INTRODUCTION

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The requirements for systemic plant virus infections differ from those of animal viruses because the plant cell wall poses a barrier that must be breached to permit initial infections, cell-to-cell movement, and entry into and exit from the vasculature. Localized spread from primary infection foci requires that plant viruses move to adjacent cells through the intercellular channels formed by plasmodesmata (for reviews, see references 11, 35, and 37). Consequently, most plant viruses have evolved dedicated genes that function to mediate cell-to-cell transit. A considerable body of evidence now exists indicating that many of these viruses encode genes that function mechanistically to increase the permeability of the plasmodesmata and to facilitate transport of bound viral nucleoprotein complexes through the plasmodesmata to adjacent cells (1, 17, 30, 31, 41, 61).

Tobacco mosaic virus (TMV) is the prototype of those viruses that encode a single cell-to-cell movement protein, and the movement processes described for TMV have provided valuable models for analysis of the infection processes of other viruses (28, 49). In many viruses, a single movement protein facilitates cell-to-cell transit by a series of incompletely defined steps during which the protein encapsidates the viral genome and associates with cytoplasmic membranes, cytoskeletal elements, and cell wall proteins. These events alter plasmodesmal permeability sufficiently to permit cell-to-cell transport of putative viral nucleoprotein complexes (40). However, numerous other viruses require coordinated activities of more than one gene for local and long-distance transport. For example, in the case of the DNA-containing geminiviruses, a dual shuttle gene system mediates transit of single-stranded (ss) DNA from the nucleus, through the cytoplasm, and into and across the plasmodesmata (28, 41). Two small proteins, p8 and p9, are required for systemic infections of the cytoplasmically replicating carmoviruses (29), and the tombusviruses have two nested genes (p19 and p22) that function in different aspects of movement (55). The Tomato bushy stunt virus p22 protein appears to be the functional analog of the TMV 30-kDa movement protein, whereas the p19 protein is dispensable for systemic movement in some host backgrounds but is required in other hosts (55). Additional complexity and division of labor is observed among a diverse set of RNA viruses differing substantially in genome organization that encode movement proteins organized in a "triple gene block" (TGB). These proteins have homologs in monopartite potexviruses (30, 39) and carlaviruses (51), bipartite benyviruses (18) and pecluviruses (22), and tripartite hordeiviruses (38, 56).

Barley stripe mosaic virus (BSMV) is the type member of the hordeiviruses. The viral genome is composed of positive-sense ssRNA divided into three components designated , , and . The  and  RNAs are strictly required for replication, while RNA is required for cell-to-cell movement (27, 44). RNA encodes a replicase protein, a, that contains methyl transferase and helicase domains. RNA is bicistronic and encodes a second replicase protein, a, that is characterized by a polymerase (GDD) motif. The second protein, b, is expressed from a subgenomic (sg) RNA (20). This cysteine-rich protein is not required for replication or cell-to-cell movement per se, but mutations within the protein affect pathogenicity (12, 43).

RNA encodes a 5'-proximal coat protein (CP) separated from the TGB by a short intergenic region (19, 23). The TGB1 protein, formerly designated b, is expressed from the 2.45-kb sgRNA1 (66). This 58-kDa TGB1 protein has been purified from BSMV-infected barley and shown to bind ssRNA and double-stranded RNA, to exhibit ATPase activity, and to bind nucleotides in vitro (13). A helicase motif is also a prominent component of TGB1, and mutations of conserved amino acids within the motif abrogate cell-to-cell movement (27); however a variety of assays have failed to detect helicase activity in vitro (13). The remaining TGB proteins are expressed from the low-abundance 960-nucleotide sgRNA2 (66). TGB2, a 14-kDa protein (formerly designated d), and its low-abundance 23-kDa translational read-through protein, TGB2' (formerly designated d'), are translated by ribosomes initiating at the first AUG of sgRNA2. The 17-kDa TGB3 protein (formerly designated c) is expressed via leaky scanning through the TGB2 AUG codon of sgRNA2 (66). Sequence analyses predict that both the TGB2 and TGB3 proteins are membrane associated, as they contain two hydrophobic membrane-spanning domains separated by a hydrophilic region (38, 56). Subcellular fractionation studies have also shown that TGB2 and its read-through extension, TGB2', are associated with membrane and cell wall fractions (14, 66). Infectivity results clearly demonstrate that the TGB1, TGB2, and TGB3 proteins are each required for cell-to-cell movement in both monocot and dicot hosts (27). These results are similar to and extend those obtained with the potexviruses White clover mosaic virus (2, 30) and Potato virus X (PVX) (60), the benyvirus Beet necrotic yellow vein virus (BNYVV) (4, 18), and the pecluvirus Peanut clump virus (PCV) (22).

Only limited information is available to describe long-distance movement processes whereby viruses enter, navigate and exit the vascular system, but the mechanisms required for these events appear to be distinct from those required for cell-to-cell movement processes (7, 32). For example, efficient long-distance transport of TMV requires the CP in addition to the 30-kDa protein (40). However, a limited number of viruses encoding different classes of movement protein genes can establish efficient local and systemic infections in the absence of the CP. In the prototypical case of Tobacco rattle virus (6) and other tobraviruses encoding a single movement protein (5, 33), the CP is dispensable for long-distance movement. Other examples where local and long-distance transit do not require the CP have been reported among members of the tombusviruses (9, 54).

Considerable variation in the requirements for the CP for local and long-distance movement also exists among those viruses that require the TGB proteins for movement. In the case of BSMV, the CP is dispensable for cell-to-cell and systemic movement (27, 44). Similarly, the CP is dispensable for cell-to-cell movement of BNYVV (15) and PCV (22) and for systemic movement of the pomovirus Potato mop top virus (PMTV) (34). However, unlike BSMV and PMTV, the CP is required for vascular transport of BNYVV (53) and PCV (22). In contrast, potexviruses have an absolute requirement for the CP for cell-to-cell movement (30, 52). To begin to define the cellular interactions during the movement of BSMV, we have constructed green fluorescent protein (GFP) fusions to the amino terminus of TGB1 and have used these reporter derivatives to assess expression patterns and subcellular localization during cell-to-cell movement. In addition, protoplast experiments have been conducted to evaluate the roles of TGB2 and TGB3 in subcellular targeting of TGB1.

	MATERIALS AND METHODS

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Recombinant plasmids. Full-length ,  (42SpI), and  (42) cDNA clones derived from the BSMV ND18 strain (45) were used in this study. GFP was expressed from the  cDNA clone as a b-GFP fusion (27). Several site-directed mutations (25) described below were introduced into 42SpI or B7, a mutant of 42SpI that lacks the AUG of the CP (a) gene (47)_. Additional cDNA clones containing mutations in the RNA TGB were used in various experiments. The _2.0 cDNA clone contains a 200-amino-acid deletion in TGB1 (47), the Cla cDNA clone alters two amino acids (K₁₁  N and Y₁₂  R) in TGB2 (47), and the c-stop cDNA clone introduces a UAA at codon 72 in TGB3 (27). The  cDNA clones that contained mutations in the helicase domain of b resulted in the following amino acid changes: K₂₇₅  R (M1); K₂₇₅  A (M2); D₈₂₆ and E₈₂₇  N and Q (M3); G₈₄₈, D₈₄₉, and Q₈₅₂  A, N, and E (M4); R₃₇₇  A (M5); Q₄₆₂ and G₄₆₃  E and A (M6); R₄₉₂  A (M7); and R₃₇₇ and R₄₉₂  A and A (M5/M7) (13).

Inoculation and treatment of plants and protoplasts. RNA, -, and - transcripts prepared as described previously were used to inoculate barley (Hordeum vulgare), Chenopodium amaranticolor, and Nicotiana benthamiana plants (46) or to transfect protoplasts from the BY-2 Nicotiana tabacum cell suspension culture (62). Transfected protoplasts were incubated with cytochalasin D (25 µg/ml) or subjected to a cold treatment prior to being aldehyde fixed and visualized by epifluorescence microscopy (36). The protoplasts were also stained at 24 h posttransfection by incubation in fresh medium containing 10 µM ER-tracker blue-white DPX (Molecular Probes, Eugene, Oreg.) or 0.1 µM rhodamine B hexyl ester (Molecular Probes) for 30 min, transferred to fresh medium without the marker dyes, and visualized under epifluorescence microscopy.

Protein and RNA analyses. Infected leaves or protoplasts were harvested, extracted, and separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels prior to Western blot serological analysis (14). RNA was extracted, separated on 1% agarose gels, blotted onto nylon membranes, and hybridized with ³²P-labeled -specific or -specific probes as described previously (66).

GFP fusion protein expression and immunofluorescence. GFP expression was visualized by excitation at 450 to 490 nm and emission at 520 nm using a Zeiss Axiophot (Thornwood, N.Y.) microscope. Computer images were acquired with an Optronics 450 Color charge-coupled device camera and captured at high resolution by using a Scion CG-7 RGB framegrabber board or photographed with Kodak color film. Figures were assembled using Canvas (Deneba Software, Miami, Fla.) and Adobe Photoshop (Adobe Systems Inc., San Jose, Calif.) software. For immunofluorescence, protoplasts were fixed with aldehyde prior to incubation for 1 h at 4°C with a TGB1 polyclonal mouse antibody (1:500 dilution) (35). Following incubation, the protoplasts were washed with phosphate-buffered saline (10 mM phosphate, pH 7.4, 150 mM NaCl) and incubated with a 1:30 dilution of fluorescein-conjugated goat anti-mouse immunoglobulin G (Calbiochem, San Diego, Calif.) for 30 min at 4°C. Residual nonspecific fluorescence was removed by a second phosphate-buffered saline wash prior to microscopic observation.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

BSMV GFP-TGB1 reporter virus moves efficiently in monocot and dicot hosts. To examine the interactions of BSMV TGB1 during infection of plants and protoplasts, we engineered an amino-terminal fusion of the GFP reporter gene to TGB1 (Fig. 1A). Fusions were constructed in both wild-type RNA (.GFP:TGB1) and in the RNA mutant B7 (B7.GFP:TGB1), in which CP expression had been eliminated. The .GFP:TGB1 and B7.GFP:TGB1 derivatives were designed as alternatives to facilitate comparisons of the movement of BSMV reporters in different genetic backgrounds and to provide a high-resolution marker for subcellular localization of GFP-TGB1 during cell-to-cell movement. When the .GFP:TGB1 (CP-expressing) and B7.GFP:TGB1 (inactivated CP) RNAs were cotransfected with wild-type RNA and - into BY-2 tobacco protoplasts, RNA blotting revealed that the .GFP:TGB1 and B7.GFP:TGB1 RNA accumulation was reduced by more than 50% compared to levels of wild-type RNA or B7 RNA (Fig. 1B). To determine whether these decreases in abundance were due to a general reduction in total viral RNA replication, the RNA blots were stripped and reprobed with a -specific probe (Fig. 1B). Some batch-to-batch sample variation was observed between electroporated protoplasts. However, RNA blots hybridized with - and -specific probes revealed that similar levels of variation were observed in comparisons of RNA and sgRNA with the wild-type  or B7 RNAs and wild-type  or B7 RNAs that contained the GFP-TGB1 sequence (Fig. 1B). These results indicate that the smaller amounts of the .GFP:TGB1 and B7.GFP:TGB1 RNAs are specifically correlated with reduced RNA replication rather than reduced viral replication. However, the GFP fusions were expressed to easily detectable levels in protoplasts with antisera raised to either TGB1 or GFP (Fig. 1C).

View larger version (43K): [in this window] [in a new window]   FIG. 1.   GFP-TGB1 fusion protein expression in BY-2 protoplasts. (A) Diagram of GFP fused to TGB1 in the  RNA or the B7 RNA in which the CP AUG codon was destroyed (*) showing the relative size of the two sgRNAs. (B) Hybridization patterns of RNA from protoplasts transfected with BSMV RNAs at 22 h p.i. The RNAs were separated on agarose gels and blotted onto nylon membranes prior to being hybridized with a 32P-labeled -specific probe or a 32P-labeled -specific probe. Lane 1, mock transfection (M); lane 2, ,  (+cp), and  RNAs; lane 3, , B7 (cp), and  RNAs; lane 4, , .GFP:TGB1 (+cp), and  RNAs; lane 5, , B7.GFP:TGB1 (cp), and  RNAs. Note that the genomic (g) RNA is somewhat obscured by rRNAs, but the sgRNA is clearly visible. (C) Proteins from protoplasts transfected with BSMV RNAs at 22 h p.i. Extracts were separated on an SDS-10% (wt/vol) polyacrylamide gel prior to immunoblotting with antiserum raised to TGB1 or GFP. Lane 1, mock inoculation (M); lanes 2 and 4, protoplasts transfected with , .GFP:TGB1 (+cp), and  RNAs; lanes 3 and 5, protoplasts transfected with , B7.GFP:TGB1 (cp), and  RNAs; lanes 1, 2, and 3, protoplasts harvested at 19 h posttransfection; lanes 4 and 5, protoplasts harvested at 26 h posttransfection.

CP expression affects the virulence of the TGB1 reporter derivatives. We previously observed similar timing of infection in comparisons of the wild-type BSMV (RNA) and its RNA B7 CP derivative (47). To investigate whether the TGB1 fusions affected the infection phenotype, comparisons were carried out with the RNA (CP-expressing) and the B7 (inactivated CP) GFP-TGB1 derivatives. These comparisons revealed that the appearance of local lesions in C. amaranticolor was delayed between 24 and 36 h in the GFP-TGB1 B7 background, and these lesions were approximately 50% smaller at 6 days postinoculation (p.i.) (Fig. 2A and B). In addition, the lesions failed to spread and coalesce, and the infected leaves did not undergo abscission by 14 days p.i. as normally occurs in wild-type infections (43). In barley and N. benthamiana, a 36- to 72-h delay in development of systemic symptoms was also observed, but otherwise the infection phenotype appeared to be typical of GFP-TGB1 in a CP-expressing background and of wild-type virus infections (data not shown). These results suggest that even though the GFP-TGB1 derivatives are able to establish systemic infections in the absence of the CP, subtle interactions requiring the CP promote optimal efficiency of both cell-to-cell and systemic movement.

View larger version (79K): [in this window] [in a new window]   FIG. 2.   Symptom phenotype and protein expression in infections with .GFP:TGB1 or B7.GFP:TGB1. (A and B) C. amaranticolor leaves inoculated with  and .GFP:TGB1 (CP+) or B7.GFP:TGB1 (CP) and  RNAs at 6 days p.i. (C) Proteins from infected plants separated on an SDS-10% (wt/vol) polyacrylamide gel prior to immunoblotting with antiserum raised to TGB1 or GFP. Lane 1, plants infected with , , and  RNAs; lanes 2, 4, 6, and 8, plants infected with , .GFP:TGB1, and  RNAs (I); lanes 3, 5, 7, and 9, mock-infected plants (M). Lanes 1, 2, 3, 4, and 5, barley; lanes 6 and 7, N. benthamiana; lanes 8 and 9, C. amaranticolor.

Dicot and monocot hosts differ in the expression patterns of GFP-TGB1. For infectivity comparisons, the wild-type  RNA or .GFP:TGB1 RNA (CP-expressing background) and the  and  RNAs were inoculated onto C. amaranticolor, N. benthamiana, and barley. As was previously described in detail (27), the initial infection events differed markedly in the monocot host and the dicot hosts. The accumulation of the fusion proteins was first examined by analysis of protein extracted from infected leaves. The GFP-TGB1 fusion protein was detected in C. amaranticolor, N. benthamiana, and barley by using antiserum raised against the GFP or TGB1 protein (Fig. 2C).

View larger version (40K): [in this window] [in a new window]   FIG. 3.   Localization of GFP-TGB1 fusion protein in developing infection foci in leaves. (A) Inoculated leaf of C. amaranticolor at 24 h p.i. Bar = 25 µm. (B) Inoculated leaf of C. amaranticolor at 48 h p.i. Bar = 25 µm. (C) Inoculated leaf of C. amaranticolor at 96 h p.i. Note that the center of the lesion is necrotic and that GFP fluorescence is intense at the periphery of the foci some distance away from the necrotic center. Bar = 50 µm. (D) Inoculated leaf of C. amaranticolor with , , and b-GFP RNAs at 96 h p.i. Bar = 50 µm. (E and F) Epidermal C. amaranticolor cells at the outer edge of the infection ring at 72 h p.i. Bar = 50 µm. Inset, bar = 5 µm. Note the paired punctate foci appressed along the cell walls. (G) Inoculated leaf of N. benthamiana at 5 days p.i. Bar = 50 µm. (H) Epidermal N. benthamiana cells at 6 days p.i. Bar = 50 µm. Inset, bar = 5 µm. (I) Inoculated barley leaf at 72 h p.i. Bar = 100 µm. (J) Systemically infected barley leaf at 96 h p.i. Bar = 200 µm.

TGB1 is associated with membranes. The subcellular compartmentation of GFP-TGB1 was investigated at higher resolution by inoculating BY-2 protoplasts with wild-type  and  RNAs plus the .GFP:TGB1 RNA. GFP fluorescence first appeared 19 to 21 h p.i. and was localized to membranes around the nucleus (Fig. 4A, C, and D) and to discrete foci at the plasma membrane (Fig. 4B, C, and D). In the subsequent 18 h, little alteration in the localization of fluorescence was observed. To confirm that the GFP fusion had no effect on the localization of TGB1, immunofluorescence of BSMV-infected BY-2 protoplasts was evaluated with antiserum raised against the TGB1 protein. At 18 h p.i., antibody fluorescence against the unfused TGB1 protein was primarily localized to membranes surrounding the nuclei (Fig. 4E), and by 24 h p.i., bright spots of antibody fluorescence were also evident at the plasma membrane (Fig. 4F). The foci present at the plasma membrane were slightly more diffuse in protoplasts following immunofluorescence than was GFP fluorescence observed in GFP-TGB1-infected protoplasts. This was due to fixation of the protoplasts prior to immunofluorescence in contrast to nonfixed GFP-fluorescing protoplasts. The punctate foci at the plasma membrane appeared not to be associated with the presence of remnants of the cell wall or with newly deposited cell wall components because calcofluor white staining did not reveal any residual cell wall material (data not shown). From these results, we conclude that the subcellular localization patterns of the native TGB1 protein and the GFP-TGB1 fusion protein are indistinguishable.

View larger version (34K): [in this window] [in a new window]   FIG. 4.   Subcellular localization of GFP-TGB1 and b-GFP in BY-2 tobacco protoplasts. The protoplasts were transfected with BSMV RNAs and examined at 18 to 24 h p.i. (A to D) Protoplasts transfected with , .GFP:TGB1, and  RNAs and examined at 22 h p.i. Protoplasts under bright-field illumination (A) and GFP fluorescence (B, C, and D) are shown. Note that panel B is focused on the cell surface and the GFP fluorescence is localized to punctate foci associated with the plasma membrane, while panels C and D are focused on the interior of the cell and GFP fluorescence is accentuated at perinuclear membranes. (E and F) Protoplasts transfected with , , and  RNAs and fixed at 18 (E) or 24 (F) h p.i. and then subjected to immunofluorescence with antiserum raised to the TGB1 protein. (G) Protoplasts transfected with , , and b-GFP RNAs and examined at 22 h p.i. (H) Protoplasts transfected with PVX KDEL-GFP at 24 h p.i. (I and J) Protoplasts transfected with , .GFP:TGB1, and  RNAs and treated with rhodamine B hexyl ester chloride R6 at 22 h p.i. and examined using the fluorescein isothiocyanate (FITC) channel (I) or the rhodamine channel (J). (K and L) Protoplasts transfected with , .GFP:TGB1, and  RNAs, treated with ER-tracker blue-white DPX at 22 h p.i., and examined using the FITC channel (K) or the DAPI (4',6'-diamidino-2-phenylindole) channel (L). Bar = 30 µm.

Subcellular localization of GFP-TGB1 is affected by mutations in the TGB1 helicase motif. We previously noted that site-specific mutations in the helicase domain of TGB1 prohibited cell-to-cell movement of BSMV in planta (27). To ascertain whether subcellular localization of GFP-TGB1 containing each of these helicase mutations is altered, RNA and - and the individual .GFP:TGB1 derivatives containing mutations (M1, M2, M3, M4, M5, M6, M7, and M5/M7 [Fig. 5A]) in each of the six conserved motifs of the helicase domain were used to transfect BY-2 protoplasts. Each of these mutants exhibited pronounced alterations in the subcellular localization of GFP-TGB1. In most cases, fluorescence of the mutant fusion proteins was associated with the plasma membrane and ER membranes surrounding the nucleus, but the proportion of protoplasts exhibiting punctate foci at the plasma membrane declined precipitously (eightfold or more) compared to wild-type GFP-TGB1 (Table 1; Fig. 5B to D). In each case, full-length GFP-TGB1 fusion protein was found to be expressed in these protoplasts by Western blot analyses using antiserum raised against GFP (Fig. 5E) or TGB1 (data not shown).

View larger version (42K): [in this window] [in a new window]   FIG. 5.   Effects of mutations in the TGB1 helicase domain and the TGB2 and TGB3 proteins on subcellular localization of GFP-TGB1 in BY-2 protoplasts. (A) Amino acid sequence of the helicase domain in TGB1. The overlined amino acids labeled I to VI indicate the conserved regions that define the helicase motif (24). M1 to M7 indicate the positions of site-specific mutations engineered into these regions. (B to D)  and  RNAs and a wild-type or mutant GFP:TGB1 RNA were transfected into BY-2 protoplasts and examined at 21 to 24 h p.i. (B) Wild-type GFP-TGB1 showing punctate foci; (C and D) shift in fluorescence of GFP-TGB1 helicase mutants M5 (C) or M5/M7 (D) away from punctate foci at the plasma membrane. Similar shifts were noted with the M1, -2, -3, -4, -6, and -7 mutants. (E) Immunoblot analyses of proteins from mock-transfected protoplasts (M) or protoplasts transfected with  and  RNAs and a wild-type (wt) or a mutant GFP-TGB1 RNA (M1, -2, -3, -4, -5, -6, -7, and M5/M7) at 24 h p.i. Immunoblots were probed with antiserum raised to GFP. (F to H) RNAs  and  and GFP-TGB1 expressing TGB2 and TGB3 (F and G) or GFP-TGB1 that did not express the TGB2 and TGB3 proteins (H) were transfected into BY-2 protoplasts and examined at 21 to 24 h p.i. Bar = 30 µm.

TGB2 and TGB3 facilitate localization of TGB1 to the plasma membrane. To investigate the effects of the TGB2 and TGB3 proteins on the subcellular localization of GFP-TGB1, RNA and - plus .GFP:TGB1 RNA unable to express TGB2 and TGB3 were transfected into BY-2 protoplasts, and the localization of GFP-TGB1 was monitored by fluorescence microscopy at 22 to 24 h. Protoplasts transfected with the TGB2- and TGB3-deficient derivative exhibited a diffuse GFP-TGB1 fluorescence associated with the perinuclear ER and a drastic shift of fluorescence away from the plasma membrane (Table 2; Fig. 5F, G, and H). Similar results were obtained with derivatives expressing only TGB2 or TGB3 (data not shown).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

To examine the initial phases of infection of BSMV in monocots and dicots, we have constructed GFP fusions with TGB1 and b (27). These fusions each provide reliable indicators of the early stages of virus movement, and the GFP-TGB1 results verify and extend the differences shown by b-GFP during infections of dicot and monocot hosts. In dicot hosts, infections typically spread as circular-to-oval foci through several parenchyma cells separating the reticulate vascular tissue. In contrast, in the monocot host, single cells exhibiting green fluorescence were extremely rare, and infections quickly reached the vascular system and spread throughout the plant.

In dicots, GFP-TGB1 fluorescence was observed by 24 h p.i. in the ER and pronounced punctate foci formed along the cell walls. GFP-TGB1 also exhibited a temporal pattern of expression in dicots, as has been reported for the 42-kDa BNYVV TGB1 protein (15) and the TMV 30-kDa protein (42). This differs markedly from the constitutive cytosolic localization of b-GFP (27). The temporal expression of the TMV movement protein, P30-GFP, in N. benthamiana has been previously attributed to both enhanced protein degradation and decreased expression near the centers of the foci (42, 59). We have no direct evidence to determine whether TGB1 turns over more rapidly than b following the initial burst of synthesis. Previous time course studies of RNA synthesis in barley protoplasts suggest that the temporal b expression may in part be a consequence of early expression of sgRNA1 (67; cf. Fig. 1). In this case, sgRNA1 appeared to reach maximum abundance by 12 h p.i., whereas the genomic RNAs and sgRNA continued to increase in abundance throughout the 24-h period of analysis.

Infections in barley differed markedly from those of the dicot hosts in the timing of GFP-TGB1 fluorescence and in the patterns of expression in infected cells. GFP-TGB1 fluorescence was not evident until 72 h p.i., and no punctate foci were detected in the mesophyll or epidermal cells of infected barley leaves. GFP-TGB1 fluorescence also appeared to be relatively consistent throughout the infected tissue and could be observed for at least 7 days p.i. This suggests that GFP-TGB1 has different patterns of expression and localization in monocot and dicot cells during the initial stages of infection. However, additional experiments are required to determine whether these differences result from primary adaptations to barriers to cell-to-cell movement presented by monocots and dicots or whether they represent indirect host-specific events of little consequence to cell-to-cell movement per se.

Interestingly, the BSMV 58-kDa TGB1 protein and the TGB1 proteins from BNYVV (42 kDa), PCV (51 kDa), and PMTV (51 kDa) have variable amino-terminal extensions that are absent in the smaller (24- to 28-kDa) TGB1 proteins of the potexviruses. At present, the roles of these amino-terminal extensions of the hordeivirus, benyvirus, pecluvirus, and pomovirus TGB1 proteins is unclear. In particular, our lack of knowledge of the significance of these regions is highlighted by findings that in vitro RNA binding by the 42-kDa TGB1 protein of BNYVV (3) maps to the first 24 amino acids but, surprisingly, that site-specific mutations of the basic residues in this region fail to disrupt cell-to-cell movement (15). In contrast, mutagenesis experiments indicate that several regions of the BSMV TGB1 protein function in RNA binding. The amino-terminal region of the TGB1 protein contains elements that contribute to double-stranded RNA versus ssRNA binding in vitro (13), but the functions of these regions in RNA binding in vivo have not been evaluated. However, several deletions affecting RNA binding also compromise cell-to-cell movement (27). The amino-terminal extensions of the larger TGB1 proteins may be able to compensate in part for requirements for the CP for cell-to-cell movement of those viruses containing a smaller TGB1 protein. Analysis of the TGB1 genes of benyviruses, carlaviruses, hordeiviruses, pecluviruses, pomoviruses, and potexviruses suggests that these genes may have evolved from a common progenitor, based on the conservation of the helicase domain present in these proteins (63). However, it is also possible that during the course of evolution, the TGB1 proteins of BSMV, BNYVV, PCV, and PMTV may have acquired amino-terminal functions from diverse sources that permitted them to dispense with a CP requirement for cell-to-cell movement. Alternatively, the larger TGB1 proteins may have retained some functions that the potexvirus TGB1 proteins have lost.

The 42-kDa BNYVV (15) and 51-kDa PCV (16) TGB1 proteins are similar to the BSMV TGB1 protein in that they form punctate foci at the cell walls of infected dicot leaf cells. Specifically, the BNYVV (15) and PCV (16) TGB1 proteins localize to plasmodesmata, while the BSMV TGB1 foci are often present as paired foci that appear to traverse the walls of adjacent cells. This localization pattern contrasts with that of the smaller TGB1 proteins of the potexviruses Bamboo mosaic virus (8), Foxtail mosaic virus (50), and PVX (10) that do not localize to the cell walls in infected cells. In this regard, the potexvirus TGB1 proteins exhibit subcellular localization patterns more typical of BSMV in barley. From these results, it is evident that the TGB1-encoded proteins of hordeiviruses, benyviruses, pecluviruses, and the potexviruses differ substantially in several aspects of their function and interactions. However, the host-specific differences in cell wall associations of the BSMV TGB1 protein in monocots versus dicots complicates straightforward interpretation of results based on cytological analyses in a single host. Therefore, it is evident that more detailed analyses of TGB activities in a variety of hosts are needed to elucidate nuances of the movement processes of these viruses.

The patterns of localization of GFP-TGB1 in cells of infected plants and in protoplasts were similar in that GFP-TGB1 formed punctate foci at the cell walls and at the plasma membrane, respectively. These foci could possibly have been associated with remnants of the cortical ER at sites where the cell wall was attached. However, the foci apparently do not colocalize with cell wall fragments because calcofluor white staining failed to reveal such remnants in the vast preponderance of infected cells. The protoplast localization studies also revealed an association of TGB1 with ER membranes located in close proximity to the nucleus in the absence of TGB2 and TGB3. However, our examination of the TGB1 sequence failed to reveal known ER signal sequences. Even though BSMV TGB1 does not form substantial interactions with cytoskeletal components, its membrane associations appear to be similar to those of the TMV P30 movement protein, which behaves as an integral ER membrane protein (21, 48) despite the lack of known ER retention sequences. Although the exact mechanisms of TMV P30 and BSMV TGB1 membrane associations have not yet been resolved, it is possible that TGB1 ER localization may be mediated by interactions with host proteins that serve to target the movement protein. In this regard, a host transmembrane protein, TOM1, has been identified in Arabidopsis thaliana that interacts with the helicase domain of the TMV replicase, suggesting that this protein may function as an ER membrane anchor for the replicase (64). Although our individual helicase mutations argue against direct helicase domain interactions, multiple host contact sites might exist within the protein.

We have previously demonstrated that site-specific mutations introduced into each of the six conserved helicase motifs of the TGB1 protein abrogate cell-to-cell movement (27). Therefore, even though we were unable to detect helicase activity in vitro using purified TGB1 protein (13), each conserved element in the helicase domain is critical for cell-to-cell movement (27). Moreover, in infected protoplasts, a series of mutations in the TGB1 helicase motif each shifted the subcellular localization of GFP-TGB1 away from the plasma membrane. The mutant GFP-TGB1 proteins showed a much lower proportion of punctate foci at the plasma membrane than the wild-type protein, but the mutants still exhibited an intense membrane fluorescence. These results suggest that the helicase motif may also function to facilitate plasmodesmal interactions with TGB2 and TGB3, or with host proteins required for plasmodesma associations. The fact that both TGB2 and TGB3 are required for formation of punctate plasma membrane foci favors a model wherein TGB2, TGB3, or both proteins interact directly with TGB1. Our ongoing studies of the interactions of these proteins with a ribonucleoprotein complex formed in planta (D. M. Lawrence, J. Yu, and A. O. Jackson, unpublished data) may provide additional details to clarify this model.

Several lines of evidence also indicate that interactions of the TGB proteins of other viruses are required for cell-to-cell movement. For example, like BSMV, the TGB1 protein of BNYVV fails to form punctate foci at the cell walls of infected Chenopodium quinoa leaf cells in the absence of the TGB2 and TGB3 proteins (15). The PCV TGB1 protein is also associated with plasmodesmata in infected cells, but when TGB1 is expressed in transgenic plants, it is not localized to plasmodesmata until after virus infection (16). The TGB1 protein of PVX is able to move from cell to cell when expressed alone but is restricted to single cells when expressed in the presence of TGB2 and TGB3 or the CP, suggesting that the TGB functions may be facilitated by direct interactions (65). Interestingly, the relative levels of expression of the TGB2 and TGB3 proteins of BNYVV also appear to be important for cell-to-cell movement (4), and these results may provide evidence for interactions of TGB2 and TGB3. However, transient-expression experiments in epidermal leaf cells with the hordeivirus Poa semilatent virus-encoded TGB2 and TGB3 proteins have been interpreted to suggest that these two proteins do not form complexes, even though TGB3 assists in the targeting of TGB2 (58). Irrespective of the mechanistic aspects of movement, the available results suggest the TGB proteins can function with heterologous viruses. For example, it has been shown that limited cell-to-cell movement functions of the BSMV TGB can be replaced by the Poa semilatent virus TGB (57), and similar experiments show that the BNYVV TGB can be replaced by the PCV TGB (26). However, in the case of BNYVV, the individual TGB proteins could not be replaced by their respective PCV homologs. Currently, we are conducting experiments to determine how interactions among the BSMV TGB1, -2, and -3 proteins function in the formation of competent cell-to-cell movement complexes.