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Review TRENDS in Plant Science 33 Vol.9 No.1 January 2004 Getting the message across: how do plant cells exchange macromolecular complexes? Karl J. Oparka Cell-to-Cell Communication Programme, Scottish Crop Research Institute, Invergowrie, Dundee, UK DD2 5DA A major pathway for macromolecular exchange in plants involves plasmodesmata (PD), the small pores that connect adjoining cells. This article considers the nature of macromolecular complexes (MCs) that pass through PD and the pathways and mechanisms that guide them to the PD pore. Recent cell-biological studies have identified proteins involved in the directional trafficking of MCs to PD, and yeast two-hybrid studies have isolated novel host proteins that interact with viral movement proteins. Collectively, these studies are yielding important clues in the search for components that compose the plant intercellular MC trafficking pathway. Here, they are placed in the context of a functional model that links the cytoskeleton, chaperones and secretory pathway in the intercellular trafficking of MCs. Plasmodesmata (PD) form a major pathway for information exchange in plants, and have been the focus of several recent studies. PD have a unique architecture that permits the passage of both small solutes and certain macromolecules that, at first glance, seem too large to pass through the PD pore. For recent reviews describing the role of PD in short-and long-distance transport in plants, see Refs [1 – 4]. Non-cell-autonomous proteins Proteins that can move between plant cells have been termed non-cell autonomous proteins (NCAPs) [5]. Some proteins, such as green fluorescent protein (GFP), can pass through PD by non-selective movement (i.e. without requiring a specific interaction with components of the PD pore) [2]. Recently, evidence has begun to accumulate that some plant transcription factors might also pass between meristematic cells by simple diffusion [6], suggesting that, for some proteins, cell-to-cell movement might occur by default unless the protein is retained at a specific subcellular location [6]. However, of the many NCAPs identified to date, many appear to show selective transport through PD and also increase the size exclusion limit of the PD pore (Table 1). It is difficult to identify common motifs in these proteins that interact specifically with PD and at least some NCAPs probably interact with other cellular proteins to mediate their intercellular passage. In a recent study [5], the NCAP CmPP16 (a protein first detected in Cucurbita phloem sap [7]) was used as bait for the affinity purification of interacting proteins present within a PD-enriched cell wall fraction. A 40 kDa protein termed NCAPP1 (non-cell-autonomous pathway protein 1) was detected that was immunolocalized to the cortical Table 1. Non-cell-autonomous proteins and modifiers of plasmodesmata Protein Plasmodesmata modification Function Refs Virus Several viral MPs and CPs Groundnut rosette virus (ORF3) SEL regulation – RNA trafficking Long-distance RNA trafficking [2,4,10,11,35,36,49,56] [61] Plant Assorted transcription factors CmPP16 CmPP36 – SEL regulation SEL regulation [62,63] [7] [48] CmNACP SEL regulation HSP70 (from phloem exudate) PP1, PP2 (from phloem) SUT1 Thioredoxin h Assorted phloem proteins ISE 1 SEL regulation SEL regulation – SEL regulation SEL regulation SEL regulation – Long-distance RNA trafficking Phloem movement (when proteolytically cleaved) Long-distance developmental signaling, shoot meristem function Protein trafficking RNA interaction Protein trafficking (CC-SE only) – Protein trafficking – [63] [9] [64,65] [66] [67] [65] [68] Abbreviations: CC-SE, companion cell-sieve element; CP, coat protein; ISE, increased size exclusion; MP, movement protein; NCAP, non-cell-autonomous protein; ORF, open reading frame; SEL, size exclusion limit, ‘ –‘ indicates unknown or unclear. Corresponding author: Karl J. Oparka ([email protected]). http://plants.trends.com 1360-1385/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2003.11.001 34 Review TRENDS in Plant Science Vol.9 No.1 January 2004 endoplasmic reticulum. N-terminal deletion to the transmembrane domain of this protein produced a dominantnegative mutant that blocked the trafficking of specific NCAPs such as CmPP16 and the movement protein (MP) of tobacco mosaic virus (TMV). Transgenic tobacco plants expressing the mutant form of NCAPP1, or in which the gene encoding NCAPP1 was silenced, were compromised in their ability to regulate leaf and flower development, consistent with NCAPP1 having a role in the selective transport of key developmental proteins. NCAPP1 is probably one of several proteins used on the NCAP pathway, possibly shuttling NCAPs to the PD pore [5]. Other putative NCAP pathway proteins remain to be identified, although some of these are likely to be resident within the PD pore itself, playing a role in trafficking NCAPs through, rather than to, PD. New insights from protein –protein interactions Several recent studies have examined the interaction between viral MPs and plant proteins in an attempt to identify host factors involved in the trafficking of macromolecular complexes (MCs) to PD (Table 2). With few exceptions, plant proteins that interact with MPs can be grouped into distinct categories. Chaperones Several viral MPs have now been shown to interact with DNAJ-like chaperones, a group of small proteins belonging to the HSP40 subclass (Table 2). DNAJ proteins have a range of functions including protein import into organelles and in regulating HSP70 chaperone activity [8]. Collectively, HSP-like chaperones might play a role in the partial unfolding of proteins before their translocation through the PD pore [1]. Recently, HSP70 has emerged as a possible chaperone for trafficking endogenous MCs to PD [9]. HSP cognate 70 (HSPc70) chaperones isolated from PD-rich wall fractions and from Cucurbita phloem exudates were found to interact with PD and to modify the PD size exclusion limit. Interestingly, a cytosolic, non-phloem, HSP70 protein did not possess the necessary motif for modifying the size exclusion limit, indicating that the HSP70 identified in phloem might have a crucial role in mediating the non-cell-autonomous movement of MCs into or out of the phloem system. In a gain-of-movement function assay, the same motif, when transferred to a human HSP70 protein, allowed the human homologue to modify the PD size exclusion limit. These data provide compelling evidence for a role of HSP-like chaperones in trafficking MCs to and through PD. Interestingly, beet yellows virus (BYV) encodes in its genome a HSP70 homologue that associates with PD, and is essential for viral cell – cell movement [10]. However, the viral HSP70 does not contain a plant-like PD-interacting domain [9], suggesting that this virus might use a different pathway to interact with and traffic through PD. This example illustrates an interesting case Table 2. Plant proteins that interact with viral movement proteins Virus Host protein Putative function Refs TSWV TSWV PMTV (TGB 2) Chaperones DNAJ-like DNAJ-like DNAJ-like Chaperone (regulator of HSP70) Chaperone Chaperone [69] [18] A. Ziegler and L. Torrance (unpublished) ToMV ToMV TBSV GRV (ORF 3) Nucleus KELP MBF1 HFi22 Fibrillarin Transcriptional co-activator of pathogenesis-related protein Transcriptional co-activator Homeodomain protein (leucine zipper) Unknown TBSV (19K) REF Transcriptional co-activator, RNA export factor [14] [70] [13] M. Taliansky (unpublished) J. Uhrig et al. (unpublished) TMV TMV TMV TMV TSWV GFLV GRV (ORF 3) CaMV TMV TVCV CaMV TCV PVX (triple-gene-block-interacting proteins) Cytoskeleton Actin Tubulin Tubulin MPB2C At4/1 Vesicle trafficking KNOLLE Unknown MP 17 PD and cell periphery PME PME PME AtP8 TIPs vRNA movement vRNA movement vRNA degradation Negative regulator of MP function Limited homology to myosin or kinesin [71] [72] [17] [91] [18] t-SNARE (syntaxin) RNA and vesicle trafficking (N-terminal Rab sequence) Rab receptor (homology to rat PRA1) [32] M.Taliansky (unpublished) [73] MP –PD interaction (pectin esterification) MP –PD interaction MP –PD interaction Unknown (contains RGD motifs) Interact with b-1,3-glucanase [33,34] [33] [33] [74] [37] Abbreviations: CaMV, cauliflower mosaic virus; GFLV, grapevine fan leaf virus; GRV, groundnut rosette virus; MP, movement protein; PD, plasmodesmata; PMTV, potato mop top virus; PVX, potato virus X; TBSV, tomato bushy stunt virus; TCV, turnip crinkle virus; TMV, tobacco mosaic virus; ToMV, tomato mosaic virus; TSWV, tomato spotted wilt virus; TVCV, turnip vein clearing virus; vRNA, viral RNA; 19K, 19 kDA. http://plants.trends.com Review TRENDS in Plant Science of molecular mimicry, in which a host protein appears to have been usurped by a plant virus to fulfil its requirement for cell – cell transport. However, to date, few viruses have been shown to encode a direct homologue of a plant chaperone and it is likely that some viral groups have instead developed the ability to ‘recruit’ essential host chaperones during the infection process. Nuclear proteins Protein – protein interaction studies have revealed that some viral MPs interact with nuclear proteins to achieve successful cell-to-cell movement. In the case of DNA-based geminiviruses, the virus must enter the nucleus for replication [11] and recent studies have identified nuclear components that interact with geminivirus MPs [12]. However, the MPs of some RNA-based viruses also interact with nuclear components (Table 2), particularly transcription factors, suggesting that one mechanism by which viral MPs might achieve selective transport through PD is to commandeer host proteins that themselves have the capacity to move through PD. For example, the MP (P22) of tobacco bushy stunt virus (TBSV) interacts with a homeodomain leucine-zipper transcription factor, HFi22 [13]. The binding of HFi22 to P22 might enable directional transport of P22– RNA complexes through PD [13]. However, the binding of P22 to HFi22 might also prevent the host transcription factor from activating expression of one or more defence genes, a view that agrees with a recent suggestion that some viral MPs interact with transcriptional co-activator proteins to influence host gene expression [13,14]. Collectively, these data raise the possibility that at least some viral MPs must interact with nuclear proteins to mediate cell-to-cell virus movement. A challenge for the future will be to distinguish between those viral proteins that enter the nucleus to influence host defence genes from those that do so to interact with a nuclear MC destined for export. 35 Vol.9 No.1 January 2004 Cytoskeletal proteins The plant cytoskeleton provides an attractive route by which MCs might reach PD [15,16] and several viral MPs have been shown to interact with elements of the cytoskeleton (Table 2). However, such an association might not indicate the direction of viral MP trafficking, nor imply that the cytoskeleton directs MCs specifically to PD. Indeed, in the case of TMV, recent studies suggest that microtubules might transport MPs as part of a targeted degradation mechanism [17]. Protein – protein interaction assays have revealed a direct association of some viral MPs with myosin- or kinesin-like proteins [18], suggesting that actin –myosin-based motility might be required for trafficking some MCs to PD. In the future, it will be essential to determine the direction of MC trafficking along cytoskeletal components. In this respect, fusions of viral MPs to recently developed photo-activatable fluorescent proteins [19,20] might provide a useful approach to study MC trafficking along the cytoskeleton, as well as determining the kinetics of transport. Vesicle trafficking proteins Rabs In all eukaryotes, key regulatory proteins play a central role in determining vesicle transport specificity [21]. These include the Rab GTPases, which determine key membrane fusion events between donor and acceptor membranes [22,23]. In the case of plant viruses, one means of achieving selective transport to PD would be to ‘grab a Rab’ that is travelling to the correct subcellular location. This could occur in either of two ways: by binding directly to a Rab that traffics to PD as part of its internal cycling mechanism, or by becoming attached to a vesicular cargo that is being delivered to PD by an appropriate Rab. The large number of unique proteins in and around PD (Table 3) suggests that many of these are targeted to PD by vesiclemediated pathways. By attaching to a cargo that is being trafficked to PD, a viral MC could be delivered to the Table 3. Macromolecules found in and around plasmodesmata Macromolecule Function Comments Refs Callose Pectin Pectin methylesterase Actin Myosin Myosin VIII Centrin Calreticulin Ca-dependent protein kinase Ca-independent protein kinase 41 kDa protein 45 kDa protein PRms RTM proteins Ubiquitin PD 08 PD 09 PD 10 PD 11 Wound sealing SEL regulation Wall architecture Pectin de-esterification MP interaction SEL regulation SEL regulation SEL regulation SEL regulation SEL regulation Protein phosphorylation signaling Protein phosphorylation signaling Unknown Unknown Unknown Restrict phloem entry of tobacco etch virus Turnover of PD proteins Unknown Unknown Unknown Unknown – Enriched around PD Putative PD receptor Cytoskeletal PD component Might link desmotubule to plasma membrane Unconventional plant myosin enriched at PD Ca-binding cytoskeletal protein Ca-sequestering protein Possible involvement in MP phosphorylation MP phosphorylation Isolated from PD-enriched wall fractions Isolated from nodal walls of Chara Pathogenesis-related protein RTM1 localized to punctate spots (possibly PD) in SEs Localized in suspension cultures Unknown (contains WD-40 repeat) Homology to monodehydroascorbate reductase Homology to Rab11 Unknown protein (TIN15.3; At) [39,75,76] [77] [33,34,78] [51,79–81] [75,81,82] [53,54] [83] [84] [85] [86] [87] [81] [88] [89] [90] [28] [28] [28] [28] Abbreviations: MP, viral movement protein; PD, plasmodesmata; RTM protein, restricted tobacco etch virus movement; SEL, size exclusion limit. http://plants.trends.com 36 Review TRENDS in Plant Science ‘correct’ subcellular address. This suggests that PD might represent a specific site for vesicle recycling events. Recent studies have begun to implicate Rabs and their related proteins in MC trafficking to PD (Tables 2 and 3). Although Rab proteins are generally associated with the regulation of vesicle-mediated transport [22], they might also play a role in trafficking large ribonucleoprotein complexes to the cell periphery. For example, in the Drosophila oocyte, Rab11 has been shown to be involved in highly polarized mRNA trafficking [24]. Unlike yeast and human genomes, which have only two and three genes encoding Rab11 proteins, respectively, Arabidopsis has more than 25 such related genes [25], each with a possible different localization and function. Recently, transgenic tomato plants were produced that express an antisense Rab11 sequence [26]. The plants showed abnormal development, mimicking that seen when the expression of homeobox genes is disrupted [27], suggesting that Rab11 might be connected with the trafficking of transcription factors through PD. Recently, we have shown that the N-terminal moiety of Rab11, along with other plant cDNAs, associates strongly with PD when expressed as a cDNA– GFP fusion (Figure 1) [28]. It would be interesting in the future to determine whether Rab proteins do indeed play a role in regulating the trafficking of MCs to PD. KNOLLE The KNOLLE protein is a target-soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (t-SNARE) that belongs to the syntaxin family [29]. Specific t-SNAREs located on target membranes form part of the integral membrane trafficking machinery of eukaryotic cells and interact directly with vesicle SNARE (v-SNARE) complexes to allow specific vesicle– membrane fusions to occur [30]. During the formation of the phragmoplast, Golgi-derived vesicles are delivered to the Figure 1. The N-terminal moiety of a Rab11 protein localizes to plasmodesmata (PD) when expressed as a green fluorescent protein (GFP) fusion. The figure shows an optical stack of a single spongy mesophyll cell. PD are labelled at the end walls connecting cells (arrows). Scale bar ¼ 10 mm. Reproduced, with permission, from Ref. [28]. http://plants.trends.com Vol.9 No.1 January 2004 equatorial plane, where they fuse to form a membrane network that matures centrifugally into a disc-shaped cell plate [31]. In a recent study, the MP of grapevine fanleaf virus, which forms PD-associated tubules, was found to interact with KNOLLE in co-immunoprecipitation studies, and also to co-localize with KNOLLE at the developing cell plate [32]. The authors proposed a model in which the grapevine fanleaf virus MP is transported to specific sites in the cell, possibly by co-packaging with KNOLLE in the same Golgi-derived vesicles. The increasing number of vesicle-trafficking proteins found to interact with viral MPs (Table 2) suggests that the targeted delivery of some components to PD might occur via post-Golgi compartments. Protein mediators of vesicle trafficking and fusion (e.g. Rabs and SNAREs) would provide attractive ‘targets’ for viral MPs, allowing the directed translocation of the viral genome to PD. Peripheral proteins Pectin methylesterase Several viral MPs appear to interact with putative PD components. However, direct in situ interactions between PD proteins and viral MPs are lacking in many studies and it is probably more appropriate to refer to ‘peripheral’ proteins than to PD proteins. The MP of TMV, along with other viral MPs (Table 2), has been shown to interact with pectin methylesterase (PME) [33,34], an enzyme involved in modifying pectin-rich regions of the wall (Table 3). Conceivably, MPs only interact with PME once the MP has been transported to the PD [35]. However, another possibility is that MPs interact with PME before its delivery to the plasma membrane [1]. In this scenario, PME might function as a hijacked cargo rather than a bona fide PD receptor for MP. Provided that this interaction occurred early enough in the infection cycle, MPs could ‘piggy back’ on the host MC trafficking machinery to reach their required location. TGB12K-interacting proteins Potato virus X requires gene products of the triple-gene block (TGB), along with the viral coat protein, to mediate cell-to-cell movement [35]. Using the 12 kDa (12K) TGB protein (which increases the PD size exclusion limit [36]) as bait in a yeast two-hybrid assay, three host TGB12Kinteracting proteins (TIPs) have been identified [37]. All three TIPs interacted with b-1,3-glucanase, an enzyme involved in callose degradation. Callose is an integral component of the neck region of PD [2,3] and previous studies have shown a direct link between callose degradation and virus movement [38,39]. Thus, regulation of b-1,3-glucanase activity by viral MPs might be a means by which some viruses overcome host deposition of callose during infection [39]. Significantly, TIPs are detected in non-virus-infected material and virus infection does not affect their expression [37]. Therefore, TIP proteins might normally function to regulate callose deposition around PD. Not all viral MPs interact with TIPs, indicating that MP – TIP binding is yet another piece of a jigsaw in which specific virus – host interactions control the movement of MCs to PD. Review TRENDS in Plant Science Vol.9 No.1 January 2004 37 How is specific molecular complex trafficking achieved? The ability of viral MCs to bind cargos and/or host chaperones does not in itself guarantee successful delivery of the MC to the PD pore. Indeed, random diffusion of MCs would appear to be an inaccurate way to interact with PD, even if the chaperone– MC has an integral PD-interaction motif [40]. If many MCs do use components of the host cytoskeleton to access PD [15,16], what factor(s) bind the MC to the cytoskeleton and how is successful PD targeting achieved? Important clues have been obtained from recent studies of MC trafficking in yeast and Drosophila. or they might be spatially separated on different proteins within the pore. In the context of the putative PD docking protein, it is significant that both transcription factors and viral MPs might interact with a common PD receptor protein [48], consistent with the notion that plant viruses acquired the capacity for cell – cell movement from the plant genome [49]. The putative PD docking protein(s) awaits isolation. However, given its apparent ability to interact with a wide range of protein cargos, it might have multiple binding domains capable of forming stable protein – protein interaction platforms. Chaperone –motor associations In many animal cells, the destination of mRNAs is determined by specific sequences in their 30 untranslated regions (UTRs) that are often referred to as ‘zip codes’ [41]. Zip codes are recognized in turn by transacting proteins that determine the correct subcellular address of the mRNA [41]. Of these transacting proteins, linkers between RNA and cytoskeletal motors are key determinants of mRNA destination [41 – 43]. Many chaperonerelated proteins bind directly to a molecular motor, ensuring the delivery of the MC to the cytoskeleton. Several molecular motors, including those from the myosin, kinesin and dynein families, have been shown to interact with cellular proteins that determine transport specificity [43]. The dual function of Rab GTPases, namely their specificity for their cargo and their ability to link their cargo to the cytoskeleton, make these proteins attractive candidates for mediators of MC trafficking [41,44]. In yeast, specialized ‘adaptor proteins’ perform the function of linking motor proteins to a specific cargo [45]. In yeast, a class V myosin (myo4) has been shown to transport ASH1 RNA in a highly polarized manner (localization of ASH1 at the bud tip is required for matingtype switching [46]) via interactions with adaptor proteins known as She proteins [45]. It is estimated that several hundred different transport complexes can travel along the cytoskeleton in a single eukaryotic cell [44]. Plants possess up to 17 myosins [47], although the functions of these have yet to be determined. In the same way that yeast-two hybrid approaches have begun to identify plant proteins that interact with viral MPs, using the newly discovered motor proteins from the Arabidopsis database as bait in yeast-two hybrid studies should allow identification of many of the interacting transport complexes that are shuttled by different molecular motors in plants. Search for plasmodesmal components Several proteins have now been localized to PD (Table 3), although the list is not extensive. The paucity of known PD components is largely because it is difficult to isolate intact PD from the cell wall fraction for proteomic analyses. In our search for proteins that interact specifically with PD, we have exploited a virus-based vector expressing random cDNA– GFP fusions [28]. One of the unknown PD proteins we have isolated encodes a WD-40-repeat sequence (Table 3), a regulatory protein domain involved in protein – protein interactions [50]. The interaction of these PD-associated proteins with other plant proteins and viral proteins will be an interesting future subject area. Getting through the plasmodesmal pore Assuming that a MC has recruited the necessary machinery to target it to PD, the next major challenge is successfully to negotiate passage through the PD pore. Several criteria must be met for successful transport of the MC through PD. The first is the docking of the MC with a putative PD receptor at or within the orifice of the PD pore, the second is the successful initiation of ‘gating’ (transient increase in the size exclusion limit) of the pore, and the third is the trafficking of the MC through the pore from one cell to the next [48]. The PD components responsible for each of these functions might be present on a single protein Role for protein kinases? If components of the cytoskeleton are an integral component of PD, how are the individual elements regulated to achieve specific trafficking of MCs? Regulation of cell signalling in both plants and animals involves signal cascades that require protein phosphorylation – dephosphorylation cycles, and it is tempting to invoke a role for specific protein kinases in regulating PD function. It is known that the MPs of several viruses are phosphorylated during infection [56]. However, it is not known whether the endogenous MCs that are trafficked through PD require phosphorylation to mediate their passage. http://plants.trends.com Myosin within plasmodesmata In the structural model proposed by Robyn Overall and Leila Blackman [51], actin is depicted as running through the PD pore, closely associated with the central desmotubule. Putative myosin ‘spokes’ radiate out from the actin, physically linking it to the plasma membrane. Such a structural arrangement could create tension between the plasma membrane and the desmotubule, regulating pore aperture. Myosin has a role in generating tension between adjacent membranes in many mammalian cells [52]. Recently, myosin VIII, a unique plant unconventional myosin, has been localized to PD [53] and has been implicated in the regulation of PD function [54,55]. This myosin might be bound to the plasma membrane within PD, possibly by its C-terminal globular region [54]. However, myosin VIII also has a characteristic motor-domain region, common to all myosin motors, as well as four IQ motifs that are predicted to bind calmodulin [54]. It is therefore possible that myosin VIII functions as a Ca2þregulated molecular motor that is capable of trafficking cargo along the actin filaments that traverse the PD pore (see below). Review 38 TRENDS in Plant Science Vol.9 No.1 January 2004 To date, several protein kinases have been implicated in PD function and in MP trafficking (Table 3). Protein kinases present within PD might be involved in the phosphorylation of MCs directly, or alternatively might play a role in the phosphorylation of the chaperones and/or cytoskeletal motors that deliver them. Phosphorylation of the myosin bound to the plasma membrane within PD would provide an attractive mechanism for modulating the size exclusion limit. In many animal cells, phosphorylation by myosin-specific kinases results in the dissociation of the myosin motor from its attached membrane [43]. Interestingly, the unique C-terminus of myosin VIII, a PD-localizing myosin, contains several predicted phosphorylation sites for protein kinases A and C [54]. However, the role of phosphorylation in the regulation of this unconventional plant myosin has yet to be demonstrated. Linking structure to function Here, I propose a model that couples known structural components of PD to a potential regulatory mechanism in which protein kinases regulate the activity of key components within PD (Figure 2). It should be stressed that this model borrows heavily from recent developments in animal and yeast biology, and that other mechanisms, including passive transport for some MCs [6], remain a possibility. However, the model is intended to generate debate about how selective MC trafficking through PD P K Cell wall P D Plasmodesma Nucleus Ribonucleoprotein complex Cytoplasmic chaperone (putative Rab) Nuclear export factor Actin D PD-docking protein Golgi-derived cargo K PD-kinase Endosomal compartment Myosin TRENDS in Plant Science Figure 2. Potential trafficking pathways to and through plasmodesmata (PD). (Left) A ribonucleoprotein complex traffics from the nucleus to the cytoplasm, assisted by a nuclear export factor. A cytoplasmic chaperone then interacts with the macromolecular complex (MC) and binds it to a specific myosin motor. These interactions determine transport specificity to the PD pore. At the neck region of the pore, the MC binds to a putative docking protein, which in turn activates a myosin-specific kinase that phosphorylates the C-terminus of the myosin motor, resulting in its release from the membrane. The MC is then free to traffic via the motor domain of the myosin along actin filaments associated with the desmotubule that spans the central part of the pore. (Right) Protein cargos destined for secretion at or around PD are packaged at the trans face of the Golgi apparatus. A cytoplasmic chaperone (putative Rab protein) associates with the vesicle membrane and binds it to myosin. As above, these specific interactions determine vesicle destination. At the neck region of the PD pore, the vesicle contents are discharged by regulated exocytosis. The plasma membrane in this region, along with essential PD components, is subsequently recycled by an endocytic step and delivered to an endosomal sorting compartment. http://plants.trends.com Review TRENDS in Plant Science 39 Vol.9 No.1 January 2004 might be achieved. In this particular model, the cytoskeletal motor, not the cargo, is phosphorylated to permit MC trafficking through the pore (although it is appreciated that other protein kinases within PD might phosphorylate MCs directly). The model is based on the requirement for chaperone– motor interactions to traffic MCs in plants, a feature that has already become established for MC trafficking in yeast and mammals. In the case of MCs originating inside the nucleus (Figure 2, left), a chaperone protein (nuclear export factor) is required to transport the MC into the cytoplasm. In the cytoplasm, the MC associates with a second chaperone (putative Rab protein) that in turn binds the cargo to an appropriate motor protein. The specific interaction between chaperone and motor protein determines transport specificity and targets the cargo to PD. One of the plant-specific myosins is envisaged to link the MC – chaperone complex to the actin cytoskeleton via its motor domain. The continuity of the actin cytoskeleton from cytoplasm to PD provides a pathway for directional traffic of the MC to the PD orifice. At the PD, a docking protein binds the MC, either by attaching directly to the cargo or, alternatively, to the myosin motor protein at its C-terminus. The gating motif on the MC activates a myosin-specific kinase that phosphorylates the C-terminus of the myosin motor, resulting in its release from the membrane and a resultant increase in the size exclusion limit of the pore. The MC is then free to traffic to another cell via the motor domain of the myosin, along actin filaments that span the central pore. In this model, phosphorylation – dephosphorylation cycles of the molecular motor regulate the detachment and attachment of the MC from the plasma membrane lining the PD pore, providing a generic mechanism for PD regulation that is based on the motor protein, not the cargo. The model incorporates recent data that implicate specific vesicle trafficking events in the delivery of cargos to PD (Figure 2, right). Specific proteins destined for secretion at or around PD are packaged at the trans face of the Golgi before their delivery to the PD pore. Notice that the coordination of this vesicle-mediated transport requires a similar subset of chaperones and molecular motors to achieve specific targeting. At the PD pore, the protein cargo is discharged to the apoplast by regulated exocytosis. The model also incorporates an endocytic vesicle recycling step in which key PD components are returned to intracellular compartments for degradation. Figure 3 shows a putative pathway for MPs to PD. Several viral MPs associate with the endomembrane system early in the viral infection cycle, and several of these are predicted to have transmembrane domains [17,35,57,58]. A viral MP is shown to traffic to PD by becoming incorporated, along with its host-interacting partner, into Golgi-derived vesicles destined for secretion at PD (Figure 3). Such vesicles achieve directed trafficking to PD by binding to an appropriate Rab protein. At the PD pore, specific interactions between v-SNARE and t-SNARE complexes on the vesicle and plasma membrane, respectively, allow the vesicle membrane to fuse with the plasma membrane, leaving the MP inserted into the plasma membrane within PD. The MP might then subsequently associate with the PD trafficking machinery (Figure 3). Conceivably, viral RNA attached to the MP could be trafficked on the cytosolic face of such a transport complex. Future directions The aim of this article is to highlight areas of future research that might yield information about the pathways and mechanisms of selective MC trafficking. Future studies will gain from further isolation of PD proteins and determination of the cellular components with which they interact, particularly the chaperones and molecular motors responsible for directional trafficking to PD. This article focuses on targeted MC movement. In the future, it will be essential to identify those MCs that move between cells by nonselective movement and to distinguish between the mechanisms that underlie selective and nonselective transport through PD. The elusive protein kinases that regulate the trafficking of specific MCs remain to be Cell wall GTP GTP Plasmodesma Ribonucleoprotein complex Rab protein Movement protein Plasma membrane v-SNARE t-SNARE Golgi-derived cargo TRENDS in Plant Science Figure 3. Putative mechanism by which some viral movement proteins (MP) might use the endomembrane system to target plasmodesmata (PD). The viral MP interacts with a specific host protein via an endomembrane-spanning domain before (or during) the packaging of the cargo protein by the Golgi. A specific Rab protein then guides the vesicle to the vicinity of PD. Specific interactions between v-SNARE and t-SNARE proteins on the vesicle membrane and plasma membrane, respectively, permit the vesicle to fuse with the plasma membrane, discharging the protein cargo to the apoplast. The MP is then left associated with the plasma membrane at the neck of the PD pore. The figure also shows how a viral ribonucleoprotein complex could be trafficked to PD by its attachment to the cytosolic face of the MP. Such a ribonucleoprotein complex might subsequently interact with the PD trafficking machinery to permit selective trafficking of the viral genome. http://plants.trends.com 40 Review TRENDS in Plant Science characterized. New evidence is beginning to emerge for a role of the endomembrane system in delivering viral MPs to PD, and this important area requires further study. Determining the vesicle trafficking proteins with which viral MPs interact will provide clues leading to the dissection of the endogenous MC trafficking pathway to PD. Once a battery of PD-interacting proteins has been identified, protein imaging and analysis techniques such as FRAP (fluorescence recovery after photobleaching) [59] and FRET (fluorescence resonance energy transfer) [60] will assist in determining PD protein turnover rates, as well as establishing meaningful in situ protein – protein interactions within the PD pore. Central to our understanding of MC trafficking in plants will be the acceptance that PD are not isolated structures embedded in the cell wall but rather an integral component of an intercellular network that includes nuclear proteins, cytoskeletal proteins, motor proteins, vesicle trafficking proteins, PD-specific chaperones and protein kinases. The challenge for the future is to place the pieces of this jigsaw puzzle into a meaningful order. Acknowledgements I thank Trudi Gillespie for drawing the figures and SEERAD and the Gatsby Foundation for financial support. References 1 Jackson, D. (2000) Opening up the communication channels: recent insights into plasmodesmal function. Curr. Opin. Plant Biol. 3, 394 – 399 2 Roberts, A.G. and Oparka, K.J. (2003) Plasmodesmata and the control of symplasmic transport. Plant Cell Environ. 26, 103– 124 3 Blackman, L.M. and Overall, R.L. (2001) Structure and function of plasmodesmata. Aust. J. 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