range of different viruses from a common vector, it should be possible to determine whether the same host plasmodesmal protein(s) is responsible for movement protein recognition in each case.
Green fluorescent protein-coat protein fusions Recently, a modified PVX was described49 in which GFP was fused to the amino terminus of the viral coat protein. For the assembly of the GFP-coat protein fusion into virions, it was necessary to provide a mixed population of both free coat protein subunits and GFP-coat protein fusion subunits. The 'overcoat' virus (Fig. 4), assembled from coat protein and GFP-coat protein subunits, was over twice the diameter of the wild-type PVX and remained capable of both local and systemic movement. It was also apparent that the December 1996, Vol. 1. No. 12
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Fig. 4. Diagram indicating how the virion of potato virus X is assembledfrom: (left side of figure) free coat protein subunits (shown as spheres); and (right side of figure) both free coat protein subunits and fusion protein subunits (shown as 'paddles'), which are formedfrom green fluorescentprotein and coat protein fusion. The viral nucleic acid is shown as a coil. Courtesy of Michael Wilson.
in determining the form (capsidated or uncapsidated) in which infectious viral entities move in the phloem. Using severed aphid stylets, it should be possible to collect phloem exudate from infected plants and examine it for the presence of fluorescent virions. Such an approach may answer some long-standing questions concerning the nature of virus movement in the phloem~. In addition, several viruses are transmitted by arthropod or nematode vectorsS°: the use of GFP-tagged viruses may provide an invaluable opportunity to study their transmission.
Do fusion proteins behave like the wild-type protein?
When considering the use of fusions to GFP, it is important to consider possible majority of the GFP produced in the modified-PVX-infected interference with the normal function of the proteins. For plants was fused to the coat protein, and that the fusion example, fusion of GFP to the movement protein of TMV proteins were assembling into fibrillar aggregates within produces a protein with approximately twice the molecular infected cells. In transverse sections of the leaf, paramass (57 kDa) of the wild-type movement protein (30 kDa). crystalline aggregates (i.e. regular arrays that appear Nonetheless, the movement protein- GFP successfully targets crystalline in the electron microscope) of fluorescent virus plasmodesmata and supports intercellular movement of the were clearly visible in epidermal, palisade and mesophyll TMV vector 24. When planning movement protein-GFP fusion cells (Fig. 5). Confirmation that the virus carried a GFP 'over- strategies, the potential for successful targeting might be coat' was obtained by probing electron microscope sections improved by careful selection of the fusion site (i.e. C-terminal with polyclonal antibodies to both GFP and the virus coat or N-terminal fusions). Insertion of a linker sequence between protein49. the two fused proteins may also help to limit potentially It remains to be seen how many plant viruses can be deleterious effects of the GFP tag on the function of the target tagged with GFP. However, a strategy in which GFP is protein24. In the case of the TMV movement protein, previous located on the assembled virus, rather than in the cytosol of studies have mapped functional domains in the protein imporinfected cells, potentially extends the utility of GFP in tant for nucleic acid binding and plasmodesmal targeting', plant-virus movement studies. For example, the presence of suggesting that insertion of GFP at the C-terminus of the a fluorescent virus will be of use in systemic transport studies movement protein would be less likely to affect function than a fusion to the N-terminus. Previous case histories of GFP fusions in animal . . . . . . systems suggest that the GFP does not interfere with the function of the proteins to which it is fused26.~'m;however, each strategy must be considered carefully against the background of information available for the protein under study. In the case ofviral movement proteins, a number of questions remain to be addressed. For example, does the movement protein-GFP fusion itself traffick through plasmodesmata in the manner reported for wild-type movement proteins? Does the movement protein-GFP fusion gate plasmodesmata to the same degree as the wild-type movement protein, or does the presence of such a large protein impede the movement of macromolecules through the pore? In connection with the latter Fig. 5. Transverse section of a leaf of Nicotiana benthamiana infected with potato virus X expressing the green fluorescent protein as a coat protein fusion. The question, the microinjection of fluoresfluorescent virus is present in palisade and spongy mesophyll cells. To obtain the cent dextrans will help to discriminate precise localizationof virus, the fluorescentimage taken at 488 nm excitation was between those domains of the protein superimposed on a bright field image of the same cells. Both images were obtained essential for targeting to plasmodeswith a confocallaser scanning microscope. Scale bar represents 50 ~m. mata and those responsible for gating. Using a combination of such powerful 416
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reviews techniques it may soon be possible to dissect the functional domains of viral movement proteins. Conclusions The use of GFP in plant-virus studies is in its infancy, but it shows great promise for tracing the movement of functional plant viruses, both locally and systemically, and for unravelling some of the key events involved in the interactions between viral movement proteins and plasmodesmata. At present only a limited number of viral proteins have been fused to GFP, but initial studies suggest that the targeting functions of viral movement proteins are unaffected by the fusion. This strategy, used critically in conjunction with other cell biology techniques, has much potential in the study of virus-plasmodesma interactions in plants.
Acknowledgements We are grateful to D. Prior for preparation of the figures, to R. Beachy, B. Epel, J. van Lent and J. Wellink for access to unpublished data and to P. Palukaitis for helpful discussions. The authors' work was supported by the Scottish Office Agriculture, Environment and Fisheries Dept and by grants from the Biotechnology and Biological Sciences Research Council and Leverhulme Trust. References 1 Lucas, W.J. and Gilbertson, R.L (1994) Plasmodesmata in relation to viral movement within leaf tissues, Annu. Rev. Phytopathol. 32, 387-411 2 Gilbertson, R.L. and Lucas, W.J. (1996) Hew do viruses traffic on the 'vascular highway'? Trends Plant Sci. 1,260-268 3 Maule, A.J. (1991) Virus movement in infected plants, Crit. Rev. Plant Sci. 9, 457-473 4 Leisner, S.M. and Turgeon, R. (1993) Movement of virus and photoassimilate in the phloem: a comparative analysis, BioEssays 15, 741-747 5 Deom, C.M., Oliver, M.J. and Beachy, R.N. (1987) The 30-kilodalton gene product of tobacco mosaic virus potentiates virus movement, Science 237, 389-394 6 Tomenius, K., Clapham, D. and Meshi, T. (1987) Localization by immunogold cytochemistry of the virus-coded 30K protein in plasmodesmata of leaves infected with tobacco mosaic virus, Virology 160, 363-371 7 Atkins, D. et al. (1991) The tobacco mosaic virus 30K movement protein in transgenic tobacco plants is localised to plasmodesmata, J. Gen. Virol. 72, 209-211 8 Citovsky, V. et al. (1990) The P30 movement protein of tobacco mosaic virus is a single-strand nucleic acid binding protein, Cell 60, 637-647 9 Wolf, S. et al. (1989) Movement protein of tobacco mosaic virus modifies plasmodesmatal size exclusion limit, Science 246, 377-379 10 Dawson, W.O., Bubrick, P. and Grantham, G.L. (1988) Modifications of the tobacco mosaic virus coat protein gene affecting replication, movement and symptomatology, Phytopathology 78, 783-789 11 Wellink, J. et al. (1993) The cowpea mosaic virus M RNA encoded 48-kilodalton protein is responsible for induction of tubular structures in protoplasts, J. Virol. 67, 3660-3664 12 Wieczorek, A. and Sanfa~on, H. (1993) Characterization and subcellular localization of tomato ringspot nepovirus putative movement protein, Virology 194, 734-742 13 Dolja, V.V. et al. (1994) Distinct functions ofcapsid protein in assembly and movement of tobacco etch potyvirus in plants, EMBO J. 13, 1482-1491 14 Scholthof, H.B., Morris, T.J. and Jackson, A.O. (1993) The capsid protein of tomato bushy stunt virus is dispensable for systemic movement and can be replaced for localized expression of foreign genes, Mol. Plant-Microbe Interact. 6, 309-322 15 Taliansky, M.E., Robinson, D.J. and Murant, A.F. (1996) Complete nucleotide sequence and organization of groundnut rosette umbravirus, J. Gen. ViroI. 77, 2335-2345
16 Chalfie, M. et al. (1994) Green fluorescent protein as a marker for gene expression, Science 263,802-805 17 Chapman, S.N., Kavanagh, T. and Banlcombe, D.C. (1992) Potato virus X as a vector for gene expression in plants, Plant J. 2, 549-557 18 Dolja, V.V., McBride, H.J. and Carrington, J.C. (1992) Tagging of plant potyvirus replication and movement by insertion of ~-glucuronidase into the viral polyprotein, Proc. Natl. Acad. Sci. U. S. A. 89, 10208-10212 19 Stearns, T. (1995) The green revolution, Curr. Biol. 5, 262-264 20 Prasher, D.C. (1995) Using GFP to see the light, Trends Genet. 11, 320-323 21 Baulcombe, D.C., Chapman, S.N. and Santa Cruz, S. (1995) Jellyfish green fluorescent protein as a reporter for virus infections, Plant J. 7, 1045-1053 22 Oparka, K.J. et al. (1995) Imaging the green fluorescent protein in plants - viruses carry the torch, Protoplasma 189, 133-141 23 Epel, B.L. et al. Plant virus movement protein dynamics probed with a movement protein fused to the GFP, Gene (in press) 24 Heinlein, M. et al. (1995) Interaction oftobamovirus movement proteins with the plant cytoskeleton, Science 270, 1983-1985 25 Oparka, K.J. et al. Viral coat protein is targeted to, but does not gate, plasmodesmata during cell-to-cell movement of potato virus X, Plant J. (in press) 26 Wang, S. and Hazelrigg, T. (1994) Implications for bcd mRNA localization from spatial distribution of exu protein in Drosophila oogenesis, Nature 369, 400-403 27 Derrick, P.M., Barker, H. and Oparka, K.J. (1992) Increase in plasmodesmatal permeability during cell-to-cell spread of tobacco rattle virus from individually inoculated cells, Plant Cell 4, 1405-1412 28 Angell, S., Davies, C. and Baulcombe, D.C. (1996) Cell-to-cell movement of potato virus X is associated with a change in the size-exclusion limit ofplasmodesmata in trichome cells ofNicotiana cIevelandii, Virology 216, 197-201 29 Fujiwara, T. et al. (1993) Cell-to-cell trafficking of macromolecules through plasmodesmata potentiated by the red clover necrotic mosaic virus movement protein, Plant Cell 5, 1783-1794 30 Waigmann, E. et al. (1994) Direct functional assay for tobacco mosaic virus cell-to-cell movement protein and identification of a domain involved in increasing plasmodesmatal permeability, Proc. Natl. Acad. Sci. U. S. A. 91, 1433-1437 31 Nouiery, A.O., Lucas, W.J. and Gilbertson, R.L. (1994) Two proteins of a plant DNA virus coordinate nuclear and plasmodesmal transport, Cell 76, 925-932 32 Ding, B. et al. (1995) Cucumber mosaic virus 3a protein potentiates cell-to-cell trafficking of CMV RNA in tobacco plants, Virology 207, 345-353 33 McLean, B.G., Zupan, J. and Zambryski, P. (1995) Tobacco mosaic virus movement protein associates with the cytoskeleton in tobacco cells, Plant Cell 7, 2101-2114 34 Topp, K.S., Meade, L.B. and LaVail, J.H. (1994) Microtubule polarity in the peripheral processes of trigeminal ganglion cells: relevance for the retrograde transport of herpes simplex virus, J. Neurosci. 14, 318-325 35 van Lent, J. et al. (1991) Tubular structures involved in movement of cowpea mosaic virus are also formed in infected cowpea protoplasts, J. Gen. Virol. 72, 2615-2623 36 Wellink, J. and van Kammen, A. (1989) Cell-to-cell transport of cowpea mosaic virus requires both the 58K/48K proteins and the capsid proteins, J. Gen. Virol. 70, 2279-2286 37 Murant, A.F. and Roberts, I.M. (1971) Cylindrical inclusions in coriander leaf cells infected with parsnip mosaic virus, J. Gen. Virol. 10, 65-70 38 Lawson, R.H. and Hearon, S.S. (1971) The association of pinwheel inclusions with plasmodesmata, Virology 44, 454-456 39 Davies, C. and Symons, R.H. (1988) Further implications for the evolutionary relationships between tripartite plant viruses based on cucumber mosaic virus RNA 3, Virology 165, 216-224 40 Vaquero, C. et al. (1994) The 3a protein from cucumber mosaic virus increases the gating capacity of plasmodesmata in transgenic tobacco plants, J. Gen. Virol. 75, 3193-3197 41 Kaplan, I.B. et al. (1995) Complementation of virus movement in transgenic tobacco expressing the cucumber mosaic virus 3a gene, Virology 209, 188-199
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reviews 42 Burman, A.J., Osman, T.A.M. and Buck, K.W. (1994) Detection of the 3a protein of cucumber mosaic virus in a cell wall fraction from infected Nicotiana clevelandii plants, J. Phytopathol. 142,317-323 43 Cooper, B. and Dodds, J.A. (1995) Differences in the subcellular localization of tobacco mosaic virus and cucumber mosaic virus movement proteins in infected and transgenic plants, J. Gen. Virol. 76, 3217~3221 44 Esau, K. and Thorsch, J. (1985) Sieve plate pores and plasmodesmata, the communication channels of the symplast: ultrastructural aspects and developmental relations, Am. J. Bot. 72, 1641-1653 45 Kempers, R. et al. (1993) Plasmodesmata between sieve element and companion cell of extrafascicular phloem of Cucurbita maxima permit passage of 3 kDa fluorescent probes, Plant J. 4, 567-575 46 Monzer, J. and Kloth, S. (1991) The preparation of plasmodesmata from plant tissue homogenates: access to the biochemical characterization of plasmodesmata-related polypeptides, Bot. Acta 104, 82-84 47 Kotlizky, G. et al. (1992) An improved procedure for the isolation of plasmodesmata embedded in clean maize cell walls, Plant J. 2,623-630 48 Turner, A., Wells, B. and Roberts, K, (1994) Plasmodesmata of maize root tips - structure and composition, J. Cell Sci. 107, 3351-3361
49 Santa Cruz, S. et al. (1996) Assembly and movement of a plant virus carrying a green fluorescent protein overcoat, Proc. Natl. Acad. Sci. U. S. A. 93, 6286-6290 50 Gibbs, A. and Harrison, B.D. (1976) Plant Virology: the Principles, Edward Arnold 51 Ogawa, H. et al. (1995) Localization, trafficking, and temperature dependence of the Aequorea green fluorescent protein in cultured vertebrate cells, Proc. Natl. Acad. Sci. U. S. A. 92, 11899-11903 52 Stauber, R., Gaitanaris, G.A. and Pavlakis, G.N. (1995) Analysis of trafficking of Rev and transdominant Rev proteins in living cells using green fluorescent protein fusions: transdominant Rev blocks the export of Rev from the nucleus to the cytoplasm, Virology 213,439-449
Karl Oparka, Petra Boevink and Simon Santa Cruz are at the Unit of Plant Transport Processes, Scottish Crop Research Institute, Invergowrie, Dundee, UK DD2 5DA. Fax (K.O.): +44 1382 562 426 e-mail: [email protected]
Phloem loading and plasmodesmata Robert Turgeon Phloem loading, the active accumulation of photosynthate in minor veins, is thought to be the motivating force for translocation. Can loading take place along an entirely symplastic route, through the plasmodesmata, from mesophyll to phloem? Transport of small molecules through the plasmodesmata is apparently passive, and the concept of symplastic phloem loading thus appears to violate thermodynamic principles. Nonetheless, evidence for such a pathway in many plants has been accumulating steadily and an answer to the thermodynamic argument has been put forward. The proposed mechanism involves synthesis and trapping of raffinose and stachyose in the phloem.
p
hloem loading creates very high turgor pressure in the sieve element-companion cell complex (SE-CCC), and has generally been viewed as an essential component of Mfinch pressure flow, which is driven by the difference in pressure between source and sink 1. This concept, that the sugar concentration in the phloem is higher than in the mesophyll, and thus requires a loading step, arose in the 1930s and 1940s. The term 'phloem loading' was coined by Loomis, in 1955, who described the phenomenon as an 'endothermic pumping action '2. Since that time, physiologists have been trying to determine the physical location of the pump and its molecular nature. This is important, not only for the study of transport, but because the mechanisms that drive loading create a special structural and biochemical boundary between the phloem and the surrounding cells, defining the phloem as a specific symplastic domain. Because this is an integrating domain that permeates the entire plant, loading is often invoked as an important physiological control point (e.g. in studies of mineral nutrition ~, responses to elevated carbon dioxide 4, ozone 5and sulfur dioxide6, and resistance to virus movementT). As Geiger et al. 8 first demonstrated, the solute content of the SE-CCC in the minor veins of leaves is far higher than that of the surrounding cells and the mesophyll. It is therefore this boundary between the SE-CCC and the surrounding cells that is especially important in phloem loading. Here, the following questions are addressed: 4-1 8
December1996, Vol, 1, No. 12
• Can transport of photosynthate into the SE-CCC take place through plasmodesmata? In other words, is it possible for sugar to stay within the symplast all the way from the mesophyll to the sink? • If so, what is the mechanism of active accumulation? Plasmodesmal frequency
Early studies on phloem loading focused on the apoplastic route 1. According to the most widely accepted version of this model, sucrose diffuses between mesophyll cells through the plasmodesmata, but somewhere in the vicinity of the minor veins it enters the apoplast. It has long been thought that the site of effiux is within the vein, and is possibly the vascular parenchyma. This concept is now supported by structural evidence that, in an export-deficient maize mutant, plasmodesmata at the bundle sheathphloem parenchyma interface are aberrant and probably nonfunctional 9. From the apoplast, the sucrose is loaded into the SE-CCC, against a steep concentration gradient, by symport with protons. In this model, the plasmodesmata do not play a direct role in the loading step. In fact, the presence of plasmodesmata raises a difficult question. Why is it that sucrose, at such a high concentration, does not diffuse out of the SE-CCC and back into the mesophyll through these pores? The evolutionary solution to this problem in many species appears to have been a simple reduction in plasmodesmal frequency (Fig. 1). In plants for which evidence of
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Studying the m o v e m e n t of plant viruses using green fluorescent protein opo,,o,o,,o BoevinkandSimonSantaCruz Recent studies with green fluorescent protein (GFP) are providing n e w insights into the mechanisms of virus movement in plants. The gfp gene has been inserted into the genomes of a number of plant viruses, enabling production of either the free protein or a fusion with a virus-encoded protein. Fusion of GFP to the coat protein of potato virus X (PVX) has produced a functional virus with a GFP 'overcoat' that can be traced as it moves. Many plant viruses are known to use 2novement proteins' to assist in their transit through plasmodesmata. Movement protein-GFP fusions can be expressed from the parental virus or, in the absence of a full-length infectious clone, using an alternative expression vector. Such fusions retain their capacity to target plasmodesmata and are providing n e w information on the factors responsible for trafficking viral RNA to, and through, the plasmodesmal pore. ost plant viruses are able to move both locally (between cells) and systemically (over long distances). Local movement occurs through plasmodesmata, the small channels that interconnect higher plant cells ~-3. Systemic movement is dependent on the entry of the virus (or an infectious component of the virus) into the long-distance transport system of the phloem 2,4. However, although some viruses have the capacity to move systemically
(e.g. if introduced directly into the phloem by a feeding insect), they may not possess the ability to escape from the phloem in sink tissues. Such phloem-limited viruses 1 appear to lack the correct 'key(s)' to allow local movement out of the phloem. Other viruses have proteins that potentiate both local and systemic virus movement. For example, in tobacco mosaic virus (TMV), local movement is facilitated by a 30 kDa movement protein ~that targets plasmodesmata during virus infection~,7, binds to singlestranded nucleic acids s and 'gates' plasmodesmata to a much higher than normal size exclusion limit s. AlthoUgh TMV does not require its coat protein for local movement, this protein is apparently essential for efficient long-distance movement 10. In contrast to this, some viruses require their coat protein in addition to movement protein(s) for local movementH-l~; other viruses can dispense with the coat protein for both local and systemic movement~4,15. Fig. 1. Leaf trichome cells of Nicotiana clevelandii systemically infected with potato virus X To date, unravelling the (PVX) constructs allowing expression of the green fluorescent protein (GFP). In (a), the GFP is roles of different virusbeing released as a free protein in virus-infected cells and remains restricted to the cytosol and encoded genes in local and nucleus. In (b), the PVX vector is carrying a GFP fusion to the 3a movement protein of cucumsystemic movement has ber mosaic virus. The GFP-movement protein fusion has been targeted to plasmodesmata on been hindered by the lack of the end walls of the subapical (neck) cell during intercellular virus movement. Note the absence a suitable in vivo marker for of labelling on the lateral walls - all trichome lateral walls lack plasmodesmata. In (c), the GFP is fused to the viral coat protein of PVX to produce a functional virus with a GFP 'overcoat'4~. tracking the key elements Note that the virus adopts different morphologies in different cells. In the apical (tip) cell the of movement with adequate virus forms flexuous, spindle-shaped aggregates, while in the neck cell the aggregated virus cellular resolution. Hence forms a continuous doughnut-shaped ring. This image was reconstructed from a series of conthe introduction of GFP focal sections taken through the trichome. All the images were obtained with a confocal laser as a noninvasive reporter scanning microscope. Scale bars represent 10 ~m. in a range of systems 1~ has opened up a unique
M
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reviews opportunity to study viral trafficking in plants (a recent review 2 highlighted some of the current problems in studying this phenomenon). Here, the ways in which GFP may aid in the noninvasive detection of virus movement are reviewed.
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4
.4
Green fluorescent protein as a reporter of virus infection
,
p
The marker protein 13-glucuronidase (GUS) has previously been used as a reporter of virus infection and to study ," the roles of different viral genes in both local and systemic movement 14,17,1s. ",-" Histochemical staining of tissue infected with viruses expressing GUS . f 'll allows the virus to be localized to specific cells and tissues. However, the staining procedure is destructive and does not allow for continuous monitorFig. 2. Association of the movement protein of tobacco mosaic virus (TMV)with the ing of the viral infection. In contrast to cytoskeleton of infected Nicotiana benthamiana plants. The GFP was fused to the this procedure, detection of GFP movement protein of TMV and released as a fusion protein from the TMV vector24. depends only on excitation of the protein The cells shown are close to the infection front and depict different stages of the using light of the appropriate waveassociation of the movement protein-GFP fusion with the cytoskeleton. In some length and does not require the addition cells the fusion protein is present as discrete aggregates, but other cells lack such structures. The image was obtained using a confocal laser scanning microscope. of exogenous substrates or cofactors19,20. Scale bar represents 20 ~m. Courtesy of Roger Beachy and Denton Prior. Baulcombe et al. 21 were the first to introduce the gfp gene into a plant virus, using the potexvirus PVX. Transcription of the gfp mRNA was directed by a viral promoter It was subsequently shown that the local and systemic sequence. Following infection of plants with the modified movement of this coat protein-lacking mutant could be virus, GFP accumulated in the cytoplasm and nucleus of restored when it was inoculated onto transgenic tobacco infected cells2~,22(Fig. la). Green fluorescent protein has also expressing the PVX coat protein• Further observations claribeen expressed as the free protein using as vectors both TMV fied the role of the protein. First, microinjection studies using (Ref. 23) and cowpea mosaic virus (J. Wellink and J. van Lent, fluorescent dextran probes showed that transgenic exprespers. commun.). Detection of fluorescence in virus-infected sion of the coat protein did not alter the plasmodesmal size cells is facilitated by the high expression level of GFP when directed by viruses such as PVX and TMV. The fluorescent signal from tissue infected with either TMV or PVX is sufficiently strong to allow macroscopic imaging under long,:. "~ wavelength ultraviolet light, thereby providing a means of studying systemic :~': virus movement as the infection ~. ~" p r o g r e s s e s 21,22,24. ~" =,
.
I
•
Replacing viral proteins with green fluorescent protein The use of GFP also provides novel opportunities for studying mutant viral genomes, by the replacement of virusencoded proteins with GFP. This approach has already yielded information on the requirement of viral genes for local movement. For example, when the coat protein gene of PVX was replaced with the GFP gene 21, it was found that the resulting virus was capable of replication and GFP production, but was restricted to single cells on the leaf epidermis; this demonstrates that the coat protein of PVX is essential for local movement.
t
i
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:
2.
Fig. 3. Transverse section of a leaf ofNicotiana benthamiana infected with potato virus X expressing a fusion of green fluorescent protein (GFP) with the 3a movement protein of cucumber mosaic virus. The movement protein-GFP fusion is exclusively localized to the plasmodesma pit fields of the palisade mesophyll. The image was obtained using a confocal laser scanning microscope. Scale bar represents 20 ~m.
December 1996, Vol. 1, No. 12
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reviews exclusion limit. Second, in uninfected coat protein-containing transgenic tobacco, the protein was shown by immunolocalization to be in the cytoplasm only; however, infection of these transgenic plants with the PVX coat protein deletion mutant resulted in targeting of the coat protein to plasmodesmata (in wild-type PVX infections, the coat protein is also localized at plasmodesmata). These observations suggest that, for PVX, the coat protein is important for trafficking the viral RNA to the plasmodesmal pore, but is not directly involved in gating 25.
Fusion of green fluorescent protein to viral proteins Although the release of free GFP in cells is a valuable marker for the presence of virus and the roles of different viral genes in determining local and systemic movement, an alternative approach is to create fusions between GFP and viral proteins. This exploits one of the principal advantages of GFP as a reporter- its ability to fluoresce in fusion proteins 2~ - and opens up the possibility for noninvasive imaging of viral proteins during the infection process. Green fluorescent protein-movement protein fusions The role of the movement protein from TMV in plasmodesmal gating was initially demonstrated using transgenic plants constitutively expressing the movement protein 9. Microinjection of the mesophyll cells of these plants with fluorescently labelled dextrans and peptides demonstrated that the size exclusion limit was considerably in excess (>10kDa) of their 'basal' limit (
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on the cell wall 23,24(Fig. 2); this suggests that the interaction of the movement protein with microtubules occurs during virus infection of tissues as well as in protoplasts. The cytoskeleton (particularly microtubules) has also been shown to play a role in viral trafficking in animal systems. For example, the directional movement of herpes simplex virus in cells may be mediated by a minus-end directed motor such as cytoplasmic dynein 34. The recent studies on plant viruses, using GFP fusion approaches, have begun to open up the search for the plant host factors that interact with viral movement protein-RNA complexes and allow them to reach plasmodesmata from their sites of synthesis in the cytoplasm. The challenge now is to determine precisely how the movement protein of TMV uses the cytoskeleton to traffick the viral RNA to the plasmodesmata (if this is indeed the transport pathway) and to dissect the stages involved in the association of the movement protein-viral RNA complex with the cytoskeleton. Not all viral movement proteins necessarily associate with the plant cytoskeleton in order to locate plasmodesmata. In the case of cells infected with cowpea mosaic virus, protein tubules are assembled that span the plasmodesmal pore; these may form in situ at the plasma membrane of isolated protoplasts 3~. The formation of tubules requires a 48 kDa virus-encoded protein, and may also require hostencoded protein(s) 11(although it is not yet known if these proteins associate in any way with the cytoskeleton during viral movement). Recently, J. Wellink and J. van Lent made a GFP fusion to the C-terminus of the 48 kDa movement protein by replacing the coat protein-coding region of the virus (unpublished). The substitution prevented the virus from moving systemically (as this virus requires its coat protein for local movement3~), but GFP-tagged tubules formed on the plasma membrane of infected protoplasts. In the case of the potyviruses, pinwheel structures, composed of a virus-encoded protein, are frequently observed at or near plasmodesmata 37,3s.Little is known of the function of these structures, but they are certainly candidates for GFP fusion, which would allow their precise distribution in an advancing infection front to be studied. Noninvasive location of pinwheels would aid in the selection of infected cells for electron microscopy and for injection studies with fluorescent dextrans to determine if they have a role in plasmodesmal gating. As pointed out by Gilbertson and Lucas 2, the formation of assembly sites for movement proteins at or close to the plasmodesmal pore may obviate the need for an association of the viral movement protein with the cytoskeleton. Different viruses are likely to locate plasmodesmata in different ways, and in this respect movement protein-GFP fusions may play an important role in dissecting the pathway leading to the plasmodesmal pore.
Alternative vectors for studying movement protein targeting and function A functional analysis of the movement proteins from some viruses may be hindered either by lack of availability of a full-length infectious clone, or because of the inability to modify the viral genome without losing infectivity. Thus, although the specific protein(s) implicated in viral movement may have been identified and sequenced, it could be impossible to express these as GFP fusions from the parental virus. One approach to overcome this is to fuse the protein to GFP and direct its expression in the plant independently of the parental virus.
reviews We have been exploring the potential of PVX as an episomal vector for the expression of GFP fusion proteins; PVX has proven to be a versatile vector for the expression of foreign genes in plants and, because of its high multiplication rate, the levels of foreign gene expression can exceed those typically obtained in plants transformed with other vectors 17. Foreign proteins expressed in this manner are under the transcriptional control of a viral promoter. We have used this strategy to study the behaviour of the movement protein of TMV following its release as a movement protein-GFP fusion from the PVX vector22.Under PVX-directed expression, the TMV movement protein-GFP fusion was targeted to plasmodesmata within cells at the infection front. In addition, the cytoskeleton became labelled in many of the infected cells, confirming the observations ofHeinlein et al.24 that the movement protein of TMV associates with the cytoskeleton. These results, which are based on the use of an alternative vector, indicate that the association of the TMV movement protein with the cytoskeleton is an intrinsic property of the movement protein itself, and is not attributable to an interaction with other TMV-encoded proteins. The intercellular movement function of cucumber mosaic virus has been attributed to the 3a protein, a 30 kDa protein that shows weak homology to the TMV-encoded movement protein 39.This protein has been shown to gate plasmodesmata to allow the passage of fluorescent dextrans 32,4°and can itself traffick between cells 32. In addition, transgenic plants expressing this viral protein have been shown to potentiate the movement of viral RNA between adjacent mesophyll cells% To date, however, direct evidence that the 3a protein targets to plasmodesmata during viral movement has not been presented. Indeed, there is some evidence, from cell fractionation studies of virus-infected plants and transgenic plants expressing the 3a protein, that the 3a protein is distributed between a number of subcellular fractions 4°,42,43. Attempts to introduce a 3a-GFP fusion into cells using cucumber mosaic virus as the vector have not yet been successful (P. Palukaitis, pers. commun.). However, expression of a 3a-GFP fusion protein from the PVX vector has allowed the targeting of the fusion protein to be studied in plants (P. Boevink, K. Oparka and S. Santa Cruz, unpublished). Within developing infection sites, the fusion protein was exclusively targeted to plasmodesmata of epidermal and mesophyll cells. Palisade parenchyma cells showed striking plasmodesmal localization with the 3a-GFP fusion (Fig.3). Using this approach, the targeting of viral movement proteins to plasmodesmata can now be studied in vivo, opening up new opportunities for studying the interaction between the movement protein and plasmodesma proteins. The ability to assess movement protein targeting noninvasively raises some interesting new questions concerning movement protein function: • Do the movement proteins of different viruses target the same types of plasmodesmata when released from a common vector? • Do movement proteins discriminate between the plasmodesmata of different cell types (or the plasmodesmata within a single cell)? • Do movement proteins target the plasmodesmata associated with the cells of the phloem, particularly those 'specialized' plasmodesmata 44,45 that connect the sieve element with its companion cell? Hopefully the answers to these questions will come from studies in which movement protein-GFP targeting is mapped in
relation to the cellular anatomy of the leaf, and in particular the nature and distribution of plasmodesmata connecting different cell types. The PVX-based vector is clearly very useful as a delivery system for studying the targeting capabilities of putative movement proteins from different viruses. However, a drawback of the approach is that it is difficult to separate the movement functions of the foreign movement protein from those of the vector. An alternative approach would be to insert movement protein-GFP fusions into a PVX vector whose own movement functions have been deleted. The behaviour of the foreign movement protein could then be studied independently. In addition, the capacity of the foreign movement protein(s) to allow the local and systemic movement of the vector could be studied. The selective deletion of specific sequences from the movement protein, when fused to GFP, may yield important new information as to which domains are essential for different functions, such as association of the movement protein with host factors or targeting to plasmodesmata.
Isolation of plasmodesmal proteins involved in movement protein recognition An area of significant current interest is the development of techniques for the isolation and characterization of plasmodesma-associated proteins (PAPs). To date, a number of candidate PAPs have been isolated from cell wall fractions 464s, but the role of such proteins in plasmodesmal function has not yet been elucidated. It is possible that movement protein-GFP fusions will aid in the isolation of plasmodesmal proteins with a specific role in movement protein recognition. The movement protein of TMV is known to occur at the plasmodesmal pore (from immunolocalization) ~,7, and recent work with movement protein-GFP fusions confirms that the movement protein is targeted to plasmodesmata during virus infection 23. Also, a TMV movement protein-GFP fusion (expressed from the TMV vector) remained bound to the cell wall and retained its fluorescence following extraction of the cell contents and extensive washing (B. Epel et al., pers. commun.). In addition to immunocytochemical studies using antibodies raised against putative PAPs, PAP genes could be reinserted into an appropriate vector, such as PVX, allowing the production of PAP-GFP fusions in cells; it would then be possible to determine whether such a protein could be relocated to plasmodesmata. The use of transgenic plants expressing deletions or modified PAPs involved in movement protein recognition remains a future goal, but would provide a powerful tool in the study of movement proteinplasmodesma interactions. By expressing movement proteinGFP fusions of a range of different viruses from a common vector, it should be possible to determine whether the same host plasmodesmal protein(s) is responsible for movement protein recognition in each case.
Green fluorescent protein-coat protein fusions Recently, a modified PVX was described49 in which GFP was fused to the amino terminus of the viral coat protein. For the assembly of the GFP-coat protein fusion into virions, it was necessary to provide a mixed population of both free coat protein subunits and GFP-coat protein fusion subunits. The 'overcoat' virus (Fig. 4), assembled from coat protein and GFP-coat protein subunits, was over twice the diameter of the wild-type PVX and remained capable of both local and systemic movement. It was also apparent that the December 1996, Vol. 1. No. 12
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reviews
Fig. 4. Diagram indicating how the virion of potato virus X is assembledfrom: (left side of figure) free coat protein subunits (shown as spheres); and (right side of figure) both free coat protein subunits and fusion protein subunits (shown as 'paddles'), which are formedfrom green fluorescentprotein and coat protein fusion. The viral nucleic acid is shown as a coil. Courtesy of Michael Wilson.
in determining the form (capsidated or uncapsidated) in which infectious viral entities move in the phloem. Using severed aphid stylets, it should be possible to collect phloem exudate from infected plants and examine it for the presence of fluorescent virions. Such an approach may answer some long-standing questions concerning the nature of virus movement in the phloem~. In addition, several viruses are transmitted by arthropod or nematode vectorsS°: the use of GFP-tagged viruses may provide an invaluable opportunity to study their transmission.
Do fusion proteins behave like the wild-type protein?
When considering the use of fusions to GFP, it is important to consider possible majority of the GFP produced in the modified-PVX-infected interference with the normal function of the proteins. For plants was fused to the coat protein, and that the fusion example, fusion of GFP to the movement protein of TMV proteins were assembling into fibrillar aggregates within produces a protein with approximately twice the molecular infected cells. In transverse sections of the leaf, paramass (57 kDa) of the wild-type movement protein (30 kDa). crystalline aggregates (i.e. regular arrays that appear Nonetheless, the movement protein- GFP successfully targets crystalline in the electron microscope) of fluorescent virus plasmodesmata and supports intercellular movement of the were clearly visible in epidermal, palisade and mesophyll TMV vector 24. When planning movement protein-GFP fusion cells (Fig. 5). Confirmation that the virus carried a GFP 'over- strategies, the potential for successful targeting might be coat' was obtained by probing electron microscope sections improved by careful selection of the fusion site (i.e. C-terminal with polyclonal antibodies to both GFP and the virus coat or N-terminal fusions). Insertion of a linker sequence between protein49. the two fused proteins may also help to limit potentially It remains to be seen how many plant viruses can be deleterious effects of the GFP tag on the function of the target tagged with GFP. However, a strategy in which GFP is protein24. In the case of the TMV movement protein, previous located on the assembled virus, rather than in the cytosol of studies have mapped functional domains in the protein imporinfected cells, potentially extends the utility of GFP in tant for nucleic acid binding and plasmodesmal targeting', plant-virus movement studies. For example, the presence of suggesting that insertion of GFP at the C-terminus of the a fluorescent virus will be of use in systemic transport studies movement protein would be less likely to affect function than a fusion to the N-terminus. Previous case histories of GFP fusions in animal . . . . . . systems suggest that the GFP does not interfere with the function of the proteins to which it is fused26.~'m;however, each strategy must be considered carefully against the background of information available for the protein under study. In the case ofviral movement proteins, a number of questions remain to be addressed. For example, does the movement protein-GFP fusion itself traffick through plasmodesmata in the manner reported for wild-type movement proteins? Does the movement protein-GFP fusion gate plasmodesmata to the same degree as the wild-type movement protein, or does the presence of such a large protein impede the movement of macromolecules through the pore? In connection with the latter Fig. 5. Transverse section of a leaf of Nicotiana benthamiana infected with potato virus X expressing the green fluorescent protein as a coat protein fusion. The question, the microinjection of fluoresfluorescent virus is present in palisade and spongy mesophyll cells. To obtain the cent dextrans will help to discriminate precise localizationof virus, the fluorescentimage taken at 488 nm excitation was between those domains of the protein superimposed on a bright field image of the same cells. Both images were obtained essential for targeting to plasmodeswith a confocallaser scanning microscope. Scale bar represents 50 ~m. mata and those responsible for gating. Using a combination of such powerful 416
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reviews techniques it may soon be possible to dissect the functional domains of viral movement proteins. Conclusions The use of GFP in plant-virus studies is in its infancy, but it shows great promise for tracing the movement of functional plant viruses, both locally and systemically, and for unravelling some of the key events involved in the interactions between viral movement proteins and plasmodesmata. At present only a limited number of viral proteins have been fused to GFP, but initial studies suggest that the targeting functions of viral movement proteins are unaffected by the fusion. This strategy, used critically in conjunction with other cell biology techniques, has much potential in the study of virus-plasmodesma interactions in plants.
Acknowledgements We are grateful to D. Prior for preparation of the figures, to R. Beachy, B. Epel, J. van Lent and J. Wellink for access to unpublished data and to P. Palukaitis for helpful discussions. The authors' work was supported by the Scottish Office Agriculture, Environment and Fisheries Dept and by grants from the Biotechnology and Biological Sciences Research Council and Leverhulme Trust. References 1 Lucas, W.J. and Gilbertson, R.L (1994) Plasmodesmata in relation to viral movement within leaf tissues, Annu. Rev. Phytopathol. 32, 387-411 2 Gilbertson, R.L. and Lucas, W.J. (1996) Hew do viruses traffic on the 'vascular highway'? Trends Plant Sci. 1,260-268 3 Maule, A.J. (1991) Virus movement in infected plants, Crit. Rev. Plant Sci. 9, 457-473 4 Leisner, S.M. and Turgeon, R. (1993) Movement of virus and photoassimilate in the phloem: a comparative analysis, BioEssays 15, 741-747 5 Deom, C.M., Oliver, M.J. and Beachy, R.N. (1987) The 30-kilodalton gene product of tobacco mosaic virus potentiates virus movement, Science 237, 389-394 6 Tomenius, K., Clapham, D. and Meshi, T. (1987) Localization by immunogold cytochemistry of the virus-coded 30K protein in plasmodesmata of leaves infected with tobacco mosaic virus, Virology 160, 363-371 7 Atkins, D. et al. (1991) The tobacco mosaic virus 30K movement protein in transgenic tobacco plants is localised to plasmodesmata, J. Gen. Virol. 72, 209-211 8 Citovsky, V. et al. (1990) The P30 movement protein of tobacco mosaic virus is a single-strand nucleic acid binding protein, Cell 60, 637-647 9 Wolf, S. et al. (1989) Movement protein of tobacco mosaic virus modifies plasmodesmatal size exclusion limit, Science 246, 377-379 10 Dawson, W.O., Bubrick, P. and Grantham, G.L. (1988) Modifications of the tobacco mosaic virus coat protein gene affecting replication, movement and symptomatology, Phytopathology 78, 783-789 11 Wellink, J. et al. (1993) The cowpea mosaic virus M RNA encoded 48-kilodalton protein is responsible for induction of tubular structures in protoplasts, J. Virol. 67, 3660-3664 12 Wieczorek, A. and Sanfa~on, H. (1993) Characterization and subcellular localization of tomato ringspot nepovirus putative movement protein, Virology 194, 734-742 13 Dolja, V.V. et al. (1994) Distinct functions ofcapsid protein in assembly and movement of tobacco etch potyvirus in plants, EMBO J. 13, 1482-1491 14 Scholthof, H.B., Morris, T.J. and Jackson, A.O. (1993) The capsid protein of tomato bushy stunt virus is dispensable for systemic movement and can be replaced for localized expression of foreign genes, Mol. Plant-Microbe Interact. 6, 309-322 15 Taliansky, M.E., Robinson, D.J. and Murant, A.F. (1996) Complete nucleotide sequence and organization of groundnut rosette umbravirus, J. Gen. ViroI. 77, 2335-2345
16 Chalfie, M. et al. (1994) Green fluorescent protein as a marker for gene expression, Science 263,802-805 17 Chapman, S.N., Kavanagh, T. and Banlcombe, D.C. (1992) Potato virus X as a vector for gene expression in plants, Plant J. 2, 549-557 18 Dolja, V.V., McBride, H.J. and Carrington, J.C. (1992) Tagging of plant potyvirus replication and movement by insertion of ~-glucuronidase into the viral polyprotein, Proc. Natl. Acad. Sci. U. S. A. 89, 10208-10212 19 Stearns, T. (1995) The green revolution, Curr. Biol. 5, 262-264 20 Prasher, D.C. (1995) Using GFP to see the light, Trends Genet. 11, 320-323 21 Baulcombe, D.C., Chapman, S.N. and Santa Cruz, S. (1995) Jellyfish green fluorescent protein as a reporter for virus infections, Plant J. 7, 1045-1053 22 Oparka, K.J. et al. (1995) Imaging the green fluorescent protein in plants - viruses carry the torch, Protoplasma 189, 133-141 23 Epel, B.L. et al. Plant virus movement protein dynamics probed with a movement protein fused to the GFP, Gene (in press) 24 Heinlein, M. et al. (1995) Interaction oftobamovirus movement proteins with the plant cytoskeleton, Science 270, 1983-1985 25 Oparka, K.J. et al. Viral coat protein is targeted to, but does not gate, plasmodesmata during cell-to-cell movement of potato virus X, Plant J. (in press) 26 Wang, S. and Hazelrigg, T. (1994) Implications for bcd mRNA localization from spatial distribution of exu protein in Drosophila oogenesis, Nature 369, 400-403 27 Derrick, P.M., Barker, H. and Oparka, K.J. (1992) Increase in plasmodesmatal permeability during cell-to-cell spread of tobacco rattle virus from individually inoculated cells, Plant Cell 4, 1405-1412 28 Angell, S., Davies, C. and Baulcombe, D.C. (1996) Cell-to-cell movement of potato virus X is associated with a change in the size-exclusion limit ofplasmodesmata in trichome cells ofNicotiana cIevelandii, Virology 216, 197-201 29 Fujiwara, T. et al. (1993) Cell-to-cell trafficking of macromolecules through plasmodesmata potentiated by the red clover necrotic mosaic virus movement protein, Plant Cell 5, 1783-1794 30 Waigmann, E. et al. (1994) Direct functional assay for tobacco mosaic virus cell-to-cell movement protein and identification of a domain involved in increasing plasmodesmatal permeability, Proc. Natl. Acad. Sci. U. S. A. 91, 1433-1437 31 Nouiery, A.O., Lucas, W.J. and Gilbertson, R.L. (1994) Two proteins of a plant DNA virus coordinate nuclear and plasmodesmal transport, Cell 76, 925-932 32 Ding, B. et al. (1995) Cucumber mosaic virus 3a protein potentiates cell-to-cell trafficking of CMV RNA in tobacco plants, Virology 207, 345-353 33 McLean, B.G., Zupan, J. and Zambryski, P. (1995) Tobacco mosaic virus movement protein associates with the cytoskeleton in tobacco cells, Plant Cell 7, 2101-2114 34 Topp, K.S., Meade, L.B. and LaVail, J.H. (1994) Microtubule polarity in the peripheral processes of trigeminal ganglion cells: relevance for the retrograde transport of herpes simplex virus, J. Neurosci. 14, 318-325 35 van Lent, J. et al. (1991) Tubular structures involved in movement of cowpea mosaic virus are also formed in infected cowpea protoplasts, J. Gen. Virol. 72, 2615-2623 36 Wellink, J. and van Kammen, A. (1989) Cell-to-cell transport of cowpea mosaic virus requires both the 58K/48K proteins and the capsid proteins, J. Gen. Virol. 70, 2279-2286 37 Murant, A.F. and Roberts, I.M. (1971) Cylindrical inclusions in coriander leaf cells infected with parsnip mosaic virus, J. Gen. Virol. 10, 65-70 38 Lawson, R.H. and Hearon, S.S. (1971) The association of pinwheel inclusions with plasmodesmata, Virology 44, 454-456 39 Davies, C. and Symons, R.H. (1988) Further implications for the evolutionary relationships between tripartite plant viruses based on cucumber mosaic virus RNA 3, Virology 165, 216-224 40 Vaquero, C. et al. (1994) The 3a protein from cucumber mosaic virus increases the gating capacity of plasmodesmata in transgenic tobacco plants, J. Gen. Virol. 75, 3193-3197 41 Kaplan, I.B. et al. (1995) Complementation of virus movement in transgenic tobacco expressing the cucumber mosaic virus 3a gene, Virology 209, 188-199
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reviews 42 Burman, A.J., Osman, T.A.M. and Buck, K.W. (1994) Detection of the 3a protein of cucumber mosaic virus in a cell wall fraction from infected Nicotiana clevelandii plants, J. Phytopathol. 142,317-323 43 Cooper, B. and Dodds, J.A. (1995) Differences in the subcellular localization of tobacco mosaic virus and cucumber mosaic virus movement proteins in infected and transgenic plants, J. Gen. Virol. 76, 3217~3221 44 Esau, K. and Thorsch, J. (1985) Sieve plate pores and plasmodesmata, the communication channels of the symplast: ultrastructural aspects and developmental relations, Am. J. Bot. 72, 1641-1653 45 Kempers, R. et al. (1993) Plasmodesmata between sieve element and companion cell of extrafascicular phloem of Cucurbita maxima permit passage of 3 kDa fluorescent probes, Plant J. 4, 567-575 46 Monzer, J. and Kloth, S. (1991) The preparation of plasmodesmata from plant tissue homogenates: access to the biochemical characterization of plasmodesmata-related polypeptides, Bot. Acta 104, 82-84 47 Kotlizky, G. et al. (1992) An improved procedure for the isolation of plasmodesmata embedded in clean maize cell walls, Plant J. 2,623-630 48 Turner, A., Wells, B. and Roberts, K, (1994) Plasmodesmata of maize root tips - structure and composition, J. Cell Sci. 107, 3351-3361
49 Santa Cruz, S. et al. (1996) Assembly and movement of a plant virus carrying a green fluorescent protein overcoat, Proc. Natl. Acad. Sci. U. S. A. 93, 6286-6290 50 Gibbs, A. and Harrison, B.D. (1976) Plant Virology: the Principles, Edward Arnold 51 Ogawa, H. et al. (1995) Localization, trafficking, and temperature dependence of the Aequorea green fluorescent protein in cultured vertebrate cells, Proc. Natl. Acad. Sci. U. S. A. 92, 11899-11903 52 Stauber, R., Gaitanaris, G.A. and Pavlakis, G.N. (1995) Analysis of trafficking of Rev and transdominant Rev proteins in living cells using green fluorescent protein fusions: transdominant Rev blocks the export of Rev from the nucleus to the cytoplasm, Virology 213,439-449
Karl Oparka, Petra Boevink and Simon Santa Cruz are at the Unit of Plant Transport Processes, Scottish Crop Research Institute, Invergowrie, Dundee, UK DD2 5DA. Fax (K.O.): +44 1382 562 426 e-mail: [email protected]
Phloem loading and plasmodesmata Robert Turgeon Phloem loading, the active accumulation of photosynthate in minor veins, is thought to be the motivating force for translocation. Can loading take place along an entirely symplastic route, through the plasmodesmata, from mesophyll to phloem? Transport of small molecules through the plasmodesmata is apparently passive, and the concept of symplastic phloem loading thus appears to violate thermodynamic principles. Nonetheless, evidence for such a pathway in many plants has been accumulating steadily and an answer to the thermodynamic argument has been put forward. The proposed mechanism involves synthesis and trapping of raffinose and stachyose in the phloem.
p
hloem loading creates very high turgor pressure in the sieve element-companion cell complex (SE-CCC), and has generally been viewed as an essential component of Mfinch pressure flow, which is driven by the difference in pressure between source and sink 1. This concept, that the sugar concentration in the phloem is higher than in the mesophyll, and thus requires a loading step, arose in the 1930s and 1940s. The term 'phloem loading' was coined by Loomis, in 1955, who described the phenomenon as an 'endothermic pumping action '2. Since that time, physiologists have been trying to determine the physical location of the pump and its molecular nature. This is important, not only for the study of transport, but because the mechanisms that drive loading create a special structural and biochemical boundary between the phloem and the surrounding cells, defining the phloem as a specific symplastic domain. Because this is an integrating domain that permeates the entire plant, loading is often invoked as an important physiological control point (e.g. in studies of mineral nutrition ~, responses to elevated carbon dioxide 4, ozone 5and sulfur dioxide6, and resistance to virus movementT). As Geiger et al. 8 first demonstrated, the solute content of the SE-CCC in the minor veins of leaves is far higher than that of the surrounding cells and the mesophyll. It is therefore this boundary between the SE-CCC and the surrounding cells that is especially important in phloem loading. Here, the following questions are addressed: 4-1 8
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• Can transport of photosynthate into the SE-CCC take place through plasmodesmata? In other words, is it possible for sugar to stay within the symplast all the way from the mesophyll to the sink? • If so, what is the mechanism of active accumulation? Plasmodesmal frequency
Early studies on phloem loading focused on the apoplastic route 1. According to the most widely accepted version of this model, sucrose diffuses between mesophyll cells through the plasmodesmata, but somewhere in the vicinity of the minor veins it enters the apoplast. It has long been thought that the site of effiux is within the vein, and is possibly the vascular parenchyma. This concept is now supported by structural evidence that, in an export-deficient maize mutant, plasmodesmata at the bundle sheathphloem parenchyma interface are aberrant and probably nonfunctional 9. From the apoplast, the sucrose is loaded into the SE-CCC, against a steep concentration gradient, by symport with protons. In this model, the plasmodesmata do not play a direct role in the loading step. In fact, the presence of plasmodesmata raises a difficult question. Why is it that sucrose, at such a high concentration, does not diffuse out of the SE-CCC and back into the mesophyll through these pores? The evolutionary solution to this problem in many species appears to have been a simple reduction in plasmodesmal frequency (Fig. 1). In plants for which evidence of
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