Macromolecular Transport and Signaling Through Plasmodesmata

Macromolecular Transport and Signaling Through Plasmodesmata Manfred Heinlein* and Bernard L. Epel{ *Botanical Institute, University of Basel, Hebelstrasse 1, CH-4056 Basel, Switzerland {

Department of Plant Sciences, Tel Aviv University, 69978 Tel Aviv, Israel

Plasmodesmata (Pd) are channels in the plant cell wall that in conjunction with associated phloem form an intercellular communication network that supports the cell-to-cell and long-distance trafficking of a wide spectrum of endogenous proteins and ribonucleoprotein complexes. The trafficking of such macromolecules is of importance in the orchestration of non-cell autonomous developmental and physiological processes. Plant viruses encode movement proteins (MPs) that subvert this communication network to facilitate the spread of infection. These viral proteins thus represent excellent experimental keys for exploring the mechanisms involved in intercellular trafficking and communication via Pd. KEY WORDS: Plasmodesmata, Intercellular communication, Plant development, Protein trafficking, RNA trafficking, Virus trafficking, RNA silencing, siRNA, miRNA, Movement protein, Cytoskeleton. ß 2004 Elsevier Inc.

I. Introduction The integration of multitasking between cells and tissues in multicellular organisms demands specialized systems of intercellular and inter-tissue communication. Communication may be cell-to-cell or long-distance via vascular transport systems. Cell-to-cell communication may be direct, via channels that interconnect contiguous cells or it may be by export-import mechanisms in which transported molecules leave and re-enter neighboring cells by membrane transporter systems or by exocytosis and endocytosis. Such systems International Review of Cytology, Vol. 235 0074-7696/04 $35.00

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exist both in plants and in animals. In general, plants diVer from most animals in that plant cells are separated from each other by a substantial extracellular matrix, the cell wall, which prevents direct membrane–membrane contact. Although the cell wall represents a barrier between adjacent cells and prevents cell movement, it is not impervious. The wall matrix is porous, with pores of a radius of about 4 nm. Pores of this size limit unhindered diVusion to exported proteins with a Stokes radius (Rs) of up to 3 nm [equivalent to a globular protein of about 40 kDa (Baron-Epel et al., 1988; Carpita and Gibeaut, 1993; Carpita et al., 1997)]. In animal cells, direct cell-to-cell communication is by gap junctions, proteinaceous channels that interconnect contiguous cells and function in transport of small molecules of less that 1 kDa. In plants, cell walls prevent the formation of such proteinaceous cell-to-cell junctions. For direct cell-to-cell communication, plants use membranous tunnels termed plasmodesmata (Pd) (singular: plasmodesma) that interconnect the cytoplasm of contiguous cells. The interconnection of cytoplasm of groups of cells creates supracellular domains (Lucas and van der Schoot, 1993). Plants can regulate the cell-to-cell communication between cells in such supracellular domains by a number of means, for example, by developmental changes in Pd structure, by changes in their biochemical composition, or by changing the frequency of Pd between cells. Up-regulation or down-regulation of Pd conductivity can modify the size of a communication domain and also redefine domain boundaries, resulting in modulation of extent and quality of communication with resultant changes in intercellular and interdomain interactions (Bergmans et al., 1997; Cantrill et al., 2001; Duckett et al., 1994; Ehlers and Kollmann, 2001; Ehlers et al., 1999; Epel and Erlanger, 1991; Erwee and Goodwin, 1985; Gisel et al., 1999, 2002; Kim et al., 2002a; Lucas and van der Schoot, 1993; Ormenese et al., 2002; Pfluger and Zambryski, 2001; Rinne and van der Schoot, 1998; Rinne et al., 2001; Ruan et al., 2001; Shepherd and Goodwin, 1992a,b; van der Schoot and Rinne, 1999a,b, 1995). Recent findings indicate that the Pd system and associated phloem form cell-to-cell and long-distance communication networks that mediate the selective cell-to-cell and systemic traYcking of RNA and protein macromolecules. These macromolecules act as specific intercellular messengers that regulate plant gene expression at a level above that of an individual cell (Haywood et al., 2002; Heinlein, 2002b; Wu et al., 2002). The role for RNA as an non-cell autonomous information macromolecule is now emerging as a new paradigm in biology (Lucas et al., 2001). For example, traYcking of RNA molecules is implicated in systemic RNA silencing (Voinnet and Baulcombe, 1997; Voinnet et al., 1998), although the exact nature of the RNA signal is still not known (Mlotshwa et al., 2002; Waterhouse et al., 2001). In addition to its role in RNA silencing, RNA traYcking and its regulation appear to play an important role in plant development (Haywood et al., 2002; Vance and Vaucheret, 2001). Several mRNAs can be detected in phloem sap and may represent signaling molecules destined

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for long distance transport into young tissues (Ruiz-Medrano et al., 2001). This concept is supported by recent studies indicating that the selective transport of mRNA into the shoot apex has important roles in plant morphogenesis (Foster et al., 2002; Kim et al., 2001; Lucas et al., 2001). Importantly, several non-cell autonomous phenomena are known that imply systemic signaling but for which the nature of the signal is yet unknown. Examples for such phenomena are systemic acquired resistance [SAR (Dong, 2001)], the systemic wounding response (Pearce et al., 1991), the systemic acclimation to light (Karpinski et al., 1999), the photoperiodic induction of flowering [involving ‘‘Florigen’’ of a yet unknown nature (Colasanti and Sundaresan, 2000)], or the systemic induction of genomic instability following local pathogen infection (Kovalchuk et al., 2003). The ability of plant cells to exchange RNA macromolecules is compellingly demonstrated by the traYcking of viral genomes, allowing viruses to achieve systemic infection. The finding that systemic virus movement and systemic silencing share sensitivity to low concentrations of cadmium suggests that both processes may be related (Carr and Murphy, 2002; Goshroy et al., 1998; Ueki and Citovsky, 2001, 2002). Moreover, plant proteins with functional and structural similarity to virus-encoded ‘‘movement proteins’’ (MPs) have been reported (Xoconostle-Cazares et al., 1999). These and other studies suggest that viruses and their MPs usurp endogenous RNA traYcking pathways to spread infection. Thus, virus infection continues to provide an outstanding system to investigate the nature of Pd and the mechanism of macromolecular cell-to-cell and systemic transport (Heinlein, 2002a).

II. Structure of Plasmodesmata A. General Description Pd are membranous tunnels that span plant cell walls linking the cytoplasm of contiguous cells (Fig. 1). These quasiorganelles function in regulating direct cell-to-cell communication, forming an intercellular communication network that permits direct cytoplasmic communication either at the local level, within limited local field of cells (Ehlers et al., 1999; Erwee and Goodwin, 1985; Gisel et al., 1999; Lucas and van der Schoot, 1993; Perbal et al., 1996; Rinne and van der Schoot, 1998), or at the systemic level via the phloem vascular network (Imlau et al., 1999; Oparka et al., 1999; van Bel et al., 2002). In higher plants, these trans-wall membranous tunnels are coaxial, the outer limiting membrane consisting of the plasma membrane that is continuous from one cell to the other, while the inner coaxial membrane, historically termed the desmotubule, consists of modified tubular

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FIG. 1 A schematic model of the substructure of a simple plasmodesma. The outer coaxial membrane lining the plasmodesma consists of modified plasma membrane (red) that is continuous between adjoining cells. The cell wall sheath surrounding the Pd (blue) is devoid of cellulose and hemicellulose and is composed in part of nonesterified pectin, callose, other noncellulosic polyglucans and probably as yet uncharacterized proteins. The inner coaxial component, the desmotubule (green), consists of modified cortical ER and may be constricted with no lumen or it may be tubular with an open lumen. Particles present within the cytoplasmic sleeve may be embedded within the plasma membrane, desmotubule, or present entirely within the cytoplasmic sleeve.

endoplasmic reticulum (ER) that is continuous with the cortical ER (Ehlers and Kollmann, 2001). The coaxial ER component of the Pd may be constricted with no lumen (Fig. 2A and B) or it may be tubular with an open lumen (Fig. 2C and D). The desmotubule as seen in electron microgaphs of most plants studied appears to be constricted along the entire length and it has been suggested that it be termed appressed ER (Lucas and van der Schoot, 1993). However, in some Pd, the desmotubule may be partially or entirely dilated (Fig. 2C–F) (Glockmann and Kollmann, 1996; Robinson-Beers and Evert, 1991; Waigmann et al., 1997). Thus, since the ER Pd component is not always appressed, we will employ in this review the term desmotubule. Higher plant Pd may be simple monocoaxial tunnels, termed simple Pd, or they may be multibranched, forming a network of interconnected coaxial tunnels termed branched Pd (Ehlers and Kollmann, 2001). Branched Pd often have a central sinus-like cavern to which the branches are interconnected. The desmotubule within the sinus-like cavern is often dilated with a lumen.

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Basically, Pd may be formed in two fundamentally diVerent ways. Pd may be formed during cytokinesis by the fusion of Golgi vesicles around ER strands that traverse the dividing mother cell. Such Pd are termed primary Pd. Primary Pd are initially single-stranded, i.e., simple, but may subsequently undergo transformation to a branched form (Ehlers and Kollmann, 2001). Pd may also be formed across established cell walls. Such Pd are termed secondary Pd. Secondary Pd may initially be simple or branched. Following their formation, Pd, both primary and secondary, may undergo modifications in structure and probably in biochemical composition. Lateral fusion of adjacent Pd at the middle lamella region of the cell wall may occur (Fig. 2I) with subsequent addition of branches during wall thickening (Ehlers and Kollmann, 2001; Oparka et al., 1999; Volk et al., 1996). Pd may also be plugged or occluded during diVerentiation (Fig. 2C–F) (Ehlers and Kollmann, 2001; Ehlers et al., 1999; Kwiatkowska, 1988; Rinne and van der Schoot, 1998). Both primary and secondary Pd may span contiguous cells that were formed as a result of cytokinesis. However, all Pd that span contiguous cells of diVerent lineage are secondary, as the cell wall between them did not arise as a result of cell division (Ehlers and Kollmann, 2001). Pd structure is not uniform, but can vary between diVerent cell types within a single organism and can even vary within a single cell (van Bel et al., 1999). When diVerent cell types abut a single cell, Pd interconnecting the diVerent cell types to the common neighbor may be quite diVerent. Both primary and possibly secondary Pd may interconnect adjacent daughter cells that arose as a result of cell division while only secondary Pd can be present between cells that are of diVerent lineage. For example, along the path from the mesophyll to the sieve element in the small veins of Saccharum oYcinale leaves, a distance of about 100 nm, five morphologically diVerent types of Pd were identified (Robinson-Beers and Evert, 1991). Pd between companion cells and sieve elements are morphologically and probably functionally diVerent from Pd between mesophyll cells (Robinson-Beers and Evert, 1991).

B. Ultrastructure Although the ultrastructure of Pd has been the subject of numerous electron microscopic studies (Badelt et al., 1994; Beebe and Turgeon, 1991; Blackman and Overall, 2001; Botha et al., 1993; Ding et al., 1992b; Ehlers and Kollmann, 2001; Gunning and Overall, 1983; Overall, 1999; Overall and Blackman, 1996; Overall et al., 1982; Radford and White, 2001; Tilney et al., 1990; Turner et al., 1994; Waigmann et al., 1997), the interpretation of these studies has been the subject of debate over the past years. The debate

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FIG. 2 Electron micrographs of plasmodesmata. (A) Longitudinal and (B) transverse section of plasmodesmata between adjacent vascular parenchyma cell in mature sugarcane leaf. ER, endoplasmic reticulum. These plasmodesmata have constricted desmotubules and neck constrictions (bar ¼ 200 nm). (C) Longitudinal (bar ¼ 200 nm) and (D) glancing (bar ¼ 100 nm) section of plasmodesmata between mesophyll and bundle sheath cell. Internal sphincters (S) are present at both ends of the plasmodesma. Plasmodesmata are distinctly narrowed where they traverse the suberin lamella [SL in (C); unlabeled arrows in (D)]. Between the suberin lamella and the sphincters the desmotubule is an open convoluted tubule [in (D) darts point to open dilated portions of desmotubule between suberin lamella and sphincters). (E–G) Plasmodesmata between adjacent Krantz mesophyll cells in mature sugarcane leaf. (E) Longitudinal section of plasmodesma between adjacent cells: electron-opaque structures, internal sphincters, are present at both ends near orifices of plasmodesma. The desmotubule is open between two sphincters. (F) Transverse section of plasmodesmata at the level of the sphincter; the sphincter is electron dense and the desmotubule is constricted. (G) Transverse section of plasmodesmata at mid level showing open lumina of endoplasmic reticulum. (H) Simple plasmodesma in young cell walls in the needles of Metasequoia glaptostoboides exhibiting a funnel-shaped neck

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focuses on the eVect of diVerent fixation and contrasting procedures on the structure and spatial arrangement in the cytoplasmic sleeve and on the structure and composition of the desmotubule. Moreover, almost all studies have dealt with simple Pd. The diVerent interpretations of the electron micrographs prepared under diVerent conditions of fixation and the chemical analysis of these Pd have presented us with a variety of models, none of which or all of which may be correct to some degree, depending on the plant, the cell type, and/or the developmental stage of the tissue (Blackman and Overall, 2001; Botha et al., 1993; Ding et al., 1992b; Overall, 1999; Overall and Blackman, 1996; Overall et al., 1982; Radford and White, 2001; Tilney et al., 1991; Turner et al., 1994; Waigmann et al., 1997). High-resolution enhanced EM images of simple Pd have revealed the presence of helically arranged particles within the cytoplasmic sleeve of the Pd (Badelt et al., 1994; Botha et al., 1993; Ding et al., 1992b; Overall, 1999; Overall et al., 1982; White et al., 1994). These particles have been interpreted as being embedded in the plasma membrane (Ding et al., 1992b) and/or the desmotubule (Botha et al., 1993) or to be present entirely within the cytoplasmic sleeve (Overall, 1999). Data have been presented indicating that actin and myosin are present within the cytoplasmic sleeve (Blackman and Overall, 1998; Radford and White, 1998; Reichelt et al., 1999; White et al., 1994) and it has been hypothesized that the actin strands are wrapped helically around the desmotubule (Overall, 1999). Electron-dense spoke-like extensions have been observed radiating out from the desmotubule, apparently interconnecting the two axial membranes (Burgess, 1971; Cook et al., 1997; Ding et al., 1992b; Schulz, 1995; Tilney et al., 1991). Although the composition of these spoke-like structures has yet to be determined, it has been suggested that these spoke-like structures may represent myosin molecules attached to microfilaments (Overall and Blackman, 1996). It has been proposed that the spaces between the particles observed within the cytoplasmic sleeve form microchannels of approximately 2.5–3 nm in diameter, which thus limits the size of molecules that can traverse the Pd to small molecules of about 1 kDa (Botha et al., 1993; Ding et al., 1992b). New data that indicate that Pd can conduct macromolecules configuration and with constricted desmotubule. (I) Branched plasmodesma in young wall possibly having developed from lateral fusion of two adjacent simple plasmodesmata; the neck region is constricted and the desmotubule has no lumen along its entire length. (J, K) Longitudinal section of plasmodesmata between adjacent onion root cells not treated with DDG (J) or treated with DDG prior to tissue fixation. Plasmodesma in the nontreated sample (J) shows a raised collar and constricted neck region (arrowheads). Plasmodesmata in tissue treated with DDG prior to fixation (K) exhibit a funnel-shaped neck configuration (arrowheads). (Figures A–G from Robinson-Beers and Evert, 1991; Figures H and I from Kollmann and Glockmann, 1999; Figures J and K from Radford, Vesk, and Overall, 1998.)

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are in conflict with this hypothesis suggesting the need for a reevaluation of current models of Pd ultrastructure (see below). It is generally accepted that transport cell to cell via Pd is through the cytoplasmic sleeve, the space between the desmotubule membrane and the plasma membrane (Ding et al., 1999; Epel, 1994; Lucas and van der Schoot, 1993; Roberts and Oparka, 2003). However, an open tubular structure is present in the desmotubule of trichome cells of Nicotiana clevelandii (Waigmann et al., 1997) and in the Pd of cotton nectary papillae (Eleftheriou and Hall, 1983) and data have been presented that indicate that transport may occur within the lumen of such dilated desmotubules (Cantrill et al., 1999; Gamalei et al., 1994; Lazzaro and Thomson, 1996; Waigmann et al., 1997). Although the plasma membrane and the ER are contiguous through Pd, the composition of these membranes within the Pd is apparently modified. The lipid composition of the Pd membranes has never been determined due to the diYculty in isolating native Pd for lipid analysis, but indirect evidence suggests that these membranes have undergone modifications. Hepler (1982) showed that the mixed-stain osmium tetraoxide–potassium ferricyanide, which stains cytoplasmic ER, specifically does not stain the desmotubule. Tilney et al. (1991) in studies of the Pd of gematophytes of the fern Onoclea sensibilis presented evidence that suggested that the desmotubule in this fern is primarily proteinacious in character (Tilney et al., 1991). It was shown that if the cut surface of Pd were exposed to the detergent Triton X-100 prior to fixation, the plasma membrane limiting the plasmodesma was partially or completely solublized while the desmotubule remains intact. However, if the cut surface was exposed to papain and then fixed, the desmotubule disappeared, but the plasma membrane limiting the Pd remained intact. Waigmann et al. (1997) suggested that this might also be the case for the desmotubule in trichome cells of N. clevelandii. However, the above conclusions are in conflict with the studies by Grabski et al. (1993) and Turner et al. (1994). Turner et al. (1994) showed that membrane-solubilizing detergents removed the desmotubule and plasma membrane from Pd embedded in isolated cell walls from maize roots tips. Grabski et al. (1993) employing the technique of fluorescence redistribution after photo bleaching (FRAP) demonstrated the lateral mobility and intercellular transport capability of a number of fluorescent lipid and phospholipid analogs that partition either in the ER or plasma membrane. The results of this study suggested that the ER, but apparently not the plasma membrane, forms a dynamic communication pathway for lipids across the cell wall between connecting plant cells (Grabski et al., 1993). Collars and sphincter-like electron-opaque structures have been observed to exist both in the cell wall surrounding Pd orifices as well as internally near the Pd orifice (Fig. 2A–F and J) (Badelt et al., 1994; Cook et al., 1997; Evert et al., 1977; Olesen, 1979; Olesen and Robards, 1990; Overall, 1999; Overall

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et al., 1982; Rinne and van der Schoot, 1998; Robinson-Beers and Evert, 1991; Tilney et al., 1991). In addition, in certain species, cell wall-localized spiral structures have been observed to circle around the entire length of Pd (Badelt et al., 1994). However, the nature of these structures has been a matter of dispute (van Bel et al., 1999). The neck region of the Pd may have funnel-shaped openings or it may be constricted (Fig. 2H and K) (Badelt et al., 1994; Beebe and Russin, 1999; Blackman and Overall, 2001; Botha et al., 1993; Ding et al., 1992b; Overall, 1999; Overall et al., 1982; Radford and White, 2001; Radford et al., 1998; Robinson-Beers and Evert, 1991). Neck constrictions have been proposed to function in restricting the size of the cytoplasmic sleeve at the site of the constriction and limiting molecular diVusion. Constrictions also occur in the suberin lamella between mesophyll cells and bundle-sheath cells in mature leaves of sugarcane and many grasses (Fig. 2C) (Beebe and Russin, 1999; Robinson-Beers and Evert, 1991) and may function in regulating conductivity. Constrictions in the neck region are seen in most electron micrographs of Pd and are made up, in part, by callose, a b-1,3-glucan that is deposited just outside the plasma membrane. In many grasses, the cytoplasmic sleeve at the level of the sphincter appears filled with an amorphous electron-dense substance (Fig. 2C, E, and F). The nature and function of this substance are unclear, although it has been suggested to function in regulating Pd conductivity (Evert et al., 1996). Callose deposition may occur in response to wounding or chemical fixation (Radford et al., 1998) or in the course of normal developmental events to close oV communication (Rinne and van der Schoot, 1998). Just the act of cutting a small piece of tissue prior to either rapid freezing or placing it in fixative may be suYcient to induce a wound reaction and the formation of Pd-associated callose (Radford et al., 1998). Delmer et al. (1993) employing a monoclonal antibody (MAb) localized callose synthase to Pd in cucumber (Cucumis sativus) seedlings, and in Pd of onion (Allium cepa) epidermal cells. This MAb recognized a polypeptide of about 65 kDa found in membranes isolated from a variety of plant sources (Delmer et al., 1993). The molecular identity of this protein remains to be determined. The deposition of callose may restrict Pd transport, thereby serving as a sphincter mechanism (Fig. 2J and K). Closure is probably rapid but reopening would probably take hours. Thus, other mechanisms must be present to account for rapid modulation of Pd conductance. Surrounding the coaxial membranous component of the Pd is a specialized sheath of wall material. This Pd wall sheath may be an essential part of the Pd and has been largely ignored. This sheath is devoid of cellulose and hemicellulose and is composed in part of nonesterified pectin (Dahiya and Brewin, 2000; Roy et al., 1997; Sutherland et al., 1999), callose, other noncellulosic polyglucans, and probably as yet uncharacterized proteins. Turner et al. (1994), who studied Pd from maize root tips, concluded that the cell wall

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FIG. 3 A schematic model for nonselective regulation of plasmodesmal conductivity. (A) The cross section of a single plasmodesma embedded in a cell wall (CW; gray slanted stripes and green speckle). The cell wall sheath immediately surrounding the Pd is devoid of cellulose and hemicellulose and is composed in part of nonesterified pectin, callose, other noncellulosic polyglucans and probably as yet uncharacterized proteins. Embedded in the Pd plasma membrane (orange) are callose synthase, b-1,3-glucanase, and other Pd-associated proteins.

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sheath around the Pd was subdivided with an inner amorphous collar surrounded by a peripheral zone of callose interdispersed with fibrillar wall material. They also suggested that the carbohydrate wall sheath may be held in position by protein links (Turner et al., 1994). This wall sheath is often characterized as an electron-lucent region with sphincter-like electronopaque structures present within the specialized sheath. Fine electron-dense spokes are sometimes observed to connect the plasma membrane to the surrounding wall material. Changes in activity of wall-associated proteins in the Pd wall sleeve may play an important role in regulating Pd conductivity and need further intense study. The absence of cellulose in the Pd wall sleeve may have functional significance, rendering the wall domain more extensible. Changes in wall extensibility induced by the release of eVector molecules into the surrounding wall sheath could regulate Pd dilation. Molecular data are lacking in this regard. The Pd cell wall sheath should be regarded as a dynamic rather than a static extracellular component, possibly with essential functions in the regulation of Pd function (model presented in Fig. 3). The presence of callose seen in many studies of Pd may in many cases be an artifact due to injury caused during the preparation of the material for microscopy. To study the validity of the observation of callose deposits around Pd in the wall sheath in A. cepa L. roots, callose synthesis was inhibited by incubating the plant tissue with 2-deoxy-d-glucose (DDG), an inhibitor of callose formation, for 1 hr prior to fixation in 2.5% glutaraldehyde. The inhibition of callose formation was monitored through aniline blue-induced fluorescence of callose. The neck region of the Pd from tissue treated with DDG exhibited a funnel-shaped configuration. This was in contrast to the Pd from tissue not incubated with DDG, which exhibited constricted necks similar to those previously reported (Fig. 2J and K). Apparently, both the initial dissection and glutaraldehyde fixation induced neck constriction in Pd. The inhibition of callose formation by chemical means showed that the neck constrictions and raised collars in this area are artifacts due to physical wounding and glutaraldehyde fixation (Radford and White, 2001).

The desmotubule–ER continuum is colored gray. Actin microfilaments (MF) (red) to which are anchored motor proteins transverse Pd possibly bound to desmotubule. The net accumulation of callose is low and the cytoplasmic sleeve is open. Nontargeted macromolecules (black spheres) diVuse according to electrochemical gradient and hydrodynamic (Stokes) radius. (B) As in (A) but callose synthase is activated and callose deposits (brown weave) around the cytoplasmic sleeve and especially at the cytoplasmic annulus result in the formation of a sphincter that reduces the size of the annulus and creates a bottle neck for diVusion of macromolecules.

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C. Molecular Characterization The most direct approach to characterizing Pd composition would be to isolate Pd and analyze their composition. This has not proved an easy task. Pd are embedded in the cell wall and their release from the wall matrix and purification have proved diYcult. Such an approach requires that a clean wall fraction be obtained first and that the wall then be digested to release the embedded Pd without damaging the Pd. The isolated Pd could than be subjected to analysis to determine composition (Epel et al., 1995). A less rigorous approach, employed in a number of reports, entails isolation of proteins from isolated ‘‘clean walls,’’ generation of antibodies against the proteins, and determination by immunolocalization whether these proteins localize to Pd. This approach has led to the identification of a number of putative Pd proteins (Blackman et al., 1998; Epel et al., 1996b; Waigmann et al., 1997; Yaholom et al., 1998). Antibodies generated against a 41-kDa protein isolated from clean maize mesocotyl cell walls immunolabeled Pd in maize mesocotyls. Silver-enhanced immunogold light microscopy showed that the 41-kDa protein was associated with the cell walls of cells both in the stele and cortex. Electron microscopic immunogold labeling localized the polypeptide both to Pd and to electron-dense cytoplasmic structures that are apparently Golgi membranes (Epel et al., 1996b). The gene for the 41-kDa protein was cloned [gene bank U89897 (Katz et al., 1997)] and identified to be homologous to reversible glycosylated polypeptides (RGPs) unique to plants (Delgado et al., 1998; Dhugga et al., 1997). Pd in leaves of transgenic Nicotiana tabacum stably expressing Arabidopsis RGP fused to green fluorescent protein (GFP) were labeled, clearly identifying this protein as Pd associated (Sagai, Katz, and Epel, unpublished observations). Two monoclonal antibodies, JIM 64 and JIM 67, generated against a 130-kDa protein extracted from clean walls from maize root tips clearly labeled a plasma membrane component of trichome and mesophyll Pd of N. clevelandii (Waigmann et al., 1997). The identity of this protein has yet to be determined. The unique cellular anatomy of the green alga Chara corallina was exploited to isolate putative Pd-associated proteins. C. corallina has large internodal cells that are symplastically connected via intervening nodal complexes of smaller cells that have Pd in their cell walls. Comparison of proteins extracted from walls containing Pd (nodal complexes) with those without Pd (external internodal walls) identified four putative Pd-associated proteins. These putative Pd-associated proteins were approximately 95, 45, 44, and 33 kDa. A monoclonal antibody (MAB45/22) was raised against the 45-kDa putative Pd-associated protein (CPAP45). MAB45/22 localized to the Pd of C. corallina and, in particular, to the central cavity using

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immunogold cytochemistry. MAB45/22 also exhibited weak labeling to onion root tip and cauliflower floret Pd (Blackman et al., 1998). The identity of this protein has not yet been determined. Employing an improved method of Pd isolation, the Epel group has isolated Pd from N. tabacum and N. benthamiana, Vigna unguiculata (cowpea), and Arabidopsis thaliana. A proteomic analysis of proteins associated with the Pd fraction of Arabidopsis has identified putative Pd-associated proteins including three protein kinases, a pectin methyl esterase, a reversible glycosylated polypeptide previously identified as associated with Pd (see above), a b-1,3-glucanase, a heavy myosin-like protein, a putative TCP1 chaperonin-like protein, as well as a number of unknown proteins (Epel, unpublished observations). Future studies employing GFP fusions and immunocytology are in progress to ascertain which of these putative Pd-associated proteins are resident Pd-associated proteins. An important new approach for identifying Pd-associated proteins was recently described (Escobar et al., 2003). Libraries of random, partial cDNAs fused to the 50 or 30 end of the gene for GFP were expressed in planta using a vector based on tobacco mosaic virus (TMV). Viral populations were screened en masse on inoculated leaves using a confocal microscope. Each viral infection site expressed a unique cDNA–GFP fusion, allowing several hundred cDNA–GFP fusions to be screened in a single day. Twelve fusion proteins were identified that localized to Pd. Of these, two proteins were apparently false positive and two others showed no similarity to any known gene. Two proteins are homologous to unknown proteins (plasmodesmata 01 and 03; accession numbers T52393 and AAK93625) and one is homologous to a hypothetical protein (plasmodesmata 08; accession number AAK76647). Of the other homologous sequences recovered, one encodes an enzyme related to the ethylene biosynthesis pathway (plasmodesmata 11; accession number D96524), one encodes an enzyme for the first committed step in the nicotine biosynthesis pathway (plasmodesmata 07; accession number AAF14879), and two (plasmodesmata 09 and 12; accession numbers T06407 and AAK96519) encode proteins involved in ‘‘redox’’-based signaling reactions. Plasmodesmata 10 (accession number Q40521) contains a Rab sequence, a protein involved in vesicular transport, suggesting that the native protein may associate only transiently with Pd and that vesicular transport of the fusion protein is blocked at the acceptor membrane target stage. The significance of the targeting of these proteins to Pd and their function are at present unclear. Since the cDNA library used was of partial cDNAs many fusions may have been dysfunctional and did not target to Pd. Future screening results obtained with full-length cDNAs may reveal additional proteins that are Pd associated. Results from various immunolocalization studies have led to the identification of a number of proteins and polysaccharides as Pd associated. As

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pointed out above, changes in wall properties of the Pd wall sheath may be of major importance in regulating Pd conductivity. Callose has been shown in numerous studies to be specifically localized to the specialized wall sheath surrounding individual Pd and within pit fields containing numerous Pd. This Pd wall sheath also contains pectins. In studies on the localization of pectin, cellulose, xyloglucan, and callose in kiwi fruit (Sutherland et al., 1999), apple (Roy et al., 1997), and tomato (Casero and Knox, 1995), it was found that the wall sheath was rich in callose and pectins. In kiwi fruit Pd, xyloglucan-S and cellulose showed little or no localization in the Pd. Pectin was visualized using three diVerent methods: labeling of galacturonic acid residues, labeling of negatively charged groups, and labeling with JIM 5 (nonesterified residues) and JIM 7 (methyl-esterified) monoclonal antibodies. JIM 5, but not JIM 7, bound at the plasma membrane boundaries of Pd, indicating the presence of pectins with a low degree of esterification specifically localized in this region. In apple, Roy et al. (1997) did not observe specific localization of low-esterified pectin at the plasma membrane boundaries of the Pd. Instead, such pectin was distributed across the cell wall in the Pd regions. In tomato, JIM 5 recognized the primary pit fields, suggesting the presence of low esterified pectin. In studies of barley embryos employing specific antibodies against callose, pectins, and arabinogalactan proteins, callose was found in induced embryos and in the numerous Pd (Pulido et al., 2002). The importance of 1,3-glucanase in the regulation of Pd conductivity was shown in studies with a tobacco mutant that had decreased levels of a class 1 b-1,3-glucanase (GLU 1) generated by antisense transformation. In this mutant line, susceptibility to virus infection was decreased. Monitoring the cell-to-cell movement of dextrans and peptides revealed that the Pd size exclusion limit (SEL) was also reduced (Iglesias and Meins, 2000). These studies supported the supposition that callose turnover regulates Pd SEL. This conclusion was further supported by studies employing TMV replicons that either expressed active GLU1, inactive enzyme or the antisense GLU 1 construct. Compared with the size of local lesions produced on plants infected with virus expressing either an enzymatically inactive GLU I or a frame-shift mutant of the gene, the size of local lesions caused by infection with virus expressing active GLU I was consistently increased. Viruses expressing antisense GLU I constructs led to lesions of decreased size (Bucher et al., 2001). Further indications of the possible importance of the Pd wall sleeve in regulating traYcking was provided by studies that showed that the cell wallassociated plant enzyme pectin methylesterase (PME) of N. tabacum L. specifically binds to the movement protein encoded by TMV, turnip vein clearing virus (TVCV), and cauliflower mosaic virus (CaMV) and that PME is an RNA-binding protein (Chen et al., 2000; Dorokhov et al., 1999). The use of amino acid deletion mutants of TMV MP identified a binding domain

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that was necessary and suYcient for association with PME. Deletion of the PME-binding region resulted in inactivation of TMV cell-to-cell movement (Chen et al., 2000). These studies suggested that PME functions as a host cell receptor involved in cell-to-cell movement of TMV and that wall modifications may be of biological significance in virus spread. Using immunolocalization techniques cytoskeletal proteins have been shown to be associated with Pd (see Table I). Physiological studies suggest that microfilaments and myosin may play a functional role in controlling Pd conductivity. When fluorescent dextrans of various molecular sizes were each coinjected into tobacco mesophyll cells in the presence of specific actin filament perturbants such as cytochalasin D (CD) or profilin, dextrans up to 20 kDa (4.45 Stokes radius, see Table II) moved from the injected cell into surrounding cells within 3–5 min (Ding et al., 1996). In contrast, when such dextrans were injected alone or coinjected with phalloidin, a drug that

TABLE I Cytoskeletal Elements Associated with Pd Protein Actin/actin like

Centrin like Myosin/myosin like

Plant

Reference

Azolla pinnata

White et al., 1994

Hordeum vulgare

White et al., 1994

Nephrotepis exaltata

White et al., 1994

Nicotiana plumbaginfolia

White et al., 1994

Chara corallina

Blackman and Overall, 1998

Aesculus hippocastanum

ChaVey and Barlow, 2001, 2002

Populus tremula

ChaVey and Barlow, 2001, 2002

Pinus pinea

ChaVey and Barlow, 2002

Allium cepa

Blackman et al., 1999

Brassica oleraceae

Blackman et al., 1999

Chara corallina

Blackman and Overall, 1998

Allium cepa

Radford and White, 1998

Hordeum volgare

Radford and White, 1998

Zea mays

Radford and White, 1998; Reichelt et al., 1999

Aesculus hippocastanum

ChaVey and Barlow, 2001

Populus tremula

ChaVey and Barlow, 2001, 2002

Aesculus hippocastanum

ChaVey and Barlow, 2002

Pinus pinea

ChaVey and Barlow, 2002

Lepidum sativum

Reichelt et al., 1999

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TABLE II Stokes Radius (Rs) of Selected Dextrans and Globular Proteins Molecule Dextran 4

Mw 4000

Stokes radius (nm) 1.4

Reference Sigma, 1997

Cytochrome c

12,400

1.81

Rogers et al., 1997

Myoglobin

17,500

1.94

Rogers et al., 1997

7000

2.15

Waigmann and Zambryski, 2000 Sigma, 1997

Dextran 7 Dextran 10

10,000

2.3

Carbonic anhydrase

30,000

2.38

Rogers et al., 1997

GFP

27,000

2.82

Terry et al., 1995

Ovalbumin

43,000

3.05

Rogers et al., 1997

Dextran 20

20,000

3.3

Sigma, 1997

Albumin

67,000

3.6

Rogers et al., 1997

Glyceraldehyde-3-phosphate dehydrogenase Dextran 40 Dextran 50 Catalase Dextran 70

145,000

4.35

Rogers et al., 1997

40,000

4.5

Sigma, 1997

50,000

4.95

Sigma, 1997

248,000

5.22

Rogers et al., 1997

6.0

Sigma, 1997

70,000

stabilizes actin filaments, the fluorescent dextrans remained in the injected cells. Phalloidin coinjection slowed down or even inhibited CD- or profilinelicited dextran cell-to-cell movement. Moreover, when Nephrolepsis exaltata cells were treated with CD, Pd became greatly enlarged (White et al., 1994). This conformation change, however, did not occur in Azolla or barley roots. Support for a role of myosin together with actin in controlling Pd conductivity comes from studies with 2,3-butanedione monoxime (BDM), an inhibitor of actin-myosin motility. BDM apparently stabilizes actin filaments (Samaj et al., 2000). Pretreatment of the tissue with BDM resulted in a strong constriction of the neck region of Pd. These results indicate that a myosin-like protein may also play a role in the regulation of transport at the neck region (Radford and White, 1998). Myosin VIII, an unconventional myosin unique to plants, has been localized to primary Pd at newly formed cross walls at the stage in which the phragmoplast cytoskeleton has depolymerized and the new cell plate is beginning to mature (Reichelt et al., 1999). Immunoelectron microscopic studies localized actin in the neck region of Pd and down the length of the Pd, apparently in association with the desmotubule (Blackman and Overall, 1998; Radford and White, 1998;

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White et al., 1994). It was suggested that this distribution may allow a reversible constriction of the Pd along their entire length (Ding et al., 1996; Radford and White, 1998; Reichelt et al., 1999). Several alternate mechanisms can be envisioned. If the actin–myosin were associated only with the desmotubule, then upon F-actin depolymerization, the desmotubule would expand and the cytoplasmic sleeve would narrow, decreasing conductivity through the cytoplasmic sleeve. Since this is not what has been measured, it has been alternatively hypothesized that the helically arranged actin may be linked to the plasma membrane component of the Pd via myosin and the contraction of F-actin would constrict the aperture of the cytoplasmic sleeve (Overall and Blackman, 1996). This hypothesis envisions that in the unperturbed state, the Pd is in the constricted configuration. Dilation of the Pd would require some signal that promotes actin depolymerization. An alternate hypothesis is that the disruption of F-actin clears the cytoplasmic sleeve of the presence of F-actin and the associated myosin thus clearing the sleeve of the obstructing F-actin–myosin complex, resulting in an increase in the eVective diVusional cross section. The distance between the desmotubule and the plasma membrane is 13–20 nm (Overall, 1999; Robards, 1976). Such a gap, if free of obstructing molecules, is suYcient for the free diVusion of very large macromolecules. In the presence of actin–myosin and in the absence of callose, the cytoplasmic sleeve has an eVective width of about 7–14 nm (Overall, 1999). Under such conditions, molecules with Stokes radius of 3–6 nm, depending on plant and tissue, could move through the cytoplasmic sleeve of open Pd in the absence of callose (Stokes radius of various globular proteins and dextran probes are presented in Table II). Under conditions of actin depolymerization and the absence of callose, a soluble molecule with Stokes radius in the range of less than 5–10 nm could diVuse through the open cytoplasmic sleeve. Centrin, a calcium-binding contractile protein and a member of the EF-hand calcium-binding caltractin/cdc31 subfamily, was shown by immunogold electron microscopy to be localized to Pd in cauliflower florets with label being concentrated around the necks of Pd (Blackman et al., 1999). Centrin or caltractin, as it is also known, plays a fundamental role in microtubule-organizing center structure and function. Centrin’s subcellular location has generally been in the centrosome of interphase and mitotic cells. It is hypothesized that centrin dephosphorylates in response to increasing cytoplasmic Ca2þ leading to a rapid contraction of the centrin nanofilaments and thus resulting in a contraction of Pd at the neck region (Blackman et al., 1999). In support of this hypothesis is the report that increased cytoplasmic Ca2þ causes Pd closure (Baron-Epel et al., 1988; Holdaway-Clarke et al., 2000; Lew, 1994; Tucker, 1990; Tucker and Boss, 1996) and that a calcium-dependent protein kinase is associated with Pd (Baron-Epel et al., 1988; Holdaway-Clarke et al., 2000; Lew, 1994; Tucker, 1990; Tucker and Boss, 1996).

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Calreticulin, an ER lumenal resident calcium-binding chaperone, has also been reported to be associated with Pd (Balus˘ka et al., 1999). In an immunocytological study employing a polyclonal antibody raised against calreticulin, it was shown that in root tip cells, the antibody immunolocalized preferentially at cellular peripheries, exhibiting punctate labeling at the longitudinal walls, in addition to nuclear envelopes and cytoplasmic structures. Immunogold electron microscopy revealed Pd as the most prominently labeled cell periphery structure. Punctate labeling was restricted to meristematic cells in the root tip, suggesting that the antigen was localized only in newly formed cells prior to the maturation of the Pd. It has also been suggested that protein kinases are associated with Pd. In a maize (Zea mays L.) mesocotyl cell wall fraction, a membrane-bound, Ca2þ-dependent protein kinase (CDPK) phosphorylated wall-associated proteins. The kinase is membrane associated and is not extracted by EGTA, NaCl, up to 4 mol/liter LiCl, Triton X-100, or Na2CO3 (pH 11), but is fully extracted with SDS or 8 mol/liter LiCl. Two polypeptides with apparent molecular masses of 51 and 56 kDa cross-react with an Arabidopsis CDPK antiserum and undergo in situ Ca2þ-dependent autophosphorylation on nitrocellulose. The molecular masses of the CDPKs extracted by 8 mol/ liter LiCl from the cell wall fraction are diVerent from those extracted from the cell membrane fraction, suggesting that the wall-associated CDPK is unique to the cell wall fraction. Immunofluorescence microscopy with isolated walls localizes CDPK to discrete punctate loci in the cell wall. Isolated Pd challenged with CDPK antiserum show a pattern of crossreactivity similar to the cell wall fraction. These data suggest that the cell wall-associated CDPK is a putative Pd-associated membrane protein and may be involved in regulating Pd conductivity (Yaholom et al., 1998). In a proteomic analysis of an isolated Pd fraction from Arabidopsis three protein kinase-like proteins were identified (Epel, unpublished observations). Localization studies either with antibodies or GFP fusion proteins are needed to confirm Pd localization of one or more of these protein kinases. Immunocytochemical studies employing antibodies against gap junction proteins led to the suggestion that connexin-homologous proteins may be Pd associated. Employing aYnity-purified antibodies against two diVerent gap junction proteins, connexin-32 and connexin-43, it was shown by indirect immunogold labeling of thin sections of maize mesocotyl tissue that the maize mesocotyl Pd contain two diVerent proteins that cross-react with connexin gap junction antibodies (Yaholom et al., 1991). The connexin 32 antiserum cross-reacted with a 27-kDa maize Pd-associated protein termed PAP27 while an aYnity purified antiserum against connexin-43 labeled a 26-kDa protein termed PAP26. PAP26 immunolocalized along the entire length of the plasmodesma as well as to plasmalemma regions surrounding the plasmodesma orifice. PAP27 immunolocalized to outer regions of the Pd

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(Yaholom et al., 1991). BLAST and FASTA analysis of the Arabidopsis and rice genomes did not reveal the presence of connexin homologous genes, leading us to conclude that the antiserum did not identify connexin homologous proteins in maize and that these antisera probably cross-react with unknown unrelated proteins that contain common epitopes to regions also present in connexin proteins (Epel, unpublished observations).

III. Transport of Macromolecules Through Plasmodesmata The interpretation of electron microscopic (EM) studies of Pd structure purportedly showing the presence of narrow open channels between protein structures within the cytoplasmic sleeve led to a model in which the cytoplasmic sleeve was suYciently occluded that only small channels were open for unimpeded diVusion of molecules from cell to cell. Early microinjection studies with fluorescently labeled probes, carboxyfluorescein, Lucifer yellow, fluorescein-labeled poly-amino acids, and fluorescein-labeled dextrans led to the view that only molecules of less than about 1 kDa could diVuse through the Pd (Erwee and Goodwin, 1984; Goodwin, 1983; Terry and Robards, 1987; Tucker, 1982). Studies with inhibitors of energy metabolism (azide, cyanide, and anaerobiosis) showed that Pd apparently dilate in the presence of low ATP (Cleland et al., 1994; Tucker, 1993). These studies led to the view that Pd are held closed under normal conditions and that upon energy stress Pd dilate, permitting the passage of larger molecules. During the past few years, the belief that Pd diVusionally conduct only small molecules in their unperturbed state has been challenged by various studies showing that Pd allow diVusional traYcking of xenomacromolecules as well as numerous endogenous proteins and RNAs. In addition, there apparently exists an endogenous mechanism for facilitated traYcking of macromolecules. An early indication that Pd between companion cells (CC) and sieve elements (SE) traYc macromolecules came from a study of protein turnover in wheat sieve tubes investigated by [35S]methionine labeling and by the use of aphid stylets to sample the sieve tube contents along a source-to-sink pathway (Fisher et al., 1992). This study showed the presence of a few hundred labeled mobile phloem-sap proteins that exhibited rapid turnover. Presumably these proteins are synthesized in the CC, traYc through the Pd between the CC and SE into the SE, and unload symplastically into sink tissue along the postphloem pathway. Recent studies support this conjecture and indicate that both phloem loading and unloading may be for some proteins passive diVusional (Fisher and Cash-Clark, 2000; Imlau et al., 1999; Kempers and van Bel, 1997; Oparka et al., 1999) while for others

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assisted (Aoki et al., 2002; Balachandran et al., 1997; Ishiwatari et al., 1998; Lee et al., 2003). Recent observations indicate that Pd involved in SE/CC unloading and postphloem transport are passively permeable to macromolecules. Dextrans as large as 16 kDa (Rs ¼ 2.8 nm) following microinjection into sieve tubes were found to be mobile in the maternal postphloem pathway of wheat grains (Fisher and Cash-Clark, 2000) and to traYc between a sieve element and a companion cell in the fascicular stem phloem of Vicia faba (Kempers and van Bel, 1997). The Rs-SEL of 2.8 nm may be a lower limit, as damage, as evident from callose and P-protein plug formation, was noted following microinjection. Similarly, Pd between trichome cells of N. clevelandii conduct fluoresceinlabeled dextrans with a molecular mass of at least 7 kDa (Rs ¼ approximately 2.2 nm) introduced by pressure injection. That macromolecules can passively diVuse through Pd was conclusively demonstrated in experiments employing transgenic Arabidopsis or tobacco plants that express GFP under the control of the companion cell-specific AtSUC2 promoter (Imlau et al., 1999; Oparka et al., 1999). GFP (27 kDa, Rs ¼ 2.8 nm), which is expressed only in CC in source leaves, diVused into the SE and then moved extensively along the postphloem pathway, spreading cell to cell in a diversity of physiological sinks, including young leaves, root tips, and ovules (Imlau et al., 1999; Oparka et al., 1999). It thus appears that the Pd in the CC/SE complex can passively conduct molecules with an Rs of at least 2.8 nm. Likewise, GFP and GFP fusions, when introduced into cells either by microinjection or by ectopic expression following biolistic bombardment, passively diVuse between cells in sink leaves and in some, but not all, source leaves (Crawford and Zambryski, 2000, 2001; Epel, unpublished observations; Oparka et al., 1999; Wymer et al., 2001). Almost all microinjection studies conducted to date probably underestimated the permeability of Pd due to wounding eVects caused by the tissue preparation or injection techniques employed (Radford and White, 2001; Radford et al., 1998; Zellnig et al., 1991). Wounding causes rapid callose deposition at Pd between the cell wall and the plasma membrane, especially in the neck region, causing a narrowing of the Pd (Radford and White, 2001; Radford et al., 1998). Cytological changes were shown to occur in Abutilon nectary trichomes as a result of the microinjection of Lucifer yellow (LYCH) including the introduction of globules within the Pd (Zellnig et al., 1991). Incubation of tissues with 2-deoxy-d-glucose, an inhibitor of callose formation, increased the permeability of Pd (Radford and White, 2001). Wounding eVects are probably greater in microinjection experiments performed with mesophyll cells than with epidermal cells. Microinjection into mesophyll cells necessitates exposing the mesophyll by peeling oV the epidermis: such a procedure almost certainly induces massive injury reactions and major callose formation with a concomitant decrease in Pd passive

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conductivity. It should nevertheless be noted that in the study of Oparka et al. (1999) the microinjection into epidermal cells also probably caused some damage and may have caused callose formation that might have restricted traYcking of higher molecular weight probes (Oparka et al., 1999). Biolistic bombardment also causes damage and probably induces callose formation. However, in the case of biolistic expression of GFP, measurements are performed 16–48 hr postbombardment, possibly allowing for partial recovery of the cell to the wound stress. It has been claimed that low-pressure biolistic bombardment minimizes stress during transfection and that diVerences in movement were noted dependent on the method of particle delivery (Crawford and Zambryski, 2000, 2001; Itaya et al., 2000). It was suggested that the Pd aperture can fluctuate (Crawford and Zambryski, 2000, 2001). It was reported that as a result of low-pressure biolistic bombardment, all cells traYcked the lowmolecular-weight fluorescent probe HPTS acetate (524 Da) while a lesser number of cells traYcked GFP (27 kDa, Rs ¼ 2.8 nm) and still fewer cells traYcked cell to cell a GFP dimer (55 kDa, unknown Stokes radius). These data were interpreted to indicate that within a leaf there is a heterogeneous population of Pd. Some Pd do not conduct GFP but are ‘‘open’’ in that they can conduct small molecules (<1000 Da), other Pd show varying degrees of dilation, some being able to conduct GFP but not larger molecules (termed dilated-low), while others are highly dilated and can conduct molecules as large as a GFP dimer (Crawford and Zambryski, 2000, 2001). An alternative explanation that should be considered is that the diVerences may be the result of various degrees of damage reactions and that diVerent levels of dilation or closure are artifacts due to callose formation or other injury reactions. Intercellular transport of macromolecules was also reported for the characean plants Nitella axilliformis (Kikuyama et al., 1992) and Chara corallina (Plieth and Hansen, 1996). The Pd of these lower plants diVer from those of higher plants in that they do not have desmotubules. Fluorescent probes with molecular weights equal to or less than 45 kDa moved from the injected N. axilliformis cells to neighboring nodal and internodal cells within 24 hr after injection. In C. corallina cells, fura-dextran (10 kDa; Rs ¼ 2.3 nm) was also shown to move cell to cell within 1–10 hr postinjection. The variable lag in cell-to-cell movement was attributed to wounding shock. Larger molecular probes were not tested. The capacity of a plasmodesma to transport molecules is dependent on multiple factors. Molecules that are compartmentalized, bound to subcellular structures such as membranes, cytoskeletal structures, etc. and very large macromolecular complexes, will a priori be immobile. Nuclear-targeted proteins may be mobile if they cycle between the nuclear and cytoplasmic compartments. If a macromolecule is totally compartmentalized to the

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nucleus it will probably not be available for cell-to-cell transport. Intercellular mobility will be dependent, in part, on the coeYcient of cytoplasmic/ nuclear partitioning. For cytoplasmic soluble macromolecules, conductivity will be a function of the electrochemical gradient between cells, Stokes radius, the open cross-sectional area of the cytoplasmic annulus (or desmotubule lumen), the length of the plasmodesma, the hydrophobicity of the molecule, the molecule charge, entropy eVects due to movement though a narrow tunnel containing highly structured water, and other wall eVects. As the molecular dimensions of the macromolecule approach dimensions of the tunnel membrane, charge eVects will become more important. Such diVusion is termed ‘‘restricted diVusion.’’ The diVusional permeability coeYcient of a molecule of Stokes radius Rs for restricted diVusion through a pore of radius r is given by P ¼ (APd/l )Df(Renkin) (Paine et al., 1975) where APd is the eVective open cross-sectional area of Pd, l is the length of Pd, D is the diVusion coeYcient for free diVusion ¼ (RT/N6pZRs), R is the gas constant, T is the absolute temperature, N is Avogadro’s number, Z is the viscosity of the solvent, and Rs is the Stokes radius. f (Renkin) is the Renkin function ¼ (1  a)2 (1  2.1a þ 2.09a3  0.95a5) where a ¼ Rs/r (Renkin, 1954). Note that the permeability coeYcient is inversely correlated with the length (l ) of Pd. Thus, as the cell wall thickens, the apparent Pd permeability will decrease. For molecules with Rs much smaller than r, diVusion will be very rapid, on the order of seconds. For molecules with Rs approaching r, restricted diVusion will be slow and it would take many hours for macromolecule to reach detectable concentrations in adjacent cells (Paine et al., 1975; Terry and Robards, 1987). Many proteins that are soluble in the cytoplasm exist as homooligomers or heterooligomers; if this is the case, the Stokes radius of the oligomer will be the determining size constraint and not the monomer molecular weight of the molecule itself. At present we are not able to determine the coeYcient of conductivity of a single Pd but rather measure the combined conductivity of all Pd. Thus, one must also take into consideration that changes in cell-to-cell transport are not necessarily a function of changes in the properties of the Pd as such but may reflect changes in the number of Pd interconnecting adjacent cells. Thus, the changes in Pd conductivity observed during the sink-to-source transition may either be due to a true change in Pd properties or may reflect only the decrease in the number of Pd interconnecting cells and/or changes in the length of the Pd. Between mesophyll cells there is a major reduction in the number of Pd between cells as the leaf expands and walls separate forming air spaces (Roberts et al., 2001). This is not true for epidermal–epidermal cell contact since there is no wall separation; however, there is a major decrease in Pd between epidermal cells and mesophyll cells during the sink-to-source transition (Roberts et al., 2001). Thus, to

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determine changes in true Pd conductivity that are not due just to changes in the number of Pd, one must perform ratio measurements of conductivity using two fluorescent probes of diVerent molecular weights and diVerent fluorescent properties. Such a system will allow true changes in conductivity to be determined.

IV. Movement Proteins in Macromolecular Transport Through Plasmodesmata A. Viral Movement Proteins Movement proteins (MPs) are classically defined as plant virus-encoded factors that interact with Pd to mediate the intercellular spread of virus infection. We now know that viruses subvert an intercellular communication network that supports the traYcking of a wide spectrum of endogenous proteins and ribonucleoprotein complexes that play non-cell autonomous roles in developmental and physiological processes. Moreover, successful spread of infection appears to rely upon the ability of a virus to counteract innate defense mechanisms of the plant. Movement proteins may thus belong to a larger group of proteins that facilitates the intercellular traYcking of macromolecules through a variety of cellular functions. The first evidence for a virus-coded MP came from studies with the temperature-sensitive Ls-1 strain of TMV that is unable to move cell to cell at a restrictive temperature. Nishiguchi and colleagues (1978) demonstrated that this strain can replicate and assemble normally in leaf cells or protoplasts but cannot move cell to cell in leaves at nonpermissive temperature. The defect was correlated with slight changes in the tryptic peptide map prepared from an in vitro translated 30-kDa protein (Leonard and Zaitlin, 1982) and was later shown to be due to a single base change in the Ls-1 genome, which substituted a serine for a proline residue (Ohno et al., 1983). Plants transgenic for TMV 30-kDa protein were shown to complement for Ls-1 at nonpermissive temperatures (Deom et al., 1987; Meshi et al., 1987) and frame-shift mutations in the 30-kDa gene of the virus gave rise to cell-to-cell movement-defective TMV phenotypes (Meshi et al., 1987). Since then, this protein has been studied in detail and was reported to bind singlestranded nucleic acids (Citovsky et al., 1990, 1992), to accumulate in Pd and to increase their SEL (Atkins et al., 1991; Ding et al., 1992a; Moore et al., 1992; Oparka et al., 1997; Tomenius et al., 1987; Wolf et al., 1989), to localize to the ER and cytoskeletal elements (Heinlein et al., 1995, 1998a; McLean et al., 1995), and to be phosphorylated by cellular kinases (Atkins et al., 1991; Citovsky et al., 1993; Haley et al., 1995; Kawakami et al., 1999, 2003;

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Waigmann et al., 2000; Watanabe et al., 1992). The ability of MP to increase Pd SEL was investigated by injection of fluorescence-labeled dextrans into leaf mesophyll cells of transgenic tobacco plants expressing the protein (Wolf et al., 1989). The Pd in MP-transgenic plants allowed the intercellular diVusion of 10-kDa dextrans, whereas Pd in nontransformed plants restricted the traYcking of dextrans larger than 1.0 kDa (Wolf et al., 1989). The apparent ability of MP to increase Pd SEL was also demonstrated in wild-type plants, upon microinjection of Escherichia coli-purified protein. The injection of MP led to an increase in Pd SEL within a very short time (3–5 min) and it was suggested that MP may interact with an endogenous intercellular transport machinery (Waigmann et al., 1994). Moreover, coinjected dextrans traveled as far as 20–50 cells away from the site of injection. This observation indicated that microinjected MP itself traYcs from cell to cell, leading to modification of Pd quite distant from the injected cell. Experiments in which MP was immunolocalized following microinjection (Waigmann and Zambryski, 1995) or observed upon transient expression of GFP-tagged protein in leaf epidermal cells (Kotlizky et al., 2001) confirmed this hypothesis. However, recent studies indicated that plants and plant tissues have a mosaic nature with respect to the SELs of Pd (Crawford and Zambryski, 2000, 2001; Oparka et al., 1999; Wymer et al., 2001). This should remind us that observed SEL increases may not be caused by MP alone but that the physiological conditions of the assayed plant cells play an equally important role. Published evidence indicates that the ability of MPs to influence or modify Pd SEL is a property of many, if not all, viral MPs. To date, many other MPs, including those of red clover necrotic mosaic dianthovirus (RCNMV), alfalfa mosaic bromovirus (AMV), cucumber mosaic bromovirus (CMV), tobacco rattle tobravirus (TRV), potato potexvirus X (PVX), and the BL1 MP of bean dwarf mosaic geminivirus (BDMV), have been reported to mediate transport of large fluorescent dextrans between plant cells (Angell et al., 1996; Derrick et al., 1992; Fujiwara et al., 1993; Noueiry et al., 1994; Poirson et al., 1993; Vaquero et al., 1994). Moreover, like TMV MP, the MPs of other viruses, including tomato mosaic tobamovirus (ToMV), maize streak geminivirus (MSV), apple chlorotic leaf spot trichovirus (ACLSV), PVX, RCNMV, CMV, and BDMV, have also been observed to move from cell to cell themselves (Ding et al., 1995; Fujiwara et al., 1993; Itaya et al., 1997; Kotlizky et al., 2000; Lough et al., 2000; Noueiry et al., 1994; Satoh et al., 2000; Tamai and Meshi, 2001a). Recent studies substantiated the view that MPs can act as direct or indirect modifiers of Pd conductivity. For example, MPs are able to overcome the down-regulation of Pd SEL during plant development and thus are able to move viral RNA molecules between cells of mature leaves (Oparka et al., 1999). MPs also seem to be able to upregulate Pd that border symplastic domains, as was recently shown by the

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demonstration in transgenic tobacco plants that the CMV 3a MP fused to GFP can traYc out of the symplastically isolated SE–CC via Pd (Itaya et al., 2002). Thus, Pd-mediated traYcking of selected macromolecules can act as a mechanism for symplasmically isolated cells to communicate with one another.

B. Plant Movement Proteins Although much of our knowledge of Pd and the movement of macromolecules comes from studies on the function of viral MPs and on the spread of viral infection, it is clear that Pd are of pivotal importance for endogenous plant processes, such as signal relay, defense, or the transport of nutrients. Recent studies emphasize that intercellular communication through Pd includes the traYcking of protein and RNA macromolecules. Some of the proteins for which there is evidence for movement between cells have been previously shown to act as non-cell autonomous transcription factors during plant development (Jackson and Hake, 1997). Important examples are the maize homeobox-containing transcription factor KNOTTED 1 (KN1) of maize (Jackson et al., 1994; Kim et al., 2002b; Lucas et al., 1995), the Antirrhinum DEFICIENS (DEF) (Mezitt and Lucas, 1996; Perbal et al., 1996), FLORICAULA (FLO) (Mezitt and Lucas, 1996), and GLOBOSA (GLO) (Kragler et al., 1998) proteins, and the Arabidopsis LEAFY (LFY) (Sessions et al., 2000) and SHORT ROOT (SHR) (Helariutta et al., 2000; Nakajima et al., 2001) proteins. Other proteins with the apparent ability to spread between cells include the Cucurbita maxima phloem proteins PP2 (Balachandran et al., 1997), PP 16 (Xoconostle-Cazares et al., 1999), PP36 (Xoconostle-Cazares et al., 2000), Hsc70 chaperones (Aoki et al., 2002), as well as thioredoxin h of rice (Ishiwatari et al., 1998). Since not all transcription factors move between cells, the traYcking of the examples mentioned is likely to be actively controlled. Movement could be controlled either by restricted diVusion or, as proposed for viral MP, by active transport. Previous studies indicated that GFP can diVuse between cells unless targeted to cellular compartments or structures (Crawford and Zambryski, 2000). Thus, if diVusion of proteins would occur by default, the spread of proteins not destined for traYcking into adjacent cells could be restricted through retention, i.e., by complexing a protein with other factors. Such a retention mechanism could apply to the Arabidopsis MADS domain protein APETALA3 (AP3), the ortholog of DEF, and its heterodimerization partner PISTILLATA (PI), both of which are similar in size to GFP (Jenik and Irish, 2001) and form stable complexes with other MADS box proteins (Egea-Cortines et al., 1999; Honma and Goto, 2001; McGonigle et al., 1996; Riechmann et al., 1996). An active

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mechanism for intercellular transport, however, is suggested, for example, in the case of the KN1 protein that was reported to increase the SEL of Pd similar to viral MP, if microinjected into tobacco mesophyll cells (Lucas et al., 1995). This finding was substantiated by a recent report (Kim et al., 2002b) showing that a GFP:KN1 fusion protein expressed in Arabidopsis can traYc between the cell layers of the apical meristem, where it usually functions. In contrast, movement did not occur in the case of a smaller GFP:YFP fusion or of a fusion of GFP with the M6 mutant of KN1, which does not traYc in tobacco microinjection assays (Lucas et al., 1995). In addition to the ability to increase the SEL of Pd, the KN1 protein was also reported to cause intercellular movement of coinjected KN1 mRNA. The traYcking of Kn1 mRNA also occurred if the mRNA was coinjected with the MP of CMV (Lucas et al., 1995). The KN1 protein thus appears to have properties remarkably similar to those of viral MP and could represent a class of endogenous, MP-like proteins. The hypothesis that plants in fact do encode such proteins is also supported by experiments in which antibodies raised against the MP of RCNMV were used to identify cross-reacting proteins from pumpkin phloem exudate. The sequence of the protein termed CmPP16 showed similarity to the RCNMV MP. Moreover, by microinjection into leaf mesophyll, the protein was shown to mediate its own cell-to-cell movement and that of its mRNA. Both the protein and the mRNA were detected in phloem exudate derived from a cucumber scion grafted onto a pumpkin stock, but not in the exudate of nongrafted cucumber, indicating that CmPP16 mRNA and protein are systemically transported (Xoconostle-Cazares et al., 1999). Another interesting case is the Cucurbita maxima phloem protein CmPP36, a 36-kDa cytochrome b5 reductase whose expression is confined to companion cells. In the phloem sieve elements, only an N-terminally truncated (31-kDa) form of this protein is found. Microinjection experiments indicated that that the truncated form has the capacity to induce an increase in Pd SEL and to move cell to cell, whereas the full-length protein displayed neither activity. Apparently, proteolytic processing is required before this protein can enter the phloem long-distance translocation stream (Xoconostle-Cazares et al., 2000). Whether this type of control applies to other examples of macromolecular traYcking remains to be shown. Nevertheless, the examples mentioned indicate that plants encode endogenous MPs and suggest that viral MPs function to exploit an existing plasmodesmal mechanism for macromolecular movement. Recently, this hypothesis gained strong support by the identification of NON-CELL AUTONOMOUS PATHWAY PROTEIN1 (NtNCAPP1) that interacts with CmPP16 as well as other non-cell autonomous proteins (Lee et al., 2003). Importantly, upon expression in transgenic plants or upon cellular microinjection, a mutant of this protein interfered with the ability of CmPP16 and also of TMV MP (but

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not of the CMV MP or KN1) to induce an increase in Pd SEL. The transgenic plants expressing the mutant protein exhibited abnormal developmental phenotypes. These findings provide support for the concept that cellto-cell communication via Pd occurs in a regulated manner. Several recent studies confirmed the cell-to-cell and systemic traYcking of RNA species endogenous to the plant, including gene transcripts and RNA-based gene silencing signals (Fagard and Vaucheret, 2000; Kim et al., 2001; Lucas et al., 2001; Mlotshwa et al., 2002; Ruiz-Medrano et al., 1999; Waterhouse et al., 2001). It will be important to identify the mechanisms and MPs that support the specific transport and targeting of these RNA molecules.

V. Intercellular Transport of RNA Molecules A. Examples and Role of Intercellular RNA Transport In addition to proteins, Pd mediate the intercellular traYcking of RNA molecules. The Pd-mediated cell-to-cell movement of RNA viruses is the most compelling example. Apparently, viruses have adapted to intercellular communication pathways to transport their RNA genomes between cells and to spread infection. Examples of studies indicating the systemic spread of endogenous RNA molecules have already been mentioned and include the spread of specific RNA transcripts in C. maxima (Ruiz-Medrano et al., 1999; Xoconostle-Cazares et al., 1999) and the spread of maize KN1 mRNA (Lucas et al., 1995). Another example is the mRNA of the sucrose transporter, SUT1, which is found in enucleate sieve elements presumably following its transport from associated companion cells (Ku¨ hn et al., 1997). The systemic spread of C. maxima mRNAs was demonstrated by heterologous grafting and in situ PCR, whereas the evidence for the spread of maize KN1 mRNA is limited to microinjection assays in tobacco that involved the coinjection of labeled and in vitro-transcribed KN1 mRNA together with E. coli-produced viral MP or KN1 protein into mesophyll cells (Lucas et al., 1995). In vivo evidence and insight into the functional significance for the spread of KN1 mRNA in maize has yet to be presented. Results of recent double labeling experiments to localize KN1 mRNA and KN1 protein in the maize shoot apex rather argued against the in vivo movement of KN1 mRNA (Jackson, 2002). Thus, although the spread of protein molecules in plants appears to be a general and now widely accepted phenomenon, the evidence that the non-cell autonomous spread of gene transcripts has indeed a function remains scarce. Compelling evidence for a function of mRNA traYcking was provided in a report by Kim and colleagues (2001) that describes grafting experiments in

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tomato in which a rootstock-encoded transcript of the dominant Mouse ears (Me) leaf mutation accumulated in the meristem of the wild-type scion, where it specified a change in leaf morphology. However, although this finding is supportive for a functional role of non-cell autonomous gene transcripts, the results were only correlative and not conclusive. The morphological changes in the scion may have been caused by the traYcking of a graft-transmissible signal other than the Me transcript. Moreover, the transcript was derived from a dominant, ectopically expressed, gain-of-function KNOX-fusion gene. Thus, whether mRNA transport represents a function that is essential during development remains an open question. The answer to this question will rely on the analysis of a regulatory role of non-cell autonomous mRNA transcripts that are derived from wild-type genes rather than mutant genes. In addition to playing a role in virus movement and, potentially, in plant development, the traYcking of RNA molecules appears to have a major role in the non-cell autonomous spread of posttranscriptional RNA silencing, a nucleotide sequence-specific defense mechanism that can target both cellular and viral mRNAs. RNA silencing can be suppressed by several virusencoded proteins (Anandalakshmi et al., 1998; Beclin et al., 1998; Brigneti et al., 1998; Kasschau and Carrington, 1998; Li and Ding, 2001; PfeVer et al., 2002; Pruss et al., 1997; Silhavy et al., 2002; Vance and Vaucheret, 2001) and is closely related to RNA-mediated virus resistance and cross-protection in plants (Covey et al., 1997; Lindbo et al., 1993; RatcliV et al., 1997, 1999). Therefore, it has been suggested that RNA silencing represents a natural antiviral defense mechanism (Voinnet, 2001). RNA silencing also targets the expression of transgenes and is implicated in the control of transposable elements and in plant development (Llave et al., 2002a). The silencing mechanism appears to be directed by double-stranded RNA and is associated with the production of two classes of short 21–24 and 24–27 nt RNAs (Hamilton and Baulcombe, 1999; Hamilton et al., 2002; Mallory et al., 2002) and with a diVusible or transported sequence-specific signal that mediates the spread of PTGS through the plant (Fagard and Vaucheret, 2000; Mlotshwa et al., 2002; Voinnet and Baulcombe, 1997; Voinnet et al., 1998). Although the exact nature of the silencing signal is yet to be demonstrated, the sequence specificity of the signal is thought to arise from an RNA component (Palauqui et al., 1997). Given the precedence of the systemic transport of viral genomes and endogenous mRNA molecules, the silencing signal may be represented by a large RNA molecule, although elegant studies employing viral silencing suppressors correlated systemic spread with the presence of the 25–26 nt class short RNA (Hamilton et al., 2002). However, the debate will continue since other studies could not detect any correlation between systemic silencing and the accumulation of short or long silencingassociated RNAs (siRNAs) (Mallory et al., 2001, 2003). The spread of the

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silencing signal is likely to occur through Pd, since silencing does not progress into cells that are symplastically isolated from surrounding cells, such as guard cells (Voinnet et al., 1998). The biochemical pathway that leads to the production of siRNAs from dsRNA precursors involves the enzyme Dicer, a molecule with an N-terminal helicase and tandem C-terminal ribonuclease III domains (Bernstein et al., 2001; Hammond et al., 2000; Knight and Bass, 2001) and also Argonaute proteins, which are found in the RNA-induced silencing complex (RISC) that performs siRNA-guided RNA degradation (Hammond et al., 2000; Zamore et al., 2000). RNA silencing is part of a larger set of pathways involving small RNAs (Hannon, 2002; Voinnet, 2002). A similar biochemical pathway is involved in the processing of 22 nt long microRNAs (miRNAs) that are involved in developmental control in plants and animals. miRNAs are structurally similar to siRNAs except that they arise from structured precursor transcripts derived from miRNA genes (Pasquinelli and Ruvkun, 2002). Nearly 200 miRNA genes have been found in animals and plants (Ambros, 2001; Jones, 2002; Lagos-Quintana et al., 2001, 2002; Lau et al., 2001; Lee and Ambros, 2001; Llave et al., 2002a; Moss and Poethig, 2002; Mourelatos et al., 2002; Park et al., 2002; Reinhard et al., 2002). MicroRNAs can bind to mRNAs that have complementary sequences, and thus can regulate gene expression by modulating translation of the target mRNA (Olsen and Ambros, 1999; Wightman et al., 1993). In plants, many of the miRNAs have near perfect matches to known genes that are likely to serve as potential targets (Llave et al., 2002a; Rhoades et al., 2002). As was shown for miR171 from Arabidopsis, perfect base pair interaction can trigger site-specific mRNA cleavage by a mechanism that resembles siRNA-guided cleavage (Llave et al., 2002b). Most of the predicted miRNA target genes in plants encode transcription factors of which many are involved in various aspects of plant development. It is therefore not surprising that caf/dcl-1 and hen1 mutations, which aVect genes that encode proteins required for miRNA accumulation such as DICER-LIKE1, have strong developmental phenotypes (Kidner and Martienssen, 2003; Park et al., 2002; Reinhard et al., 2002). However, a wonderful link can be made between the developmental defects of these mutants, the failure to accumulate miRNAs, and the predicted miRNA targets. The predicted targets, PHABULOSA and PHAVOLUTA, are particularly intruiging, since they are both involved in the perception of radial positional information in the leaf primordium that determines abaxial and adaxial leaf fates (McConnell et al., 2001). Importantly, PHABULOSA exhibits asymmetric distribution with respect to the abaxial and adaxial sides of leaf primordia suggesting the possibility that PHB expression may be regulated by a gradient of non-cell autonomous miRNA molecules that traYc between cells via Pd. In fact, the overexpression in tobacco of the 25-kDa MP of PVX gives rise to radial leaves,

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a phenotype common to dominant PHB alleles in Arabidopsis (Foster et al., 2002). This finding substantiates the hypothesis that miRNAs may act in a non-cell autonomous fashion to direct asymmetric gene expression and plant development. Moreover, this finding also suggests that proper miRNA function may be perturbed in tissues infected by viruses. Indeed, infection of Arabidopsis by turnip mosaic virus (TuMV) or expression of its RNA silencing suppressor (P1/Hc-pro) (Anandalakshmi et al., 1998; Brigneti et al., 1998; Kasschau and Carrington, 1998) was shown to trigger developmental defects and to inhibit miRNA-guided cleavage of mRNA targets encoding members of several families of transcription factors (Kasschau et al., 2003). Thus, interference with miRNA-guided functions may contribute to the production of disease symptoms by plant viruses and may also explain why certain viruses cause developmental abnormalities during infection.

B. Viruses for Studying Cellular RNA Transport Mechanisms The cellular mechanism of RNA transport through Pd is likely to depend on plant factors that stabilize, chaperone, and target the RNA to the Pd, as well as on factors that modify Pd and support the translocation process. Although a general role of RNA as an informational macromolecule for cell-to-cell and systemic signaling is apparent (Lucas et al., 2001), viruses and also viroids represent the prevailing model to study the cellular mechanism of RNA transport. These systems provide the advantage that both the exact nature of the transported RNA and the functional significance for transport are known. Importantly, viroids do not encode any protein, indicating that the spread of their circular RNA genomes relies on an existing cellular machinery for intercellular RNA transport. The movement of the much larger viruses depends, in addition, on the aforementioned virus-encoded MPs. These proteins provide a direct key to address the molecular nature of cellular mechanisms that facilitate viral and endogenous nucleic acid transport. As will be summarized in the following section, viruses use diVerent mechanisms for movement though Pd. Subsequent sections will then concentrate on the movement of RNA viruses as they reveal insights into the cellular mechanism of RNA transport. 1. Mechanisms Used by Viruses for Cell-to-Cell Movement Viruses use diVerent mechanisms for movement through Pd. For instance, cell-to-cell transport of several ssRNA viruses [i.e., como-, nepo-, olea-, alfamo-, bromo-, and trichoviruses (Grieco et al., 1999; Kasteel et al., 1996, 1997a; Ritzenthaler et al., 1995; Satoh et al., 2000; van Lent et al., 1991; Wieczorek and Sanfacon, 1993; Zheng et al., 1997)], ssDNA viruses

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[i.e., tospoviruses (Storms et al., 1995)], and dsDNA plant viruses [i.e., caulimoviruses (Kasteel et al., 1996; Perbal et al., 1993) and badnaviruses (Cheng et al., 1998)] involves a ‘‘tubule-guided’’ transport of mature virions through Pd that are structurally modified by a tubule made of virus-encoded MP (Kasteel et al., 1997b; Wellink et al., 1993). In contrast, other RNA viruses (including tobamo-, diantho-, beny-, tobra-tombus-, and hordeiviruses) are believed to move from cell to cell in the form of a ribonucleoprotein complex (vRNP). Viruses belonging to this group do not require CP (Dawson et al., 1988; Siegel et al., 1962; Takamatsu et al., 1987) and the movement process does not involve major changes in Pd structure. Pd of TMV MP-transgenic plants contain fibrous material that can be labeled with anti-MP antibodies (Ding et al., 1992a; Moore et al., 1992). These fibers may be comparable to the tubular arrangement of MP-containing fibers that have been observed to form across intercellular junctions in MP-transgenic cyanobacteria (Heinlein et al., 1998b). Some other viruses, including CMV (Suzuki et al., 1991), tobacco etch potyvirus (Dolja et al., 1994, 1995), and several potexviruses (Chapman et al., 1992; Foster et al., 1992; Sit and AbouHaidir, 1993) do not form tubules but do require CP for cell-to-cell movement. In these cases, however, the role of the CP in the movement processes is not well understood. For example, the mechanism for the spread of potexviruses is controversial. Studies on the potexvirus white clover mosaic virus (WCIMV) led to the suggestion that movement occurs in the form of a vRNP, i.e., similar to TMV (Lough et al., 2000). However, previous studies on PVX demonstrated that CP was cotransported along with the spread of infection and correlated with the presence of fibrillar material, immunoreactive with virion-specific antisera, within the Pd. These findings rather indicated that the movement of PVX occurs in the form of virions. Certainly, the movement mechanism is tubule independent and thus distinct from the movement via tubules of, for example, nepoviruses and comoviruses (Santa Cruz et al., 1998). It has been suggested that a tubule-independent mechanism may be specifically adapted for the transport of filamentous virion particles (Santa Cruz et al., 1998). In fact, several filamentous viruses have been localized to the plasmodesmal channel (e.g., Esau et al., 1967; Weintraub et al., 1976). However, whether size and shape of virions act as determinants for a tubule-guided versus a tubule-independent mechanism remains to be seen. 2. Movement of Viruses Containing the Triple Gene Block Members of several virus families (potex-, carla-, hordei-, allexi-, fovea-, beny-, pomo-, and pecluviruses) encode three ‘‘movement proteins’’ organized in a ‘‘triple gene block’’ (TGB). TGB-encoded proteins are referred to as TGBp1, TGBp2, and TGBp3, according to the position of their genes

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(Solovyev et al., 1996a). Mutational analyses of infectious cDNA clones of virus genomes demonstrated that none of the three TGB proteins is essential for replication and that each in some manner enhances virus movement (Beck et al., 1991; Gilmer et al., 1992; Herzog et al., 1998; Petty and Jackson, 1989; Petty et al., 1990). Although several potexviruses need the CP for movement, there is no correlation between the presence of a TGB and the requirement of CP for movement, since members of the hordei- and benyvirus groups were shown not to require the CP for cell-to-cell movement (Petty and Jackson, 1989; Quillet et al., 1989). The particular roles of each of the TGBs in viral movement have been studied most extensively by using PVX and other representative viruses as model systems (Morozov and Solovyev, 2003). The TGBp1 proteins bind single-stranded nucleic acids (Bleykasten et al., 1996; Donald et al., 1997; Kalinina et al., 1996, 2001; Lough et al., 1998; Rouleau et al., 1994) and have RNA helicase activity in vitro (Kalinina et al., 2002). The proteins also bind NTP and have Mg2þ-dependent NTPase activity (Bleykasten et al., 1996; Donald et al., 1997; Kalinina et al., 1996; Rouleau et al., 1994; Solovyev et al., 1999), which is required for helicase activity (Kalinina et al., 2002). Structures composed of viral RNA and TGBp1 were isolated from hordeivirus-infected plants (Brakke et al., 1988), suggesting that the RNAbinding activity of TGBp1 is responsible for formation of movementcompetent vRNPs. Since PVX and other potexviruses require the CP for movement, the TGBp1 of these viruses may either interact with vRNPs that also contain CP (Lough et al., 1998, 2000) or may bind virions for transport through Pd (Atabekov et al., 2000; Santa Cruz et al., 1998). The potexviral TGBp1 is able to interact with Pd and to increase their SEL but requires TGBp2 and TGBp3 for its targeting to Pd (Angell et al., 1996; Lough et al., 1998, 2000; Morozov et al., 1999; Yang et al., 2000). Mutational analysis of the protein revealed that its ability to dilate Pd is correlated with ATP binding and/or hydrolysis rather than with helicase activity. However, since the protein is cotranslocated with viral RNA through Pd it is possible that the helicase activity of the protein supports movement through the pore by unwinding the viral genome. It has been proposed that during or in advance of this process, the protein may also displace cellular proteins to facilitate the formation of a movement-competent complex (Morozov and Solovyev, 2003). Several studies involving cellular fractionation or in vivo expression of GFP fusion proteins have indicated that the TGBp2 and TGBp3 proteins are associated with cellular membranes (Cowan et al., 2002; Donald et al., 1993; Gorshkova et al., 2003; Morozov et al., 1991; Niesbach-Klosgen et al., 1990). Whereas TGBp2 tends to interact with ER (Cowan et al., 2002; Solovyev et al., 2000) as well as with motile vesicles (Solovyev et al., 2000), TGBp3 associates with peripheral membrane bodies in the vicinity of the cell wall

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and Pd (Cowan et al., 2002; Gorshkova et al., 2003; Solovyev et al., 2000). When coexpressed with TGBp3, TGBp2 is retargeted to peripheral bodies that are similar to the bodies observed in cells expressing TGBp3 alone (Solovyev et al., 2000; Zamyatnin et al., 2002). The TGBp2 and TGBp3 proteins of potato mop-top virus (PMTV) interact in the yeast two-hybrid system (Cowan et al., 2002), but the interaction between TGBp2s and TGBp3s is rather mediated by structural features than by protein sequence. For example, a TGBp3 derived from one virus can target a TGBp2 derived from a diVerent virus (Solovyev et al., 2000), and TGBp3 of poa semilatent virus (PSLV) was shown to target even totally unrelated membrane-bound MPs to peripheral bodies (Zamyatnin et al., 2002). Based on the observation that the presence of homologous TGBp2 and TGBp3 are required for the targeting of TGBp1 to Pd (Erhardt et al., 2000; Lauber et al., 2001; Lawrence and Jackson, 2001; Lough et al., 1998, 2000; Solovyev et al., 1999), it was proposed that TGBp2 and TGBp3 play a role in the intracellular delivery of TGBp1-formed transport-competent vRNPs to Pd (Morozov and Solovyev, 2003). Recent reports indicated that the TGB proteins have additional functions in supporting the spread of infection. The TGBp1 protein of PVX has been shown to inhibit the spread of the systemic RNA silencing signal and thus appears to be capable of supporting virus movement indirectly by interfering with RNA silencing as a plant defense reponse (Voinnet, 2001; Voinnet et al., 2000). The TGBp2 of PVX was reported to facilitate movement of GFP between adjacent epidermal cells (Tamai and Meshi, 2001b), suggesting that this protein is able to manipulate Pd SEL. Consistently, the same protein was recently shown to interact with TIP, a host protein regulator of the callosedegrading enzyme b-1,3-glucanase (Fridborg et al., 2003). Studies with TMV demonstrated that coexpression of this enzyme during infection enhances the spread of virus, presumably by increasing the conductivity of Pd upon removal of callose (Bucher et al., 2001). Thus, it appears possible that PVX infection accelerates the turnover of callose in the cell wall near Pd to facilitate movement. 3. Movement of TMV In contrast to the TGB viruses, TMV employs only one virus-encoded MP to facilitate cell-to-cell spread (Deom et al., 1987). Moreover, intercellular spread is CP independent (Dawson et al., 1988; Siegel et al., 1962; Takamatsu et al., 1987), establishing that the virus spreads in a nonvirion form. Both in vitro and in vivo experiments provided evidence that the MP of this virus supports viral RNA movement through the formation of a ribonucleoprotein complex (vRNP). This protein was shown to bind both RNA and ssDNA in vitro resulting in protein:RNA complexes with a thin and elongated

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appearance (Citovsky et al., 1990, 1992). The estimated diameter of the complex was determined to be 2.0–2.5 nm (Citovsky et al., 1992). Studies by atomic force microscopy indicated that MP binds RNA in a biphasic mode in which a lower MP:RNA ratio gives rise to a ‘‘bead on a string’’ structure and a higher MP:RNA ratio results in a higher density of MP in the complex and the conversion of the ‘‘bead on a string’’ structure to a ‘‘thick string’’ structure in which clusters of MP may be formed through cooperative interactions (Kiselyova et al., 2001). The in vivo formation of a vRNP was supported by reports indicating that complexes containing vRNA and MP could be isolated from TMV-infected plants but not from plants inoculated with virus encoding a temperature-sensitive MP and extracted at nonpermissive temperature (Dorokhov et al., 1983, 1984). Moreover, complexes formed between vRNA and recombinant MP in vitro were shown to be nontranslatable both in vitro and also in vivo, upon electroporation into protoplasts. In contrast, translation and replication occurred in planta, suggesting that the vRNP undergoes modification upon passage through Pd (Karpova et al., 1997). Treatment of complexes in vitro with protein kinase C or with cell wall–associated kinase alleviated translational inhibition in protoplasts, suggesting the involvement of MP phosphorylation (Karpova et al., 1999). Moreover, similar to the MPs of other viruses, the MP of TMV moves between cells upon microinjection, and also mediates the transport of coinjected nucleic acids (Ding et al., 1995; Fujiwara et al., 1993; Noueiry et al., 1994; Waigmann et al., 1994). Experiments involving microinjection of N. clevelandii trichome cells provided evidence that MP associates directly with the molecule to be transported, rather than by activation of trans-acting factors. These experiments demonstrated that microinjected b-glucuronidase (GUS) can move between trichome cells only when fused to MP but not when coinjected with MP as a free protein (Waigmann and Zambryski, 1995). Although it is possible that GUS may form immobile oligomeric complexes when injected as a free protein, the experiments suggest that MP binds vRNA in vivo to mediate its transport. However, available evidence to date for the in vivo formation of a vRNP remains rather circumstantial and awaits further and more direct support by in vivo experiments. The cell-to-cell transport of the proposed vRNP certainly depends on host cell components, on factors whose identity remains to be revealed, and on the presence of MP. Even in young (sink) leaves that have Pd able to transport macromolecules, MP is required for virus spread (Oparka et al., 1999), suggesting that vRNA movement depends on MP and probably on additional MP-interacting host functions. First attempts to localize MP in infected cells and to identify intercellular targets of the protein employed immunoelectron microscopy (Atkins et al., 1991; Meshi et al., 1992; Moore et al., 1992; Tomenius et al., 1987) and biochemical fractionation using virus-infected

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tissues and MP-transgenic plants (Deom et al., 1990; Moore et al., 1992; Moser et al., 1988). According to these studies, the MP was present in cell wall– and plasma membrane–rich fractions and was localized to branched Pd. Later biochemical analyses suggested that the MP is associated with the microsomal fraction as an integral membrane protein (Reichel and Beachy, 1998), a notion that became further supported by biochemical studies using E. coli-purified protein that led to the suggestion that MP contains two protease-insensitive a-helical trans-membrane domains (Brill et al., 2000). More insight into in vivo associations of MP with host cell factors became possible with the introduction into plant biology of GFP of Aequorea victorea (Baulcombe et al., 1995; HaseloV and Amos, 1995; Niedz et al., 1995). At this time, CP-deficient TMV derivatives expressing the MP as a functional MP:GFP fusion protein (TMV–MP:GFP) (Epel et al., 1996a; Heinlein et al., 1995) were developed that enabled the analysis of infection sites as well as examination of the subcellular localization and function of MP during vRNA replication and movement in living plant leaf tissue (Heinlein et al., 1995, 1998a; Padgett et al., 1996). Infection in leaves of susceptible Nicotiana species produced radially expanding fluorescent infection sites. The leading edge of theses sites reflects the leading front of the spreading infection, as was shown by experiments involving manual incisions to the leaf lamina. These incisions if made just beyond the leading edge of fluorescence interrupted further spread of infection but allowed further spread if made just behind the leading fluorescent cells (Oparka et al., 1997). These results also indicated that in virally infected tissue MP does not move cell to cell far ahead of infection. In contrast, in the absence of infection, when MP was either microinjected (Waigmann and Zambryski, 1995; Waigmann et al., 1994) or transiently expressed (Kotlizky et al., 2001), it spread extensively cell to cell. These findings support the idea that during viral infection, newly synthesized MP associates with vRNA during replication and spreads in the form of an infectious vRNP complex rather than as free protein. However, since the MP is able to accumulate in Pd and to modify their SEL in the absence of infection [e.g., as shown in transgenic plants (Ding et al., 1992a; Moore et al., 1992)], it appears conceivable to suggest that a portion of the MP may move one or two cells ahead of infection to modify Pd in yet noninfected cells in order to prepare them for the spread of newly synthesized vRNPs (Fig. 4). The infection sites caused by TMV–MP:GFP appear in the form of fluorescent rings, whereas infection sites of virus constructs producing free GFP driven by the CP subgenomic promoter (TMV–
C-GFP) expand in the form of slightly smaller but highly fluorescent disks (Padgett et al., 1996; Sze´ csi et al., 1999). These diVerences in the appearance of infection sites are consistent with fluorimetric measurements using infected protoplasts, which demonstrated that MP:GFP accumulated only transiently during infection, with a peak at about 24 hr postinfection (hpi) (Epel et al., 1996a).

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FIG. 4 A model summarizing potential functions of TMV MP during intercellular spread of its RNA genome from newly infected cells into noninfected cells. The top part of the figure shows three cells of which the one in the center illustrates events in newly infected cells. Replication of viral RNA and synthesis of viral proteins occur in association with the ER. During this early stage of infection the MP is strongly produced and associates with viral RNA to form a vRNP

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This pattern of accumulation is probably due to the specific degradation of MP since treatment of virus-infected protoplasts with inhibitors of the 26S proteasome led to accumulation of ubiquitinylated MP, but not of ubiquitinylated replicase or CP (Reichel and Beachy, 2000). An analysis of the time course of MP:GFP targeting was obtained by the sequential examination by fluorescence microscopy of cells within infection sites produced by TMV–MP:GFP in N. benthamiana leaves with cells at the leading front of the infection site representing the earliest stages of the infection and progressively more inner cell layers in the infection site representing progressively later stages of the infection (Heinlein et al., 1995, 1998a; Oparka et al., 1997). During early stages of the infection, MP:GFP accumulates in Pd and also transiently associates with the ER. Later on, the protein accumulates in ER-associated inclusion bodies and associates with microtubules. Finally, MP:GFP fluorescence disappears from all locations except from Pd. Similar associations were observed in cells infected with the related tomato mosaic tobamovirus Ob (Heinlein et al., 1995; Padgett et al., 1996). TMV constructs that produce lower levels of MP:GFP were not aVected in movement eYciency as was expected, since no more than 2% of the amount of MP produced during TMV infection is suYcient for the maximum rate of virus spread in the inoculated leaf (Arce-Johnson et al., 1995). In cells of weakly fluorescent infection sites of such viruses, Pd were still strongly fluorescent, whereas fluorescence associated with microtubules and bodies was faint, if detectable at all (Heinlein et al., 1998a), implying that

particle. The particles, which may contain ER membranes, motor proteins, ribosomes, and other factors, associate with the cytoskeleton that targets the complex to Pd. A fraction of the MP may target Pd by infection (viral RNA)-independent mechanisms, for example, by association with pectin methylesterase (PME) that is secreted. The Pd at the leading edge have an increased SEL (GO!), permitting the spread of the vRNP complex into noninfected cells. Upon movement through the channel and association with ER, the vRNP disassociates to allow translation and replication of the viral RNA. Free MP may spread through Pd along with the vRNP and the secretory pathway may again support the targeting of the protein to Pd, allowing the MP to increase the SEL of Pd of the now newly infected cell. As infection continues, the function of MP to facilitate RNA transport may change to a function to inhibit RNA transport. As shown in the cell on the left, the ability of MP to increase Pd SEL is down-regulated (STOP!). Perhaps in association of BP2C, a microtubule-associated protein, the proteins form nondynamic complexes with microtubules, presumably to inhibit microtubule-dependent defense responses. At this stage, large quantities of CP are produced to protect viral RNA also through encapsidation. Putative events at the level of Pd are illustrated by the panels in the middle of the figure. During early stages of infection, MP associates with Pd wall-associated host factors and dilates the Pd channel by a yet unknown function. Actin microfilaments and myosin motors located in Pd support the transport of the vRNP complex into the neighboring cell. Late during infection, the MP is posttranslationally modified and is no longer able to dilate the Pd channel. The viral RNA is encapsidated into virions and is no longer transported.

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high levels of accumulation of MP:GFP in bodies and on microtubules are not required for the spread of infection (Heinlein et al., 1998a). The inclusion bodies, which may be derived from cortical ER to which MP localizes very early during infection (Heinlein et al., 1998a; Ma´ s and Beachy, 1999), contain replicase (Heinlein et al., 1998a) and viral RNA (vRNA) (Ma´ s and Beachy, 1999) and thus have been proposed to represent sites of virus replication and protein synthesis. Earlier studies have shown that TMV replication complexes, and also PVX replication complexes, copurify with membrane extracts from infected cells (Doronin and Hemenway, 1996; Nilsson-Tillgren et al., 1974; Osman and Buck, 1996; Ralph et al., 1971; Watanabe and Okada, 1986; Young and Zaitlin, 1986; Young et al., 1987). Membranes are also the site of replication of other viruses, such as brome mosaic virus (Restropo-Hartwig and Ahlquist, 1996), tobacco etch virus (Schaad et al., 1997), peanut clump virus (Dunoyer et al., 2002), grapevine fanleaf virus (Ritzenthaler et al., 2002), and poliovirus (Bienz et al., 1994). Association of virus replication with membranes may provide a means to configure the replication complex (Osman and Buck, 1996, 1997), or to compartmentalize it in order to coordinate and regulate eYcient virus translation, replication, and movement, and also to protect the virus against the innate defense responses of the host. Alternatively, it may be that inclusion bodies function not as a protective mechanism for the virus, but rather in response to innate defense reactions. Such responses are exemplified by Mx proteins that function as mediators of innate resistance to RNA viruses in animals and humans by trapping and sorting viral components to subcellular locations where they become unavailable for further virus propagation (Haller and Kochs, 2002). Several reports provide evidence suggesting that the formation of inclusion bodies from infected ER might be dispensable for replication and movement. For example, evidence has been presented indicating that bodies do not form in the absence of MP (Ma´ s and Beachy, 1999; Reichel and Beachy, 1998). Yet, TMV mutants that lack MP replicate normally (Meshi et al., 1987). Moreover, a TMV derivative encoding a mutant but functional MP:GFP was reported to cause infection in N. benthamiana leaves despite the absence of MP:GFP-containing inclusion bodies (Boyko et al., 2000c). Protoplasts infected with another TMV derivative that encodes an epitope-tagged wildtype MP do not show the presence of MP-containing inclusion bodies upon staining the cells with anti-HA antibody, suggesting that MP:GFP-containing inclusion bodies observed in TMV–MP:GFP-infected cells may be caused by the GFP moiety or the prolonged expression profile of the fusion protein (Boyko and Heinlein, unpublished observations). However, independent of whether the ER-derived inclusion bodies have a role during infection, the ER network has important functions. Supporting cell biological evidence came from studies showing that viral RNA of an MP-deficient viral construct

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localized to ER. This suggests that ER association is an intrinsic property of vRNA and/or replicase (Ma´ s and Beachy, 1999) and is also in line with the initiation of cellular infection as well as replication in association with the ER. Recent studies indicate that the viral replicase coding region has a role in cell-to-cell spread of the virus (Hirashima and Watanabe, 2001). This observation may be consistent with the proposal that the ER-resident replicase participates in movement by conveying replicated viral genomes to the MP for vRNP complex formation. The colocalization of MP with microtubules (Heinlein et al., 1995) and also with microfilaments (McLean et al., 1995) suggested the involvement of cytoskeletal elements in plasmodesmal targeting and cell-to-cell movement of the vRNP (Carrington et al., 1996; Heinlein et al., 1995; Zambryski, 1995). This hypothesis is in agreement with observations in many diVerent biological systems that the coordinated activities of cytoskeleton components are responsible for the specific transport of RNAs, as well as the anchoring of RNAs at their final locations (e.g., Arn and Macdonald, 1998; Bassell and Singer, 1997; Bassell et al., 1999; Gavis, 1997; Hazelrigg, 1998; Hovland et al., 1996; King et al., 1999; Kloc et al., 2001; Oleynikov and Singer, 1998; Wilhelm and Vale, 1993). In vivo studies using TMV derivatives encoding functional, dysfunctional, and temperature-sensitive mutants of MP fused to GFP confirmed that the ability of MP to associate with microtubules is a functional requirement for the intercellular spread of infection (Boyko et al., 2000a,b,c, 2002; Kotlizky et al., 2001). Using infected protoplasts and a combination of antibody labeling and in situ hybridization procedures, Ma´ s and Beachy (1999) showed that vRNA localizes to microtubules in a MP-dependent manner. A subsequent study, again in protoplasts, demonstrated the mislocalization of vRNA in cells expressing a mutant MP [TAD5 (Kahn et al., 1998)] that binds vRNA but fails to associate with microtubules (Ma´ s and Beachy, 2000). These studies suggested a role of MP in mediating the association of vRNA with microtubules. The association of MP with microtubules appears to be direct and has been observed in protoplasts and mammalian cells transfected with MP-encoding DNA constructs (Boyko et al., 2000a; Heinlein et al., 1998a; Kotlizky et al., 2001), indicating that neither virus infection nor any plant-specific host factor is required for association. The binding of MP to microtubules also occurs in vitro (Ashby, 2003), and in vivo experiments using temperaturesensitive mutants indicated a potential role of tubulin mimicry in this association (Boyko et al., 2000a). The highly fluorescent in vivo complexes seen in late infection appear to be in a nondynamic state as they resist the treatment with cold, freezing, and thawing, as well as high concentrations of calcium and sodium salts (Boyko et al., 2000a). Similarly in transfected mammalian cells, the MP:microtubule complex resists treatments with cold as well as with high millimolar concentrations of microtubule-disrupting agents

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such as colchicine or vinblastine (Heinlein and Beachy, unpublished observations). The formation of the fluorescent, highly stabilized, and apparently nondynamic MP:GFP-associated microtubule complexes was observed at 12–24 hr postinfection (Heinlein et al., 1998a; Padgett et al., 1996) subsequent to virus replication and spread into adjacent cells, a process that occurs within a time frame of 4 hr or less (Boyko et al., 2000b). Thus, it appears likely that the late appearing highly fluorescent MP:GFP:microtubule complex is not involved in vRNA traYcking. The functional complex must be present in cells at the leading edge of infection and must be formed and active within the short time in which infection proceeds into adjacent cells. This notion is supported by leaf lamina incision experiments that demonstrated that cells at the leading front of the spreading fluorescent infection site are suYcient to permit further spread of the virus (Oparka et al., 1997). Moreover, as mentioned above, the decoration of microtubules with high amounts of MP:GFP is dispensable for movement (Gillespie et al., 2002; Heinlein et al., 1998a; Toth et al., 2002) and very low levels of MP expression suYce to permit the spread of infection (Arce-Johnson et al., 1995). Preliminary observations in leading edge cells of infection sites caused by attenuated TMV–MP:GFP constructs that produce normal levels of MP:GFP but show delayed cell-to-cell movement suggest an association of MP:GFP with particles that translocate along cytoskeletal tracks (Boyko and Heinlein, unpublished observations). This observation is reminiscent of RNA-containing particles reported in RNA transport and localization in neurons (Knowles et al., 1996; Kohrmann et al., 1999; Muslinov et al., 2002; Tiruchinapalli et al., 2003), oligodendrocytes (Ainger et al., 1993), fibroblasts (Sundell and Singer, 1990), Drosophila embryos (Ferrandon et al., 1994; Januschke et al., 2002; MacDougall et al., 2003), and Xenopus oocytes (Forristal et al., 1995; Kloc and Etkin, 1995). Microtubule-dependent transport of RNA granules in these systems appears to depend on the activity of microtubule motor proteins (Carson et al., 1997; MacDougall et al., 2003; Severt et al., 1999; Wilkie and Davis, 2001). Although at present it is impossible to suggest a similar mechanism, an involvement of microtubule motors in TMV RNA transport would be consistent with the demonstrated role of microtubules and motor proteins in the cytoplasmic transport of certain animal viruses (Do¨hner et al., 2002; Leopold et al., 2000; Mabit et al., 2002; McDonald et al., 2002; Moss and Ward, 2001; Rietdorf et al., 2001; Sodeik, 2000; Sodeik et al., 1997; Suomalainen et al., 1999) and other cases of mRNA transport (Bloom and Beach, 1999; Brendza et al., 2000; Carson et al., 1998; Hays and Karess, 2000; Januschke et al., 2002; MacDougall et al., 2003; Schnorrer et al., 2000). In this context, it also appears noteworthy to mention that expression of MP in mammalian cells interferes with the microtubule-nucleating function of the centrosome, an

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eVect that was correlated with the removal of centrosomal g-tubulin (Boyko et al., 2000a). Although further studies are required to investigate a role of g-tubulin during infection in plant cells, this finding may be related to the involvement of g-tubulin and of another g-tubulin ring complex protein in the localization of bicoid RNA during Drosophila oogenesis (Schnorrer et al., 2002). Like plant cells, the developing oocyte lacks a centrosome or microtubuleorganizing center (MTOC) (at least during midoogenesis, when the transport of diVerent RNAs to the anterior and posterior poles of the oocyte occurs). The reorientation or new establishment of specific microtubule-nucleating sites by g-tubulin complexes may represent a critical component required for RNA transport in centrosome-lacking cells. A reorganization of microtubules and a relocation of the microtubule-organizing center to cell–cell contacts is involved in the formation of a ‘‘virological synapse’’ and cell-to-cell transmission of human-T-lymphotropic virus (HTLV-1) (Derse and Heidecker, 2003; Igakura et al., 2003), a retrovirus related to human immunodeficiency virus (HIV). Thus, it is tempting to speculate that a microtubule reorganizing activity of MP may be involved in forming a ‘‘virological synapse’’ at the site of Pd. Although a role for microtubules in the traYcking of vRNA granules may be an appealing hypothesis, it is important to consider that MP-interacting microtubules may have additional or alternative roles. It has been proposed that microtubules may also function to target the MP to the proteasome for degradation (Gillespie et al., 2002; Padgett et al., 1996; Reichel and Beachy, 1998). Moreover, MP-interacting microtubules may support virus movement indirectly, for example, by interference with the traYcking of the signal involved in systemic RNA silencing that is triggered as a defense against viral infection (Voinnet, 2001). As mentioned earlier, a precedent for such activity was demonstrated for the TGBp1 protein of PVX that has the capacity to interfere with the spread of silencing signal (Voinnet et al., 2000). It appears intriguing that expression of the TGBp1 of WC1MV in transgenic plants supported the spread of the silencing signal into the meristem that usually restricts the entry of signal (Foster et al., 2002). Based on this finding, it is tempting to speculate that microtubules and Pd may be part of a general RNA transport pathway that can be manipulated by MPs in ways that either support or inhibit RNA transport. In such a scenario, the MP may permit RNA particle formation and microtubulemediated transport during early infection whereas during late stages of infection it would form the nondynamic MP:microtubule complex to inhibit transport-dependent defense responses (Fig. 4). In considering various models for vRNA movement, one also has to include a potential role of the ER. Since MP and replication complexes are

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associated with the ER (Heinlein et al., 1998a; Moore et al., 1992; Reichel and Beachy, 1998) and since the ER is continuous through Pd, it is appealing to suggest that vRNA movement occurs in association with this membrane compartment. Microtubules are known to segregate and distribute the elements of the ER (Lee et al., 1989; Terasaki et al., 1986). Thus, it is conceivable that MP transports vRNA by mediating contact between ER-associated replicase complexes and microtubules for transport. The ‘‘particles’’ observed at the leading edge of infection may represent vRNA and MPcontaining subdomains of ER or ER-derived vesicles that are translocated along microtubules. This possibility is supported by the observation that the subcellular localization of MP is greatly aVected by treatment of cells with the secretory pathway inhibitor Brefeldin A (Heinlein et al., 1998a). A role of secretory vesicles in the cell wall targeting of a viral MP has been demonstrated by elegant studies on the MP of tubule-forming grapewine fanleaf virus (GFV) (Laporte et al., 2003). Membranes also play a role in the movement of TGB-containing viruses (Cowan et al., 2002; Gorshkova et al., 2003; Solovyev et al., 2000; Zamyatnin et al., 2002). A precedent for the hypothesis that vRNA may be transported by membrane vesicles is provided by genomic RNA molecules of murine leukemia virus (MLV) that are transported to the plasma membrane by endosomal vesicles (Basyuk et al., 2003). Thus, the nature of the observed ‘‘particles’’ derserves further study. It is also possible that microtubule-mediated RNA transport localizes vRNA to the Pd-associated ER domain. The microtubule-dependent targeting of RNA molecules to specific ER domains has been reported in animal systems (Deshler et al., 1997; Kloc et al., 2002; Okita et al., 1994; Wickham et al., 1999) as well as in plants (Li et al., 1993; Okita and Choi, 2002). Once localized to an ER domain near Pd, the actin and myosins present in Pd may transport the vRNP complex further along the desmotubule. This idea is supported by the tight association of the ER in plants with actin (Allan and Brown, 1988; Hawes and Satiat-Jeunemaitre, 2001; Karchar and Reese, 1988; Lichtscheidl et al., 1990; Quader et al., 1987) and by the presence of actin and myosin in Pd (Blackman and Overall, 1998; Radford and White, 1998; Reichelt et al., 1999; White et al., 1994). An involvement of actin in the MP-mediated movement of vRNA is conceivable also because MP was reported to interact with actin (McLean et al., 1995). Since actin and microtubule filament systems cooperate (Goode et al., 2000; GriYth and Pollard, 1978; Langford, 1995), a mechanism in which microtubules first transport the vRNP to the cellular cortex and microfilaments then take over to drive the translocation of the complex through Pd can be considered. A very similar mechanism has been proposed for the spread of vaccinia virus in mammalian systems (Rietdorf et al., 2001). The observation that treatment of TMV infection sites in leaves with microtubule-disrupting agents failed to inhibit the progression of virus

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infection (Gillespie et al., 2002) (Heinlein and Beachy, unpublished observations) has led to the suggestion that microtubules are dispensable for movement. On the other hand, one must be aware that plant microtubules in nondividing cells can be quite resistant to disruption (Akashi et al., 1990; Cleary and Hardham, 1988), and that actin microfilaments may complement microtubule function (Goode et al., 2000). An integrated model of how MP may facilitate the movement of vRNA is shown in Fig. 4. It is proposed that upon synthesis, during early infection, MP binds vRNA, participates in the formation of potentially ER-containing vRNPs complexes, and mediates their interaction with molecular microtubule motors. A portion of MP that does not bind vRNA is targeted to Pd and increases their SEL. Subsequent to the spread of infection, the SEL is down-regulated (Derrick et al., 1990; Oparka et al., 1997). With the synthesis and accumulation of CP, newly synthesized vRNA encapsidates, resulting in free MP, which then accumulates on microtubules and forms a nondynamic complex that inhibits microtubule-dependent transport. Both the inhibition of cytoskeleton dynamics and the down-regulation of Pd may represent viral strategies to protect spread of innate defense responses of the host. But how are all these diVerent functions of the MP regulated? The most probable scenario is that MP undergoes multiple posttranslational modifications, most likely by protein kinases and phosphatases. Several studies with TMV have demonstrated that its MP is phosphorylated in vivo (Citovsky et al., 1993; Haley et al., 1995; Kawakami et al., 1999; Waigmann et al., 2000; Watanabe et al., 1992) and provided evidence that phosphorylation alters the function of the protein (Karger et al., 2003; Kawakami et al., 1999, 2003; Waigmann et al., 2000). In addition, cell wall–associated as well as ER-associated kinases have been identified that recognize the MP as a substrate (Citovsky et al., 1993; Karger et al., 2003; Waigmann et al., 2000). A role of phosphorylation during initial stages of infection is suggested by findings indicating that phosphorylation alleviates the ability of MP to repress translation of vRNA upon movement through Pd (Karpova et al., 1997, 1999). Late during infection, the phosphorylation of MP may cause the down-regulation of the Pd-modifying activity of the protein, since negatively charged amino acid substitutions that mimic phosphorylation of C-terminal amino acids were shown to inactivate the ability of MP to increase the SEL of Pd (Waigmann et al., 2000). Other studies suggest that phosphorylation occurs as a defense response of the host against the virus (Karger et al., 2003). Stabilized microtubule complexes appear to contain several diVerentially charged isoforms of MP (Ashby, 2003), which may suggest a role of phosphorylation in the microtubule-associated functions of this protein. There is also evidence that MPs of other viruses are targets of phosphorylation (Desvoyes et al., 2002; Matsushita et al., 2002b,

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2003; Sokolova et al., 1997) supporting the general importance of this posttranslational modification. A proteomic and functional analysis of MP during the infection cycle is needed to confirm this hypothesis and establish what changes are occurring to the MP during its ‘‘life cycle’’ and how each posttranslational alteration changes the properties of this protein. 4. Viral Movement is a Complex Phenomenon One of the hallmark features of viral MPs is their ability to mediate transport of other viruses, even if they are derived from animals (Dasgupta et al., 2001). For example, nonhost resistance of a plant against a given virus can be overcome by the presence of a ‘‘helper virus’’ (Atabekov and Taliansky, 1990), or by the permanent or transient expression of a foreign MP (Cooper et al., 1995; Deom et al., 1987; Fenczik et al., 1995; Hilf and Dawson, 1993; Holt and Beachy, 1991; Huppert et al., 2002; Morozov et al., 1997; Nejidat et al., 1991; Ryabov et al., 1999; Solovyev et al., 1996b). The host-specific ability of MPs to support the transport of heterologous virus species may be explained by their non-sequence-specific nucleic acid binding activity (e.g., Citovsky et al., 1990, 1991; Fujita et al., 1998; Osman et al., 1992; Shoumacher et al., 1992; Soellick et al., 2000). Viral synergism, however, is a widespread and multifaceted phenomenon that can aVect virus replication, movement, as well as other pathogenicity factors. Several MPs and other viral proteins known to have a role as host-specific disease symptom determinants and/or in the spread of virus infection turn out to also have specific functions in the suppression of defense responses, such as RNA silencing (Anandalakshmi et al., 1998; Beclin et al., 1998; Brigneti et al., 1998; Kasschau and Carrington, 1998; Li and Ding, 2001; PfeVer et al., 2002; Silhavy et al., 2002; Voinnet, 2001; Voinnet et al., 2000). The PVX TGBp1 protein exemplifies that a role in the suppression of silencing can apply even to a well-characterized MP (Voinnet et al., 2000). It is important to note that viral proteins that suppress silencing also inhibit miRNA pathways that cause the degradation of developmentally important mRNAs (Kasschau et al., 2003; Voinnet, 2002). The resulting ectopic expression of important transcription factors is likely to contribute to the production of disease symptoms. In addition, this virus-induced feature may function as an additional mechanism to increase the susceptibility of the plant to the infecting virus. Although MPs are defined as plant virus-encoded factors that interact with Pd and/or vRNA (DNA) to mediate the intercellular spread of virus infection and although MPs are essential for movement, we need to be aware that the successful movement of viruses is a rather complex phenomenon in which several viral and host functions act in concert to facilitate eYcient viral spread.

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5. Movement Protein-Interacting Factors To obtain a more basic understanding of how a virus spreads, we must develop tools to identify host proteins that are subverted by the virus to facilitate spread. This is a new frontier with little direct information. Current information on host proteins comes from studies of MP:host protein interactions. The known MP-interacting factors are summarized in Table III. TIP1, TIP2, and TIP3 were identified in a yeast two-hybrid screen using the 12-kDa TGBp2 protein of PVX as bait (Fridborg et al., 2003). These proteins appear to increase the susceptibility of plants by serving as bridging factors to the host callose-degrading enzyme b-1,3-glucanase, a factor previously implicated in viral movement. Several studies have shown that the expression level of this enzyme is positively correlated with the eYciency of virus spread (Bucher et al., 2001; Iglesias and Meins, 2000). The DNA-J-like proteins were identified in a yeast two-hybrid screen using as bait the MP of tomato spotted wilt virus (TSWV) (Soellick et al., 2000; van Bargen et al., 2001). The interaction of MPTSWV with these DNA-J-type proteins, proteins that act as cochaperones (Hsp40) of 70-kDa heat shock proteins (HSPs) (Hsp70) in the translocation of proteins across membranes (Bukau and Horwich, 1998; Pelham, 1986; Pilon and Schekman, 1999), suggests a role of HSP in viral movement. Their known role during virus replication, gene expression, and assembly, as well as in microtubule assembly and function has been reviewed (Liang and MacRae, 1997). A potential role of chaperones in macromolecular movement through Pd is indicated by a study that suggests that transport through the Pd channels may involve a degree of protein unfolding (Kragler et al., 1998; Sullivan and Pipas, 2001) and by the observation that in cells at the leading front of virus infection there is a transient induction of Hsp70 expression (Havelda and Maule, 2000; Whitham et al., 2003). Moreover, beet yellows closterovirus (BYV) encodes an Hsp70 homologue (Hsp70h) that functions as one of the viral MPs (Peremyslov et al., 1999), localizes to Pd (Medina et al., 1999), and binds to microtubules (Karasev et al., 1992). The Hsp70h protein associates with the tail domain of the viral capsid and is involved in capsid formation and stability as well as in virion movement (Alzhanova et al., 2001). Hsp70h also recruits another viral factor (p20) that appears to have a specific role in mediating long-distance, phloem-mediated, movement for systemic spread (Prokhnevsky et al., 2002). It has been proposed that following the targeting of virions to Pd, which may involve the microtubule-binding activity of Hsp70h, the protein anchors in the channel via an intrinsic Pd localization signal. In analogy to the mechanical role proposed for Hsp70 in the translocation of proteins into the ER (Alzhanova et al., 2001), the virion may then be translocated through the channel by virtue of mechanical force generated by this protein. It was recently reported that Hsp70-related chaperones are

TABLE III MP-Interacting Proteins Protein Tubulin

Plant

(Putative) function

Nicotiana species

Microtubules

Virus

Reference

TMV

Boyko et al., 2000a; Heinlein et al., 1995; McLean et al., 1995

Actin

Tobacco protoplasts

Microfilaments

TMV

McLean et al., 1995

BP2C

Tobacco

Microtubule adapter

TMV

Kragler et al., 2003

PME

Tobacco

Cell wall modification

TMV, CaMV, TVCV

Dorokhov et al., 1999; Chen et al., 2000

KELP

Brassica campestris

Transcriptional coactivator

ToMV

Matsushita et al., 2001

MBF1

Tobacco

Transcriptional coactivator

ToMV

Matsushita et al., 2002a

DNA J-like

Arabidopsis, Tobacco

Chaperone

TSWV

Soellick et al., 2000

DNA J-like

Lycopersicon esculentum

Chaperone

TSWV

van Bargen et al., 2001

Atp8

Arabidopsis

Unknown, RGD motifs

TCV

Lin and Heaton, 2001

At-4/1

Arabidopsis

Unknown

TSWV

Lin and Heaton, 2001

MP17

Arabidopsis

Pd modification

CaMV

Huang et al., 2001

TIP1, TIP2, TIP3

Arabidopsis

Interaction with b-1,3-glucanase

PVX

Fridborg et al., 2003

Phloem sap proteins

Melon, squash

Unknown

TMV, CMV

Shalitin and Wolf, 2000

2bip

Tobacco

Unknown

CMV

Ham et al., 1999

Homeodomain protein

Tobacco

Transcription factor

TBSV

Desvoyes et al., 2002

AtNSI

Arabidopsis

Acetyltransferase

CbLCV

McGarry et al., 2003

NtNCAPP1

Tobacco

Pd modification

CmPP16

Lee et al., 2003

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present at Pd and in phloem sap of C. maxima. Moreover, recombinant CmHsc70 proteins with ATPase activity were shown to spread between cells upon microinjection (Aoki et al., 2002). These findings support a general role of heat shock proteins in facilitating macromolecular transport through Pd. Pectin methylesterase (PME), a cell wall enzyme with important roles in plant physiology (Micheli, 2001), may be another important host factor that apparently interacts with the MP of TMV and other tobamoviruses, playing a role in transport through Pd. PME was shown to bind to the MP of two tobamoviruses in an in vitro blot overlay assay and to an MP on an aYnity column (Chen and Citovsky, 2003; Chen et al., 2000; Dorokhov et al., 1999). An interaction between the MP and this enzyme could be involved in increasing the SEL of Pd by changing the structural state of cell wall pectins that are enriched in microdomains surrounding the channels (Morvan et al., 1998). Another possibility is that the MP binds to this enzyme as a carrier for cell wall-targeted transport via the secretory pathway (Chen et al., 2000). The MP of TMV also interacts with a protein termed MBP2C. This microtubule-associated protein appears to be involved in the formation of MP:microtubule complexes late in infection (Kragler et al., 2003). Several other proteins have been identified that interact with specific viral MPs and continuing studies are required to elucidate their specific function during infection. The protein NtNCAPP1 may function in conjunction with the endogenous phloem resident protein CmPP16 or together with TMV MP to modify the SEL of Pd. This protein is associated with the ER at the cell plate as well as near Pd and may function as a shuttle for some non-cell autonomous proteins to the plasmodesmal microchannel (Lee et al., 2003).

VI. Concluding Remarks Intercellular communication and macromolecular traYcking via Pd play essential roles during plant development and in the orchestration of systemic reactions against challenging environmental factors. Despite this importance, the mechanism of Pd-mediated intercellular communication remains poorly understood. Plant viruses by virtue of their MPs hijack and modify underlying host mechanisms to potentiate their own traYcking and provide probes and models to analyze the mechanisms involved in intercellular communication. At present, we have only a rudimentary understanding of how endogenous mRNA molecules (Lucas et al., 2001; Okita and Choi, 2002) and silencing signals (Mlotshwa et al., 2002; Waterhouse et al., 2001) target to endomembranes and cytoskeletal machinery and how these molecules are transported

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to the cortical region of the cell, target to Pd, and traYc to diVerent tissues of the plant. With respect to these endogenous RNA molecules, it will be important to identify the MPs that facilitate their traYcking. Of particular importance will be research aimed at understanding the mechanisms that control the expression and traYcking of miRNAs that are likely to produce gradients of target gene expression in asymmetric plant tissues. One of the important questions that need to be addressed is how macromolecules are transported to and through Pd. Although TMV may represent an important model, further analysis will have to show whether implicated components such as the cytoskeleton or ER have a general role in Pd targeting of RNPs. Of major importance is the further elucidation of the structure, composition, and regulation of Pd. Compositional information may be derived from mass spectroscopic analysis of purified Pd preparations. Major eVorts are under way in a number of laboratories to identify Pd-associated proteins. A novel approach is provided by high-throughput viral-based genomics that also allows the identification of factors that only transiently interact with Pd (Escobar et al., 2003). More research is also needed to understand the mechanism by which macromolecular complexes such as viruses, RNPs, and proteins are transported through the Pd channel. Apparently, a number of mechanisms may be involved. Finally, it appears that MPs originally identified as proteins that interact with Pd to facilitate viral spread may function indirectly by diverse mechanisms, including the suppression of gene silencing and miRNA pathways. As we learn more about these diverse functions we will also gain novel insight into various aspects of plant system biology and will be able to design novel strategies by which we can protect agricultural crops against viral pathogens.

Acknowledgments This article was written with financial support from the United States–Israel Binational Agricultural Research and Development Fund (BARD) (Award IS-3222-01C), the Israel Science Foundation (Award 723/00-17.1), and the Swiss National Science Foundation (Award 631-65953.01).

References Ainger, K., Avossa, D., Morgan, F., Hill, S. J., Barry, C., Barbarese, E., and Carson, J. H. (1993). Transport and localization of exogeneous myelin basic protein mRNA microinjected into oligodendrocytes. J. Cell Biol. 123, 431–441. Akashi, T., Kawasaki, S., and Shibaoka, H. (1990). Stabilization of cortical microtubules by the cell wall in cultured tobacco cells. Planta 182, 363–369.

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