Cell-to-Cell and Phloem-Mediated Transport of Potato Virus X: The Role of Virions

Correspondence to: Simon Santa Cruz, ssanta{at}scri.sari.ac.uk (E-mail), 44-1382-562426 (fax).

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

Movement-deficient potato virus X (PVX) mutants tagged with the green fluorescent protein were used to investigate the role of the coat protein (CP) and triple gene block (TGB) proteins in virus movement. Mutants lacking either a functional CP or TGB were restricted to single epidermal cells. Microinjection of dextran probes into cells infected with the mutants showed that an increase in the plasmodesmal size exclusion limit was dependent on one or more of the TGB proteins and was independent of CP. Fluorescently labeled CP that was injected into epidermal cells was confined to the injected cells, showing that the CP lacks an intrinsic transport function. In additional experiments, transgenic plants expressing the PVX CP were used as rootstocks and grafted with nontransformed scions. Inoculation of the PVX CP mutants to the transgenic rootstocks resulted in cell-to-cell and systemic movement within the transgenic tissue. Translocation of the CP mutants into sink leaves of the nontransgenic scions was also observed, but infection was restricted to cells close to major veins. These results indicate that the PVX CP is transported through the phloem, unloads into the vascular tissue, and subsequently is transported between cells during the course of infection. Evidence is presented that PVX uses a novel strategy for cell-to-cell movement involving the transport of filamentous virions through plasmodesmata.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

Accumulating evidence demonstrates that both cellular and viral proteins can move between cells via plasmodesmata. To date, most of the experimental evidence demonstrating intercellular protein trafficking has been obtained from studies using recombinant viral movement proteins (MPs) microinjected into plant cells (Fujiwara et al. 1993 ; Noueiry et al. 1994 ; Waigmann et al. 1994 ; B. Ding et al. 1995 ). However, the finding that two maize proteins, the transcription factor KNOTTED (Lucas et al. 1995 ) and the pathogenesis-related protein PRms (Murillo et al. 1997 ), can also move between cells indicates that intercellular protein trafficking may be of more general relevance in plant biology and has rekindled the debate about whether plant virus movement functions represent stolen cellular functions (reviewed in Maule 1994 ; Mezitt and Lucas 1996 ).

A further example of intercellular transport of macromolecules is the supply of protein from companion cells to the enucleate conducting cells, the sieve elements, which possess no protein-synthesizing capability (Fisher et al. 1992 ; Nakamura et al. 1993 ; Sjolund 1997 ). The intimate relationship between sieve elements and companion cells is central to the transportation of photoassimilates and other macromolecules, including viruses, throughout the plant. The plasmodesmata connecting sieve elements to companion cells are structurally distinct from other plasmodesmata (Leisner and Turgeon 1993 ; Van Bel and Kempers 1997 ) and have a size exclusion limit (SEL) (Kempers et al. 1993 ; Kempers and Van Bel 1997 ) substantially greater than the SEL of plasmodesmata between either epidermal (Derrick et al. 1990 ) or mesophyll (Wolf et al. 1989 ) cells. In terms of susceptibility to virus infection, plasmodesmata at the companion cell–sieve element interface are particularly important because they provide the last line of defense against systemic virus movement.

For a plant virus to establish a systemic infection, a series of productive interactions between the pathogen and host must occur. The most basic requirement is that the plant can support the replication of the viral genome. Second, the virus must interact with the host to move to and through plasmodesmata into adjacent cells. For mechanically transmitted viruses, such as potato virus X (PVX), infection initiates in epidermal cells and spreads by cell-to-cell movement via mesophyll and bundle sheath cells to the phloem parenchyma and companion cells. For long-distance movement to occur, the virus must then pass into the conducting sieve elements. Once in the phloem, virus translocation is entirely passive and is determined by the pathway of photoassimilate translocation through the plant (Leisner et al. 1992 ; Leisner and Turgeon 1993 ; Roberts et al. 1997 ). The steps involved in virus entry into the sieve elements, and subsequent exit, are very poorly understood; however, the availability of mutants, most notably coat protein (CP) mutants, that can move from cell to cell but not systemically suggests that phloem-dependent transport is likely to involve specific interactions separate from those involved in cell-to-cell movement (Dawson et al. 1988 ; Xiong et al. 1993 ; Dolja et al. 1994 , Dolja et al. 1995 ; Ding et al. 1996 ).

That cell-to-cell movement requires the participation of virus-coded proteins has been known for some time (Nishiguchi et al. 1978 , Nishiguchi et al. 1980 ). More recent research has begun to unravel the complex functions of the tobacco mosaic virus (TMV) MP in targeting plasmodesmata (Tomenius et al. 1987 ), modifying the plasmodesmal SEL (Wolf et al. 1989 ), binding single-stranded nucleic acids (Citovsky et al. 1990 , Citovsky et al. 1992 ), interacting with the cytoskeleton (Heinlein et al. 1995 ), and trafficking nucleic acid between cells (Waigmann et al. 1994 ). In contrast to the well-characterized role of the TMV MP, far less is known about the PVX cell-to-cell movement process. PVX is the type member of the potexviruses and falls into a larger grouping of viruses that require the products of three overlapping open reading frames, the triple gene block (TGB), for cell-to-cell movement (Petty and Jackson 1990 ; Beck et al. 1991 ; Jupin et al. 1991 ). Despite their central role in cell-to-cell movement, no clear evidence exists for a role of the PVX TGB proteins in two key functions that have been ascribed to the TMV MP. First, neither the PVX 25-kD (Davies et al. 1993 ) nor the PVX 8-kD (Hefferon et al. 1997 ) TGB protein shows plasmodesmal localization. Second, the binding of RNA by the PVX 25-kD protein is extremely weak and, unlike the interaction between the TMV MP and RNA, can be disrupted by very low salt concentrations (Citovsky et al. 1990 ; Kalinina et al. 1996 ). One feature of TMV infections that is also seen with PVX is an increase in the plasmodesmal SEL of infected cells (Waigmann et al. 1994 ; Oparka et al. 1996 , Oparka et al. 1997 ). For PVX, the ability to increase the plasmodesmal SEL is attributed to the 25-kD TGB protein, although other viral proteins may also be involved (Angell et al. 1996 ). In addition to the TGB proteins, PVX also has an absolute requirement for the CP for intercellular movement (Chapman et al. 1992a , Chapman et al. 1992b ; Baulcombe et al. 1995 ). In fact, the only potexvirus protein to date that has been shown to localize to plasmodesmata is the viral CP (Rouleau et al. 1995 ; Oparka et al. 1996 ).

Although the precise role of the CP in cell-to-cell movement of PVX remains unknown, the presence of a fibrillar material in plasmodesmata of infected cells has suggested the possibility that intercellular movement of PVX requires virions (Allison and Shalla 1974 ; Oparka et al. 1996 ). In support of this suggestion, an analysis of PVX CP mutants showed that mutations preventing particle assembly correlated with a failure of virus to accumulate in inoculated leaves (Chapman et al. 1992a ).

In this study, we investigated the potential role(s) of the PVX CP in plasmodesmal modification and virus transport. Microinjection of fluorescently labeled dextrans into epidermal cells infected with movement-deficient PVX mutants was used to probe the plasmodesmal SEL. Short- and long-distance movement of the CP was investigated by using both microinjection of fluorescently labeled CP and CP transgenic plants. Data are presented on immunogold localization of PVX virions in plasmodesmata at different cell interfaces, and the possibility that cell-to-cell movement of PVX differs from previously described movement mechanisms is discussed.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

TGB Proteins but Not the CP Induce Plasmodesmal Gating
To determine the role of the PVX CP and TGB proteins in the modification of the plasmodesmal SEL and cell-to-cell transport of PVX, three modified viruses, illustrated in Figure 1, were inoculated onto leaves of Nicotiana edwardsonii. The mutant PVX.GFPCP carries the green fluorescent protein (gfp) gene in place of the cp gene and is unable to move from cell to cell (Baulcombe et al. 1995 ). PVX.GFP–CP^mut2A expresses a GFP–CP fusion protein, fails to form virions, and also is unable to move from cell to cell (Santa Cruz et al. 1996 ). After manual inoculation, both PVX.GFPCP and PVX.GFP–CP^mut2A (Figure 2E) give rise to infections that are restricted to single epidermal cells (Baulcombe et al. 1995 ; Santa Cruz et al. 1996 ). The mutant PVX.GFPTGB, which carries a deletion between nucleotides 4945 to 5500 that disrupts all three TGB open reading frames (Figure 1), is also restricted to single infected cells (Figure 2G). In experiments using GFP-tagged mutants, the movement-competent virus PVX.GFP–CP (Santa Cruz et al. 1996 ) was used as a control (Figure 2A).

View larger version (21K): [in this window] [in a new window]   Figure 1. Organization of the Wild-Type PVX Genome and Modified PVX Genomes. Shown are schematic representations of viral genomes. Untranslated regions are shown as lines, and open reading frames are boxes that include either the size of the predicted translation product in kilodaltons or the name of the encoded gene. PVX represents the genome of wild-type PVX. PVX.GFP–CP, PVX.GFPCP, PVX.GFP–CPmut2A, and PVX.GFPTGB represent the modified forms of PVX used in this analysis. Arrowheads show the positions of the foot and mouth disease virus 2A peptide in the translational fusion of GFP to CP. Closed arrowheads indicate the functional 2A sequence that cleaves cotranslationally to generate free GFP-2A and CP; the open arrowhead indicates the mutant 2A sequence, which translates to give a GFP–2A–CP fusion protein.

View larger version (88K): [in this window] [in a new window]   Figure 2. Confocal Scanning Laser Microscopy of Fluorescence in Cells Infected with GFP-Tagged PVX and Cells Injected with Texas Red–Labeled Probes. (A) to (D) Injection of Texas Red–labeled 10-kD dextran into noninfected and PVX.GFP–CP—infected epidermal cells. (A) shows the GFP signal from infected cells at 3 days after inoculation, with the position of injected epidermal cells marked with asterisks. In (B), the same field of view as shown in (A) shows Texas Red fluorescence. In (C), the noninfected control cell (lower left in [B]) shows that dextran is confined to the injected cell, whereas in (D), an infected cell (upper right in [B]) shows that the dextran probe has moved to adjacent cells. Bar in (A) = 250 µm for (A) and (B); bar in (C) = 100 µm for (C) and (D). (E) and (F) Injection of PVX.GFP–CPmut2A—infected cell with Texas Red–labeled 10-kD dextran. (E) shows the GFP signal from an infected cell. In (F), the injected dextran probe has moved out of the injected cell into surrounding cells. Bar in (E) = 100 µm for (E) and (F). (G) and (H) PVX.GFPTGB-infected cell (G) injected with the 10-kD dextran probe. (H) shows Texas Red–labeled dextran confined to the injected cell. Bar in (G) = 100 µm for (G) and (H). (I) Uninfected epidermal cell injected with Texas Red–labeled CP. No movement of the CP was detected 10 min after injection. Bar = 100 µm. (J) and (K) GFP fluorescence in cells infected with PVX.GFP–CP. (J) shows epidermal cells with punctate, wall-localized fluorescence (arrowheads); an amorphous inclusion body, which is characteristic of PVX infection, is indicated with an arrow. (K) shows punctate fluorescence at the pit fields between mesophyll cells (arrowheads). The inset shows colocalization of GFP and callose (red fluorescence) in the pit field. Bars in (J) and (K) = 25 µm; bar in the inset to (K) = 5 µm.

To assess the effect of the mutations on plasmodesmal modification, inoculated leaves were detached from plants ~40 hr after inoculation and imaged on a confocal laser scanning microscope for cells expressing GFP. Epidermal cells infected with each of the movement-deficient PVX mutants were microinjected with a Texas Red–labeled 10-kD dextran (Oparka et al. 1996 , Oparka et al. 1997 ) and monitored for escape of the fluorescent probe into adjacent cells. The results, summarized in Table 1, demonstrate that in uninfected cells and cells infected with the TGB deletion mutant PVX.GFPTGB, dye was restricted to the injected cells (Figure 2C and Figure 2H). In contrast, cells infected with either of the movement-deficient mutants PVX.GFP–CP^mut2A (Figure 2F) and PVX.GFPCP (data not shown) or the movement-competent control PVX.GFP–CP (Figure 2B and Figure 2D) allowed rapid movement of the dye into adjacent cells.

Injected Viral CP Does Not Traffic between Cells
The injection of fluorescently labeled dextran into infected cells showed that modification of the plasmodesmal SEL occurred in the absence of the PVX CP; however, a role of the viral CP in trafficking between cells could not be ruled out. To address this possibility, CP was prepared from disassociated virions, labeled in vitro with the fluorescent dye Texas Red, and microinjected into N. edwardsonii leaf epidermal cells. In 13 injections, the labeled CP was always contained within the injected cell and failed to move into adjacent cells over the 10-min examination period (Figure 2I).

Localization of the GFP–CP Fusion Protein to Plasmodesmata
Immunoelectron microscopy of PVX-infected tissue that was probed with antiserum raised against the viral CP had shown the consistent association of antibody labeling with plasmodesmata (Oparka et al. 1996 ). In our study, we also found GFP localized to punctate sites on cell walls in tissue infected with PVX.GFP–CP (Figure 2J and Figure 2K). The wall-associated GFP colocalized precisely with callose, as detected by probing PVX.GFP–CP—infected tissue with antibody raised against ß-1,3-glucan (Figure 2K), indicating the presence of the GFP–CP fusion protein either in or close to the plasmodesmal pore.

Ultrastructure of Plasmodesmata in PVX-Infected Tissue
Previous studies had shown that the plasmodesmata of PVX-infected cells frequently contained a fibrillar material that was of a size similar to that of the virus particles, suggesting that cell-to-cell transport of PVX may involve virions (Allison and Shalla 1974 ; Oparka et al. 1996 ). In this study, we looked in more detail at the plasmodesmata connecting PVX-infected cells. Representative plasmodesmata from cells infected with wild-type PVX are shown in Figure 3. In Figure 3A, which shows an unlabeled section, fibrillar material is clearly visible both within and extending from a plasmodesma. Plasmodesmata labeled with antiserum raised against the PVX CP are shown in Figure 3B to D. Gold label was consistently associated with the fibrillar material. In Figure 3C, both longitudinal and transverse sections of the fibrils are apparent.

View larger version (178K): [in this window] [in a new window]   Figure 3. Ultrastructure of Plasmodesmata from PVX-Infected Tissue. Shown is electron microscopy of plasmodesmata between PVX-infected cells. (A) Longitudinal section of plasmodesma showing fibrils both within and extending from the pore (open arrows). (B) to (D) Plasmodesmata showing labeling with antiserum raised against the viral CP. In (B) and (D), fibrils are clearly visible within the plasmodesmal pores (open arrows) and are closely associated with gold labeling. In (C), fibrils are visible in the transverse section (arrow). Bars in (A) to (D) = 100 µm.

Preparation of a Virion-Specific Antiserum
To confirm that the fibrils seen in plasmodesmata of PVX-infected cells were virus particles, we prepared antiserum that specifically recognized virions. The polyclonal antiserum raised against PVX, which recognizes both disassociated CP subunits and assembled virus particles, was cross-absorbed against CP subunits to generate an antiserum recognizing virion-specific epitopes. We conducted several tests to demonstrate that the cross-absorbed antiserum retained the ability to recognize virions but lacked any affinity for disassociated CP subunits. The affinity of the cross-absorbed antiserum for virions was demonstrated by immunogold labeling of aggregated PVX.GFP–CP particles in the cytoplasm of infected cells (Figure 4A). To determine whether the cross-absorbed antiserum retained any affinity for the disassociated CP, an ELISA-based test and immunoblotting (protein gel blotting and slot blotting) were used. The ELISA-based test showed that the antiserum retained no detectable affinity for the disassociated CP subunits, whereas affinity for intact virions was reduced to approximately one-half of the level seen with the unfractionated antiserum (data not shown).

View larger version (74K): [in this window] [in a new window]   Figure 4. Analysis of the Cross-Absorbed Antiserum by Immunogold Labeling of Virions and Protein Gel Blotting of the CP. (A) An infected cell with an aggregate of PVX.GFP–CP virions heavily labeled with gold after probing ultrathin sections of infected leaf tissue with the cross-absorbed antiserum. Bar = 500 nm. (B) Protein gel blot of PVX CP (lanes 1 and 4, 25 ng; lanes 2 and 5, 100 ng; lanes 3 and 6, 250 ng) probed with either polyclonal antiserum raised against PVX (lanes 1 to 3; antiserum diluted 1:25,000) or the same antiserum after cross-absorption against disassociated CP subunits (lanes 4 to 6; antiserum diluted 1:250). Numbers at left indicate relative molecular mass standards (x1000).

Figure 4B shows a protein gel blot of purified PVX CP probed with either the total polyclonal antiserum or the cross-absorbed antiserum. Whereas 25 ng of PVX CP was detected by the polyclonal antiserum at a dilution of 1:25,000, the cross-absorbed antiserum at a dilution of 1:250 failed to detect 250 ng of PVX CP. Slot blot analysis of the CP and virion immobilized on a nitrocellulose membrane and probed with either the total antiserum or the cross-absorbed antiserum also demonstrated that the cross-absorbed antiserum preparation was unable to bind disassociated CP subunits (data not shown).

Localization of PVX Virions to Plasmodesmata
The cross-absorbed antiserum was used to probe ultrathin sections of N. benthamiana tissue infected with wild-type PVX. Figure 5A to C show plasmodesmata labeled with the virion-specific antiserum in cells at or very close to the infection front, as determined by the presence of virus particles and/or virus-induced laminate inclusions. Although the overall level of labeling was greatly reduced (cf. Figure 5A to C with Figure 5E to G), the fractionated antiserum was highly specific for aggregates of virus in the cytoplasm and plasmodesmata and did not give any labeling in uninfected tissue (data not shown). Another feature of the plasmodesmata of infected cells was their distended appearance (e.g., Figure 5A) in both the neck region and central cavity when compared with plasmodesmata from uninfected tissues (data not shown). In addition, the desmotubule of PVX-infected plasmodesmata was seldom discernible in either longitudinal or transverse sections of infected plasmodesmata.

View larger version (129K): [in this window] [in a new window]   Figure 5. Electron Microscopy and Immunogold Localization of the PVX CP in Plasmodesmata. (A) to (C) Virion-specific antiserum resulting in gold labeling of mesophyll plasmodesmata in cells infected with wild-type PVX. (A) shows plasmodesmata in longitudinal section; (B) and (C) show transverse sections. Note the distended appearance of the neck region of the plasmodesmata shown in the longitudinal section in (A). Bars = 250 nm. Figure 5. (continued). (D) to (G) Sections probed with unfractionated polyclonal antiserum showing gold labeling of plasmodesmata within a minor vein. In (D), the structure of a class V vein is shown. Arrows numbered 1, 2, and 3 indicate the cell interfaces shown in (E), (F), and (G), respectively. Bar in (D) = 5 µm; bars in (E) and (F) = 250 nm; bar in (G) = 200 nm. (H) Interface between two mesophyll cells. Bar = 500 nm. The arrows in (E) and (H) show gold-labeled plasmodesmata. In (G), there is no gold labeling at the interface between the companion cell and sieve element. b, bundle sheath; c, companion cell; m, mesophyll; p, phloem parenchyma; s, sieve element; x, xylem.

The CP Does Not Localize to Plasmodesmata in Cells within the Minor Vein Bundle Sheath
To determine whether plasmodesmal localization of the CP occurs at all cell boundaries in the movement pathway from epidermis to phloem, inoculated leaves of N. benthamiana infected with PVX.GFP–CP were processed for electron microscopy and immunogold labeling with antiserum raised against the PVX CP. Figure 5D shows a typical class V vein infected with PVX.GFP–CP; Figure 5E to H illustrate plasmodesmata at different cellular interfaces. Gold labeling was seen at all cell interfaces along the pathway from epidermis to the minor vein bundle (data not shown and Figure 5H). Within the minor vein bundle, gold label was seen in plasmodesmata between bundle sheath cells and companion cells and between bundle sheath cells and phloem parenchyma (Figure 5E and Figure 5F). The only plasmodesmata in which the CP antigen was not detected were those at the interface between companion cells and sieve elements (Figure 5G) and those between phloem parenchyma and companion cells (data not shown).

The CP Is Translocated in and Unloaded from the Phloem
Because the PVX CP is an absolute requirement for cell-to-cell movement, establishing whether the CP is essential for phloem-mediated transport was not possible. As an alternative, we investigated whether the CP was able to move through the phloem using grafts between transgenic N. benthamiana rootstocks expressing the PVX CP and nontransformed N. benthamiana scions. Previous experiments had shown that transgenic N. tabacum plants expressing the viral CP were able to rescue the movement-deficient phenotypes of both PVX.GFPCP and PVX.GFP–CP^mut2A (Oparka et al. 1996 ; Santa Cruz et al. 1996 ). After inoculation of PVX.GFP–CP, PVX.GFPCP, or PVX.GFP–CP^mut2A onto leaves of the transgenic N. benthamiana rootstocks, cell-to-cell movement of PVX.GFP–CP and both of the mutants was observed. Cell-to-cell movement of all three viruses in leaves of transgenic plants occurred at approximately two-thirds the rate at which PVX.GFP–CP moved on nontransformed N. benthamiana, as determined by the expansion of fluorescent infection foci (data not shown). In contrast, inoculation of the CP transgenic plants with PVX.GFPTGB resulted in single-cell infections (data not shown). Systemic movement of both PVX.GFPCP and PVX.GFP–CP^mut2A was first detected as small fluorescent flecks, which were associated with the class III veins of noninoculated leaves on transgenic rootstock plants, from 12 days after inoculation. Subsequent cell-to-cell movement away from the vascular tissue led to a pattern of infection that, although slightly delayed, resembled the normal progress of systemic infection by PVX (Figure 6A and Figure 6B) (Baulcombe et al. 1995 ; Roberts et al. 1997 ).

View larger version (52K): [in this window] [in a new window]   Figure 6. Long-Distance Movement and Phloem Unloading of Viral Mutants on Transgenic N. benthamiana Expressing the PVX CP. (A) A grafted plant showing GFP fluorescence in a transgenic rootstock systemically infected with PVX.GFP–CPmut2A. The nontransformed scion is outlined, and the graft union is shown by a white rectangle. Figure 6. (continued). (B) The leaf of a transgenic rootstock systemically infected with PVX.GFP–CPmut2A showing virus distributed across the leaf lamina. (C) The leaf of a nontransformed scion systemically infected with PVX.GFP–CPmut2A. Note that the virus is closely associated with the major vein network. (D) to (F) Magnified images of nontransgenic leaves systemically infected with PVX.GFPCP (D) and PVX.GFP–CPmut2A ([E] and [F ]) showing the restriction of the virus to the vicinity of class III veins. In (F), the virus has unloaded from phloem and entered mesophyll and epidermal cells. In (D) to (F), xylem has been allowed to import Texas Red to aid vizualization of the vascular tissue. Bar in (D) = 250 µm; bar in (E) = 500 µm; bar in (F) = 200 µm.

The appearance of virus in the nontransgenic scion tissue was delayed in comparison to systemic movement into transgenic rootstock leaves; however, from 15 days after inoculation, GFP was detected in leaves of the nontransgenic scions. Unloading of virus from the phloem of the nontransgenic scion tissue was seen in four of four plants inoculated with PVX.GFP–CP^mut2A and four of six plants inoculated with PVX.GFPCP. The most significant difference between the progress of the infection in systemically infected leaves above and below the graft junction was that after virus unloading from the phloem, virus infection of nontransgenic scion tissue failed to progress significantly into the mesophyll, and the infection remained restricted to cells adjacent to the class III veins (cf. Figure 6B and Figure 6C). Microscopic examination of systemically infected nontransgenic scion leaves infected with either PVX.GFPCP or PVX.GFP–CP^mut2A (Figure 6D to F) illustrates more clearly the extent to which virus had escaped from the vascular tissue. In general, the distribution of PVX.GFPCP was more restricted to cells close to the phloem than was PVX.GFP–CP^mut2A (cf. Figure 6D and Figure 6E). However, both PVX.GFP–CP^mut2A (Figure 6F) and PVX.GFPCP (data not shown) were detected in mesophyll and epidermal cells. To determine whether the nontransgenic tissue was infected with the inoculated mutants and to rule out the presence of recombinant viral genomes, infected tissue was used to prepare inoculum to back-inoculate nontransformed plants. In all cases, back-inoculations resulted in single-cell infections, as determined by GFP fluorescence, and the inoculated plants did not show any visible symptoms of infection in noninoculated leaves (data not shown).

Virus Distribution around Class III Veins of Systemically Infected Leaves
To examine in greater detail the pattern of virus distribution in systemically infected leaves, tissue showing the presence of GFP was taken from both below and above the graft union and prepared for electron microscopy. Figure 7 shows electron microscopic analysis of sections probed with antiserum raised against either the CP or the 25-kD TGB protein. Figure 7A and Figure 7B show representative cross-sections illustrating the distribution of the two viral proteins in the vicinity of class III veins of nontransgenic scion tissue infected with PVX.GFPCP and PVX.GFP–CP^mut2A, respectively. Despite the obvious presence of virus-induced laminate inclusions that are characteristic of PVX infection (Shalla and Shepard 1972 ) and heavy labeling with the antiserum raised against the 25-kD protein in PVX.GFPCP-infected scion tissue (Figure 7C), no CP antigen was detected in infected cells (Figure 7D). This lack of CP labeling in scion tissue contrasts with the labeling seen in rootstock tissue systemically infected with PVX.GFPCP in which immunogold labeling of CP is clearly visible (cf. Figure 7D and Figure 7E). The distribution of viral antigens in PVX.GFP–CP^mut2A—infected scion tissue is illustrated in Figure 7B. In cells close to the phloem, viral CP was detected in PVX.GFP–CP^mut2A—infected tissue (Figure 7F), whereas farther from the site of phloem unloading, no labeling of the CP was observed, even though the virus-induced laminate inclusions were clearly visible (Figure 7G).

View larger version (123K): [in this window] [in a new window]   Figure 7. Immunogold Labeling of PVX-Infected Cells after Phloem Unloading. (A) and (B) Sections viewed under a light microscope showing the distribution of viral antigens in infected cells (as determined from immunoelectron microscopy of serial sections) around infected class III veins. (A) was infected with PVX.GFPCP; (B) was infected with PVX.GFP–CPmut2A. Figure 7. (continued). Uninfected cells are unshaded (1), cells labeled with the antiserum raised against the PVX 25-kD protein are lightly shaded (2), and cells labeled with the antiserum raised against the CP are heavily shaded (3). x, xylem vessels. (C) to (G) Electron microscopy of ultrathin sections prepared from systemically infected tissues. (C) to (E) show cells systemically infected with PVX.GFPCP. (C) shows labeling in nontransgenic scion tissue probed with the antiserum raised against the 25-kD protein. Gold label is seen on the laminate inclusion structures typical of PVX infections. (D) shows an infected nontransgenic cell probed with the antiserum raised against the CP. No label is seen despite the presence of laminate inclusion structures. In (E), an infected transgenic cell shows clear labeling, with the antiserum raised against the CP around a laminate inclusion. In (F), in cells close to the phloem, nontransgenic cells infected with PVX.GFP–CPmut2A show clear labeling, with the antiserum raised against the CP. Farther from the phloem, no CP labeling is seen, despite the obvious presence of PVX-induced inclusions (G). c, companion cell; s, sieve element. Bars in (C) to (G) = 1 µm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

Two distinct mechanisms for viral cell-to-cell movement have been described (reviewed in Maule 1991 ; Carrington et al. 1996 ). One strategy is typified by TMV. It is independent of the CP (Siegel et al. 1962 ; Takamatsu et al. 1987 ) and requires a single MP that can traffic between cells (Waigmann et al. 1994 ). A second well-described movement strategy used by the comoviruses and nepoviruses is CP dependent and involves the transport of virions through virus-induced tubules that span the cell walls of adjacent cells (Wellink and Van Kammen 1989 ; Van Lent et al. 1990 ; Wieczoreck and Sanfacon 1993 ). Other plant viruses, including cucumber mosaic virus (Suzuki et al. 1991 ), tobacco etch virus (Dolja et al. 1994 , Dolja et al. 1995 ), and several potexviruses (Chapman et al. 1992a ; Forster et al. 1992 ; Sit and AbouHaidir 1993 ), are also known to depend on the CP for cell-to-cell movement; however, the exact role of the CP in the movement processes of these viruses has not yet been determined. The CPs of potexviruses and potyviruses, both of which encapsidate viral RNA as flexuous rods, have been shown to be structurally related (Dolja et al. 1991 ). The recent demonstration that for the potyviruses, tobacco vein mottling virus (Rodriguez-Cerezo et al. 1997 ), and pea seed-borne mosaic virus (A. Maule, personal communication), the CP localizes to plasmodesmata raises the possibility that the potexviruses and potyviruses may share some aspects of their movement processes.

The results presented here demonstrate that the increase in the plasmodesmal SEL seen in PVX-infected tissue requires one or more of the TGB proteins and is completely independent of the CP. The failure of the CP Texas Red conjugate to move from injected epidermal cells is consistent with the observation that in the absence of infection, the CP in transgenic plants shows no affinity for plasmodesmata (Oparka et al. 1996 ) and indicates that the PVX CP lacks the intrinsic trafficking ability associated with characterized movement proteins (Fujiwara et al. 1993 ; Noueiry et al. 1994 ; Waigmann et al. 1994 ; B. Ding et al. 1995 ). These results show that for PVX, plasmodesmal modification alone is insufficient to permit cell-to-cell movement of virus and that the PVX CP does not play an active role in the modification of plasmodesmata.

This localization of the CP to the plasmodesmal pores implies an association of CP and viral RNA during the cell-to-cell transport process, either as virions or some other form of ribonucleoprotein complex. The possibility that virions are present in plasmodesmata is consistent with the observation that flexuous fibers are frequently seen within the pore. To determine whether virus particles were present in plasmodesmata, the polyclonal antiserum raised against PVX virions was cross-absorbed against intact and denatured CP subunits in an attempt to generate a virion-specific antiserum. Several assays confirmed that the cross-absorbed antiserum retained an affinity for virions but was unable to bind disassociated CP subunits. Using this antiserum, we were able to demonstrate that PVX virions are present in the plasmodesmata connecting infected cells. Moreover, antibody labeling was seen in plasmodesmata of cells at the leading edge of the infection, suggesting that this localization is biologically significant and does not reflect cytological changes at a late stage in the infection process. The localization of virions in plasmodesmata would also explain the bright GFP signal detected from plasmodesmata in cells infected with the fluorescent virus PVX.GFP–CP (see Figure 2J and Figure 2K).

All cell interfaces between the epidermis and bundle sheath showed the CP localized to plasmodesmata. Within the vascular bundle of the minor veins, the CP was localized at the bundle sheath–phloem parenchyma and the bundle sheath–companion cell interfaces but was not detected in plasmodesmata connecting either phloem parenchyma and companion cell or companion cell and sieve element. The lack of labeling at the phloem parenchyma–companion cell boundary is unlikely to be of great significance because in members of the genus Nicotiana, the bundle sheath cells within the minor veins contact companion cells directly (X.S. Ding et al. 1995 ). More important was the complete absence of detectable CP, and also fibrillar material, in the specialized plasmodesmata connecting companion cells to sieve elements. It is these plasmodesmata through which the virus must pass to gain access to the long-distance transport pathway. The lack of CP labeling at this key gateway to systemic movement could be due to the fact that the virus crosses these plasmodesmata in a form different from that used at other cell boundaries (i.e., in non-virion form). Alternatively, the large SEL of the plasmodesmata at the companion cell–sieve element interface (Kempers et al. 1993 ; Kempers and Van Bel 1997 ) may permit the relatively free passage of intact virus particles and lack the tendency to block seen at other plasmodesmata.

That the PVX CP can enter, move through, and exit the phloem is clearly demonstrated by the results obtained from the grafting experiments in which cell-to-cell movement within the nontransgenic scion tissue of the mutants PVX.GFPCP and PVX.GFP–CP^mut2A was dependent on CP synthesized in the transgenic rootstock. Movement of the PVX CP outward from the phloem is shown by the progress of the infection in the scion tissue into mesophyll and epidermal cells. This ability of the CP to move between cells is not an intrinsic property of the CP itself but a consequence of the infection process. The "freezing" of the progress of infection to cells within and immediately adjacent to the class III veins, as shown in Figure 6C, emphasizes the conclusion of a previous study that the phloem unloading of PVX in N. benthamiana occurs predominantly from this branched veinal system (Roberts et al. 1997 ).

That the infection stops abruptly after phloem unloading indicates that movement of the mutant viruses seen in the nontransgenic tissue did not reflect the presence of recombinant viral genomes that had acquired a functional CP. This point was also confirmed by the back-inoculation experiments that showed only single-cell infections, with no evidence of the recombinant virus lacking the gfp gene but expressing the functional CP. The restriction of the infection to within a few cells of the phloem presumably reflects the fact that the virus simply runs out of sufficient CP for particle assembly to occur. The low amount of CP that is unloaded from the phloem in nontransgenic scion tissue infected with PVX.GFPCP is evident from the failure to detect PVX CP by immunoelectron microscopy. Even in scion tissue infected with PVX.GFP–CP^mut2A, the CP was only detected in cells within the phloem bundle. The inability of the antiserum raised against the CP to detect the assembly-deficient GFP–CP fusion protein in infected cells most probably reflects the low affinity of the antiserum for the unassembled CP (S. Santa Cruz, unpublished data). Also, the immunodominant domain at the N terminus of the CP (Baratova et al. 1992 ) is masked by the fusion with GFP. However, the presence of infected mesophyll and epidermal cells indicates that in the course of infection, the CP had been transported through a number of plasmodesmata. One unresolved question relating to the cell-to-cell transport of the CP is whether the CP from disassembled virions is recycled to encapsidate progeny virus or whether virus entering an already infected cell can be passed straight into the transport pathway and moved to an adjacent cell without entering the cycle of disassembly, replication, and encapsidation.

The data presented here suggest that the dependence on CP for intercellular movement of PVX reflects a requirement for encapsidation of the viral RNA before cell-to-cell transport and that it is the intact virion that is transported through plasmodesmata. This is consistent with a previous study of PVX (Allison and Shalla 1974 ) and also with reports of other filamentous viruses localizing to the plasmodesmal pore (Esau et al. 1967 ; Weintraub et al. 1976 ; discussed in Lucas et al. 1990 ). Intercellular movement of the filamentous PVX virion implies a mechanism distinct from the CP-independent transport process used by TMV. In addition, the absence of virus-induced tubules in the plasmodesmata of PVX-infected cells indicates that the PVX movement process differs from the movement via tubules of nepovirus and comovirus virions.

The necessary plasmodesmal modification required to support transport of PVX virions (diameter of 13 nm; Koenig and Leseman 1989 ) would be greater than the modification needed for transport of the TMV RNA–MP complex (diameter of 2 nm; Citovsky et al. 1992 ) but less than the dilation required to allow passage of icosohedral viruses, such as the comovirus cowpea mosaic virus (diameter of 20 to 24 nm; Van Kammen and de Jager 1978 ). The apparent lack of a desmotubule and the distorted appearance of plasmodesmata in infected cells suggest that the PVX movement process may involve removal from the plasmodesmal pore of internal membranes and proteins. This could allow the opening of a passage that even in the constricted neck region would have a diameter of >20 nm (Olesen and Robards 1990 ) between the inner leaflets of the plasma membrane lining the plasmodesmal pore. The challenge now is to understand how the proteins encoded by the TGB function in the movement process, both in the modification of the plasmodesmal SEL and in their possible role in the transport of PVX virions to and/or through plasmodesmata.

	METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

Virus, Viral cDNA Clones, and Plant Inoculation
Wild-type potato virus X (PVX) strain UK3 was propagated on Nicotiana benthamiana. Sap from infected plants, diluted 1:100 (w/v) in 50 mM sodium borate, pH 8.2, was used to inoculate aluminium oxide–dusted leaves. Plasmids pTXS.GFP–CP, pTXS.GFP–CP^mut2A, and pTXS.GFPCP (Figure 1) have been described previously (Baulcombe et al. 1995 ; Santa Cruz et al. 1996 ). The plasmid pTXS.GFPTGB was created by deleting the sequence in pTXS.GFP–CP between an ApaI restriction site at nucleotide 4945 and an EcoNI restriction site at nucleotide 5550 (Figure 1). All plasmids were digested with SpeI before in vitro transcription by using an mMessage mMachine T7 RNA polymerase kit (Ambion, Austin, TX), according to the manufacturer's instructions, and a template concentration of 0.2 µg/µL. The transcription reactions were inoculated directly onto aluminium oxide–dusted leaves by using 5 µL of reaction product per leaf. Infections established with transcripts from the plasmids described above are referred to by replacing the prefix pTXS with the prefix PVX.

Preparation of Fluorescently Labeled Probes
The Texas Red–labeled 10-kD dextran probe was purchased from Molecular Probes (Eugene, OR). Because of the significant levels of low molecular weight contaminants and unconjugated dye in the dextran preparation, it was necessary to filter the dye by using a Microcon-10 membrane exclusion filter (Amicon, Beverly, MA) with a nominal 10-kD cutoff. The absence of unconjugated dye was confirmed by thin-layer chromatography, as described previously (Oparka et al. 1996 , Oparka et al. 1997 ). Aliquots were stored at -20°C until required for microinjection. Freeze–thaw cycles were avoided to prevent accumulation of breakdown products. Labeling of the coat protein (CP) with Texas Red was performed with disassociated CP subunits prepared according to the method of Goodman 1975 . The absence of either intact or fragmented virions in the CP preparation was confirmed by electron microscopic analysis of poly-L-lysine–treated carbon support films that had been incubated with the CP preparation, as described by Roberts 1986 . Disassociated CP subunits were labeled using a Texas Red protein conjugation kit, according to the manufacturer's instructions (Molecular Probes). Unconjugated dye was removed by centrifugation of the sample through a Micro Bio-Spin 6 column (Bio-Rad), and the labeled CP was dialyzed against 10 mM Sörensen's phosphate buffer (Na₂HPO₄/KH₂PO₄, pH 7.2). The concentration of Texas Red–labeled CP was checked by PAGE alongside known quantities of PVX CP, and samples were adjusted to a concentration of 0.25 mg/mL. Labeled CP was stored at 4°C and used within 2 days of preparation.

Microinjection
Microinjection of the Texas Red–labeled dextran and CP was performed as described previously (Oparka et al. 1996 ), using a modified pressure probe to prevent vacuolar rupture during impalement (Oparka et al. 1990 ). Injected cells were observed for 10 min after injection and scored for the presence or absence of fluorescent label in cells adjacent to the injected cell. The injections of 10-kD dextran to epidermal cells infected with the movement-deficient mutants were, as far as was possible, performed blind (i.e., without the operator knowing which mutant was infecting the target cells).

Labeling of Xylem with Texas Red
In some experiments, to allow the vascular tissue to be seen easily under the confocal laser scanning microscope, the petioles of detached leaves were immersed in a solution containing 0.1 mg/mL of 3-kD Texas Red dextran (Molecular Probes), as described by Roberts et al. 1997 .

Immunolocalization of ß-1,3-Glucan (Callose)
Callose was localized in epidermal and mesophyll tissues, essentially as described in Oparka et al. 1997 . Rabbit antibody raised against ß-1,3-glucan (Genosys Biotechnologies, Cambridge, UK) was used to probe sections prepared from PVX.GFP–CP—infected leaves. Callose was detected by labeling the primary antibody, using an anti–rabbit antibody conjugated to CY3 (Sigma Immunochemicals, Poole, UK).

Detection of Green Fluorescent Protein in Leaves and Whole Plants
Plants were illuminated with long-wavelength UV light (365 nm) by using a Blak-Ray hand-held lamp (UV Products, Upland, CA). Photographs were taken on Kodachrome PKL 200 daylight film with a green x1 filter (Hoya, Japan), as described previously (Oparka et al. 1996 ; Roberts et al. 1997 ).

Detection of CY3, the Green Fluorescent Protein, and Texas Red by Confocal Laser Scanning Microscopy
Detached leaves were viewed with an MRC 1000 confocal laser scanning microscope (Bio-Rad) to image the green fluorescent protein (GFP), CY3, and Texas Red fluorescence. The imaging procedures used have been described in detail previously (Baulcombe et al. 1995 ; Oparka et al. 1997 ; Roberts et al. 1997 ).

Plant Material and Grafting
Plants (N. benthamiana and N. edwardsonii) were grown from seed in a heated greenhouse and used for experiments when they were between 20 and 30 days old. Grafting experiments were performed using transgenic N. benthamiana expressing the PVX CP (the generous gift of T. Lough and R. Forster, Horticulture and Food Research Institute of New Zealand, Auckland, New Zealand). Grafts were performed using a standard wedge graft technique, as described by Garner 1979 . N. edwardsonii was used for microinjection experiments because the leaf lamina is flat and epidermal cells can be easily injected.

Preparation of the Antiserum Raised against the 25-kD Protein
Recombinant 25-kD protein was expressed in Escherichia coli BL21(DE3) pLysS as a fusion to thioredoxin from the expression vector pET32a (Novagen, Madison, WI) and purified according to the supplier's instructions. Purified protein was diluted to a concentration of 1 mg/mL in 50 mM sodium phosphate, pH 7.0, and mixed with an equal volume of Freund's incomplete adjuvant. Intramuscular injection of a New Zealand White rabbit was performed at 2-week intervals using 1 mL of antigen. Serum was prepared from sample bleeds collected 6 weeks after the first immunization and used for immunogold labeling, as described below.

Preparation and Analysis of Virion-Specific Antiserum
Polyclonal antiserum raised against PVX particles (generously provided by I. Barker, Central Science Laboratories, York, UK) was passed through a HiTrap NHS-activated Sepharose column (Pharmacia, Uppsala, Sweden) to which PVX CP had been coupled. The PVX CP subunits, prepared as described above, were dialyzed against 50 mM Sörensen's buffer, pH 7.2, and coupled to the column, according to the manufacturer's instructions. The column flow-through was purified to give an IgG fraction by using an E-Z-Sep kit, according to the manufacturer's instructions (Pharmacia), and equilibrated in 50 mM Sörensen's buffer, pH 7.2. The affinity of the cross-absorbed antiserum for virions was tested by immunoelectron microscopy of PVX.GFP–CP—infected tissue, as described below. The affinity of the cross-absorbed antiserum for disassociated CP subunits was tested by using SDS-PAGE of purified PVX CP, followed by protein gel blotting and probing of blots with either the total polyclonal antiserum or the cross-absorbed antiserum. An ELISA-based test, using microtiter plates coated with either 1 µg of CP or 1 µg of virion, was also used to determine the affinity of serial dilutions of the total polyclonal antiserum and the fractionated antiserum for both CP and virion.

Immunoelectron Microscopy
Leaf tissues were fixed and embedded in epoxy resin for immunogold labeling, as described by Fasseas et al. 1989 . Ultrathin sections on nickel grids were labeled using antiserum to either the PVX CP or PVX 25-kD proteins, followed by goat anti–rabbit gold conjugate (15 nm; GAR-G; Amersham). To examine the cellular distribution of PVX after its unloading from the phloem, leaf veins were examined under the confocal laser scanning microscope to detect the first appearance of the GFP-tagged virus. Areas of leaf with veins showing GFP were then excised with a razor blade and processed for electron microscopy, as described above.

	FOOTNOTES

² Current address: Axis Genetics PLC, Babraham, Cambridge CB2 4AZ, UK.

	ACKNOWLEDGMENTS

We are grateful to Tony Lough and Richard Forster for providing transgenic N. benthamiana plants expressing the PVX CP. We thank Peter Nettleton and Tracy Fitzgerald for preparing the antiserum raised against the 25-kD protein and Ian Barker for providing antiserum raised against PVX. We also acknowledge George Duncan for assistance with the electron microscopy and Paul Donaghy for antibody fractionation. We thank Peter Palukaitis for helpful discussions. The Scottish Crop Research Institute is grant-aided from the Scottish Office Agriculture Environment and Fisheries Department. A.G.R. was the recipient of a postgraduate research award from Dundee University.

Received November 19, 1997; accepted January 26, 1998.

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