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First published online December 17, 2004; 10.1105/tpc.104.027821 © 2005 American Society of Plant Biologists
Two Plant–Viral Movement Proteins Traffic in the Endocytic Recycling Pathway
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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). It is now clear that plant viruses have evolved different strategies for cell-to-cell movement that involve one or more virus-encoded movement proteins (reviewed in Carrington et al., 1996; Morozov and Solovyev, 2003). In this report, we focus on the conserved group of three partially overlapping reading frames encoding the triple gene block (TGB) of movement proteins.
The TGB module is a conserved genetic element found in several different viral genera that infect both monocotyledonous and dicotyledonous plants. Two groups of TGB-containing plant viruses have been distinguished (Morozov and Solovyev, 2003): the hordei-like (Group 1; typified by the Hordeivirus Barley stripe mosaic virus; BSMV) and the potex-like (Group 2; typified by the Potexvirus Potato virus X; PVX). In this work, we examine the TGB of the soil-borne Pomovirus Potato mop-top virus (PMTV), a Group 1 TGB virus that is transmitted by the plasmodiophorid Spongospora subterranea, a protozoan parasite that invades root hairs and potato tuber epidermal cells (Braselton, 1992, 1995; Arif et al., 1995).
The first of the TGB proteins (TGB1) is a sequence nonspecific RNA binding protein that contains seven NTPase motifs typical of a viral helicase. In Group 1 viruses, TGB1 contains an additional N-terminal domain of variable mass (
20 to 40 kD), whereas in Group 2 viruses, TGB1 has a molecular mass of 25 kD, encoding only the helicase domain. The sequence of the second protein (TGB2; 12 to 14 kD) of both groups contains two predicted transmembrane helices. The N and C termini are predicted to be located in the cytoplasm, and there is a conserved hydrophilic motif (G-G-x-Y-R/K-D-G) in the intervening loop. The TGB2 of PMTV was shown previously to bind RNA in Northwestern gel blots (Cowan et al., 2002).
Unlike TGB2, TGB3 proteins are more heterogeneous both in size and sequence. For example, the TGB3 proteins of hordeiviruses and pomoviruses have a mass of 17 to 21 kD, and the amino acid sequences contain two predicted transmembrane helical domains (with N and C termini inside), a conserved Cys residue motif (H-x3-C-x-C-x2-C) in the N terminus, and a conserved motif (Y-Q-D-L-N) in the intervening loop. Potexvirus TGB3s are smaller (7 to 8 kD), and the sequences contain a single hydrophobic (transmembrane) domain within the C terminus that is predicted to be cytoplasmic.
The TGB proteins are essential for virus movement. A major difference between the two TGB-containing virus groups is the requirement for coat protein (CP) for movement. Group 2 viruses require the CP, whereas in viruses belonging to Group 1, the CP is not required for cell-to-cell movement. In hordeiviruses and pomoviruses, the CP is also dispensable for long-distance movement (Petty and Jackson, 1990; Savenkov et al., 2003). These observations indicate differences in the composition of the ribonucleoprotein (RNP) movement complex between the two groups. Clearly, if the viral CP is dispensable for movement of the Group1 viruses, then the RNP cannot move in the form of intact virions. In the potexvirus group, two models have been proposed, in which movement may occur as intact virions (Santa Cruz et al., 1998) or, alternatively, as a nonvirion RNP complex (Lough et al., 2000). PVX CP does not modify the size exclusion limit (SEL) of PD but is translocated with the infectious material (Santa Cruz et al., 1998). The TGB1 protein of PVX and White clover mosaic virus can move cell to cell independently of the other TGB proteins and is also translocated with viral RNA (Lough et al., 2000; Yang et al., 2000). In biolistic bombardment experiments, in which TGB proteins and CP were transiently expressed in cells to complement the corresponding defective genes in PVX clones, TGB1 and CP rescued mutant virus movement across several cell layers, but TGB2 and TGB3 only facilitated movement of defective virus to the adjacent cells. This indicates that these proteins were not themselves transported between cells, presumably because of their strong association with cellular membranes (Lough et al., 2000).
Recently, PVX TGB2 has been shown to increase the SEL of PD, enabling the passage of free green fluorescent protein (GFP) between infected cells (Tamai and Meshi, 2001). Also, fusions of GFP to PVX TGB2 and TGB3 were shown to move to adjacent cells when the proteins were expressed from plasmids bombarded onto Nicotiana benthamiana leaves (but not in five other host plants tested). The proteins also moved to adjacent cells when bombarded onto transgenic N. tabacum expressing TGB1 (Krishnamurthy et al., 2003).
BSMV (Group 1) TGB1 was associated with perinuclear endoplasmic reticulum (ER) membranes and punctate foci at the plasma membrane when expressed from a BSMV-based vector in tobacco BY-2 protoplasts. In the absence of TGB2 and TGB3, fluorescence was only associated with perinuclear ER and not plasma membranes (Lawrence and Jackson, 2001a). Similarly, punctate spots that colocalized with callose were seen in the walls of cells expressing Beet necrotic yellow vein virus (Group 1) TGB1 in the presence of TGB2 and TGB3 (Erhardt et al., 2000). The punctate spots are thought to be plasmodesmata.
Previously, we studied the TGB proteins of PMTV and described their subcellular localizations when expressed as N-terminal fusions to GFP from a Tobacco mosaic virus (TMV)–based vector (Cowan et al., 2002). In these experiments, both the PMTV GFP-TGB2 and GFP-TGB3 fusions were associated with cellular endomembranes, particularly the ER network and membranes surrounding the nucleus. Fluorescent spots and aggregates were seen to move along the ER and transvacuolar strands, and stationary aggregates were seen at the periphery of the cells. In addition, GFP-TGB3 labeled opposing pairs of fluorescent spots across neighboring cell walls that suggested localization at, or near, plasmodesmata.
The above findings are in general agreement that the movement proteins of several viruses may associate with the endomembrane system (see Oparka, 2004) and are in line with the reported localization of TGB proteins of Poa semilatent hordeivirus (PSLV; Solovyev et al., 2000) and PVX (Krishnamurthy et al., 2003; Mitra et al., 2003), which in transient expression assays showed an association with the ER network or ER-derived structures at the cell periphery (Solovyev et al., 2000; Zamyatnin et al., 2002). Mutations in the predicted transmembrane domains of PVX TGB proteins that disrupt membrane binding inhibited the association with ER and virus movement (Krishnamurthy et al., 2003; Mitra et al., 2003). In studies with PSLV, coexpression of the two proteins revealed that they colocalized in the cell and that TGB3 redirected localization of TGB2 from the ER network to the peripheral bodies (Solovyev et al., 2000). TGB3-assisted targeting to the periphery has also been shown with unrelated proteins, such as PVX TGB2 and the 6-kD movement protein of Beet yellows closterovirus (Solovyev et al., 2000; Zamyatnin et al., 2002). Also, evidence for the association of PSLV TGB3 with plasmodesmata has been obtained from experiments with GFP-TGB3 transgenic plants in which colocalization with callose deposits was observed (Gorshkova et al., 2003). Recent studies have shown that on transient expression of PMTV GFP-TGB1, fluorescence was seen in the cytosol and nuclei, whereas when GFP-TGB1 was coexpressed with TGB2 and TGB3, the fluorescence appeared in peripheral bodies close to the plasma membrane (Zamyatnin et al., 2004). Furthermore, the same authors showed that when GFP-TGB1 was expressed alone, fluorescence was confined to the transfected cell, but when GFP-TGB1 was coexpressed with TGB2 and TGB3, fluorescence moved into the adjacent cells.
It is generally accepted that the TGB1 protein interacts with RNA and is transported, as part of the RNP complex, out of the cell and long distance throughout the plant. The TGB2 and TGB3 proteins facilitate RNP intracellular transport, but the details of this process are not well understood, and the respective roles of these two integral membrane proteins are the focus of this report. Our current hypothesis is that TGB2 and TGB3 act as endomembrane anchors, where they bind viral RNP to direct its transport to plasmodesmata. We have conducted a detailed investigation of the subcellular pathways involved in trafficking of the PMTV TGB2 and TGB3 to the cell periphery. Expression of GFP-tagged and monomeric red fluorescent protein (mRFP)–tagged TGB proteins was examined in wild-type and transgenic plants in which a range of specific subcellular compartments were either stained with specific markers or labeled with GFP. In addition, the interaction of TGB2 and TGB3 with host factors was investigated. We dissect the distinctive roles of each protein and provide several lines of evidence to show that the TGB2 and TGB3 proteins of PMTV interact with components of the endocytic pathway. The data are discussed in the light of the emerging evidence for endocytosis in plants.
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RESULTS |
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In these experiments, identical results were obtained regardless of whether the fusion proteins were expressed under the control of P35S, or from TMV, or whether the TMV vector had a functional or defective MP, except that infection was confined to a single cell when expressed from the TMV vector with defective MP (see Supplemental Figure 2 online).
Later, TGB2 Fusion Protein Labels Motile Vesicular Structures
A characteristic feature of the expression cycle of cells bombarded with TGB2 fusion proteins was the appearance of a motile population of vesicle-like structures with a size range of 0.5 to 4 µm (Figure 1H). Many of these vesicles were associated with the plasma membrane and seemed to bud from it at discrete locations in the periphery of the cell. Many of the vesicles appeared to move by cytoplasmic streaming, and many became closely associated with the nuclear envelope (Figure 1I). To determine whether the labeled vesicles were present within the cytoplasm, or located within the vacuole, P35S-mRFP-TGB2 plasmid DNA was cobombarded into cells along with P35S-GFP as a cytosolic marker. In these cases, the mRFP-TGB2–labeled vesicles were clearly surrounded by cytoplasm (Figure 1J). When the GFP-TGB3 or mRFP-TGB3 was expressed independently from either P35S or the TMV-based vectors, they were never found to label the vesicular structures.
TGB2 Recruits TGB3 to Vesicles
In cobombardment experiments with P35S-GFP-TGB3 and P35S-mRFP-TGB2, the colocalization of the two proteins in the vesicles was clearly visible (Figure 1K to 1M). The GFP-TGB3 seemed to be more evenly distributed throughout the vesicle, whereas mRFP-TGB2 strongly labeled the membrane (Figures 1N to 1P). At high magnifications, the vesicles often appeared to be beaded, containing small hot spots on their surfaces (Figures 1N to 1P).
Expression of TGB Fusion Proteins in PMTV-Infected Cells Reveals Recruitment of mRFP-TGB3 to Vesicles
To investigate whether the subcellular localization of TGB fusion proteins was affected by the presence of a replicating virus, we bombarded P35S-mRFP-TGB2 or P35S-mRFP-TGB3 onto leaves that had been manually inoculated with PMTV preparations. Tissue print immunoassays were done on the inoculated leaves after imaging to confirm that the inoculated leaves had a uniform distribution of virus-infected cells. The presence of virus was also confirmed by RT-PCR. In these experiments, the presence of replicating virus made no difference to the subcellular localization of mRFP-TGB2 (in five independent experiments). By contrast, the presence of replicating virus did affect the subcellular localization of mRFP-TGB3. Red fluorescence was seen as before in ER membranes and motile spots, but also in vesicles, which were never seen in noninfected cells. This result indicates that the virus-expressed TGB2 recruited mRFP-TGB3 to the vesicles (Figure 2A). In these experiments, neither of the fluorescent fusion proteins moved out of the bombarded cells, indicating that neither the presence of viral RNA nor other virus-expressed TGB proteins enabled the intercellular movement of the membrane anchored TGB2 or TGB3.
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; Roberts et al., 2001). To examine whether the stationary structures were located at plasmodesmata, plasmid P35S-mRFP-TGB3 was bombarded onto leaves of transgenic plants expressing TMV MP-GFP (Figures 2D to 2F). These colabeling experiments showed that many of the stationary mRFP-TGB3 deposits were precisely colocalized with plasmodesmata (yellow signal in Figure 2F), whereas other deposits were not. Thus, at least a subpopulation of TGB3 fusion protein appears to become stably associated with plasmodesmata. In addition, unlike the MP of TMV, which can be seen to target plasmodesmata at the leading edge of the infection (Oparka et al., 1997), when GFP-TGB3 was expressed from a TMV-based vector containing functional MP, labeling of plasmodesmata occurred inside the infection site, consistently approximately two to three layers away from the leading edge (data not shown). These results suggest that for PMTV, accumulation at plasmodesmata occurs relatively late in the infection cycle and is not influenced by the presence of the TMV MP. In experiments in which GFP-TGB2 was expressed from the TMV vector in N. benthamiana epidermal cells, no labeling of plasmodesmata was observed. In experiments where P35S-mRFP-TGB2 was cobombarded with P35S-GFP-TGB3, no association with the punctae representing plasmodesmata was observed, but dual-labeled stationary structures were seen at the cell periphery (Figure 2G).
Plasmolysis Causes Removal of TGB Fusion Proteins from the Cell Periphery
In onion (Allium cepa) epidermal cells expressing mRFP-TGB2 and mRFP-TGB3 fusions, the localization of the fluorescent proteins was similar to that seen for transient or viral expression in N. benthamiana plants. Fragments of ER were seen throughout the cells and in the perinuclear region, and a mobile vesicle population was frequently labeled with the TGB2 fusion protein (data not shown). mRFP-TGB3 was also seen at peripheral punctae, possibly associated with plasmodesmata (Figure 2H). When the cells were incubated in water, there was no alteration in the localization of fluorescence, but there was a distinct alteration in plasmolysed cells that had been incubated in a sugar solution (Figures 2I to 2L). In both mRFP-TGB2 and mRFP-TGB3–expressing cells, fluorescence became predominantly cytosolic after plasmolysis (Figures 2I and 2K) and was retained inside the protoplast as it retracted from the cell wall (Figures 2J and 2L). No fluorescence was maintained at the cell periphery for either protein, indicating that neither TGB fusion protein was embedded within plasmodesmata.
TGB2 and TGB3 Fusions Both Increase the SEL of Plasmodesmata
In biolistic bombardment experiments in which mRFP-TGB2, mRFP-TGB3, or mRFP was coexpressed in the same cell together with GFP-sporamin, both TGB proteins increased the SEL of plasmodesmata, allowing GFP-sporamin to move to adjacent cells (Figures 2M and 2N). The numbers of cells in which the initial bombarded source cell was surrounded by one or more fluorescent cells were recorded (Table 1). In N. benthamiana cells, there were significantly more cells surrounding the target cells expressing mRFP-TGB2 (28%) or mRFP-TGB3 (35%) than for mRFP alone (4%) (Figures 2M to 2O, respectively). The results from N. tabacum also indicated that both TGB proteins increased the plasmodesmatal SEL, but the numbers of cells showing gating were fewer (23% for TGB2 and 18% for TGB3). We never observed movement of mRFP-TGB2 or -TGB3 from the initial target cell in any of these experiments. An analysis of deviance showed that the small difference in the proportion of cell clusters found between the TGB2 and TGB3 fusion protein treatments within a species (i.e., 28 and 35% for N. benthamiana or 23 and 18% for N. tabacum) was not significant, but it confirmed that the difference between the two species was significant. Therefore, the fusion proteins facilitated a greater increase in plasmodesmatal SEL in N. benthamiana epidermal cells than N. tabacum epidermal cells.
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) showed that the granular structures that moved along the ER were not colocalized with Golgi bodies (see Supplemental data 3 online). In addition, treatment of these latter plants with 100 µg mL–1 brefeldin A (BFA), a concentration that in our experiments caused the Golgi bodies to be reabsorbed into the ER network (data not shown), showed no effect on the movement of either the mRFP-TGB2 or mRFP-TGB3 granules or vesicles. The subcellular distribution and movement of fluorescent TGB in cells infiltrated with water was similar to that of untreated cells.
The TGB Containing Vesicles Are Derived from the Plasma Membrane
Because many of the TGB2-labeled vesicles were associated with the plasma membrane, we examined whether the vesicle membrane was derived from the plasma membrane by staining the latter with FM4-64 (Ueda et al., 2001; Bolte et al., 2004). Within 30 min of application to N. benthamiana leaves, the FM4-64 predominantly labeled the plasma membrane (Figure 4D), and after 45 min, an internalized vesicle population was apparent (Figure 4E), showing that this population is present in uninfected cells and is not induced by overexpression of heterologous proteins. Subsequently, N. benthamiana cells expressing GFP-TGB2 were treated with FM4-64, and after 1 to 3 h, dye-labeled vesicles were observed that also showed discrete labeling with GFP-TGB2 (Figures 4A to 4C). Noticeably, not all FM4-64–labeled vesicles contained TGB2 fusion protein, suggesting that only a subpopulation of vesicles contained the internalized TGB2 protein. FM4-64 staining of cells expressing GFP-TGB2 revealed highly dynamic, tubular structures from which the colabeled vesicles were derived (data not shown). These complex structures are similar to those described for the early endosome compartment found in animal cells (Gruenberg, 2001).
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). When cobombarded with P35S-mRFP-TGB2, some but not all of the vesicles were shown to contain both mRFP-TGB2 and GFP-Ara7 (Figures 4G to 4I), and some labeling of the plasma membrane by mRFP-TGB2 is also visible. As in previous experiments (see Figure 1I), many of the vesicles were delivered to the vicinity of the nuclear membrane, which showed conspicuous labeling with mRFP-TGB2 but not GFP-Ara7 (Figure 4J). Next, we bombarded cells with GFP-Ara7, followed by FM4-64 staining of the plasma membrane. After application of FM4-64, a distinct vesicle population was observed that showed precise colocalization of GFP-Ara7 and FM4-64 (Figures 4K and 4L). At high magnifications, these vesicles were seen to be highly pleiomorphic and often contained a subpopulation of Ara7/FM4-64–labeled vesicles (Figure 3L, inset). The above data confirm that TGB2 fusion protein does not induce the formation of vesicles in bombarded cells, but rather becomes localized to a pre-existing population of Ara7-positive vesicles.
TGB2 Interacts with a Tobacco-DnaJ Protein Belonging to the Receptor-Mediated Endocytosis-8 Family of Endosomal Trafficking Proteins
To identify host factors that might interact with the TGB proteins, we searched a library of tobacco leaf proteins. The proteins were expressed from colonies induced on replica plates, and in vitro interactions with purified recombinant TGB2 were analyzed using overlay blots. Approximately 105 library clones were screened on the filters. Very few clones displaying strong interactions were visible, indicating specificity of binding. The positive clones were rescued from the noninduced replica plates, and plasmid DNA from five clones was sequenced. Four of the five sequences were identical, and BLAST searches (Altschul et al., 1997) revealed significant sequence identity (E-value = 5e-90) to a DnaJ domain–containing Arabidopsis thaliana protein (GenBank accession number AAC32237) belonging to the Receptor-Mediated Endocytosis-8 (RME-8) family (Zhang et al., 2001) of proteins (Figure 5). The other sequence shared sequence identity to an unknown protein from pea (Pisum sativum) (data not shown).
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, 2000; Santa Cruz et al., 1998; Erhardt et al., 2000; Lawrence and Jackson, 2001a, 2001b; Zamyatnin et al., 2004). Previous results also suggest that cell-to-cell movement may be regulated by the relative amounts of TGB2 and TGB3 expressed in the cell (Bleykasten-Grosshans et al., 1997; Yang et al., 2000; Tamai and Meshi, 2001). The genetic arrangement where TGB proteins are expressed from overlapping reading frames may well facilitate the regulation of translation, but this arrangement complicates investigations of the intracellular movement of the TGB2 and TGB3 when expressed from the homologous virus. Moreover, TGB proteins are expressed at low levels and are not readily detectable in virus-infected plants. Therefore, in several studies, the role and functions of TGB2 and TGB3 proteins have been investigated using microinjection or biolistic bombardment to introduce plasmids for transient expression of fluorescent fusion proteins (Solovyev et al., 2000; Zamyatnin et al., 2002, 2004; Krishnamurthy et al., 2003; Mitra et al., 2003). In addition, expression of fusion proteins from viral vectors has provided important information on the function and localization of viral and plant proteins (Blackman et al., 1998; Boevink et al., 1998; Ueda et al., 2001). In this work, we used transient P35S-driven expression plasmids and TMV-based vectors (with or without a functional MP) to investigate localization of GFP- and mRFP-tagged fusion proteins in epidermal cells. Throughout these experiments, the same pattern of intracellular fluorescent localization was observed regardless of the expression system or whether the proteins were tagged with mRFP or GFP. The same pattern of expression was seen in the PMTV hosts N. benthamiana and N. tabacum and the nonhosts Arabidopsis and A. cepa. Moreover, when expressed in PMTV-infected cells, in the presence of viral RNA and virus-encoded proteins, there was no change in the distribution of mRFP-TGB2, whereas mRFP-TGB3 was recruited to vesicles by the virus-expressed TGB2. Thus, although the TGB fusion proteins were not expressed from a PMTV-based vector, we believe that the subcellular localizations of the TGB fusion proteins are representative of their native distribution.
The TGB2/3 Complex Traffics on the Actin-ER Network
The results described in this article show a close association of TGB2 and TGB3 fusion proteins with the ER and perinuclear membranes, and the fusion proteins were colocalized when expressed in the same cell. There was a consistently early association of TGB fusion proteins with the ER network followed by the appearance of motile granular structures. The small motile bodies induced by both TGB fusion proteins (and in which they were colocalized) moved quickly along the ER network and through cytoplasmic strands. We think these motile granules may transport viral RNP complex to the plasmodesmata. No association of TGB fusion proteins was seen with microtubules when the former were introduced into microtubule-labeled plants. Furthermore, oryzalin (which completely fragmented the microtubules as described in Gillespie et al., 2002) had no effect on the subcellular distribution of the TGB fusion proteins. By contrast, movement of the small granules was aligned with actin filaments when expressed in GFP-hTalin plants (where GFP is fused to the F-actin binding domain of human talin; Takemoto et al., 2003), and such movement was completely inhibited by treatment of cells with latrunculin, which disrupted the F-actin–labeled filaments. In higher plants, the cortical ER network is intricately linked to underlying actin cables that function as tracks for Golgi movements (Boevink et al., 1998). It remains possible that the TGB fusion proteins could pass through the trans-Golgi network en route to plasmodesmata. However, BFA had no influence on the localization or movement of the labeled proteins, and the TGB-containing bodies did not colocalize with the Golgi when the latter were labeled with a Golgi-GFP marker (see Boevink et al., 1998). We suggest, therefore, that the TGB2/3 complex uses the actin-ER network to facilitate movement to plasmodesmata. A similar conclusion was reached for the movement of PSLV TGB3 based on colocalization with ER markers at the cell periphery (Zamyatnin et al., 2002; Gorshkova et al., 2003).
Our results show that the TGB3 fusion protein contains the signal for plasmodesmatal targeting. However, colocalization of this protein with the TMV MP (expressed transgenically as a MP-GFP fusion; Roberts et al., 2001) occurred a few cell boundaries behind the leading edge of infection, and mRFP-TGB3 only targeted a subpopulation of those plasmodesmata labeled with TMV MP. Plasmolysis experiments also indicated that TGB3 did not enter the plasmodesmal pore when transiently expressed in onion cells. Both TGB3 and TGB2 fusion proteins remained associated with the plasma membrane and did not form strong links with the cell wall, a finding similar to that reported by Gorshkova et al. (2003) for PSLV TGB3. Therefore, we suggest that at the leading edge of an infection, TGB3 targets the neck region of plasmodesmata but does not enter the plasmodesmal pore. Unlike mRFP-TGB3, TGB2 fusion proteins did not localize to plasmodesmata and mRFP-TGB3 did not appear to label the punctae thought to represent plasmodesmata when coexpressed with GFP-TGB2, although both fusion proteins were clearly colocalized in larger bodies at the periphery of the cell. Colocalization of TGB2 and TGB3 to peripheral bodies at the plasma membrane has been observed when PSLV TGB2 and TGB3 were coexpressed in the same cell (Solovyev et al., 2000; Zamyatnin et al., 2002). This finding may reflect a regulatory role for TGB2. The arrangement of the genetic module provides a mechanism for regulation where (in the systems studied) TGB2 and TGB3 are expressed from the same subgenomic RNA, with TGB3 probably translated by a leaky scanning mechanism (reviewed in Morozov and Solovyev, 2003). Also, the finding that cell-to-cell movement of PVX TGB1 is inhibited in the presence of transgenically expressed TGB2 and TGB3 provides some evidence for a regulated movement process (Yang et al., 2000).
Although TGB2 does not apparently contain a plasmodesmal targeting signal, both TGB2 and TGB3 fusion proteins were able to increase the SEL of plasmodesmata. However, unlike the PVX proteins, no movement of the PMTV proteins to adjacent cells was detected in the permissive host N. benthamiana (Krishnamurthy et al., 2003). Neither did they move out of PMTV-infected cells. Recently, it was shown that PVX TGB2 could interact with TIP, a host factor that interacts with ß-1,3 glucanase, which may serve to regulate SEL of plasmodesmata by callose degradation (Fridborg et al., 2003).
Mutation in the Conserved YQDLN Motif Affects Localization of TGB3
The sequence of TGB3 contains a conserved YQDLN motif in the cytoplasmic domain. This sequence may contain a putative Tyr-based sorting signal based on the motif YXX
(where X is any amino acid residue, and represents an amino acid with bulky hydrophobic side chain; Marks et al., 1997). Such motifs are found in the cytoplasmic domains, usually the cytoplasmic tails of transmembrane proteins and are involved in internalization and targeting of proteins to subcellular compartments, such as endosomes, lysosomes, or other organelles (Marks et al., 1997; Bonifacino and Dell'Angelica, 1999). This motif is found in several animal viruses that enter cells by endocytosis (Sieczkarski and Whittaker, 2002), and mutagenesis studies on Varicella-zoster virus glycoprotein B showed that two such Tyr-based motifs in the cytoplasmic tail operated to internalize and to traffic the protein to the Golgi (Heineman and Hall, 2001). Recently, Tyr-based motifs have been noted to occur in the MP of Grapevine fanleaf virus and in the MPs of several nepoviruses (Laporte et al., 2003). In this study, the mutation YQDLN to GQDGN caused a change in subcellular localization of TGB3, eliminating the ER localization and the presence of small motile granules. Disruption of this motif in PSLV TGB3 by insertion of four amino acids (RSTD) between the D and L residues abolished localization to the cell periphery (Solovyev et al., 2000). In our experiments, the mutation also affected the targeting to plasmodesmata and efficiency of plasmodesmal gating. The role that this motif plays in the formation and/or sorting of compartments containing the viral MP complex is the subject of current research in our laboratory.
A Role for the Endocytic Pathway in Retrieving TGB Proteins?
FM4-64 is widely used as a marker for the endocytic pathway (Vida and Emr, 1995; Ueda et al., 2001; Grebe et al., 2003; Bolte et al., 2004). It is a lipophilic stain that fluoresces when inserted into the outer leaflet of lipid bilayers, entering cells by endocytosis from the plasma membrane and subsequently labeling the membranes of many intermediate compartments and small organelles, including Golgi stacks and prevacuolar compartments and finally the vacuolar membrane (Bolte et al., 2004). Staining of the TGB2-containing vesicles with FM4-64 revealed that they were derived, at least in part, from components of the plasma membrane and indicates that they are components of the endocytic pathway. The TGB2 fusion protein was seen in these motile vesicles later in the expression cycle, and when coexpressed in the same cell, TGB2 and TGB3 fusion proteins became colocalized to the same structures. Significantly, GFP- or mRFP-TGB3 were never seen to associate with vesicle-like structures when expressed independently and continued to accumulate at the cell periphery. Thus, one of the functions of TGB2 may be to remove excess TGB3 once the former has successfully located the viral RNP to the plasmodesmal pore.
Colocalization of mRFP-TGB2 with GFP-Ara7, a Rab5 ortholog from Arabidopsis, is another indicator that these vesicles represent a component of a plant internal recycling compartment, possibly the endosome (Ueda et al., 2001). Rab5 GTPase is a key regulator of homotypic vesicle fusion in early endosomes (Stenmark et al., 1994; Zerial and McBride, 2004) and may be distributed within discrete domains within the endosomal membrane. Zerial and McBride (2004) have suggested that the Rab5 domain may be the gateway into the early endosome. In plants, Ueda et al. (2001) have suggested that Ara7 locates to the early/late endosome and regulates membrane fusion in the early endocytic pathway.
We also found that TGB2 is needed for TGB3 to enter the endocytic pathway. Both proteins localized to discrete hot spots of fluorescence on vesicles, indicating that they may bind to functionally distinct domains of the same vesicle. This dot and ring structure is very similar to that reported for Ara6 and Ara7 by Ueda et al. (2001). The plant endocytic pathway is not fully described, and doubts continue to be expressed as to the true nature of endosomal compartments in plants (Bolte et al., 2004; Geldner, 2004). However, in animal cells, the early endosome is a dynamic organelle that acts as a sorting station for recycling receptors and their ligands (Holstein, 2002), and enveloped RNA viruses exploit the endocytic sorting mechanisms to facilitate budding (Raiborg et al., 2003). Transport along the recycling pathway depends on a functional actin cytoskeleton (Ayscough, 2004). TGB2 and TGB3 were shown previously to self-interact and also to interact with each other in the yeast two-hybrid system (Cowan et al., 2002). Therefore, it is possible that these interactions are important for homotypic vesicle fusion and multimerization of sorting signals that would improve the efficiency of internalization (Arneson and Miller, 1995).
TGB2 Interacts with RME-8, an Endocytic DnaJ Chaperone
The unique binding of TGB2 to a DnaJ-like plant chaperone belonging to the RME-8 family is further evidence that TGB2 may function in endocytic recycling. The RME-8 proteins do not share sequence similarity outside the J domain, they were first identified from Caenorhabditis elegans and are required for both fluid-phase and receptor-mediated endocytosis (Zhang et al., 2001; Chang et al., 2004). RME-8 is ubiquitous in cells and functions in an early trafficking event before delivery of endocytosed cargo to the lysosomal compartment. In C. elegans, RME-8 localizes to the limiting membrane of large endosomes (Zhang et al., 2001). Recently, Chang et al. (2004) showed that, in Drosophila melanogaster, the J-domain of RME-8 interacts with Hsc70 to function as an essential cochaperone to control clathrin-dependent endocytosis. This function has also been demonstrated with the RME-8 protein auxilin (Ungewickell et al., 1995). The vesicular transport model for the function of endosomes predicts that they are stable cellular compartments from which endosomal contents move from one compartment to another by means of small transport vesicles (Gruenberg and Howell, 1989; Gruenberg and Maxfield, 1995). RME-8 was discovered in a genetic screen for mutants defective in endocytosis in C. elegans (Grant and Hirsh, 1999) and is ubiquitous among several cell types. Using an RME-8:GFP reporter under control of the rme-8 promoter, Zhang et al. (2001) found that RME-8 was localized strongly to vesicles ranging in size from 0.5 to 4.0 µm in diameter, which accumulated the fluid-phase endocytosis marker BSA. These vesicles are remarkably similar to those shown here labeled with TGB2 and Ara7 (e.g., Figures 3J to 3M). To date, there are very few known markers for the endocytic pathway in plants (Bolte et al., 2004), and it will be interesting to determine whether other Rab and RME proteins locate to the structures shown here.
Our results show that the TGB fusion proteins are inserted in the membranes of endosomal vesicles, but there are different possible destinations after entry to the endocytic pathway. For example, proteins could be recycled from early endosomes back to the plasma membrane (either directly or via intermediate compartments), or they could be targeted to the vacuolar pathway for degradation in the lytic vacuole (Holstein, 2002; Bolte et al., 2004). It is possible that association of TGB fusion proteins with membranous compartments in the endocytic pathway indicates that they are being targeted to prevacuolar compartments and eventual degradation in lysosomes, possibly as part of a plant defense mechanism to eliminate virus movement, or triggered as a consequence of prolonged protein overexpression. However, we favor the hypothesis that they are being recycled to the plasma membrane for the following reasons: (1) We do not see the TGB fusion proteins associated with multivesicular bodies or structures characteristic of late endosomes (reviewed in Holstein, 2002) nor any labeling of vacuolar membranes; (2) if the proteins were directed to lysosomes, then it is not clear why the fusion protein–labeled vesicles accumulate in clusters around the nuclear envelope; (3) if the proteins were targeted for degradation, we might expect to see similar localizations with the other overexpressed proteins, such as GFP, mRFP, GFP-sporamin, or GFP-TGB3 or mRFP-TGB3 expressed independently, which does not occur.
The association of PMTV TGB proteins with the endocytic pathway is unique and has not been reported in similar published studies of other plant–viral TGB proteins. However, a recent report suggests similar vesicles are also produced in PVX GFP-TGB2 transgenic plants (Mitra and Verchot-Lubicz, 2004). It is possible that the endocytic pathway is involved in the natural transmission of PMTV, which is transmitted in nature by the soil-borne plasmodiophorid S. subterranea, an intracellular parasite that infects the cells of potato tubers and roots. PMTV is carried within vector zoospores, but it is not known how the virus is acquired or transmitted. The contents of the primary zoospore are injected directly into host cytoplasm, which then develops into a multinucleate plasmodium (Braselton, 1992, 1995). The plasmodium is separated from the host cell by a single unit membrane, so it is conceivable that PMTV requires an endocytic/exocytic mechanism for vector transmission. If so, a similar mechanism might be predicted to occur in other TGB containing soil-borne viruses, such as Beet soil-borne virus, Beet necrotic yellow vein virus, and Peanut clump virus, which are transmitted by the plasmodiophorids Polymyxa betae and Polymyxa graminis.
Positive strand RNA viruses use host cellular membranes for replication and in several plant viruses; for example, TMV (Más and Beachy, 1999), Cowpea mosaic virus (Carette et al., 2002), Brome mosaic virus (Restrepo-Hartwig and Ahlquist, 1996), and Tobacco etch virus (Schaad et al., 1997) replication is associated with ER derived membranes. TMV and Peanut clump virus replication is thought to occur in close association with perinuclear ER membranes (Más and Beachy, 1999; Dunoyer et al., 2002). Some viruses induce the formation of spherules or invaginations of the membrane that contain the RNA template, replicase, and other replication-associated factors on the cytoplasmic face. The spherules bring together the replication factors to enhance replication and separate them from possible host defense factors. The nascent RNA strands are thought to exit from the neck of the spherules and presumably then interact with components of the movement complex (see reviews in Ahlquist et al., 2003; Noueiry and Ahlquist, 2003; Salonen et al., 2005). The fact that PMTV TGB proteins are associated with ER and perinuclear membranes may indicate that they, too, are located close to sites of positive strand RNA synthesis. The TGB-associated vesicles are not thought to contain replication complexes because they appear to form at the plasma membrane and their membrane is not derived from ER, but we cannot rule out the possibility that PMTV replicates on endosomal membranes and that another function of TGB2 is to help recruit such membranes.
A Model for PMTV TGB Functions
A schematic model to interpret our results is shown in Figure 4M. The model depicts different phases of movement we would hypothesize from our results, namely trafficking to plasmodesmata and recycling of membrane anchored components. TGB2 and TGB3 colocalize in the same small motile granules that move along the actin-ER network and are targeted to the neck of plasmodesmata by TGB3. The granules dock at the neck of the plasmodesmal pore, and the TGB2/3 proteins function in tandem to increase the SEL of the pore. At present, it is unclear why both TGB2 and TGB3 possess the capacity to gate plasmodesmata, although studies of other TGB viruses have reported that both these proteins possess gating capacity (Krishnamurthy et al., 2003; Mitra et al., 2003). At or near the plasmodesmal pore, we hypothesize that there is a transfer of the TGB2/3 complex from the ER to the plasma membrane (see also Oparka, 2004) and delivery of viral RNP complex, which would move through the plasmodesmal pore. After delivery, the TGB2 and TGB3 proteins are recycled together through the endocytic pathway. This model is also consistent with recent data showing that TGB2 and TGB3 are required for localization of GFP-TGB1 to the periphery of the cell and movement into adjacent cells (Zamyatnin et al., 2004).
The Link between Endocytosis and Viral Movement
We believe our findings for TGB2 and TGB3 are relevant to the native distribution of the TGB proteins because they have been validated in the presence of infectious virus. In the future, the recycling model must be tested to determine whether viral MPs are recycled to the cell interior to resume the trafficking of viral RNA complexes (under control of the virus) or whether they are targeted to the vacuole for subsequent degradation by the plant cell (under control of the host). The above model is designed to stimulate such experimentation and to initiate further studies of the relationship between plant–viral MPs and the plant endocytic recycling pathway. To date, entry of viruses into plant cells by the endocytic pathway has not been documented (Hull, 2002), although the mechanism for entry of plant viruses into tissues with no symplastic connection, such as embryos or zoospores, is unknown. However, the use of the endocytic pathway for virus entry is common in the animal kingdom (Sieczkarski and Whittaker, 2002). Our observations that the TGB2/3 complex of PMTV may use the endocytic pathway raises important questions for virus movement in plants and lends further support to the growing body of evidence that viral MPs may interact with components of the plant endomembrane recycling pathway, including Rabs, syntaxins, and DnaJ-based chaperones (reviewed in Oparka, 2004). Such components are likely to be the elusive host factors alluded to in many viral movement models (Oparka, 2004).
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. These clones were used to prepare the TMV-based clones with defective MP. The respective plasmid DNA was digested with ApaI, followed by treatment with T4 DNA polymerase and religation. This procedure causes a frame-shift mutation in the TMV MP, resulting in movement-incompetent clones.
Transient Expression Plasmids
The TGB3 and GFP genes were amplified separately from the TMV-GFP-TGB3 vector template, and the products of each reaction were combined in an overlap extension PCR (Higuchi et al., 1988) to fuse the TGB3 gene to the 3' terminus of the GFP sequence. The GFP-TGB3 fragment was ligated into plasmid vector pRTL2 (Restrepo et al., 1990), which expresses GFP-TGB3 under the control of the 35S promoter (P35S). Similarly, overlap extension PCR was used to clone the TGB2 and TGB3 genes as fusions to the 3' terminus of mRFP. The TGB2 and TGB3 genes were amplified from a plasmid containing the PMTV RNA2 sequence (Cowan et al., 2002), and mRFP was amplified from plasmid pmRFP1 (Clontech, Palo Alto, CA). The fusions were ligated into pRTL2 as above.
Two mutant constructs were prepared from the P35S-GFP-TGB3 using the QuikChange kit (Stratagene, La Jolla, CA) where the TGB3 protein sequence -Y89QDLN93 was modified to YAAAA or GQDGN. GFP-Ara7 was produced by PCR amplification of Ara7 cDNA and cloned into pVKH 18-En6 (Batoko et al., 2000) so that GFP is expressed as an N-terminal fusion.
Plant Material
Several transgenic plants were used for colabeling experiments, including the following: Nicotiana tabacum, TMV-30K-GFP (TMV MP-GFP) (Reichel and Beachy, 2000); N. benthamiana, SmRS.GFP-TUA6 (tua-GFP) (Ueda and Hashimoto, 1999); N. benthamiana, HDEL-GFP (erGFP) (Carette et al., 2000); Arabidopsis thaliana, sialyl transferase-GFP (Boevink et al., 1998); A. thaliana, GFP-hTalin (Takemoto et al., 2003). Plants were grown from seed and maintained at 28°C in a Snijder climatic cabinet (Snijder, Tilburg, Holland) with a photoperiod of 16 h. Light intensity was 400 µE m–2 s–1. Plants were used for experiments when they were between 15 and 60 d old.
Microscopic Imaging of Intact Leaves
Intact leaves, infected with the different vectors expressing GFP- or mRFP-TGB2 and TGB3 fusions, were fixed to microscope slides using double-sided adhesive tape (Sellotape GB, Dunstable, UK). Confocal images were obtained using a Leica TCS SP spectral confocal laser-scanning microscope (CLSM; Leica Microsystems, Heidelberg, Germany) with Leica water-dipping lenses. GFP was excited at 488 nm and emissions collected at 500 to 530 nm. mRFP was excited at 568 nm and emissions collected at 580 to 600nm. For dual imaging of fusions with both fluorescent proteins, images were taken sequentially to avoid bleed-through of signals (Leica TSC Confocal Systems user manual). Post-acquisition processing of images was done using Adobe Photoshop 7.0 software (Mountain View, CA).
Preparation of Infectious Transcripts and Plant Inoculation
Run-off transcripts were synthesized from viral vectors using a T7 transcription kit (Ambion, Austin, TX). For manual inoculation, transcripts were rubbed onto aluminium oxide-dusted N. benthamiana leaves. Two and 4 d postinoculation, inoculated leaves were detached from plants and examined under the CLSM. At this time, infection sites containing the virus vector with a functional MP covered an area of between 20 and 100 epidermal cells.
Microprojectile Bombardment
Plasmid DNAs and transcripts from viral constructs were introduced into source leaf epidermal cells by particle bombardment using a Handgun essentially as described by Gal-On et al. (1997). Briefly, 2 µg of plasmid DNA or RNA transcript was mixed with 5 µL of ethanol and 0.55 mg of 1-µm gold micro carrier (Bio-Rad Laboratories, Hercules, CA) in ethanol to give a final volume of 18 µL. The nucleic acid-gold mixture (2 µL) was applied to a discharge assembly (13-mm Plastic Swinney Filter Holder; PALL Gelman Laboratory, Ann Arbor, MI) and eth
