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Keywords:

  • actin;
  • endoplasmic reticulum;
  • microtubule;
  • plasmodesmata;
  • targeting;
  • tobacco mosaic virus

Abstract

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and methods
  6. Acknowledgments
  7. References
  8. Supporting Information

Fluorescence recovery after photobleaching (FRAP) was used to study the mechanism by which fluorescent-protein-tagged movement protein (MP) of tobacco mosaic virus (TMV) is targeted to plasmodesmata (PD). The data show that fluorescence recovery in PD at the leading edge of an infection requires elements of the cortical actin/endoplasmic reticulum (ER) network and can occur in the absence of an intact microtubule (MT) cytoskeleton. Inhibitors of the actin cytoskeleton (latrunculin and cytochalasin) significantly inhibited MP targeting, while MT inhibitors (colchicine and oryzalin) did not. Application of sodium azide to infected cells implicated an active component of MP transfer to PD. Treatment of cells with Brefeldin A (BFA) at a concentration that caused reabsorption of the Golgi bodies into the ER (precluding secretion of viral MP) had no effect on MP targeting, while disruption of the cortical ER with higher concentrations of BFA caused significant inhibition. Our results support a model of TMV MP function in which targeting of MP to PD during infection is mediated by the actin/ER network.

Tobacco mosaic virus (TMV), type member of the tobamoviruses, is one of the most extensively studied viral pathogens of plants. Despite over 100 years of research into the interaction of TMV with its hosts, the mechanism by which TMV moves between plant cells remains uncertain (1–6). However, it has been speculated that during coevolution with host plants, viruses ‘hijacked’ one or more host proteins for trafficking molecules through plasmodesmata (PD). As a result, studies of the interaction of TMV with host plants are of particular relevance because they form the basis for models of macromolecular trafficking. TMV, in common with a wide range of viruses, encodes a viral movement protein (MP) that is involved in a number of interrelated functions. These include targeting and accumulation within PD (7–10), an increase in the size exclusion limit (SEL) of the PD pore (9,11,12) and the ability to bind to single-stranded RNA (13). Although the MP accumulates within PD (14), and increases the SEL of the PD pore in transgenic plants (11), it appears that during a natural viral infection, the ‘gating’ of PD is transitory and is restricted to the leading edge of infection (9), while the loss of gating function possibly involves the phosphorylation of the MP within the PD pore (15).

In recent years, considerable attention has focused on the mechanism by which the TMV MP is targeted to PD, although data remain conflicting as to the precise intracellular pathway used by MP to reach the PD pore. Over the past decade, a series of reports have shown the association of the TMV MP with elements of the cytoskeleton, including actin (16), but more predominantly, microtubules (MTs) (17–23), although the latter is under spatiotemporal control, being more pronounced several cell layers behind the leading edge of the infection. In keeping with the growing body of evidence that suggests that MTs may play a general role in RNA trafficking in eukaryotic cells (24–27), a number of studies have correlated MP binding to MT with the efficiency of TMV spread (21,22,28,29). Support for a role of MTs in the intracellular trafficking of MP was obtained by Boyko et al. (21) who identified a conserved tobamovirus MP sequence with homology to a tubulin motif and from observations that TMV mutants with point mutations in the putative tubulin-binding domain of the MP showed reduced cell-to-cell spread. Also, a mutant form of MP, MPNT-1, which interferes with the association of MP–green fluorescent protein (MP–GFP) with MTs, reduced the trafficking of MP through PD, although targeting of MP to PD was not reduced (30). However, in a previous report, we examined the putative role of MTs in delivery of the TMV MP to PD and found that viral cell-to-cell movement was unimpeded by MT inhibitors, such as colchicine or oryzalin, or by silencing of the α-tubulin (TUA) gene (23,31). Furthermore, a TMV vector expressing a DNA-shuffled MP gene (32) that showed enhanced cell-to-cell movement showed greatly reduced association with MTs (23,33), suggesting that MTs are not essential for TMV cell-to-cell movement. While MTs may not play a direct role in the TMV cell-to-cell movement process, the pathway of MP to the PD pore remains unresolved. It has been shown that the TMV MP interacts with a number of intracellular components during infection. For example, an early event during TMV infection is the association of MP with the cortical endoplasmic reticulum (ER) (18,20,23,34), leading to the suggestion that the ER might provide the functional pathway for MP targeting to PD (23,34).

One of the major problems in identifying the very early stages in the trafficking of MP to the PD pore lies in the examination of static images during the infection cycle. Thus, early events in the targeting process are assumed to be reflected by events at the leading edge of TMV infection site (17,19,21–23,31) or by the first observable events in infected protoplasts (16,20,35). These limitations are exacerbated by the fact that GFP and similar fluorophores, which have been used extensively as tags for the viral MP, may take several hours to mature (36,37), during which time the earliest events in the infection process are likely to have occurred or are unobservable due to the imposed detection limits of the fluorescent reporter. To overcome this problem, we used the technique of fluorescence recovery after photobleaching (FRAP) to selectively photobleach MP–GFP-labeled PD at the leading edge of the infection site, and subsequently followed the recovery of fluorescence within the PD, reflecting movement of new fluorescent protein into the photobleached area (38). Fluorescence recovery in PD was significantly affected by pharmacological agents that disrupted the ER and/or the actin cytoskeleton, and strongly inhibited by the less specific metabolic inhibitor azide. In contrast, disruption of MTs had no effect on the trafficking of MP to PD. Our results support the view that the TMV MP is targeted to PD by means of the cortical ER/actin network.

Results

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and methods
  6. Acknowledgments
  7. References
  8. Supporting Information

FRAP studies of TMV MP targeting to PD

In leaves of Nicotiana benthamiana, the distribution of MP across an expanding infection site produced by a coat-protein-containing TMV vector expressing a MP–GFP fusion (TMV.MP–EGFP.CP) was confirmed to be similar to that of previous studies (17–19,23). At the leading edge of infection, GFP labeling was first detected in wall-associated punctae (Figure 1A, arrows), shown previously to coincide with PD (9).

image

Figure 1. The appearance of Nicotiana epidermal cells under control conditions and treated with sodium azide or BFA. In all images, GFP-associated fluorescence is colored green and chlorophyll-associated autofluorescence is colored blue. A) Maximum projection of a stack of confocal images taken at the leading edge of a TMV.MP–EGFP.CP lesion on Nicotiana benthamiana showing the targeting of MP to PD (arrows) and the punctate labeling of intracellular structures under control conditions. Bar = 25 μm. B) Alexa 488 phalloidin staining of actin in N. benthamiana under control conditions. Bar = 25 μm. C). ER–GFP transgenic plants under control conditions. Bar = 25 μm. D). STtmd–GFP transgenic plants under control conditions showing dispersed Golgi stacks. Bar = 25 μm. E). TUA–GFP plants showing the MT cytoskeleton under control conditions. Bar = 25 μm. F) TUA–GFP transgenic plants, 4 days after Agrobacterium inoculation for expression of YFP-HDEL. Under control conditions, the MTs (green) do not associate with the ER (magenta) in the infected cell (lower left). Bar = 10 μm. G) Targeting of MP to PD and the labeling of intracellular structures at the leading edge of a TMV.MP–EGFP.CP lesion after treatment with 0.02% sodium azide. Bar = 10 μm. H) ER–GFP transgenic plants following treatment with sodium azide. Bar = 25 μm. I) Alexa 488 phalloidin staining of actin in N. benthamiana following disruption by treatment with sodium azide. Bar = 25 μm. J) STtmd–GFP transgenic plants showing Golgi bodies after treatment with sodium azide. Bar = 25 μm. K) TUA–GFP transgenic plants showing MTs and the concentration of GFP at discrete sites in the presence of sodium azide. Bar = 25 μm. L) TUA–GFP transgenic plants expressing YFP-HDEL after treatment with sodium azide. TUA–GFP fluorescence (green) in a noninfected cell (left) forms a similar pattern to ER-associated YFP-HDEL fluorescence (magenta) in the neighboring cell indicating possible accumulation of GFP–tubulin at the ER vertices. Bar = 10 μm. M) STtmd–GFP transgenic plants treated with 10 μg/mL BFA showing a faint ER network (arrow) and fluorescent aggregates due to absorption of the Golgi bodies into the ER. Bar = 10 μm. N) ER–GFP transgenic plants after treatment with 10 μg/mL BFA showing the ER network and fluorescent aggregates. Bar = 10 μm. O) ER–GFP transgenic plants showing complete disruption of the ER network in the presence of 100 μg/mL BFA. Bar = 25 μm. P) STtmd–GFP transgenic plants showing complete disruption of the Golgi bodies in the presence of 100 μg/mL BFA. Bar = 25 μm. YFP-HDEL, ER-targeted yellow fluorescent protein.

Leaves infected with TMV.MP–EGFP.CP were infiltrated with either water (control) or a range of chemical agents prior to assessment of FRAP as described in Materials and Methods. PD showing MP–GFP localization at the leading edge of infection (see Figure 1A) were photobleached with laser light at 488 nm. Following photobleaching under control conditions (water infiltration), the level of fluorescence within PD returned to about 40% of its original value within a period of 40 min (Figure 2A, B; Table 1). Following water infiltration, the actin network (Figure 1B), motile reticulate ER network (Figure 1C; Supplemental Video 1), motile Golgi bodies (Figure 1D; Supplemental Video 2) and MTs (Figure 1E) were as expected for unperturbed tissue (39,40). Under control conditions, there was little apparent interaction between the MTs and ER (Figure 1F), with the ER tubules aligning independently from the MTs.

image

Figure 2. FRAP of PD in Nicotiana leaf epidermal cells. A) FRAP of PD under control conditions and in the presence of azide. Graph of time versus fluorescence recovery as a proportion of the prebleached value showing that treatment with 0.02% sodium azide (dashed line) resulted in significant inhibition (**significant at 1% level) relative to the control (solid line). (B and C) PD at the leading edge of a TMV.MP–EGFP.CP lesion on Nicotiana benthamiana before and after photobleaching. Images were recorded prior to, immediately following, and 10, 20, 30 and 40 min after photobleaching the circled PD. Leaf tissue was pretreated for 2 h prior to bleaching with water (control) (B) or 0.02% sodium azide (C). Bar = 5 μm. (D–I) Graphs of time versus fluorescence recovery as a proportion of the prebleached value. Treatment with 10 μg/mL BFA (dashed line) (D) showed no significant difference compared with the control (solid line), in contrast to treatment with 100 μg/mL BFA (dashed line) (E), which resulted in significant inhibition (**significant at 1% level) relative to the control (solid line). The actin-depolymerizing agent, latrunculin (F) (25 μm, dashed line), results in significant inhibition (*significant at 5% level) relative to the control (solid line). Treatment with the actin-disrupting agent, cytochalasin (G) (0.1 mg/mL, dashed line), results in significant inhibition (*significant at 5% level) relative to the control (solid line). Treatment with the MT-depolymerizing agent, colchicine (H) (500 μm, dashed line), or with the MT-disrupting agent, oryzalin (I) (20 μg/mL, dashed line), showed no significant difference compared with the control (solid line).

Table 1.  FRAP of PD
InhibitorProportion FRAPp valueNumber of replicates required to detect changeNumber of replicates
  1. Proportion FRAP indicates the fluorescence intensity, expressed as a proportion of the prebleach value (mean ± 95% confidence interval) of PD for a range of inhibitors 40 min after bleaching. Significant differences between inhibitor treatments and control, based on analysis of variance, are presented. The number of treatment replicates required to detect a 20% difference and the actual number of replicates examined are shown.

Control0.41 ± 0.08 19
Azide0.10 ± 0.060.01611
BFA (10 μg/mL)0.42 ± 0.10ns922
BFA (100 μg/mL)0.23 ± 0.100.01914
Cytochalasin0.27 ± 0.080.05814
Latrunculin0.28 ± 0.050.05913
Colchicine0.46 ± 0.07ns1418
Oryzalin0.40 ± 0.08ns1010

Sodium azide severely inhibits MP targeting and disrupts cell structure

When tissue infected with TMV.MP–EGFP.CP was treated with the metabolic inhibitor sodium azide prior to FRAP experiments, fluorescence recovery was greatly reduced in PD relative to water-treated controls (Figure 2A,C; Table 1). Azide led to approximately an 80% reduction in FRAP within PD. Near the leading edge of a TMV.MP–EGFP.CP infection site treated with azide, MP accumulated at the vertices of the ER in a similar way to control tissue except that in certain areas the size of these accumulations was slightly increased, coincident with the disruption in the structure of the ER (Figure 1G). Further away from the infection front, the localization of MP to MT was unaffected by treatment with azide (Supplemental Figure 1A). In uninfected tissue treated with azide, the ER stopped moving (Supplemental Video 1) and many of the ER tubules had apparently withdrawn and accumulated at the ER vertices (Figure 1H). Azide treatment also resulted in the disruption of the actin cytoskeleton (Figure 1I) with only short actin filaments being observed. The Golgi bodies, although still visible as discrete stacks, stopped moving (Figure 1J; Supplemental Video 2). Although many MTs were still visible within the azide-treated tissue, GFP–tubulin, presumed to be in the form of unassembled subunits, was concentrated in certain regions (Figure 1K). Images obtained of dual-labeled tissue were consistent with the hypothesis that GFP–tubulin was concentrated around ER vertices (Figure 1L).

Disruption of the ER, but not inhibition of vesicle-mediated secretion, results in a reduction of MP accumulation in PD

To determine whether recovery of fluorescence within PD is dependent on the cortical ER, leaf tissue was treated with the fungal toxin Brefeldin A (BFA) (41). To monitor the effect of BFA on the secretory system, transgenic plants expressing a sialyl-transferase–GFP fusion (STtmd–GFP) were used (42). When this tissue (Figure 1D) was treated with 10 μg/mL BFA, the Golgi bodies were reabsorbed into the ER within 1 h of treatment (Figure 1M, arrow), leading to a faint GFP fluorescence in the ER network and in a number of irregularly shaped aggregates. Treatment of transgenic plants that expressed an ER-targeted form of GFP (ER–GFP) (Figure 1C) with the same concentration of BFA resulted in slight disruption to the ER, although the reticulated network was still present (Figure 1N), along with similar irregular aggregates as seen in the STtmd–GFP plants. In contrast, treatment of ER–GFP plants with 100 μg/mL BFA resulted in severe disruption of the cortical ER network (Figure 1O), with the majority of the ER tubules being absent. This high concentration of BFA resulted in the complete disruption of the Golgi bodies in the STtmd–GFP transgenics, with only aggregates remaining (Figure 1P). TUA–GFP plants expressing a fusion of GFP to α-tubulin (23) treated with 100 μg/mL BFA showed intact MTs and similar aggregations of GFP (Supplemental Figure 1B). The larger size of MP–GFP clusters at the leading edge of TMV.MP–GFP.CP lesions was once again consistent with the increased accumulation of ER at the vertices (Supplemental Figure 1C). However, there was no apparent effect of 100 μg/mL BFA on actin structure (Supplemental Figure 1D).

Analysis of the FRAP data at each of these concentrations showed that disruption of the cortical ER network (using 100 μg/mL BFA; Figure 2E; Table 1), but not secretion (using 10 μg/mL BFA; Figure 2D; Table 1), was required to reduce significantly trafficking of MP into PD. This suggests that transport along the membrane of the ER network, without passage of MP through the secretory system, may be sufficient for MP targeting to PD.

Disruption of the actin, but not the MT, cytoskeleton reduces MP targeting to PD

Treatment of tobacco epidermal cells with the actin-depolymerizing drug latrunculin resulted in the complete disappearance of actin (Figure 3A), while treatment with the actin-disrupting agent cytochalasin B resulted in severe fragmentation of the actin cytoskeleton (Figure 3B). Because the structure and movement of the ER and Golgi bodies are dependent on actin, treatment with either latrunculin or cytochalasin B resulted in contraction of the ER to the vertices (Figure 3C,D) and cessation of movement of both the ER (Supplemental Video 1) and Golgi bodies (Supplemental Video 2; Figure 3E,F). As with the azide-treated tissue, treatment of TUA–GFP plants with either latrunculin (Figure 3G) or cytochalasin B (Figure 3H) resulted in some GFP–tubulin accumulation, probably at the ER vertices (Supplemental Figure 1E).

image

Figure 3. The appearance of Nicotiana epidermal cells after treatment with latrunculin, cytochalasin B, colchicine or oryzalin. Alexa 488 phalloidin staining of actin, showing complete disruption of the actin network after treatment with 25 μm latrunculin (A) and severe fragmentation in the presence of 0.1 mg/mL cytochalasin B (B). Bar = 25 μm. ER–GFP transgenic plants showing alteration to the ER network in the presence of latrunculin (C) or cytochalasin B (D). Bar = 25 μm. STtmd–GFP transgenic plants showing Golgi bodies after treatment with latrunculin (E) or cytochalasin B (F). Bar = 10 μm in (E) and 25 μm in (F). TUA–GFP transgenic plants showing MTs after treatment with latrunculin (G) or cytochalasin B (H) and the accumulation of GFP at discrete sites. Bar = 25 μm. TUA–GFP transgenic plants showing the disruption to MTs in the presence of either 500 μm colchicine (I) or 20 μg/mL oryzalin (J). Bar = 25 μm. Anti-β-tubulin Cy3 conjugate labeling of Nicotiana benthamiana leaf epidermis tissue treated for 2 h with colchicine showing a fragmented network (K) and showing a disrupted network after treatment with oryzalin (L). Bar = 10 μm.

When tissue infected with TMV.MP–EGFP.CP was treated with latrunculin, accumulation of MP–GFP in PD was significantly reduced (Figure 2F; Table 1). Similar results were obtained with cytochalasin (Figure 2G; Table 1). Once again, there was little change in the distribution of MP–GFP at the edge of a lesion other than that consistent with increased ER accumulation at the vertices (Supplemental Figure 1F).

In marked contrast, treatment of tissue with the MT-depolymerizing drug colchicine showed no significant difference to water-treated controls with respect to fluorescence recovery in PD (Figure 2H; Table 1). In addition, no significant differences were observed with the MT-disrupting drug, oryzalin (Figure 2I; Table 1). Treatment with either colchicine (Figure 3I) or oryzalin (Figure 3J), respectively, resulted in the partial or complete disappearance of MTs in TUA–GFP transgenic plants, whereas an intact MT network could be observed in water-treated tissue (Figure 1E). Labeling of N. benthamiana with antibodies specific for β-tubulin showed an intact network of MTs in water-treated tissue (Supplemental Figure 1G), short fragments of MTs in colchicine-treated tissue (Figure 3K) and completely disrupted MTs in oryzalin-treated tissue (Figure 3L), confirming that the inhibitors had an identical mode of action on all MTs present, irrespective of whether the MTs were GFP labeled or not. Similarly, treatment with either colchicine (Supplemental Figure 1H) or oryzalin (not shown) resulted in the disappearance of MTs in TUA–GFP plants at the edge of a TMV.MP–mRFP.ΔCP lesion in contrast to untreated tissue (Supplemental Figure 1I). However, the presence of MP on MTs closer to the center of a lesion appeared to prevent their disruption (Supplemental Figure 1J) presumably due to some stabilization or masking of the MTs by MP binding. There was no obvious effect on the actin cytoskeleton (Supplemental Figure 1K,L), ER network (Supplemental Figure 1M,N) or Golgi bodies (Supplemental Figure 1O,P) when treated with either colchicine or oryzalin.

Discussion

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and methods
  6. Acknowledgments
  7. References
  8. Supporting Information

Although GFP is a powerful tag for MP studies (43), real-time imaging of MP–GFP kinetics at the true leading edge of infection is hindered by the maturation time of the GFP fluorophore, which can take a period of hours even in rapidly maturing GFP variants (36,37). It is therefore likely that some of the very early events in MP trafficking to and through PD will already have occurred before the MP–GFP fusion becomes detectable. Although MP–GFP accumulation in PD has been reported to be a very early event in TMV infection (18,19,23,44), it is also possible that viral RNA trafficking to adjoining cells has occurred prior to maturation of the fluorophore. However, in a previous study of TMV infection, it was shown that excision of unlabeled cells at the leading edge of an infection site prevented virus movement beyond these cells, suggesting that accumulation of MP within PD is indeed an early event in the infection process (9). As levels of mobile MP–GFP in the cell may be too low for detection by confocal microscopy, we adopted an FRAP approach to examine the kinetics of MP targeting to PD in TMV-infected cells.

FRAP studies

Following photobleaching, MP–GFP fluorescence was restored in PD, which reached 40% of prebleach levels within 40 min. The recovery rate is considerably slower than the rate observed for the recovery of photobleached Golgi bodies [in the order of 100 seconds (45)], intralumenal diffusion as reported for the ER (46), or the remodeling and diffusion of plant ER membranes (47) and other membrane-bound plant compartments such as chloroplast stromules (48). However, in our experiments, the amount of MP available for movement to the PD was considerably lower at the infection front than in the systems analyzed above, which could explain the slow recovery rates.

An early role for the actin/ER network in MP trafficking

The cortical ER network in plants is intimately associated with the actin cytoskeleton (39,49,50), the latter providing a cytoskeletal scaffold for the movement of specific macromolecular complexes, including the Golgi bodies, along the ER in cortical regions of the cell (39,51,52). Previous studies of MP targeting during TMV infection have implicated both the cortical ER (34) and the actin cytoskeleton (16) in MP trafficking to PD, based on an association of MP with both of these components at various stages of the infection cycle (18,23). To examine the role of the ER/secretory system in MP trafficking to PD, we used the drug BFA. At low concentrations, this drug has been shown to block vesicle-mediated secretion in plant cells and to cause the Golgi bodies to become reabsorbed back into the ER (41). As some viral MPs have been suggested to be delivered to PD by Golgi-body-derived vesicles (53), we were interested to determine whether this might be a feasible pathway for trafficking of the TMV MP. Following confirmation that low BFA concentrations (10 μg/mL) caused the Golgi bodies to disappear, but did not severely disrupt the ER, subsequent monitoring showed that fluorescence recovery at PD was unaffected relative to untreated controls, indicating that Golgi-body-mediated secretory events are not required for MP delivery to PD.

In contrast, at the higher BFA concentration (100 μg/mL), the ER was severely disrupted (54) and fluorescence recovery into PD was significantly reduced. Similarly, MP trafficking into PD was significantly decreased by inhibitors of the actin cytoskeleton, cytochalasin B and latrunculin. While causing complete disruption of the actin cytoskeleton, these compounds also affect the structure and movement of the ER. Although it is possible to disrupt the ER structure and maintain the actin cytoskeleton, disruption of the actin scaffold leads to considerable alteration of the overlying ER network. These data therefore suggest that MP trafficking to PD requires components of the ER network closely linked to a functional actin network.

Although it has been claimed that TMV MP lacks an apparent ER signal peptide (2,55), it does behave as an integral membrane protein (34,56,57). In this context, it has been proposed that the interaction between MP and either PME or calreticulin might provide the MP with the ER-targeting function it requires (6,58). It has also been suggested that MP may use an endogenous non-cell-autonomous protein (NCAP) pathway for targeting to PD (59). Mutations in the transmembrane domain of NCAPP1 (non-cell-autonomous pathway protein 1), normally localized to the cortical ER, have been shown to block trafficking of specific NCAPs including TMV MP and CmPP16.

Treatment with either inhibitor of actin function or high concentrations of BFA resulted in some recovery of fluorescence. It has recently been shown that diffusion of protein within the ER membrane is reduced but not abolished by treatment with latrunculin (47). We therefore propose that disruption of the ER results in accumulations of ER membrane at the vertices some of which may associate closely with PD sites. Therefore, under treatment with inhibitors, MP may still be able to diffuse along the ER membranes to the PD at these sites. Whether the MP actively moves along the ER with independent motility using molecular motors, or whether it simply moves with the flow of the cortical ER membrane, the latter showing significant mobility within the cortical layer, remains to be determined (47). Thus, the MP may require only a temporary insertion into the ER membrane, with subsequent actin-driven ER flow, or even diffusion within the ER membrane, carrying it to the vicinity of PD. The proposed role of the ER in the targeting of MP to PD may explain the apparent contradiction between the present work and the recent observations of Prokhnevsky et al. (60) that TMV MP targeting can take place in the presence of actin and myosin inhibitors. This accumulation occurs over a time period in excess of 18 h and may therefore involve diffusion of MP within the ER membrane into PD.

We found that fluorescence recovery was more severely inhibited by treatment of cells with azide than with any of the other treatments used. Because the disruption of cell structure by azide was apparently similar to treatment with either cytochalasin B or latrunculin, that is, disruption of actin filaments and cessation of ER and Golgi body movement, this would suggest that additional factors may be involved. Azide functionally depletes energy levels within cells indicating that one or more energy-dependent processes, for example, phosphorylation of the MP (61), are required for trafficking of viral MP to or its accumulation within PD. Previous studies have suggested that energy depletion (62,63) or actin inhibitors (64) dilate rather than close PD. Our results do not contradict these studies as the observed effects would reflect transport to (or trapping within) PD, rather than movement through the PD pore.

Taken together, the FRAP data obtained for PD indicate that MP traffics to PD in the ER membrane through an actin-dependent, energy-dependent mechanism and does not require Golgi-body-mediated secretion or MTs. This is consistent with the observation that movement of TMV viral replication complexes, labeled with GFP, is inhibited by latrunculin (31) and the recent demonstration that the TMV replication complex, along with the 126-kD protein, a constituent of the viral replication complex, traffics along microfilaments (65). Similarly, it has recently been shown that the actin/myosin network is involved in the targeting of a viral Hsp70 homolog to PD (60).

MTs and MP trafficking

MP trafficking to PD at the leading edge of infection was unaffected by MT inhibitors (oryzalin and colchicine). During later stages of the infection process, we observed that the association of MP with MTs protects the MTs against disruption by either oryzalin or colchicine as also recently shown by Ashby et al. (66). However, we confirmed that disruption of filaments incorporating TUA–GFP does occur at the infection front (66). Although plant MTs may require high concentrations (in the mm range) of colchicine to cause depolymerization, the dinitroaniline herbicide oryzalin has been shown to be more potent and cause the disappearance of most MTs when used at nanomolar concentrations (67). It has been contested that tua–GFP is not a reliable marker for MT and that MT might not be completely disrupted by chemical treatment (68). In an attempt to counter this suggestion, we showed, in parallel experiments, that colchicine and oryzalin, especially the latter, resulted in a marked disruption of MTs when both α-tubulin (indicated by observation of TUA–GFP transgenic plants) and β-tubulin (revealed by antibody labeling) were imaged. If MTs are involved in the early trafficking of MP to PD, we contend that such severe treatment, even if not disrupting every MT, should have resulted in significant differences in the FRAP measurements, which were not observed.

In conclusion, we contend that at the leading edge of a TMV infection, the targeting of MP to PD requires components of the ER network closely linked to a functioning actin network and does not involve MTs.

Materials and methods

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and methods
  6. Acknowledgments
  7. References
  8. Supporting Information

Plant material

N. benthamiana plants and Nicotiana transgenic plants were grown from seed and maintained under glasshouse conditions with a day length of 16.5 h, minimum daytime temperature of 28°C and minimum nighttime temperature of 22°C. Supplementary lighting was provided below a daytime threshold of 250/Wm2, and sun screening was used above 400/Wm2. Plants were used for experiments when they were 30–35 days old. Nicotiana tabacum transgenic plants were used only when N. benthamiana lines were not available.

ER–GFP N. benthamiana plants expressing m-gfp5-ER were kindly provided by D. Baulcombe (54).

N. tabacum plants expressing the sialyl-transferase transmembrane domain fused to GFP (STtmd–GFP) as a Golgi body marker, created in a similar way to STtmd–mRFP plants (42), were a gift from C. Hawes and A. Andreeva (Oxford Brookes University, UK).

N. benthamiana plants expressing a fusion of GFP to Arabidopsis α-tubulin (TUA–GFP) were constructed as described previously (23).

Construction of viral vectors and infection of plants

TMV.MPWT–GFP.CP was produced as described previously (23). Derivatives of TMV.MPWT–GFP.CP were constructed in which the gene for GFP was replaced with those for EGFP (Clontech Laboratories Inc., Palo Alto, CA, USA) or mRFP (69). Fluorescent-protein-coding sequences were amplified from plasmid templates with Expand High Fidelity PCR System (Roche, Mannheim, Germany) using primers that introduced AvrII sites at the 5′ end of the coding sequences and XhoI sites after the termination codons. The amplification products were digested with AvrII and XhoI, and inserted between the same sites of TMV.MPWT–GFP.CP. The EGFP derivative was used as a coat protein containing vector, while the mRFP derivative was further modified to delete the coat protein gene by digestion with BsiWI and KpnI, releasing a fragment containing the coat protein subgenomic promoter, coat protein gene and 3′ untranslated region, and insertion of a BsiWI/KpnI fragment from pTMV004 (70) comprising just the 3′ untranslated region.

Infectious transcripts were prepared, reassembled with TMV coat protein and inoculated onto plants as described previously (23). Inoculated plants were grown under glasshouse conditions for 4 days with a day length of 16.5 h, minimum daytime temperature of 33°C and minimum nighttime temperature of 28°C.

Transient expression in plants

ER-targeted yellow fluorescent protein (YFP)constructs (71) were provided by C. Hawes and colleagues (Oxford Brookes University), electroporated into Agrobacterium tumefaciens GV3101 and transiently expressed in plants as described by Latijnhouwers et al. (42).

Imaging of fluorescent proteins

All imaging was conducted using either a Leica TCS-SP1 CLSM (FRAP of PD; Leica Microsystems, Heidelberg GmbH, Germany) or a Leica TCS-SP2 AOBS (GFP, mRFP, YFP). Unless otherwise stated, images were obtained using a Leica HCX APO × 63/0.90w water-dipping lens; GFP was imaged using 488-nm excitation with emission collected between 500 and 530 nm. mRFP, when in conjunction with GFP, was imaged sequentially by excitation at 561 nm and emission collected at 590–630 nm. Chlorophyll-associated autofluorescence was imaged by excitation at 488 nm, with the emission collected between 650 and 700 nm. GFP, when used in conjunction with YFP, was imaged by excitation at 488 nm with emission collected between 500 and 515 nm, the emission from chlorophyll-associated autofluorescence being collected between 650 and 700 nm, with sequential collection of the YFP signal by excitation at 514 nm with emission collected between 535 and 545 nm.

FRAP of PD

PD that were labeled with TMV MP–EGFP at the leading edge of an infection in the abaxial epidermis were imaged at × 1 zoom using 488-nm excitation at 20% laser intensity and 500- to 535-nm emission, prior to, immediately following, and 10, 20, 30 and 40 min after being photobleached. The photobleach procedure involved increasing to × 32 zoom, increasing the laser intensity from 20 to 100% using the Acousto Optical Tunable Filter and scanning three times at medium speed. Control leaves were infiltrated with distilled water, while treated leaves were infiltrated by means of the abaxial surface and floated on chemical treatment for the indicated time prior to imaging (see below).

Chemical treatments

For actin disruption, leaf tissue was treated with either 25 μm latrunculin B [Calbiochem, San Diego, CA, USA; 2.5 mm stock in dimethyl sulphoxide (DMSO)] or 0.1 mg/mL (≈200 μm) cytochalasin B (Sigma-Aldrich, St Louis, MO, USA; 10 mg/mL stock in DMSO) for 2 h.

For MT disruption, leaf tissue was treated with either 500 μm colchicine (Sigma-Aldrich; 50 mm aqueous stock) or 20 μg/mL (57 μm) oryzalin (Dow Elanco, Letcombe Regis, UK; 50 mg/mL stock in acetone) in water with 0.08% ethanol for 2 h.

BFA (Sigma-Aldrich; 10 mg/mL stock in methanol) was used at two concentrations, either low [10 μg/mL (35.7 μm) for 1 h] or high [100 μg/mL (357 μm) for 2 h].

Sodium azide, 0.02% (3 mm) (VWR International Ltd, Lutterworth, UK; 10% stock in water) was infiltrated and incubated for 2 h.

All stock solutions were stored at −20°C except sodium azide, which was maintained at room temperature, and working solutions were freshly prepared before each use.

Analysis of PD photobleaching data

The fluorescence intensity associated with each photobleached PD and a neighboring nonbleached PD was measured. To allow for changes in the focal plane, or additional photobleaching during imaging, the fluorescence intensity of the bleached PD was adjusted by the factor of change in fluorescence of the nonbleached PD. The corrected fluorescence intensities were then expressed as a proportion of the prebleached value. A power test was used to determine the number of replicates required to detect a 20% difference between the amount of fluorescence recovery under control conditions and under each chemical treatment (Table 1). The relationship between fluorescence intensity and time was best described by an exponential relationship of the form a + bc t. Analysis of Variance was performed on two parameters (a and b) following a natural log transformation, to identify treatments that resulted in significant differences in FRAP from the control. The results are summarized in Table 1.

Immunolabeling of β-tubulin

N. benthamiana leaf tissue was infiltrated with water (control), colchicine or oryzalin for 2 h as described above, prior to being cut into 4- × 1-mm sections and fixed overnight in 4% formaldehyde, 0.1% glutaraldehyde in PEMT buffer (100 mm Piperazine-NN′-bis-2-ethanesulphonic acid (PIPES), 5 mm EGTA, 2 mm MgSO4, 0.05% Triton-X-100, pH 6.8 with KOH). After washing in PEMT, the sections were freeze shattered and treated with monoclonal anti-β-tubulin Cy3 conjugate (Sigma-Aldrich Co. Ltd, Gillingham, Dorset, UK) diluted 1:100, essentially as described by Wasteneys et al. (72). Cy3 labeling was imaged on the Leica TCS-SP2 (excitation 561 nm, emission 570–590 nm) using a Leica HCX PL APO × 63/1.2w water-immersion lens.

Labeling of actin with Alexa 488 phalloidin

Leaf tissue was mounted on plastic holders and abraded, under water, using fine abrasive paper (Wetordry P1200) to leave only the abaxial epidermis. The tissue was incubated essentially as described previously (73) in MBS (3-maleimidobenzoic acid N-hydroxy-succinimide ester) extraction buffer (Nonidet P-40 replaced by Igepal; Sigma-Aldrich Co. Ltd) for 1.75 h before transfer to fresh buffer containing 1.65 × 10−7 m Alexa 488 phalloidin for 0.75 h. Alexa 488 phalloidin-stained actin was imaged by excitation at 488 nm with emission collected at 500–530 nm. Where necessary, leaf tissue was treated with inhibitor for the indicated time prior to abrasion and maintained in extraction buffer plus inhibitor during the staining procedure.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and methods
  6. Acknowledgments
  7. References
  8. Supporting Information

The authors thank F. Brandizzi for advice on FRAP techniques. This work was supported by the Scottish Executive Environment and Rural Affairs Department (grants SCR/0611 and SCR/0612). Recombinant viruses and transgenic plants were contained in glasshouses under SEERAD license GM/224/2005.

References

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and methods
  6. Acknowledgments
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and methods
  6. Acknowledgments
  7. References
  8. Supporting Information

Video 1: Time series of ER movement. Series of 30 images, taken at 1s 164ms intervals, of ER-GFP epidermal cells showing normal ER movement in tissue pre-treated for 2h with water (control), 500μM colchicine or 20μg ml−1 oryzalin, and the disrupted structure and lack of movement in tissue treated with 0.02% sodium azide, 25μM latrunculin or 0.1mg ml−1 cytochalasin B.

Video 2: Time series of Golgi apparatus movement. Series of 30 images, taken at 1s 164ms intervals, of STtmd-GFP epidermal cells showing the normal movement of Golgi stacks in tissue pre-treated for 2h with water (control), 500μM colchicine or 20μg ml−1 oryzalin, and the lack of movement in tissue treated with 0.02% sodium azide, 25μM latrunculin or 0.1mg ml−1 cytochalasin B.

Figure 1: Appearance of cell organelles in Nicotiana epidermal cells. In all images GFP-associated fluorescence is coloured green and chlorophyll-associated autofluorescence is coloured blue.

(A) Appearance of a sodium azide-treated TMV.MP-EGFP.CP lesion on Nicotiana benthamiana away from the leading edge showing that azide does not alter the accumulation of MP on MTs. Bar = 25μm.

(B) TUA-GFP plants showing MTs and apparent accumulation of GFP into aggregates after treatment with 100μg ml−1 BFA. Bar = 25μm.

(C) Maximum projection of a stack of confocal images taken at the leading edge of a TMV.MP-EGFP.CP lesion on Nicotiana benthamiana (left) showing the targeting of MP to PD, formation of clusters and accumulation on MT after treatment with 100μg ml−1 BFA. Bar = 25μm.

(D) Alexa 488 phalloidin staining of actin, showing an intact actin network after treatment with 100μg ml−1 BFA. Bar = 25μm.

(E) TUA-GFP transgenic plants, 4 days after Agrobacterium inoculation with a strain carrying a binary for YFP-HDEL expression. When treated with cytochalasin B, GFP fluorescence (green) in a non-infected cell (left) forms a similar pattern to ER-associated fluorescence (magenta) in the neighbouring cell (upper right) indicating possible accumulation of GFP-tubulin at the ER vertices. Bar = 25μm.

(F) Maximum projection of a stack of confocal images taken at the leading edge of a TMV.MP-EGFP.CP lesion on Nicotiana benthamiana (lower left) showing the targeting of MP to PD and the formation of clusters after treatment with latrunculin. Bar = 25μm.

(G) Anti-ß-tubulin Cy3 conjugate labeling of Nicotiana benthamiana leaf epidermis under control conditions showing an intact MT network. Bar = 10μm.

(H) and (I) TUA-GFP transgenic plants infected with TMV.MP-mRFP.ΔCP. At the edge of the infection the presence of MP clusters (magenta) (darts) does not prevent the complete disruption of the MTs (green) following treatment with 500μM colchicine (H). Under control conditions (I) MP clusters (magenta) (darts) at the edge of the infection can be seen, as well as MTs (green) (arrows). Bar = 25μm.

(J) Maximum projection of a stack of confocal images of a TMV.MP-EGFP.CP lesion on Nicotiana benthamiana showing that the association of MP with MT prevents the disruption of MT when treated with 500μM colchicine. Bar = 25μm.

(K) and (L). Alexa 488 phalloidin staining of actin, showing no significant change after treatment with colchicine (K) or oryzalin (L). Bar = 25μm.

(M) and (N) ER-GFP transgenic plants showing no obvious alteration to the ER network in the presence of colchicine (M) or oryzalin (N). Bar = 25μm.

(O) and (P) STtmd-GFP transgenic plants treated with colchicine (O) or oryzalin (P) showing no significant effect on Golgi stacks. Bar = 25μm.

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tra_523_sm_8_1bfig1.jpg514KSupporting info item
tra_523_sm_8_1bmov1.mov2425KSupporting info item
tra_523_sm_8_1bmov2.mov2076KSupporting info item

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