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Plant Cell. Dec 2005; 17(12): 3513–3531.
PMCID: PMC1315385

Localization of the Tomato Bushy Stunt Virus Replication Protein p33 Reveals a Peroxisome-to-Endoplasmic Reticulum Sorting PathwayW in Box

Abstract

Tomato bushy stunt virus (TBSV), a positive-strand RNA virus, causes extensive inward vesiculations of the peroxisomal boundary membrane and formation of peroxisomal multivesicular bodies (pMVBs). Although pMVBs are known to contain protein components of the viral membrane-bound RNA replication complex, the mechanisms of protein targeting to peroxisomal membranes and participation in pMVB biogenesis are not well understood. We show that the TBSV 33-kD replication protein (p33), expressed on its own, targets initially from the cytosol to peroxisomes, causing their progressive aggregation and eventually the formation of peroxisomal ghosts. These altered peroxisomes are distinct from pMVBs; they lack internal vesicles and are surrounded by novel cytosolic vesicles that contain p33 and appear to be derived from evaginations of the peroxisomal boundary membrane. Concomitant with these changes in peroxisomes, p33 and resident peroxisomal membrane proteins are relocalized to the peroxisomal endoplasmic reticulum (pER) subdomain. This sorting of p33 is disrupted by the coexpression of a dominant-negative mutant of ADP-ribosylation factor1, implicating coatomer in vesicle formation at peroxisomes. Mutational analysis of p33 revealed that its intracellular sorting is also mediated by several targeting signals, including three peroxisomal targeting elements that function cooperatively, plus a pER targeting signal resembling an Arg-based motif responsible for vesicle-mediated retrieval of escaped ER membrane proteins from the Golgi. These results provide insight into virus-induced intracellular rearrangements and reveal a peroxisome-to-pER sorting pathway, raising new mechanistic questions regarding the biogenesis of peroxisomes in plants.

INTRODUCTION

Plus-sense, single-stranded RNA viruses infect most eukaryotes and are the predominant class of viruses that infect plants. Tombusviruses belong to a family of positive-strand RNA plant viruses (Tombusviridae) that possess relatively small genomes (~4800 nucleotides) and include Cymbidium ringspot virus (CymRSV), Cucumber necrosis virus (CNV), Carnation Italian ringspot virus, and Tomato bushy stunt virus (TBSV), the last of which is probably the best studied in terms of its genome replication and recombination (reviewed in White and Nagy, 2004). The TBSV genome contains five open reading frames (ORFs) (Figure 1). ORF1 encodes a 33-kD auxiliary replication protein (p33), and ORF2 encodes a 92-kD RNA-dependent RNA polymerase (p92) and is produced by the translational read-through of the p33 amber stop codon. Both p33 and p92 are translated directly from the viral genome in infected cells and interact as membrane-bound components of the RNA replication complex (K.B. Scholthof et al., 1995; Rajendran and Nagy, 2004). The remaining three ORFs in the TBSV genome encode a coat protein of 41 kD (ORF3), a 22-kD protein required for cell-to-cell movement of the virus (ORF4), and a 19-kD protein that functions as a suppressor of virus-induced gene silencing (ORF5) (H.B. Scholthof et al., 1995).

Figure 1.
Diagram of the TBSV Genome.

TBSV can infect a variety of plant species, and in all cases the most conspicuous cytopathological feature of infected cells is the presence of multivesicular bodies (MVBs) derived from peroxisomes (reviewed in Martelli et al., 1988). These novel intracellular structures (referred to herein as peroxisomal multivesicular bodies [pMVBs]) form initially by a progressive inward vesiculation of the boundary membrane of preexisting peroxisomes, resulting in the organelle's interior (matrix) housing up to several hundred spherical to ovoid vesicles of 80 to 150 nm in diameter. Eventually, the boundary membrane of individual pMVBs also produces one or more large, spherical, and vesicle-containing extrusions that fold back and engulf portions of the cytosol, yielding doughnut-shaped or sometimes C-shaped pMVBs that no longer resemble the peroxisomes from which they are derived.

Although the progressive structural reorganization of peroxisomes into pMVBs in TBSV-infected cells has been relatively well documented, the functional role of these complex membranous compartments and the molecular mechanism(s) underlying their biogenesis are largely speculative. For instance, because pMVBs are frequently observed to be in close association with segments of the endoplasmic reticulum (ER), this endomembrane compartment has been proposed as the membrane source for the numerous vesiculation events that occur at the peroxisomal boundary membrane during TBSV infection (Martelli et al., 1988). It has also been proposed that the small vesicles that are formed within pMVBs are the sites of TBSV RNA replication, because these structures can incorporate tritiated uridine (Appiano et al., 1983, 1986) and could serve to protect nascent viral transcripts from host cell RNases. Consistent with this premise, both p33 and p92 are membrane-associated proteins; thus, the TBSV replication complex is likely anchored onto the internal vesicles of pMVBs (K.B. Scholthof et al., 1995). However, how nascent p33 and p92 are initially targeted to peroxisomal membranes and participate in pMVB formation during the TBSV infection process has not been resolved. In fact, the only significant insights to these events have come from studies of homologs of p33 in the related CymRSV and CNV. For instance, modified infectious clones of CymRSV were used to demonstrate that the N-terminal portion (including the two predicted membrane-spanning domains) of p33 is essential for the formation of pMVBs (Burgyan et al., 1996; Rubino and Russo, 1998). The same N-terminal region of CymRSV and CNV p33s was also shown to contain the targeting information essential for their sorting to peroxisomal membranes in Saccharomyces cerevisiae, although a specific targeting sequence in either protein was not defined (Navarro et al., 2004; Panavas et al., 2005).

We are interested in understanding all aspects of the TBSV life cycle, including how this virus exploits peroxisomes as the sites for viral RNA replication and what this might tell us about plant peroxisome biogenesis in general. Previously, we and others showed that newly synthesized peroxisomal membrane proteins (PMPs) are sorted either directly to peroxisomes from the cytosol or indirectly to peroxisomes by way of specialized regions of the ER, known as peroxisomal endoplasmic reticulum (pER), and pER-derived vesicles (reviewed in Trelease and Lingard, 2005). Here, we provide evidence that the TBSV replication protein p33 makes use of a novel peroxisome-to-pER vesicle-mediated sorting pathway. Using a combination of fluorescence and electron microscopy, as well as site-directed mutagenesis experiments, we show that the trafficking and molecular targeting signals of p33 are far more complex than previously suggested, especially with respect to the involvement of pER. Overall, these results using p33 as an investigative tool have important implications for our overall understanding of pMVB biogenesis and the biogenetic link between peroxisomes and pER via vesicle transport in plants.

RESULTS

Peroxisomes in TBSV-Transformed BY-2 Cells Are Altered in Terms of Their Distribution and Morphology, Contain Both p33 and p92, and Serve as the Sites of Viral RNA Synthesis

To determine the subcellular localization of p33, we initially examined the protein in plant cells transformed with TBSV cDNA. Toward that end, a plasmid containing the full-length cDNA of the TBSV genome that can launch autonomous TBSV replication (pHST20; Scholthof, 1999) was introduced by biolistic bombardment into tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) suspension-cultured cells, serving as a model system for studying protein sorting in vivo (Nagata et al., 1992). Expressed p33 along with its translation read-through product, p92, were localized using fluor-conjugated secondary antibodies bound to IgGs raised against a synthetic peptide that corresponds to an amino acid sequence in both proteins (Figure 1, asterisk). Antibody specificity for both p33 and p92 was confirmed by immunoblotting of protein extracts from BY-2 protoplasts transformed with TBSV RNA (Figure 2A).

Figure 2.
Localization of p33 and p92 Replication Proteins in TBSV-Transformed BY-2 Cells.

Figure 2B is a grouping of representative micrographs illustrating that introduced p33 and p92 colocalized precisely with the endogenous peroxisomal matrix protein catalase in individual TBSV-transformed BY-2 cells at 2, 4, 24, and 48 h after bombardment. These time-course results also revealed dramatic and progressive alterations in peroxisome distribution and morphology. For instance, at 2 and 4 h after bombardment, most of the peroxisomes in TBSV-transformed cells were larger in size and fewer in number compared with peroxisomes in neighboring nontransformed cells. Close inspection revealed that these globular-like peroxisomes consisted of several individual peroxisomes (Figure 2B, inset), suggesting that they formed by coalescence of preexisting organelles.

Changes in peroxisome morphology in TBSV-transformed cells were even more pronounced at 24 and 48 h after bombardment; p33 and p92 colocalized with catalase in several large globular and elongated peroxisomal structures that ranged in size up to 30 μm in length, were typically restricted to the perinuclear region of the cell, and had a more diffuse appearance compared with the distinct aggregated peroxisomal structures observed at earlier time points (Figure 2B). In contrast with peroxisomes, the morphology and distribution of other subcellular organelles in TBSV-transformed cells, including mitochondria, ER, plastids, and Golgi, were unaltered (Figure 2C).

To determine whether the novel peroxisomal structures in TBSV-transformed BY-2 cells also contained double-stranded RNA (dsRNA) intermediates produced during viral replication (White and Nagy, 2004), cells were dual-labeled with anti-p33/p92 IgGs and monoclonal antibodies raised against dsRNA. Figure 2D shows that at 24 h after bombardment, dsRNA in a TBSV-transformed cell localized exclusively to distinct portions of the globular p33- and p92-containing peroxisomes, indicating that viral RNA synthesis occurred at these sites, presumably pMVBs. Neither mock transformations nor omission of anti-p33/92 and/or anti-dsRNA antibodies yielded immunofluorescence (data not shown).

p33 Expressed Individually in BY-2 Cells Is Localized Initially to Aggregated Peroxisomes and Then to a Subdomain of the ER Concomitant with the Formation of Peroxisomal Ghosts

Similar to the results presented in Figure 2B, p33 expressed on its own colocalized with endogenous catalase in individual and aggregated (globular) peroxisomes at the earlier stages of expression and sorting (i.e., 2 and 4 h) (Figure 3A), suggesting that p33 targeted directly from its site of synthesis in the cytosol to peroxisomes. By 24 h after bombardment, however, p33 localized to two distinct subcellular compartments: globular peroxisomes that had continued to increase in size, and a reticular network that was distributed throughout the entire cell (Figure 3A). The lack of endogenous catalase in this p33-containing reticulum suggests that it is not formed by a marked distension or induced elongation/tubulation of globular peroxisomes but instead is a separate subcellular compartment(s).

Figure 3.
Localization of p33 in BY-2 Cells.

At 48 h after bombardment, p33 remained localized to a diffuse reticular network as well as to several globular structures, although the latter were generally smaller than those observed at 24 h after bombardment and, thus, were more difficult to discern because of the pervasive nature of the reticular fluorescence (Figure 3A). Interestingly, p33-transformed cells at 48 h after bombardment also were devoid of endogenous catalase (cf. catalase staining in neighboring nontransformed cells in Figure 3A). Similar results were observed when these p33-transformed cells were immunostained for the endogenous peroxisomal matrix enzyme isocitrate lyase (data not shown).

The eventual loss of peroxisomal matrix enzymes in cells expressing p33 was unexpected and suggested that the peroxisomes in these cells were degraded (e.g., pexophagy) (Farre and Subramani, 2004). Alternatively, the integrity of the peroxisomal boundary membranes in these cells may have been compromised such that the small globular structures observed at 48 h after bombardment were remnants of peroxisomes that possessed membrane proteins but lacked matrix proteins, similar to the so-called peroxisomal ghosts described in yeast and mammalian cells having mutations in peroxisomal matrix protein import (reviewed in Purdue and Lazarow, 2001). To test these possibilities, p33-transfromed cells at 48 h after bombardment were examined for the presence or absence of two well-characterized PMPs, ascorbate peroxidase (APX) and a 22-kD PMP (PMP22) (Lisenbee et al., 2003a; Murphy et al., 2003, and references therein).

For dual-labeling experiments with p33 and APX, a myc epitope tag was appended to the C terminus of p33 (p33-myc). p33-myc resembles wild-type p33 in that it localized to both a diffuse reticulum and several small globular structures within the same cell at 48 h after bombardment (Figure 3B, a), confirming that the addition of the myc epitope to p33 does not disrupt its normal subcellular sorting. Endogenous APX was also readily immunodetectable in a p33-myc–transformed cell, and this resident PMP colocalized with p33-myc in the same small globular structures (Figure 3B, a and b, closed arrowheads), indicating that, although lacking endogenous matrix enzymes, these structures contain APX and likely other PMPs (i.e., peroxisomal ghosts). Interestingly, endogenous APX in the same transformed cell also colocalized with p33-myc in the reticular network (Figure 3B, c and d). By contrast, endogenous APX in neighboring nontransformed cells localized at its steady state only to normal individual peroxisomes, as expected (Mullen et al., 1999) (Figure 3B, b).

Transiently expressed PMP22 was also immunodetectable in p33-cotransformed cells, and similar to endogenous APX, the subcellular localization of PMP22 in these cells was substantially altered. That is, although N-terminal myc-tagged PMP22 (myc-PMP22) expressed on its own localized exclusively to normal, individual catalase-containing peroxisomes, as expected (Murphy et al., 2003) (Figure 3B, e and f), coexpression of myc-PMP22 and p33 resulted in both proteins localized to the same small globular peroxisomal ghosts and reticular network (Figure 3B, g and h).

Given the distinct morphology of the reticular compartment to which p33 (and endogenous APX and coexpressed PMP22 in p33-transformed cells) localizes and the fact that at least some plant PMPs, such as APX (Mullen et al., 1999; Nito et al., 2001; Lisenbee et al., 2003a) and PEX16 (Karnik and Trelease, 2005), sort indirectly to peroxisomes by way of pER, we speculated that p33 was also targeted to ER, or a portion thereof. Figure 3C, a and b, show that the reticular fluorescence patterns attributable to expressed p33 and concanavalin A–stained ER did not colocalize in the same cell at 48 h after bombardment. A lack of colocalization was observed also between p33 and the endogenous ER resident protein calreticulin (data not shown). By contrast, p33 colocalized entirely with coexpressed chloramphenicol acetyltransferase (CAT) fused to the 36 C-terminal residues of APX (CAT-APX) (Figure 3C, c and d). This portion of APX includes the targeting signals responsible for sorting this PMP to peroxisomes via pER; thus, when CAT-APX is overexpressed, the fusion protein localizes throughout its entire sorting pathway, including pER (Mullen et al., 1999, 2001; Mullen and Trelease, 2000).

Sorting of p33 from Peroxisomes to pER Is Not Affected by Cycloheximide or Cold Treatment but Is Disrupted by a Dominant-Negative Mutant of ADP-Ribosylation Factor1

Although the data presented in Figure 3 are indicative of p33 sorting initially from the cytosol to peroxisomes and then to pER, it is also possible that newly synthesized p33 sorted directly to pER at later time points (e.g., 12 h after bombardment) by default, as a result of the dramatic alterations in peroxisomal integrity caused by p33 (over)expression and/or saturation of the host cell machinery responsible for PMP (p33) targeting and integration. To address this possibility, p33-transformed cells at 4 h after bombardment were incubated with the protein synthesis inhibitor cycloheximide. We selected 4 h for these experiments because p33 localizes specifically to peroxisomes at this time (Figure 3A), and observations of p33 in pER in cells that were subsequently incubated with cycloheximide would be convincing evidence that the protein sorts from peroxisomes to pER.

Figure 4A shows that expressed p33 at 12 h after bombardment localized to both globular peroxisomes and pER in either the absence or presence of cycloheximide, indicating that the peroxisome is an intermediate sorting site for p33. In a positive control experiment, CAT-APX expressed in the absence of cycloheximide for 12 h localized throughout its sorting pathway, including (aggregated) peroxisomes and pER, as expected (Mullen et al., 1999) (Figure 4A). In the presence of cycloheximide, however, CAT-APX accumulated only in peroxisomal aggregates, consistent with the sorting of this PMP to peroxisomes via pER. Additional control experiments with the Golgi-localized SNARE, BS14a (green fluorescent protein [GFP]-BS14a), confirmed the effect of cycloheximide on protein synthesis and protein sorting in transformed BY-2 cells (see Supplemental Figure 1 online).

Figure 4.
Effect of Cycloheximide, a Dominant-Negative Mutant of Arf1, and Cold Shock on p33 Localization in BY-2 Cells.

The sorting of p33 from peroxisomes to pER was also analyzed using a dominant-negative mutant of the ADP-ribosylation factor1 (Arf1Q71L) that inhibits multiple steps in the secretory protein transport pathway by disrupting the formation of coat protein complex I (COPI) coated vesicles (Pepperkok et al., 2000; Takeuchi et al., 2002). Specifically, we tested whether blocking COPI vesicle transport with Arf1Q71L changed the steady state localization of coexpressed p33. Figure 4B shows that at 24 h after bombardment, p33 coexpressed with wild-type Arf1 did not alter the viral protein's normal localization to (globular) peroxisomes and pER (cf. with the localization of p33 expressed on its own at 24 h after bombardment in Figures 3A and 3B). By contrast, coexpression of p33 with Arf1Q71L altered p33's localization at 24 h after bombardment from peroxisomes and pER to entirely peroxisomes (Figure 4B), indicating that Arf1 is necessary for sorting p33 from peroxisomes to pER. Similarly, localization of the ER marker protein RFP-HDEL was altered when coexpressed with Arf1Q71L (Figure 4B), consistent with COPI being involved in the retrieval of escaped reticuloplasmins from the Golgi back to the ER (Denecke, 2003).

To test whether p33 sorts initially to pER and then to peroxisomes, but does so in such a transient manner that, unlike CAT-APX, it is not readily detectable in pER, we examined the localization of p33 at early time points after bombardment (i.e., 4 h) in transformed cells incubated at 14°C. Cold treatment, among other cellular effects, disrupts protein transport from the ER by inhibiting ER-derived vesicle formation (Bar-Peled and Raikhel, 1997; Boevink et al., 1999; Phillipson et al., 2001). Thus, if p33 sorts to peroxisomes by way of the ER (pER), then cold treatment of p33-transformed cells should adversely affect this sorting step and p33 should, at least partially, accumulate in pER. However, as shown in Figure 4C, the sorting of p33 was identical when cells were maintained 4 h after bombardment at either 14 or 25°C (i.e., p33 localized exclusively to peroxisomes at both temperatures). Similar results were observed when p33-transformed cells were incubated for 4 h after bombardment at 8°C (data not shown). By contrast, sorting of GFP-BS14a from ER to Golgi was disrupted in cells incubated at 14°C (Figure 4C).

p33 Expressed in Tobacco Leaves Is Localized to the Boundary Membranes of Aggregated Peroxisomes and Novel Cytosolic Vesicles

As noted in the Introduction, TBSV causes progressive rearrangements of peroxisome boundary membranes that eventually give rise to the formation of pMVBs (Martelli et al., 1988). Figures 5A to 5C illustrates the results of a series of replicate experiments that served as a comparative control for this study. Nicotiana benthamiana plants were rub-inoculated with TBSV RNA, and systemically infected leaves were analyzed by transmission electron microscopy. The representative sectional view of the individual mesophyll cell in Figure 5A shows a cluster of five large (3 to 5 μm) doughnut-shaped pMVBs, each consisting of an extended membranous appendage encircling or engulfing a large portion of the cytosol. Numerous 40- to 170-nm-diameter vesicles are present within the lumen of each of these pMVBs, either separate from or, on occasion, connected to the boundary membrane (Figure 5B, inset), suggesting that they formed by membrane invagination and vesiculation. Occasionally, pMVBs were also found in close association with tubular membranous structures that resembled the ER (Figure 5C), consistent with the presumption that a biogenetic link exists between these two compartments (Navarro et al., 2004). All other subcellular organelles were unaltered in TBSV-infected cells. Controls, including mock-rub-inoculated plants and wild-type untreated plants, did not yield any noticeable changes in peroxisomal ultrastructure (see the wild-type peroxisomes shown in Figures 5K and 5L).

Figure 5.
Electron and Fluorescence Microscopic Analysis of Tobacco Mesophyll Cells Transformed with TBSV or p33-GFP.

Based on the immunofluorescence results shown in Figure 3A, the large globular peroxisomal structures in p33-transformed BY-2 cells at 24 h after bombardment are most likely pMVBs; thus, p33 appears to be sufficient in forming pMVBs in the absence of other TBSV proteins. However, testing this hypothesis directly at the ultrastructural level using transiently transformed BY-2 cells was difficult because of the low frequency of cell transformation (0.5%) achieved by biolistic bombardment. To circumvent this problem, p33 was expressed transiently in tobacco leaves via Agrobacterium tumefaciens infiltration, a method that yields transformation frequencies of nearly 100% (Wroblewski et al., 2005). In addition, two GFP fusion proteins were used, GFP-SKL (GFP linked to the C-terminal tripeptide SKL, a prototypic matrix peroxisomal targeting signal) and p33-GFP (full-length p33 linked to the N terminus of GFP), as vital markers that provided a convenient means of assessing changes in peroxisome morphology and distribution in infiltrated leaf tissue before processing for electron microscopy. Three days after Agrobacterium infiltration, GFP-SKL exhibited a punctate fluorescence pattern consistent with this protein being localized to matrices of individual peroxisomes (Figure 5D). By contrast, p33-GFP exhibited a torus and globular fluorescence pattern indicative of localization to the boundary membranes of a cluster of aggregated peroxisomes (Figure 5F, inset), similar to the localization of wild-type p33 to aggregated peroxisomes in BY-2 cells at 4 h after bombardment (Figure 3A).

Consistent with these fluorescence microscopy results, electron microscopic analysis of mesophyll cells transformed with p33-GFP revealed that peroxisomes were highly aggregated, consisting of clusters of 3 to 8, and up to 15, individual organelles that varied in size from 200 to 1500 nm in diameter (Figures 5H to 5J). The interiors of these aggregated peroxisomes were as devoid of vesicles as those observed in pMVBs (cf. Figures 5B and 5H) and were only slightly more structured and electron dense than the matrices of wild-type peroxisomes in mesophyll cells either infiltrated with Agrobacterium containing empty binary vector (Figure 5K) or nontransformed (Figure 5L). On the other hand, the boundary membranes of aggregated peroxisomes in p33-GFP–transformed cells were largely distorted, with contours that gave a rippled or scalloped complexion and numerous evaginations (Figures 5H and 5J, arrowheads), suggesting that nascent vesicles are externalized into the cytosol. Consistent with this finding, collections of small (40 to 150 nm), single membrane-bound vesicles were located in the cytosol immediately adjacent to the aggregated peroxisomes (Figures 5H to 5J, asterisks). Similar cytosolic vesicles were detected in mesophyll cells transformed with wild-type p33 (data not shown) but were not observed in nontransformed cells (Figure 5L), cells transformed with vector alone (Figure 5K), or TBSV-infected cells containing pMVBs (Figures 5A to 5C). Thus, although p33 is responsible for the formation of the novel cytosolic vesicles, they are not topologically equivalent to those formed during the biogenesis of a so-called classical pMVB.

Immunogold labeling of the p33-GFP–transformed samples represented in Figure 5 was performed to determine the precise subcellular localization of the viral fusion protein. Virtually all of the gold particles decorated the periphery (membrane) of the novel cytosolic vesicles and their neighboring aggregated peroxisomes, as well as the matrix of these peroxisomes (Figure 6A). All other subcellular structures in these p33-GFP–transformed cells in which the total surface area was several orders of magnitude larger than that of the aggregated peroxisomes and their surrounding vesicles displayed significantly lower frequencies of gold particle labeling (Figures 6B and 6C), equivalent to that observed when samples were incubated with preimmune serum (Figure 6D).

Figure 6.
Localization of p33-GFP in Tobacco Mesophyll Cells.

The N-Terminal Half of p33 Possesses Both Peroxisome and pER Targeting Information

To define the targeting information responsible for sorting p33 to various subcellular compartments (i.e., peroxisomes and pER), we conducted a series of targeting experiments using chimeras consisting of different portions of p33 fused to CAT serving as a passenger protein (Figures 7A and 7B). Although CAT alone localized only to the cytosol of a transformed BY-2 cell, p33-CAT, consisting of full-length p33 fused to the N terminus of CAT, localized exclusively to aggregated (globular) peroxisomes. These data for p33-CAT are consistent with the peroxisomal localization of wild-type p33 in BY-2 cells at the same time point (i.e., 4 h after bombardment; Figure 3A), indicating that the CAT moiety does not affect p33 sorting. Similar to p33-CAT, p33 1–156-CAT, consisting of residues 1 to 156 of the p33 N terminus, including the N-terminal hydrophilic domain, transmembrane domains (TMDs) 1 and 2, and their intervening hydrophilic loop sequence appended to CAT, localized to globular peroxisomes. CAT-p33 131–296, however, was cytosolic (data not shown), suggesting that, unlike the N-terminal half of p33, the C-terminal half of the protein lacks sufficient targeting information.

Figure 7.
Localization of p33-CAT Fusion Proteins in BY-2 Cells.

p33-CAT fusion constructs consisting of smaller portions of the p33 N-terminal 1 to 156 amino acid domain also targeted to peroxisomes, albeit in an inefficient manner (i.e., both p33 1–103-CAT and p33 104–156-CAT localized to the cytosol and only partially colocalized with endogenous catalase in globular and individual peroxisomes) (Figure 7). By contrast, p33 1–75-CAT, which includes most of the 82–amino acid N-terminal hydrophilic domain of p33, did not target to peroxisomes but instead targeted entirely to pER, as indicated by its colocalization with coexpressed GFP-APX, a fusion protein that, like CAT-APX (Figure 3B), includes the 36 C-terminal amino acids of APX and serves as a well-defined marker for pER while en route to peroxisomes (Lisenbee et al., 2003b). Smaller portions of the N-terminal 75 amino acid residues of p33 were not sufficient in sorting CAT to pER or to peroxisomes (data not shown).

To define precisely the regions within the N-terminal half (residues 1 to 156) of p33 responsible for its targeting to peroxisomes and pER, we examined amino acid sequences within the protein that resemble a prototypic membrane peroxisomal targeting signal (mPTS) present in most other PMPs (i.e., a stretch of several positively charged residues adjacent to at least one TMD) (reviewed in Trelease, 2002). We also focused on sequences of the p33 homologs from CymRSV and CNV that include the protein's two TMDs and adjacent hydrophilic domains and that were necessary for targeting to peroxisomes in yeast cells (Navarro et al., 2004; Panavas et al., 2005). Alignment of the deduced amino acid sequences for p33 from TBSV, CymRSV, and CNV (Figure 8A) revealed a high degree of identity, including two positively charged domains (residues 76 to 80 and 124 to 130; shaded in gray in Figure 8A) that resembled a prototypic mPTS and that were located within the sequences implicated in the sorting of CymRSV and CNV p33s to peroxisomes.

Figure 8.
Localization of Modified Versions of p33 in BY-2 Cells.

To test whether these two positively charged domains in TBSV p33 conferred necessary peroxisomal targeting information, all of the basic residues at each region (underlined, -KRRQR- and -RPSVPKK-) or at both regions were replaced with noncharged Gly residues, and the efficiency with which the resulting mutant proteins sorted to peroxisomes at 4 h after bombardment was compared with that of wild-type p33 (Figures 8B and 8C). Unlike wild-type p33, which localized only to (globular) peroxisomes at 4 h after bombardment (Figure 3A), p33-K76R77R78R80ΔG, p33-R124K129K130ΔG, and p33-K76R77R78R80R124K129K130ΔG localized to both globular peroxisomes (Figure 8C, a to f) and pER, as indicated by their partial colocalizations with endogenous catalase (Figure 8C, a to f), and coexpressed CAT-APX (data shown for p33-K76R77R78R80R124K129K130ΔG only; Figure 8C, g and h). These results indicate that although -K76R77R78R80- and -R124K129K130- in p33 are essential for its efficient sorting to peroxisomes, additional targeting information exists within the protein.

Because the N-terminal half of p33 is sufficient for sorting CAT to peroxisomes (p33 1–156-CAT; Figure 7), any peroxisomal targeting information in p33, in addition to regions 76 to 80 and 124 to 130, is likely located within this portion of the protein. Indeed, a further inspection of the p33 N terminus revealed two dibasic motifs (-K5R6- and -K11K12-; boxed in Figure 8A) that resembled nonprototypic mPTSs identified previously in the N termini of Arabidopsis thaliana and mammalian PMP22s (Brosius et al., 2002; Murphy et al., 2003). These PMP22 mPTSs are considered unique from the more common basic cluster mPTS because they consist of a smaller stretch of positively charged residues and are not immediately adjacent to a TMD. Substitutions at both of these dibasic motifs in p33 with Gly residues resulted in the mutant protein (p33-K5R6K11K12ΔG) being only partially colocalized with endogenous peroxisomal catalase (Figure 8C, i and j), indicating that this region is essential for efficiently sorting p33 to peroxisomes. Notably, p33-K5R6K11K12ΔG, unlike the other p33 mutants described above, also partially localized to the cytosol, rather than to pER (i.e., p33-K5R6K11K12ΔG did not colocalize with coexpressed CAT-APX in pER) (see Supplemental Figure 2 online), suggesting that this region also contains (overlapping) targeting information necessary for sorting p33 to pER.

The partial targeting of p33-K5R6K11K12ΔG to peroxisomes was not unexpected, because at least two other peroxisomal targeting elements within the protein (i.e., -KRRQR- and -RPSVPKK-) were left intact. Partial localization of p33 to peroxisomes was also observed when either of these two regions was mutated in the context of p33-K5R6K11K12ΔG (i.e., p33-K5R6K11K12K76R77R78R80ΔG and p33-K5R6K11K12R124K129K130ΔG both localized to peroxisomes and the cytosol; data not shown). Only when all three of the regions essential for efficient peroxisomal targeting of p33 were altered was the resulting mutant protein (p33-K5R6K11K12K76R77R78R80R124K129K130ΔG) no longer targeted to peroxisomes but instead accumulated entirely in the cytosol (Figure 8B, k and l). Similar results were observed when the same mutations were introduced into p92 (data not shown), indicating that both protein components of the TBSV replication complex were targeted to peroxisomes in an identical manner.

We further investigated the putative pER targeting signal within the -K5R6K11K12- region of p33 by replacing each pair of basic residues with Gly residues (underlined, -KRMIWPKK-). Similar to p33-K5R6K11K12ΔG, p33-K11K12ΔG localized to peroxisomes and pER (Figure 8C, m to p), but p33-K5R6ΔG localized to peroxisomes and cytosol (Figure 8C, q to t). Similar substitutions of -K5R6- with Gly residues in the context of the pER-localized fusion protein p33 1–75-CAT (Figure 7) also resulted in the mutant (p33 1–75K5R6ΔG-CAT) being mislocalized entirely to the cytosol (data not shown). Together, these data are consistent with the -K5R6- region of p33 being essential for sorting to pER.

Disruption of p33 Targeting Signals Negatively Affects the Replication of TBSV-Defective Interfering RNAs

The identification of distinct targeting sequences within p33 prompted us to investigate whether or not p33's function in the replication of viral RNAs was impaired when it was mislocalized within the cell. Toward this end, in vivo trans-complementation assays (Oster et al., 1998; Fabian and White, 2004) were used to quantify viral RNA accumulation after infection under conditions in which either p33 only was mutated or both p33 and p92 were mutated.

Figure 9A shows the results of an analysis of viral RNA replication with a wild-type p92 and different mutant versions of p33 containing Gly substitutions in either one or combinations of two or all three of the regions necessary for efficient peroxisomal and/or pER targeting (Figure 8) (i.e., -K5R6K11K12-, -K76R77R78R80-, and -R124K129K130-). Viral RNA replication was assessed by detection and quantification of the accumulation of a small viral RNA replicon, DI-72, which requires both p33 and p92 for its replication (White and Morris, 1994). Compared with cells expressing wild-type p33, those producing mutated forms of p33 with modifications in one or more of the three targeting regions showed progressively reduced DI-72 RNA levels (Figures 9A and 9C). Indeed, virtually no DI-72 RNA was detected in cells expressing the triple mutant p33-K5R6K11K12K76R77R78R80R124K129K130ΔG.

Figure 9.
RNA Gel Blot Analysis of Progeny Viral RNAs Isolated from Cucumber Protoplasts Inoculated with Various Mutant p33 RNA Transcripts.

In Figure 9B, the replication of DI-72 was investigated under conditions in which both p33 and p92 contained the targeting mutations described above. With the exception of mutant p33/p92-K76R77R78R80ΔG, the p33/p92 mutants showed similar or more pronounced decreases in DI-72 RNA accumulation levels than were observed for the p33 mutants (Figure 9C). Moreover, DI-72 RNAs accumulated least in all double mutants, the triple mutant, and the corresponding single p33 mutant that possessed alterations within the region responsible for pER targeting (i.e., -K5R6K11K12-). These results indicate that robust viral RNA replication requires proper peroxisome and/or pER targeting, and they are consistent with the concept that such targeting contributes to the proper assembly of the viral RNA replication complex.

DISCUSSION

For viruses to successfully replicate within host cells, they must manipulate and coordinate a variety of complex cellular pathways. As a result, viral infection often leads to an enhancement of events within the cell that are otherwise intractable to study; thus, viruses and individual viral proteins can serve as unique tools to investigate these cellular processes. Therefore, we used TBSV as a means to investigate peroxisome biogenesis, a little understood event, specifically by focusing on the replication protein p33, which was demonstrated previously to be essential for pMVB formation (Burgyan et al., 1996; Rubino and Russo, 1998). Our findings support the notion that p33 successfully appropriates different peroxisomal protein sorting pathways to facilitate viral replication, including a previously unknown peroxisome-to-pER sorting pathway.

p33 Causes Vesiculation of the Peroxisomal Boundary Membrane

TBSV p33 expressed on its own caused the pronounced accumulation of novel peroxisome-associated cytosolic vesicles (Figure 5). These vesicles contained p33 (Figure 6), were dispersed among populations of aggregated peroxisomes, and most likely arose from the peroxisomal boundary membrane, as also suggested for the novel vesicles observed in plant cells transformed with CymRSV p33 (Bleve-Zacheo et al., 1997). Because peroxisomes normally undergo outward vesiculation or budding, albeit at a low frequency (Jedd and Chua, 2002), it appears that p33 usurps the host cell molecular machinery needed to accomplish peroxisome budding. However, the identity of these host cell components and how they contribute to peroxisome vesiculation remain to be determined.

The outward vesiculation of the peroxisomal boundary membrane caused by p33 expressed on its own provides a reasonable explanation for the relocalization of this viral protein, and of host cell PMPs, from peroxisomes to pER (Figure 3B). The redistribution of APX and PMP22 (along with p33) from their steady state localization in peroxisomes (Mullen et al., 1999; Murphy et al., 2003) to pER is likely attributable to their incorporation into the peroxisome-derived vesicles that, after release into the cytosol, traffic to and fuse with pER. The outward vesiculation of peroxisomal membranes may also explain the formation of peroxisomal membrane remnants or ghosts in p33-transformed cells (Figure 3B); vesiculation may compromise the organelle's integrity such that the endogenous matrix constituents (e.g., catalase and isocitrate lyase) are either degraded and/or diluted in the cytosol to below immunodetection. Indeed, transient expression of a peroxisomal matrix marker protein (RFP-SKL) in p33-cotransformed BY-2 cells resulted in the marker protein being completely mislocalized to the cytosol at later time points (e.g., 48 h; data not shown), suggesting that peroxisomal matrix constituents, unlike p33 and host cell PMPs, are not incorporated into peroxisome-derived vesicles destined for pER.

p33 Uses a Peroxisome-to-pER Sorting Pathway in Plants

All plant PMPs examined seem to be synthesized in the cytosol on free polyribosomes and sorted either directly to peroxisomes or indirectly to peroxisomes via pER and pER-derived vesicles (reviewed in Trelease and Lingard, 2005). One of the most remarkable findings of this study is that p33 sorted initially from the cytosol to peroxisomes and then to pER, presumably via peroxisome-derived vesicles. Although one could argue that the peroxisome-to-pER sorting of p33 was the result of protein overexpression, results from several sources suggest otherwise. For instance, in contrast with APX, which at early time points after biolistic bombardment of BY-2 cells is readily detectable in pER while en route to peroxisomes (Mullen et al., 1999) (Figure 4), p33 was not detectable in pER at similar times, even when cells were cold-treated to block ER (pER) protein transport (Figures 3A and and4C).4C). p33 also did not insert into purified microsomal membranes in vitro (data not shown), whereas APX does so in an efficient manner (Mullen et al., 1999), indicating that pER is not an authentic sorting site for newly synthesized p33 in the cytosol. Furthermore, experiments with cycloheximide (Figure 4A) and a dominant-negative mutant of Arf1 (Arf1Q71L) (Figure 4B) yielded the most convincing evidence that p33 localized in peroxisomes is eventually sorted to pER.

Although our conclusion for peroxisome-to-pER sorting of TBSV p33 is different from that drawn by Navarro et al. (2004) for the sorting of CymRSV p33, these two proteins probably use similar intracellular sorting pathways. Navarro and coworkers (2004) reported that CymRSV p33 localized exclusively to peroxisomes when expressed on its own in wild-type S. cerevisiae but was localized to ER in a yeast mutant lacking peroxisomes as a result of a disruption in the gene encoding the PMP receptor/chaperone Pex19p. The authors suggested that CymRSV p33 accumulated in ER in Pex19 mutant cells either because ER served as a preperoxisome sorting site or because, in the absence of peroxisomes, sorting to ER occurred simply by default, as is commonly observed for yeast PMPs (Hettema et al., 2000). We favor the latter explanation and also propose that differences observed in sorting p33 in plant and yeast cells are attributable to differences in the biogenetic relationship between ER and peroxisomes in these two organisms (reviewed in Trelease and Lingard, 2005). Thus, CymRSV p33 might not target to ER from peroxisomes in yeast simply because yeast do not possess this sorting pathway or because the plant viral protein cannot use the yeast variant of this pathway.

Properties of the p33 Peroxisomal Membrane Targeting Signal

A comprehensive analysis of the targeting information in p33 revealed that at least three distinct regions within the N-terminal half of the protein (i.e., -K11K12-, -K76R77R78R80-, and -R124K129K130-) constitute the mPTS and were necessary for its initial sorting from sites of syntheses in the cytosol to peroxisomes (Figures 7 and and8).8). One of these regions, -K76R77R78R80-, along with a C-terminal region (-R213R214R215R216-), were previously proposed to be the targeting elements responsible for the sorting of CymRSV p33 to peroxisomes in yeast (Navarro et al., 2004). However, neither of these regions was demonstrated experimentally to be necessary and sufficient for the peroxisomal targeting of full-length CymRSV p33, although substitution of the -R213R214R215R216- with Ala residues prevented peroxisomal targeting of a minimal CymRSV p33-GFP fusion protein (Navarro et al., 2004). In our study, gain-of-function and loss-of-function experiments demonstrated that the C-terminal half of TBSV p33 possessed no peroxisomal targeting information (Figure 7). Although the relative position (and number) of targeting elements within TBSV and CymRSV p33 appear to be inconsistent, these types of mutagenic studies must be interpreted carefully. Aberrant protein folding and/or function, rather than disruptions in key components of peroxisomal targeting signals, can result in subcellular mislocation. This caveat is especially relevant with respect to the putative C-terminal targeting element within CymRSV p33 (Navarro et al., 2004), as this and adjacent regions of the protein are likely involved in RNA binding (Panaviene et al., 2003) and p33–p33/p92 protein–protein interactions (Rajendran and Nagy, 2004).

The mPTS of p33, like most mPTSs, is relatively long, consisting of the N-terminal 156 amino acid residues, including the three proposed targeting regions and both putative TMDs. All of the p33-CAT fusion proteins that contained shorter fragments of the p33 N terminus were either not sorted to peroxisomes or inefficiently sorted to peroxisomes (i.e., partially mislocalized to the cytosol). Furthermore, disruptions to any of the individual targeting regions within the mPTS resulted in p33 being partially mislocalized to the cytosol or pER (Figures 8B and 8C) as well as inactive in terms of its replicase function (Figure 9). Thus, the three targeting regions and two TMDs within the mPTS appear to cooperate to ensure the fidelity of the complex multistep process involved in the correct targeting, membrane integration/assembly, and functionality of p33, as proposed previously for the role of multiple targeting elements in the biogenesis of other PMPs (Jones et al., 2001; Wang et al., 2001; Murphy et al., 2003).

p33 Possesses a pER Targeting Signal That Resembles an Arg-Based Motif Responsible for the COPI-Dependent Retrieval of Resident ER Membrane Proteins

In addition to a mPTS, p33 possesses a pER targeting signal consisting, in part, of a pair of N-terminal positively charged residues (-K5R6-). Extensive mutational analysis revealed at least two important functional properties of the p33 pER targeting signal. First, the signal overlaps with the N-terminal peroxisome targeting element whose core component includes the -K11K12- region, as sorting of p33-K5R6ΔG to peroxisomes was inefficient (Figures 8B and 8C). Second, the pER targeting signal functions from either the cytosol or peroxisomes, but it does so in a context-dependent manner. For instance, in the case of p33 1–75-CAT, the pER targeting signal was sufficient for sorting to pER directly from the cytosol, whereas in the context of full-length p33, the protein was first sorted to peroxisomes (via the mPTS) before the signal could redirect it to the pER. This also explains why mutations to any one or more of the three peroxisomal targeting elements in p33 resulted in the modified protein(s) being partially localized to pER after only 4 h of expression (Figure 8), yet wild-type p33 localized exclusively to peroxisomes at the same time point; these mutations diminish the overall efficiency of the mPTS and thereby allow the pER targeting signal to function outside its normal context and mediate sorting directly from the cytosol to pER. Such a contextual dependence of the pER targeting signal makes it difficult to elucidate how it functions in an integrative manner with the protein's mPTS, as noted previously for the overlapping pER (ER) targeting signal and mPTS in APX (Mullen and Trelease, 2000) and Pex15p (Elgersma et al., 1997).

To date, several targeting signals responsible for the localization of membrane proteins to the ER have been identified, including cytosolically exposed C-terminal dilysines and aromatic amino acid–enriched motifs and N-terminal or internally positioned Arg residues (Pelham, 2000). In each case, these targeting signals function in the retrieval of membrane proteins from the Golgi back to the ER by serving as recognition sites for specific cytosolic receptor factors, including COPI (Aniento et al., 2003). Because a vesicle transport pathway is most likely responsible for sorting p33 from peroxisomes to pER, the p33 pER targeting signal might function in a manner similar to those involved in COPI-directed transport. There are several striking similarities between the properties of the -K5R6- region in p33 and the Arg-based ER retrieval motif, a position-independent signal with a flexible consensus sequence (e.g., -R-R/K- and -R/K-X-R-) that functions in a COPI-dependent manner in evolutionarily diverse organisms (Schutze et al., 1994; Zerangue et al., 2001; Yuan et al., 2003). Consistent with these observations, we showed that replacement of -K5- with -R- in p33, yielding a prototypic diarginine ER retrieval motif, preserved its sorting to pER (see Supplemental Figure 3A online). Furthermore, substitution of the N terminus of Arabidopsis α-glucosidase I, a type II membrane protein that possessed a diarginine ER retrieval motif (Gillmor et al., 2002), with the N terminus of p33, including the -K5R6- region, preserved the ER localization of an α-glucosidase I–GFP fusion protein (see Supplemental Figures 3B and 3C online). COPI also appears to be involved in the sorting of p33 from peroxisomes to pER, because Arf1Q71L caused coexpressed p33 to accumulate exclusively in (aggregated) peroxisomes (Figure 4B). These results suggest that the Arf1 mutation prevented the outward vesiculation of the peroxisomal boundary membrane, as expected based on the established role of this protein in Golgi-derived vesicle budding (Aniento et al., 2003).

The implication that Arf1 and COPI are involved in peroxisome vesiculation and peroxisome-to-pER sorting of p33 is not without precedent. Rat Pex11p contains a C-terminal dilysine ER retrieval motif that can bind COPI, and although overexpression of Pex11p causes a proliferation of peroxisomes, cells with defects in the coatomer vesicle coat possess peroxisomes with pronounced morphological changes (e.g., tubulation and elongation), consistent with impairment in peroxisome vesiculation (Passreiter et al., 1998). These data led the authors to propose a dynamic model for the role of Pex11p and COPI in peroxisomal vesiculation in which the recruitment of COPI to the peroxisome membrane is mediated by Pex11p and probably other peroxisomal components (Anton et al., 2000). Nascent peroxisome-derived vesicles either form into new mature peroxisomes or function in the return of ER resident proteins and/or peroxisomal substrates that previously escaped from the ER via ER-derived vesicles.

Modifications and extensions of the Anton et al. (2000) model are necessary to incorporate new and previous data on the possible roles of p33 during pMVB formation. Nascent p33 in TBSV-infected cells is targeted directly from the cytosol to peroxisome membranes (via a mPTS), where it is assembled along with p92 (Rajendran and Nagy, 2004) and viral RNA (Panaviene et al., 2003) to form a functional replication complex. This interaction of p33 and p92, and possibly other viral and/or host cell factors, leads to the inward vesiculation of the peroxisomal boundary membrane during the initial stages of pMVB biogenesis. Because the amount of p33 in infected cells is ~20-fold higher than that of p92 (K.B. Scholthof et al., 1995), excess p33 could also function in a manner similar to that proposed for Pex11p, mediating COPI-dependent budding of the peroxisomal boundary membrane. The resulting peroxisome-derived vesicles containing p33 (and other peroxisomal substrates such as early peroxins that function at steady state in the ER [e.g., Arabidopsis Pex10p; Flynn et al., 2005]) would then be transported to pER, where, by some unknown means, the viral protein would modulate the trafficking (via pER-derived vesicles) of additional membrane constituents required for the increase in surface area during pMVB biogenesis. Although p33 did not accumulate in pER in TBSV-infected cells under steady state conditions (Figure 2B), disruption of the pER targeting signal in p33 (and p92) in the context of the full-length TBSV genome resulted in the virus being inactive in terms of its replicase function (Figures 9B and 9C). Thus, the sorting of p33 to pER appears to be essential for viral replication, a premise consistent with electron micrographs of TBSV-infected plant cells showing pMVBs frequently connected to ER strands that were previously suggested to be involved in pMVB biogenesis (Martelli et al., 1988) (Figure 5C). It is possible that, similar to mammalian Pex11p (Passreiter et al., 1998), only a small amount of p33 is sorted from peroxisomes to pER and/or that p33 is readily incorporated into nascent pER-derived vesicles and, thus, localized mostly at steady state in pMVBs in TBSV-infected cells.

Although this is a hypothetical model, the mechanism is appealing because it incorporates pertinent research results and interpretations of the current working models for the formation of pMVBs and peroxisome biogenetic pathways in diverse organisms, particularly in plants. Properly conceived and executed experiments are now needed to help resolve the role(s) of p33 in the context of the wild-type virus and to provide direct evidence for the intracellular trafficking of this viral protein and how it functions with, or is modulated by, other proteins in TBSV-infected cells to control distinct membrane vesiculation events at peroxisomes/pMVBs and pER, in addition to its role as an essential component of the viral RNA replication complex. These types of studies will clearly have an impact on our understanding of the complex means by which p33 along with other TBSV proteins modify their cellular environment to facilitate viral replication. Meanwhile, this model not only serves as a basis on which to analyze unexplored aspects of the cellular processes underlying TBSV replication, it also reinforces the notion of how viruses can serve as a useful tool for studying new avenues of peroxisome biogenesis, including the biogenetic link between ER and peroxisomes.

METHODS

Recombinant DNA Procedures and Reagents

Standard recombinant DNA procedures were performed as described by Sambrook et al. (1989). Restriction enzymes and other DNA-modifying enzymes were purchased from New England Biolabs, and custom synthetic oligonucleotides were obtained from either Invitrogen Life Technologies, Sigma-Aldrich, or the University of Guelph Laboratory Services. PCR site-directed mutagenesis of plasmid DNA was performed using a GeneAmp PCR system 2400 programmable thermal controller (Perkin-Elmer Canada) and the QuikChange site-directed mutagenesis kit according to the manufacturer's recommendations (Stratagene). Isolation of DNA fragments and plasmids was performed using Qiagen reagents. All DNA constructs were confirmed by fluorescent dye-terminator cycle sequencing at the University of Guelph Molecular Supercentre using an Applied Biosystems Prism 377 automated sequencer.

Construction of Plasmids

A complete description of all plasmids used in this study and a list of the sequences of oligonucleotide primers used in plasmid constructions are available in the online version of this article (see supplemental protocols).

Tobacco BY-2 Cell Cultures, Microprojectile Bombardment, and Immunofluorescence Microscopy

Tobacco (Nicotiana tabacum) BY-2 suspension-cultured cells were maintained and prepared for biolistic bombardment as described previously (Banjoko and Trelease, 1995). Transient transformations were conducted using 10 μg of plasmid DNA (or 5 μg of each plasmid DNA for cotransformations) and a PDS1000 biolistic particle delivery system (Bio-Rad Laboratories). After bombardment, BY-2 cells were incubated at either 25 or 14°C (cold shock) in the dark for 2 to 48 h to allow for expression of the introduced gene(s) and intracellular protein sorting. Bombarded cells were then fixed in 4% (w/v) formaldehyde, incubated with 0.1% (w/v) pectolyase Y-23 (ICN Canada), and permeabilized with 0.3% (v/v) Triton X-100 (Sigma-Aldrich) (Mullen et al., 2001). Cycloheximide (Sigma-Aldrich) was dissolved in BY-2 transformation buffer (Banjoko and Trelease, 1995) to a final concentration of 100 μM as described previously (Imanishi et al., 1998). Briefly, cells at 4 h after bombardment were scraped from filter papers, resuspended in buffer plus or minus cycloheximide, spread on filter papers, and incubated for an additional 8 h before fixation.

Fixed and permeabilized BY-2 cells were processed for immunofluorescence microscopy as described by Trelease et al. (1996). Primary and dye-conjugated secondary antibodies and sources were as follows: mouse anti-myc (clone 9E10) and rabbit anti-myc IgGs (Berkeley Antibody Company); mouse anti-α-tubulin IgGs (clone DM 1A) and rabbit anti-CAT IgGs (Sigma-Aldrich); mouse anti-CAT hybridoma medium (provided by S. Subramani, University of California, San Diego); rabbit anti-cottonseed catalase IgGs (provided by Richard Trelease, Arizona State University; Kunce et al., 1988); rabbit anti-cottonseed APX IgGs (provided by R. Trelease; Corpas et al., 1994); mouse monoclonal anti-tobacco catalase hybridoma medium (Princeton University Monoclonal Antibody Facility; Chen et al., 1993); mouse anti-maize β-ATPase (provided by Tom Elthon, University of Nebraska; Luethy et al., 1993); mouse anti-dsRNA hybridoma medium (clone K2) (provided by Wolfgang Nellen, Kassel University; Schönborn et al., 1991; Bonin et al., 2000); goat anti-mouse and goat anti-rabbit Alexa 488 IgGs (Molecular Probes); and goat anti-mouse Cy3 IgGs and goat anti-rabbit rhodamine red-X IgGs (Jackson ImmunoResearch Laboratories). Rabbit anti-p33 IgGs raised against a synthetic peptide corresponding to the p33 amino acid sequence VEPARELKGKDGEDLLTGSR (residues 184 to 203) and purified using a p33 peptide-Sepharose–linked column were generated by Bethyl Laboratories. For staining ER in BY-2 cells that were also labeled with antibodies, 5 μL of a 1 mg/mL stock solution of concanavalin A conjugated to Alexa 594 or Alexa 394 (Molecular Probes) was added to the cells during the final 20 min of a 60 min incubation with the secondary antibodies.

Fluorescent images of labeled BY-2 cells were acquired using an Axioskop 2 MOT epifluorescence microscope (Carl Zeiss) equipped with a Zeiss 63X Plan Apochromet oil-immersion objective and a Retiga 1300 charged-couple device camera (Qimaging). Images were deconvolved and/or adjusted for brightness and contrast (Northern Eclipse 7.0; Empix Imaging) and then composed into figures using Adobe Photoshop 8.0. All fluorescent images of cells shown in individual figures are representative of >50 transformed cells from at least two independent transformation experiments.

Rub-Inoculation and Agrobacterium Infiltration of Tobacco Leaves

Nicotiana benthamiana plants stably transformed with pMAT037/GFP-SKL were grown in chambers at 21°C with a 12-h-light/12-h-dark cycle, and 5 d before rub-inoculation plants were transferred to a laboratory bench top with lower light conditions to facilitate the infection process (Scholthof, 1999). Rub-inoculations (including mock inoculations) were performed with six- to eight-leaf-stage plants and 5 μg of TBSV RNA transcripts diluted in 30 μL of RNA inoculation buffer (Scholthof, 1999). Approximately 5 to 8 d after inoculations, tissues from systemically infected upper leaves (Burgyan et al., 1996) were processed for electron microscopy.

For transformations of tobacco cv Xanthi using Agrobacterium tumefaciens infiltration, the binary expression plasmids pMAT037/p33-GFP, pMAT037/p33, pMAT037/GFP-SKL, and pMAT037 were introduced independently into Agrobacterium (strain LBA4404) using the freeze-thaw method of Höfgen and Willmitzer (1988). Transformed Agrobacterium were grown overnight (Höfgen and Willmitzer, 1988), resuspended in 10 mM MgCl2 and 40 μM acetosyringone (Sigma-Aldrich), and then infiltrated into the upper epidermis of six- to eight-leaf-stage tobacco plants using a 10-cc syringe as described previously (Grimsley, 1995). Infiltrated tobacco plants were incubated for 2 or 3 d at the same growth conditions as those used before the transformation procedure (i.e., 21°C with a 12-h-light/12-h-dark cycle). Approximately 1-cm2 peels of the lower epidermis of Agrobacterium-infiltrated leaf tissue or 1-cm2 pieces of whole leaf tissue infiltrated with Agrobacterium were examined using an epifluorescence microscope or processed for electron microscopy, respectively.

Electron Microscopy and Immunogold Labeling

TBSV RNA-infiltrated N. benthamiana leaves and Agrobacterium-infiltrated tobacco leaves were processed for transmission electron microscopy by fixing tissue pieces in 4% (v/v) glutaraldehyde (Fisher Scientific) and 1% (v/v) acrolein (Sigma-Aldrich) in 0.05 M KPO4 buffer, pH 7.2, for 2 h in a vacuum chamber and then at 4°C overnight. Samples were then postfixed overnight at 4°C in 1% (w/v) osmium tetroxide (Fisher Scientific), dehydrated in a graded series of ethanol (50 to 100% [v/v]), and embedded in Spurr's medium-grade epoxy (Spurr, 1969). Thin sections were cut with a glass or diamond knife, mounted on copper grids, and poststained with 2% (w/v) uranyl acetate and Reynold's lead citrate (Venable and Coggeshall, 1965) for 10 and 3 min, respectively. All images were acquired at 80 kV with a Phillips CM10 transmission electron microscope.

Tobacco leaf material was processed for transmission electron microscopy immunogold labeling as described above except that postfixation in osmium tetroxide was omitted and thin sections were mounted onto nickel grids. Sections were pretreated with saturated sodium meta-periodate followed by incubation in a blocking buffer consisting of 3× PBS, 0.05% (w/v) Gly, 0.05% (v/v) Tween 20 (Fisher Scientific), and 0.5% (w/v) BSA. After incubation in blocking buffer, samples were incubated for 30 min in primary antibodies (rabbit anti-p33 IgGs or preimmune sera) diluted in blocking buffer, washed a second time in blocking buffer, and then incubated for 30 min with secondary antibodies (goat anti-rabbit IgGs conjugated to 5-nm gold particles; Sigma-Aldrich) also diluted in blocking buffer. Sections were then washed in 1× PBS, washed with water, air-dried, stained with uranyl acetate (Greenwood et al., 2005), and viewed using a Phillips CM10 transmission electron microscope.

Isolation and Inoculation of BY-2 and Cucumber Protoplasts

Protoplasts prepared from either 3-d-old BY-2 cells (Komoda et al., 2004) or 6- to 8-d-old cucumber (Cucumis sativus cv Straight 8) cotyledons (Jones et al., 1990) were quantified by bright-field microscopy using a hemocytometer. Purified protoplasts were electroporated with 5 μg of viral RNA transcripts as described previously (White and Morris, 1994). All TBSV or TBSV-derived transcripts described in Figure 9 were generated in vitro via transcription of SmaI-linearized template DNAs and using the Ampliscribe T7 RNA polymerase transcription kit (Epicentre Technologies) (Oster et al., 1998). Isolated transcripts were quantified spectrophotometrically and their integrity verified by agarose gel electrophoresis.

After electroporation, BY-2 protoplasts (~1.0 × 106) were pelleted by centrifugation (500g for 5 min), resuspended in 1 mL of BY-2 culture medium (Banjoko and Trelease, 1995), and incubated overnight at 26°C in the dark. The next day, protoplasts were centrifuged as described above and resuspended by gentle pipetting in an equal volume of 2× SDS-PAGE loading buffer. Aliquots of total protein were separated on 12% SDS-polyacrylamide gels and electroblotted onto Hybond nitrocellulose (Amersham Biosciences). Membranes were incubated with anti-p33/p92 antiserum (1:2000) and goat anti-rabbit antiserum conjugated to horseradish peroxidase (1:10,000), and immunoreactive proteins were visualized using a Western Lighting kit (Perkin-Elmer) along with a Molecular Dynamics Storm PhosphorImager (Amersham Biosciences).

Cucumber protoplasts (~3 × 105) after electroporation were incubated in a growth chamber under fluorescent lighting at 22°C for 24 h and processed for RNA gel blot analysis as described below.

RNA Gel Blot Analysis of Viral RNAs

RNA was extracted from total nucleic acids harvested from cucumber protoplasts 24 h after inoculation as described previously (White and Morris, 1994). Aliquots of isolated RNA were separated in nondenaturing 1.4% (w/v) agarose gels, and viral RNAs were detected by electrophoretic transfer to nylon (Hybond-N; Amersham Biosciences) followed by incubation with a 32P-end-labeled P9 oligonucleotide probe complementary to the 3′ terminal 23 nucleotides of the TBSV genome (White and Morris, 1994). Labeled RNAs were visualized and quantified using a phosphor imager (Amersham Biosciences).

Accession Numbers

GenBank/EMBL accession numbers for TBSV proteins, including p33 and p92, as well as other proteins described in this study are as follows: TBSV p33 (NP_062898), TBSV p92 (NP_062897), CymRSV p33 (NP_613261), CNV p33 (AAA42903), Arabidopsis PMP22 (NP_192356), cottonseed APX (T09845), Arabidopsis Golgi GDP mannose transporter (NP_178987), pea glutathione reductase (CAA62482), Arabidopsis BS14a (NP_191376), Arabidopsis α-glucosidase I (NP_176916), and Arabidopsis Arf1 (AAA32729).

Supplemental Data

The following materials are available in the online version of this article.

  • Supplemental Figure 1. Localization of GFP-BS14a in Cycloheximide-Treated Tobacco BY-2 Cells.
  • Supplemental Figure 2. p33-K5R6K11K12ΔG Colocalizes with CAT-APX in Aggregated Peroxisomes, but Not in pER, in Tobacco BY-2 Cells.
  • Supplemental Figure 3. p33 Contains an Arg-Based ER Retrieval Motif.

Supplementary Material

[Supplemental Data]

Acknowledgments

We thank our colleagues for their generous gifts of antibodies and plasmids used in this study. We also thank Robb Flynn and Brian Ellis for providing N. benthamiana and N. tabacum seeds, respectively, Rosa Di Leo for constructing pRTL2/X-myc, Chris Trobacher for constructing pRTL2/RFP-HDEL, and Preetinder Dhanoa for constructing monomeric GFP cassette vectors and pRTL2/GFP-BS14a and for guidance in experiments involving BS14a. We are also grateful to Kelly Wakeling for constructing several p33-CAT plasmids, Kimberley Gibson for assistance with sectioning material used for electron microscopy, Ian Smith for his guidance with Adobe Photoshop, and Baozhong Meng, Annette Nassuth, John Dyer, Peter Kim, Derek Bewley, and members of our laboratories for their helpful discussions during the course of this work and for their comments during the preparation of the manuscript. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) to J.S.G., K.A.W., and R.T.M. K.A.W. holds a Tier II Canada Research Chair, A.W.M. was supported by an Ontario Graduate Scholarship, M.R.F. was supported by an NSERC Postgraduate Scholarship, and K.A.W. and R.T.M. are recipients of Ontario Premier's Research in Excellence Awards.

Notes

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Robert T. Mullen (ac.hpleugou@nellumtr).

W in BoxOnline version contains Web-only data.

Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.105.036350.

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