Viral Movement Proteins as Probes for Intracellular and Intercellular Trafficking in Plants

Abstract

The ability of viruses to cross the cellulosic cell wall to propagate infection throughout a plant has been a long-standing puzzle in plant cell biology and virology. Contemplated from the perspective of the topology of the plant cell and the plant as an integrated structure of the differentiated tissue types, and given the existence of apoplastic and symplastic domains, the solution was not apparent. Plasmodesmata were the obvious conduits for moving between cells—or were they? Conventional wisdom held that plasmodesmata were too small for the transport of nucleic acids, no less virus particles. Consideration of this problem was advanced once two major biases were discarded: (1) the static view of plasmodesmata as simply being boring holes in the plant cell wall (Robards and Lucas, 1990; Beebe and Turgeon, 1991; Lucas et al., 1993); and (2) the assumption, despite the fact that moving cell to cell across a wall is a fundamentally unique aspect of plant virus infection that, as shown for animal viruses, encapsidated virus particles would move from cell to cell within the plant. Our first insights into this process came from studies on the movement protein (MP) encoded by the RNA-containing tobacco mosaic virus (TMV; the 30-kD protein) and red clover necrotic mosaic virus (RCNMV; Deom et al., 1987; Wolf et al., 1989; Citovsky et al., 1990; Ding et al., 1992a; Fujiwara et al., 1993; Giesman-Cookmeyer and Lommel, 1993; Waigmann et al., 1994). These MPs could increase the size exclusion limits (SEL) of plasmodesmata when expressed in transgenic plants or introduced by microinjection into leaf mesophyll cells, and it appeared that cell-to-cell spread of infection could occur in the absence of virus particles. Based on these findings and additional data, it was suggested that complexes of viral RNA and MP move from cell to cell. As described in a number of excellent recent reviews (Hull, 1991; Lucas and Gilbertson, 1994; Maule, 1994; Carrington et al., 1996; Lartey and Citovsky, 1997; McLean et al., 1997), these findings focused much attention and a flurry of activity on the ability of different viral-encoded MPs to affect plasmodesmal gating, the potential existence of plasmodesmal targeting sequences within these MPs, and the use of MPs as tools for identifying components of plasmodesmata. The continued investigation of the function of MPs, encoded by a number of very different viruses, from the perspective of what the virus must accomplish—namely, to coordinate replication of the virus genome and the vectorial movement of the progeny genomes from the infected cell into adjacent cells—has expanded this focus to include using MPs to unravel the fundamental mechanisms by which macromolecular transport is directed and integrated within and between plant cells. MPs not only use plasmodesmata but they can modify and at times can even intercept these intercellular channels, depending on whether they function in fully differentiated or developing cells (Thomas and Maule, 1995; Kasteel et al., 1996; Ward et al., 1997). It is not merely coincidental that quite specific and distinct interactions occur between the cortical endoplasmic reticulum (ER) and the MPs encoded by TMV or the DNA-containing bipartite geminivirus squash leaf curl virus (SqLCV) (Sanderfoot and Lazarowitz, 1995; Ward et al., 1997; Heinlein et al., 1998; Mas and Beachy, 1998; Reichel and Beachy, 1998). These distinct interactions provide new approaches for identifying and investigating the functional importance of subdomains within the ER (Reuzeau et al., 1997b; Staehelin, 1997). The discovery that there are specific associations of the TMV 30-kD protein with microtubules and actin filaments complements studies on the role of the cytoskeleton in directing transcytoplasmic movement and regulating plasmodesmal function (Heinlein et al., 1995; McLean et al., 1995). Finally, studies of the MPs encoded by SqLCV, which replicates in the nucleus, provide the additional dimension of MP function in transport and a unique to the of and the of in plant cells (Sanderfoot and Lazarowitz, 1996; et al., 1996; Ward and Lazarowitz, the unique potential of viral MPs for are within cells in a directed and integrated and for the in these a plant viruses must move from cell to cell and the which they move to a infection and process that plant viruses the by the plant cell a by was first from plant virus a in MP function can and progeny virus particles. these progeny not move of the leaf to infection and and 1990; the of MPs the cell wall to plant virus which that MPs must function to fundamentally cell and this The of the that MPs would cell structure to the cell wall to virus has been by studies on the cell-to-cell movement of a number of plant These studies that viral MPs and in the to the viral genome the cell wall and Gilbertson, 1994; Carrington et al., 1996; McLean et al., 1997). that distinct viruses this problem in not depending on the cell the of the cell wall is by interactions of MPs and viral components with the and These specific interactions are to the viral genome and move it in a directed to the cell wall for TMV and the of RNA viruses that mesophyll cells, a and not protein for cell-to-cell is for infection and Lommel, 1995; Ding et al., The MP encoded by virus can RNA in et al., Giesman-Cookmeyer and Lommel, and increase the of plasmodesmata in mesophyll cells. was shown by studies in transgenic plants the MP et al., 1987; Wolf et al., and by microinjection of the MP with into tobacco leaf mesophyll cells et al., 1993; Waigmann et al., 1994). The TMV MP has been to plasmodesmata in studies of transgenic plants et al., 1991; Ding et al., 1992a; et al., in plants infected with a TMV virus or virus that the 30-kD protein as a to the protein the protein is to in the cell wall that to plasmodesmata when by and et al., et al., the plasmodesmata not to in by the 30-kD These studies to a shown in the TMV MP as a to the viral genome and it to the MP the and movement of the viral genome into adjacent cells and Gilbertson, 1994; McLean et al., 1997). studies of MPs encoded by mosaic virus and MP encoded by the or encoded by the geminivirus mosaic virus using approaches of in nucleic microinjection of or nucleic with the MP or into transgenic plants that expressed the MP et al., 1994; Ding et al., 1995; and to the that MPs would in a and Gilbertson, 1994). The of MPs expressed in and from into leaf mesophyll cells has been for of these viral-encoded and a view of plasmodesmata. of MP and the of virus in to the of MP function and the integrated interactions of MPs with viral and components in different cell to a are from the investigation of MP function in the of virus infection using a of for and MPs in cells and plants (Heinlein et al., 1995; and Lazarowitz, 1995; et al., 1997b; et al., 1997; Ward et al., 1997; et al., 1998; Reichel and Beachy, 1998; et al., 1998; Ward and Lazarowitz, of plants infected with viruses that or for or and studies that in to the is for cell-to-cell movement of the viral genome and the role of MP in viral into the in et al., 1997; et al., 1998). using the TMV protein that on plasmodesmal are to the of infected cells et al., 1997; et al., for MP with viral RNA is to move microtubules from of viral replication and protein to additional viral at ER viral with cortical actin complexes to cell wall and plasmodesmata. is a protein that viral genomes from the to the with the complexes in the and is to these the and the cell wall into adjacent cells. these adjacent cells, complexes would and the viral to the to a new of replication and and These viruses in to MP for cell-to-cell The viral encapsidated in virus that to from plasmodesmata. endoplasmic movement viral RNA viral for MP with viral RNA is to move microtubules from of viral replication and protein to additional viral at ER viral with cortical actin complexes to cell wall and plasmodesmata. is a protein that viral genomes from the to the with the complexes in the and is to these the and the cell wall into adjacent cells. these adjacent cells, complexes would and the viral to the to a new of replication and and These viruses in to MP for cell-to-cell The viral encapsidated in virus that to from plasmodesmata. endoplasmic movement viral RNA viral RNA viruses from a of that mesophyll cells a MP and for cell-to-cell It that an encapsidated of the viral RNA genome or virus from cell to and this movement is by the of that from the cell wall to adjacent cells Kasteel et al., 1996; and 1993; et al., 1994; et al., 1995). of from infected plants the MP encoded by a number of viruses, virus mosaic virus mosaic virus and and viruses to these to particles et al., Kasteel et al., 1993; and 1993; et al., 1994; et al., 1995). The from the of infected mesophyll cells at or what to plasmodesmata that a in the and are as a has components encapsidated in and 1998). number of viruses, as and for cell-to-cell movement but not move there is no between and the transport of virus particles. is the major as has been suggested in studies of et al., to It is to that as a RNA virus would to transport protein with viral from cell to cell to replication cells. are to these using cells that the MP of and et al., 1993; et al., 1993; et al., 1995; Kasteel et al., to and to the of these and that plasmodesmata or unique plant cell are for is for the of the particles within the et al., 1997). in which plants are with RNA components of that for MP that as as MP are for the cell-to-cell spread of the virus infection et al., 1998). Based on of these it is that virus particles of and these viruses with move cell to cell and plasmodesmata. to components that are with the for these viruses to with or cell components et al., 1997). with studies on the MPs encoded by TMV and (Heinlein et al., 1998; McLean et al., 1995; Ward et al., 1997; Reichel and Beachy, 1998; of viruses, the and a of that are for cell-to-cell and movement et al., 1990; et al., 1991; et al., of these viruses, as for cell-to-cell as as The of the been in for and of plants with or viruses that can or a a for the that in to the is for cell-to-cell movement et al., 1995; et al., 1998; et al., 1997; et al., 1998). studies in infected plants and and of plants with a that to studies in transgenic plants that that not affect plasmodesmal et al., et al., 1998). viruses that a studies in with or a in the the protein that the protein plasmodesmal of was in the absence of the protein et al., studies that to the protein is in viral in the et al., using a within the plasmodesmata of cell from the to the and infection protein can to between cells et al., 1998). encapsidated particles move cell to cell what to but in the absence of The in with the function in movement has to a role in to this movement being to plasmodesmata et al., in the on with the are in plasmodesmata between cells and or cells but not in plasmodesmata that cells and the is for transport of the virus et al., 1998). It was suggested that in a or that the of these plasmodesmata is that a of virus particles is not at these et al., 1998). to this studies on the MP and of which for cell-to-cell to the that as a of and viral RNA and that viral in the et al., 1998). is for the transport of plant viruses, and it of to for that are not of virus particles in the to a The bipartite two MPs, but not for cell-to-cell or These viruses in the nucleus, and it is this aspect that two MPs and or with distinct to move the viral genome (Sanderfoot and Lazarowitz, the viral genome and it between the and with in transcytoplasmic movement and to move the viral genome across the cell wall and into adjacent cells The MPs encoded by the been in the of and has been in in tobacco as has the of as a protein and Lazarowitz, 1995; et al., 1996; Ward and Lazarowitz, has been to of and cells in infected of and and to the when expressed in tobacco et al., 1994; and Lazarowitz, 1995; and Lazarowitz, with role in moving the viral has the of a protein in et al., 1994). studies in which the MPs encoded by were into tobacco leaf mesophyll cells with nucleic the that cell to cell et al., 1998). of into tobacco leaf mesophyll cells that it to the TMV 30-kD protein and MPs in plasmodesmal and moving cell to cell et al., 1994). As with of in the of virus infection a not is to unique that from and cross the of developing cells in and and these not plasmodesmata they cross the cell wall et al., 1997). The of these studies that to the viral genome between the and and that complexes in the and these to and across the cell and into adjacent cells, the complexes would and the viral genome to the to new of replication and infection (Sanderfoot and Lazarowitz, The cytoskeleton is a and actin with a of within and cells, from the of to the directed of cell and cortical microtubules can to and 1994; Staehelin, 1997; et al., 1998; et al., 1998). of the cell and of the cell cell wall in cells, of ER and the of are only of the shown to microtubules and actin and 1996; Staehelin, 1997; et al., 1998; and 1998). The cytoskeleton is in the genomes of animal viruses that in the and to the et al., et al., 1994; et al., 1997; et al., 1998). it that specific associations between viral MPs and the cytoskeleton in the transport of viral of TMV MP with in between and the TMV MP can to with microtubules in infected tobacco from the cell for of TMV MP with in between and the TMV MP can to with microtubules in infected tobacco from the cell for of the cytoskeleton in directing as as virus and viral to plasmodesmata was first described for As shown in between and the TMV MP with microtubules in infected tobacco from the cell is by and of the with and two to microtubules (Heinlein et al., 1995; McLean et al., 1995). was to that MP with in et al., 1995). It been shown that plasmodesmata actin et al., and studies in the of that microtubules suggested that actin filaments in plasmodesmal Ding et al., Based on these it was to that microtubules and actin complexes of MP with viral RNA to and plasmodesmata (Heinlein et al., 1995; Carrington et al., plant cells, cells, microtubules are in the cortical at the cell and in with ER and et al., 1990; et al., TMV MP and the protein are with cortical and as by and (Heinlein et al., 1998; Mas and Beachy, 1998). the of MP with microtubules was the to of infection and to with of the ER Heinlein et al., 1998; Mas and Beachy, 1998). was first in or adjacent to plasmodesmata and in with that with microtubules was not to the targeting of MP to plasmodesmata et al., with is to the that with microtubules a to of MP of infection et al., 1996; Heinlein et al., 1998). The cortical ER in plants is in a number of in cell and as suggested by specific of the ER and for between the ER and the and of the plant cell and wall and et al., 1990; et al., Staehelin, 1997; et al., 1998; and in this of animal and plant cells is that the ER is not a reticulum but subdomains (Reuzeau et al., 1997b; Staehelin, 1997). to the role of the ER in virus movement between cells is the between cortical ER and cortical actin and the that the structure of the plasmodesmata et al., Ding et al., The is with the and there are between the ER and the actin cytoskeleton in plant cells 1997; et al., 1998). this with the that the ER can plant cell and and with plant cell and the ER is too a for virus to The TMV 30-kD protein and with the cortical ER in a very specific but distinct et al., 1997; Heinlein et al., 1998; Mas and Beachy, 1998; Reichel and Beachy, 1998). The distinct ER associations for MP would to the different cell infected by It has been that the an role in RNA virus studies of the replication of RNA-containing plant as as animal viruses an of virus replication with cell and 1990; 1996; 1997). RNA virus for TMV and been from infected or cells in with et al., 1987; and 1996; and and in studies that replication in with ER and and recent studies on tobacco virus provide for the targeting of the replication of this to ER et al., 1997). It is to that the of replication complexes and and in the a role in viral movement within and between plant cells. this it is to the of viral MPs with and transgenic plants that the TMV 30-kD the MP is in that and ER et al., recent the of the protein was by of infected and The was to at a number of in the cell. These the cortical and ER (Heinlein et al., 1998; Reichel and Beachy, as as plasmodesmata et al., 1996; et al., to at or the of infected cells, which were to the ER is to the (Heinlein et al., 1998; Reichel and Beachy, 1998). MP is in of ER that of with different of to the infection (Heinlein et al., 1998). The of the of the TMV MP with the ER is not fully but it has been that this is with of or not MP from ER and Beachy, 1998). the ER with of the and the MP is to with the 1994). of from infected that the of the MP is to and Beachy, 1998). of these to the that MP is with the with the of the protein being on the of the unique to the of TMV infection with ER has the of transgenic tobacco plants in which is to the of the of the ER by et al., of these plants by TMV of the ER in the to of this of ER are and Beachy, 1998). the is at or the cell adjacent to plasmodesmata. The with to the and that MP as as et al., in the of the and the ER to the of a cortical reticulum and Beachy, 1998). there is of of MP with ER throughout the on ER within or plasmodesmata. the TMV MP to and the plasmodesmata the has to fully the of to that this the It is that MP is within on cortical and at the and in that are to the and plasmodesmata. It is not that of MP with ER is to the cell-to-cell spread of on these it is to that the spread of TMV from cell to as a of of the replication or complexes MP and viral to ER and transport of the complexes to adjacent cells. infection is to the in and squash plants et al., 1994; Ward et al., 1997; and Lazarowitz, studies to unique that from and cross the of developing cells in infected of of from and squash infected with a that that the virus is moving in The the cortical ER it is to the and these with the ER protein et al., 1997). These findings to the that these are from the to for plant viruses, no particles are within or with the with not being for infection of and squash et al., 1995). of with the ER has been in cell studies and in tobacco and the is to with ER and from infected plants et al., 1997). when expressed from a in cells or as a to the in tobacco as shown in is to the cortical et al., 1994; and Lazarowitz, 1995). to the cortical The to in an with cortical as shown in of with and is to ER throughout the of the that with but only in the cortical of with the cortical ER is distinct from for the TMV MP with ER as from the TMV 30-kD protein is when at and actin and not with or in it with microtubules or actin filaments from infected plants or Ward and Lazarowitz, the the functional of the the use of in cells, are to this the of this geminivirus moving in developing cells, the infected plant and studies that of being in cells to intercept the by which plasmodesmata are with the cortical ER to in the wall that are virus specific by of are to the this would a in the topology of associations in the has the of a protein and Lazarowitz, and an an of is and the and studies two of as for ER (Sanderfoot and Lazarowitz, 1996; Ward and Lazarowitz, the that with the of or this that complexes would with adjacent the with the wall by a by in these to the cross would an could this MPs, and are et al., and studies are with in the of or regulating (Sanderfoot and Lazarowitz, of and in and were expressed in tobacco as to the were at different times and and were by and was with a or and was with a expressed to the to and in the cell at to the cortical the in which it is when expressed and Lazarowitz, 1995). is from the to the cortical when with and that and in the cortical and in the cell at of the cell for and no with but is in the of the cell the of and an protein in which within the were to were only to using in the when with for of of and in and were expressed in tobacco as to the were at different times and and were by and was with a or and was with a expressed to the to and in the cell at to the cortical the in which it is when expressed and Lazarowitz, 1995). is from the to the cortical when with and that and in the cortical and in the cell at of the cell for and no with but is in the of the cell the of and an protein in which within the were to were only to using in the when with for of fundamental aspect of cell and is the of transport complexes 1995). can for of small and nucleic as are across these by an process that is and and the of specific sequences on the being and 1996; and 1997; 1997). a of on the that with the or and additional to and 1997; 1997; et al., 1998). These which to at and transport across the been and in animal cells and and are being in plant cells and 1995; and 1997). Our of has that of in to the of at that in and of the to the of that of 1995). this has been by using of animal cells or by into the of cells or and for in a et al., 1995; et al., 1995; et al., been in a number of from and in the protein encoded by the virus in the from and and in protein protein et al., 1995; et al., 1995; et al., these are sequences of to in which the are for function et al., 1995; et al., 1995; et al., to not to structure they can the ability to from the when to the or of a has not been in plant cells. the of replication for the with genomes an MP in the of a protein to move the viral genomes from and to the The of and has been to a for investigating in plant cells (Sanderfoot and Lazarowitz, 1996; et al., 1996; Ward and Lazarowitz, to the were for and an ER and in this by and by using a using an of and that to with (ER) in the cortical to the were for and an ER and in this by and by using a using an of and that to with (ER) in the cortical was first shown to in in cells and tobacco (Sanderfoot and Lazarowitz, 1995; et al., expressed as a to the to in tobacco with from the to the cortical with and the in the and directing it to the cell and Lazarowitz, 1995; et al., The was to when and were in cells. between the and to movement a specific and to the cortical and the two MPs to move the viral genome et al., 1994; and Lazarowitz, 1996; et al., 1998). is at times of no with but is in the that the (Sanderfoot and Lazarowitz, of the to plant cell specific it not occur in cells (Sanderfoot and Lazarowitz, 1996; et al., in and been in and when into the These studies and the of to the of functional for and (Sanderfoot and Lazarowitz, was shown to in in the of the a bipartite between and and a between and of which are for function (Sanderfoot et al., studies that the of a for to with (Sanderfoot et al., this within the of has been by of to and by with and that these are to the cortical when with in tobacco (Sanderfoot et al., 1996; Ward and Lazarowitz, this has been to an within of the that the within and and Lazarowitz, of of to was and protein was for with when the was expressed in the or of tobacco The was that with an from the would from the to the cortical by only they an and Lazarowitz, These studies a of the in as being for of from the the which the in of the in this and the of from the to the cortical of tobacco in the of Ward and Lazarowitz, the from the was shown to fully with and and Lazarowitz, of is for function in and can to the of that a The that can between the and of cells, and that an that the could from in tobacco that the is and the not fully viral the of and a in for investigating in plant and animal cells. The of viruses and components are tools for investigating fundamental and in the they been in of and animal The of has an on plant to the focus on using plant viruses as a to plant cell structure and role in a process that is unique to plant viruses, the infection of a when with the of the cell MPs in are the of fundamental of and intercellular of of which unique to plant cells. with studies in leaf mesophyll cells has been a to of the of that in cells and the potential of viral-encoded MPs as for plant cell structure and function the of that to MP function and in cells in the of the virus the to unravel of the of viral genome replication and movement are It is these studies that directed and to attention to the and and in the of viruses, to and and number of the interactions between viral and cell components been and the of and cell to these are and the mechanisms in and The continued of new in and use of the to in cells, and using with and to these and in of the process of virus movement and the for plant cell there plasmodesmal targeting sequences in viral Based on to and the of there was a that plasmodesmal targeting sequences would they they to as it that viral replication complexes and MPs specific associations with subdomains of the cortical ER cortical microtubules and actin which are with plasmodesmata and to components these intercellular this the of and and identifying ER subdomains and interactions The of and interactions between viral replication or movement complexes and the ER provide new insights into the existence and function of ER subdomains within the cell. and targeting sequences in the and investigation of function and interactions with components and a for investigating the of in plant cells, and the of in the plant cell viruses in mesophyll cells the from plasmodesmata to the for cell-to-cell movement of viral the the of the these simply the two of the with the movement of viruses as and in these viral to the on the in which the virus It is a of cell to of wisdom for the and for the and for a as and as The of from which a for at the of and of the of and for of and the Heinlein for and Ward for in has been by and