Control of Tobacco mosaic virus Movement Protein ... - Plant Physiology

Control of Tobacco mosaic virus Movement Protein Fate by CELL-DIVISION-CYCLE Protein481[W][OA] Annette Niehl, Khalid Amari, Dalya Gereige 2, Katrin Brandner, Yves Mély, and Manfred Heinlein* Institut de Biologie Moléculaire des Plantes, Unité Propre de Recherche 2357 Centre National de la Recherche Scientifique, Université de Strasbourg, 67000 Strasbourg, France (A.N., K.A., D.G., K.B., M.H.); Botanisches Institut der Universität Basel, 4056 Basel, Switzerland (A.N., K.A., M.H.); and Laboratoire de Biophotonique et Pharmacologie, Unité Mixte de Recherche 7213 Centre National de la Recherche Scientifique, Université de Strasbourg, Faculté de Pharmacie, 67401 Illkirch, France (Y.M.)

Like many other viruses, Tobacco mosaic virus replicates in association with the endoplasmic reticulum (ER) and exploits this membrane network for intercellular spread through plasmodesmata (PD), a process depending on virus-encoded movement protein (MP). The movement process involves interactions of MP with the ER and the cytoskeleton as well as its targeting to PD. Later in the infection cycle, the MP further accumulates and localizes to ER-associated inclusions, the viral factories, and along microtubules before it is finally degraded. Although these patterns of MP accumulation have been described in great detail, the underlying mechanisms that control MP fate and function during infection are not known. Here, we identify CELL-DIVISIONCYCLE protein48 (CDC48), a conserved chaperone controlling protein fate in yeast (Saccharomyces cerevisiae) and animal cells by extracting protein substrates from membranes or complexes, as a cellular factor regulating MP accumulation patterns in plant cells. We demonstrate that Arabidopsis (Arabidopsis thaliana) CDC48 is induced upon infection, interacts with MP in ER inclusions dependent on the MP N terminus, and promotes degradation of the protein. We further provide evidence that CDC48 extracts MP from ER inclusions to the cytosol, where it subsequently accumulates on and stabilizes microtubules. We show that virus movement is impaired upon overexpression of CDC48, suggesting that CDC48 further functions in controlling virus movement by removal of MP from the ER transport pathway and by promoting interference of MP with microtubule dynamics. CDC48 acts also in response to other proteins expressed in the ER, thus suggesting a general role of CDC48 in ER membrane maintenance upon ER stress.

Plant viruses are obligate intracellular pathogens that replicate in association with host membranes (Laliberté and Sanfaçon, 2010) and subvert host intraand intercellular trafficking pathways to achieve cellto-cell and systemic spread (Harries and Ding, 2011; Niehl and Heinlein, 2011). In the case of the wellstudied Tobacco mosaic virus (TMV), viral replication factories form on membranes of the endoplasmic reticulum (ER; Heinlein et al., 1995, 1998). As the plant 1 This work was supported by grants from the Human Frontier Science Program Organization (RGP0022/2006) and the Swiss National Science Foundation (3100A0–111916 and 31003A–124940) and by a postdoctoral fellowship of the German Academic Exchange Service to K.B. 2 Present address: Unité Mixte de Recherche, Santé de la Vigne et Qualité du Vin, Institut National de la Recherche AgronomiqueUniversité de Strasbourg, 28 rue de Herrlisheim, 68021 Colmar cedex, France. * Corresponding author; e-mail manfred.heinlein@ibmp-cnrs. unistra.fr. 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.plantphysiol.org) is: Manfred Heinlein ([email protected]). [W] The online version of this article contains Web-only data. [OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.112.207399

ER is continuous between cells through plasmodesmata (PD; Ding et al., 1992), this membrane network provides a direct pathway for the spread of replicated virus from the replication sites in infected cells into the ER network of noninfected cells. The spread of plant viruses depends on virus-encoded movement proteins (MPs; Deom et al., 1987; Lucas, 2006). The MP of TMV facilitates the cell-to-cell passage of the infectious particle by forming a ribonucleoprotein complex with the viral RNA (Citovsky et al., 1990) and by increasing the size exclusion limit of PD (Wolf et al., 1989). During the course of infection, as well as when ectopically expressed, the MP associates with PD, the ER/actin network, and microtubules (Heinlein et al., 1995, 1998; Reichel and Beachy, 1998; Wright et al., 2007; Sambade et al., 2008; Hofmann et al., 2009; Boutant et al., 2010; Peña and Heinlein, 2012; Supplemental Fig. S1). Shortly after infection of a new cell, the MP localizes to small, mobile, ER-associated particles proposed to play a role in PD targeting of the viral RNA (Boyko et al., 2007; Sambade et al., 2008). Similar small, mobile MP particles are observed early upon ectopic expression of the protein. These particles colocalize with RNA and undergo stopand-go movements in association with the ER (Sambade et al., 2008). The particle movements pause at microtubule proximal sites and their detachment requires microtubule polymerization (Sambade et al.,

Plant PhysiologyÒ, December 2012, Vol. 160, pp. 2093–2108, www.plantphysiol.org Ó 2012 American Society of Plant Biologists. All Rights Reserved.

2093

Niehl et al.

2008). These observations suggest that the interaction with the microtubule system plays a critical role in the maturation and ER-mediated delivery of infectious viral RNA particles to PD during early infection stages. Consistently, tobacco (Nicotiana tabacum) mutants with reduced microtubule dynamics exhibit reduced TMV movement (Ouko et al., 2010). Following virus movement, the previously infected cell further accumulates MP at the ER, a process that coincides with the formation of large ER inclusions that contain viral replicase and viral RNA in addition to MP and likely function as virus factories (Heinlein et al., 1998; Más and Beachy, 1999). In mature form, these inclusions may represent the so-called viroplasms or X-bodies described in the classical literature (Bawden and Sheffield, 1939; Esau and Cronshaw, 1967; Hills et al., 1987). Their formation is associated with rearrangements of the ER membrane and likely mediated by the accumulated MP since the inclusions diminish and reconstitute a native ER structure when MP becomes degraded by the 26S proteasome (Reichel and Beachy, 1998, 2000). Transfected cells accumulate MP in similar inclusions as those formed during infection, indicating that accumulated MP is indeed necessary and sufficient to form inclusions in association with the ER (Reichel and Beachy, 1998; Supplemental Fig. S1). Following accumulation of MP in virus factories, the infected cells accumulate the MP also along microtubules (Heinlein et al., 1998). The accumulation of MP in virus factories and on microtubules in cells behind the leading front of infection is dispensable for virus movement (Heinlein et al., 1998; Boyko et al., 2000a). At these late infection stages, the virus factories may enable the virus to produce high virion titers (Laliberté and Sanfaçon, 2010; Tilsner et al., 2012), and the subsequent accumulation along microtubules may play a role in withdrawing MP from the cell-to-cell communication pathway (Curin et al., 2007) and in stockpiling MP prior to degradation (Padgett et al., 1996; Gillespie et al., 2002). The molecular mechanisms that guide the MP to the ER and subsequently to microtubules during infection are not known. The MP is a hydrophobic protein that behaves like a membrane-integral or tightly membraneassociated protein in differential fractionation experiments and contains two predicted transmembrane domains (Reichel and Beachy, 1998; Brill et al., 2000, 2004) involved in ER association (Fujiki et al., 2006). The association with microtubules depends on MP amino acids 1 to 213 required for MP function (Kahn et al., 1998; Boyko et al., 2000b, 2000c, 2002; Kotlizky et al., 2001). Moreover, certain amino acid exchange mutations known to affect the function of MP in virus movement in a temperature-sensitive manner also affect the ability of MP to interact with microtubules (Boyko et al., 2007, 2000b). Interestingly, these mutations cluster together in a short domain of 25 amino acids showing a structural similarity with the M-loop of tubulin involved in tubulin-tubulin interactions (Boyko et al., 2000b; Waigmann et al., 2007). Importantly, this M-loop similarity domain overlaps with the predicted 2094

transmembrane domain (Brill et al., 2000, 2004) thus suggesting that the association of MP with membranes or microtubules is an alternative event that may depend on specific posttranslational modifications or specific folds of MP. However, although the different subcellular localizations of MPs during the course of infection indicate directional transport of MP from the ER to microtubules and may indicate different folds and functions of the protein when associated with these different subcellular components, the mechanism that controls the subcellular localization and, thus, the fate and function of MP is not known. Here, we identify CELL-DIVISION-CYCLE protein48 (CDC48), named p97/VCP (Valosin-containing protein) in mammals and Cdc48p in yeast (Saccharomyces cerevisiae), as a cellular factor regulating MP subcellular accumulation patterns. CDC48 functions are well characterized in mammalian and yeast systems but remain poorly investigated in plants. Yeast and mammalian CDC48s are essential, conserved chaperones involved in diverse cellular processes by controlling protein fate through extraction of substrates from membranes or complexes (Tsai et al., 2002; Meusser et al., 2005; Römisch, 2005; Rumpf and Jentsch, 2006; Schrader et al., 2009; Eisele et al., 2010; Meyer et al., 2012; Yamanaka et al., 2012). We show that virus infection leads to the induction of Arabidopsis (Arabidopsis thaliana) CDC48 isoforms and demonstrate a function of CDC48 in ER maintenance upon ER stress conditions. We further demonstrate that CDC48 interacts with MP and that CDC48 activity is required for MP degradation. Interaction of CDC48 with MP depends on the MP N terminus, which is required for degradation of the protein, for PD localization and microtubule accumulation of MP, and for function of MP in cell-to-cell transport of the viral RNA. Overexpressed CDC48 shifts MP subcellular localization from ER inclusions to microtubules, suggesting that CDC48 extracts the MP from ER-associated inclusions, where it accumulates in midstages of infection, to the cytosol, where it accumulates along microtubules during late infection stages. Moreover, overexpression of active, but not inactive, CDC48 inhibits virus movement. Our data demonstrate that a CDC48-dependent pathway leading to the clearance of ER-associated protein inclusions exists in plants, that plant viral MPs are substrates for this pathway, and that this pathway determines viral protein fate during infection. We suggest that CDC48-mediated extraction of MP from the ER is part of a plant defense response to remove MP from the ER, the compartment the virus uses for replication and movement. RESULTS Virus Infection Induces Expression of CDC48B in Arabidopsis

First evidence for a role of CDC48 during TMV infection came from pull-down experiments using Plant Physiol. Vol. 160, 2012

Plant CDC48 Controls Viral Movement Protein Fate

recombinant MPTMV:His6 and tobacco BY2 suspension cell extract (Brandner et al., 2008), in which we identified a putative tobacco CDC48 (National Center for Biotechnology Information [NCBI] accession no. 98962497) specifically retained by MPTMV:His6 (Supplemental Table S1). Studies in yeast and mammals have shown that CDC48 plays a role in the retrotranslocation of proteins from the ER to the cytosol during ER-assisted protein degradation (ERAD; Ye et al., 2001; Römisch, 2005; Rumpf and Jentsch, 2006). Although CDC48 function in plants is much less understood, there is evidence that a CDC48-dependent, ERAD-like pathway for the degradation of misfolded ER proteins also exists in plants (Di Cola et al., 2001; Müller et al., 2005; Aker and de Vries, 2008; Marshall et al., 2008). As TMV and related tobamoviruses replicate in association with the ER and infection leads to the formation of ER-associated inclusions that accumulate MP in advance of MP degradation, we suspected that CDC48 activity might play a role in regulating MP fate. To test this further, we first analyzed the expression of the three different Arabidopsis CDC48 isoforms during infection with the TMVrelated Oilseed rape mosaic virus (ORMV). In contrast to TMV, ORMV readily infects Arabidopsis and induces strong disease symptoms in this host (Aguilar et al., 1996; Mansilla et al., 2009). Indeed, CDC48B (At3g53230) was strongly induced in Arabidopsis plants undergoing systemic infection (Fig. 1A). CDC48B was induced at two tested time points after virus inoculation (7 d post inoculation [dpi] and 14 dpi), whereas the more highly expressed CDC48A (At3g09840) gene (Supplemental Fig. S2) was only moderately induced and CDC48C (At5g03340) transcript levels remained largely constant

at both time points. The virus-induced induction of CDC48B is in agreement with our independent microarray analysis of transcript levels upon ORMV infection (Hu et al., 2011; Supplemental Table S2) and other publicly available microarray data describing CDC48 transcript levels in the presence of different environmental stresses (https://www.genevestigator. com/gv/index.jsp and http://www.weigelworld.org/ resources/microarray/AtGenExpress/), indicating that CDC48B represents the main stress-responsive CDC48 isoform in Arabidopsis. CDC48B Functions in ER Maintenance upon ER Stress

Virus infection has been shown to induce ER stress and the expression of ER-resident chaperones to enhance the protein folding capacity in the ER (He, 2006; Ye et al., 2011). To verify whether also tobamoviruses induce ER stress, we analyzed the expression levels of the ER luminal chaperones BINDING PROTEIN3 (BIP3) and CALRETICULIN-2 (CRT) in Arabidopsis plants systemically infected with ORMV. Indeed, in virus-infected compared with mock-inoculated samples, expression of both chaperones was induced 8.0 6 5.4-fold and 10.5 6 7.4-fold, respectively (Fig. 1B). Comparison of expression levels of ER stress-responsive chaperones, lectins, and transcription factors in biologically independent Arabidopsis transcriptome data of ORMV-infected and mock-inoculated plants (Hu et al., 2011) further validated this finding (Supplemental Table S2) and thus indicated the induction of ER stress and enhanced protein quality surveillance in virusinfected tissues. The observed induction of CDC48B

Figure 1. Arabidopsis CDC48 and ER-resident chaperones are induced upon ORMV infection. A, qRT-PCR analysis of Arabidopsis CDC48 expression in ORMV-infected and mock-treated Arabidopsis leaf samples harvested at 7 and 14 dpi, respectively. Data represent mean relative fold changes in transcript levels in virus-infected compared with mock-inoculated plants. Error bars show SD. At least four biological replicates were analyzed per treatment and time point. Each replicate represents a pool of four plants. Induction of CDC48A and CDC48B expression upon infection is statistically significant (P # 0.01, Student’s t test) for both time points. B, qRT-PCR analysis of the luminal ER stress marker genes BIP3 and CRT2 at 14 dpi. Data represent mean relative fold changes in transcript levels in virus-infected compared with mock-inoculated samples and reveal an induction of both genes upon infection (P # 0.07, Student’s t test). Error bars show SD. Three biologically independent samples were analyzed per treatment. Each sample represents a pool of four plants. Plant Physiol. Vol. 160, 2012

2095

Niehl et al.

may thus be linked to the induction of ER stress in virus-infected tissues. To address a general role of CDC48B in relation to ER stress in vivo, we expressed the protein in fusion to GFP or red fluorescent protein (RFP) and analyzed its subcellular localization in Nicotiana benthamiana epidermal cells by confocal fluorescence microscopy. We found the protein localized in the cytosol, at the

nuclear envelope, and in the nucleoplasm (Fig. 2A). When the protein was fused to fluorescent proteins at its N terminus, the subcellular localization was similar to that of the C-terminally fused protein except that the nucleocytoplasmic localization was weaker. In the cortical cytoplasm, the protein showed a clear localization adjacent to the ER labeled with the coexpressed ER marker GFP:HDEL (Fig. 2B; Supplemental Movie S1).

Figure 2. CDC48B functions in ER membrane maintenance upon ER stress. A, N. benthamiana epidermal cells transiently expressing Arabidopsis CDC48B:RFP (left) and RFP:CDC48B (right). In central view of the cell, the protein is seen localized in the cytosol, at the nuclear membrane, and in the nucleoplasm. B, N. benthamiana epidermal cell transiently expressing Arabidopsis CDC48B:RFP and the ER marker GFP:HDEL. In the cortical cytoplasm, CDC48B:RFP localizes in the vicinity of the cortical ER network. Upon expression of CDC48B:RFP, the ER network remains intact. C, N. benthamiana epidermal cell transiently expressing Arabidopsis CDC48BE308Q: RFP (CDC48BQE:RFP) together with the ER stressinducing marker GFP:HDEL. Upon expression of CDC48BQE:RFP, in which the minor ATPase activity is missing, the ER network remains intact. D, N. benthamiana epidermal cell transiently expressing Arabidopsis CDC48BE581Q:RFP (CDC48BEQ:RFP) together with the ER stress-inducing marker GFP: HDEL. Upon expression of the dominant-negative mutant protein CDC48BEQ:RFP lacking the major ATPase activity, the ER retracts to the vertices and forms aggregates. E, N. benthamiana epidermal cell transiently expressing Arabidopsis CDC48BE308Q,E581Q:RFP (CDC48BQQ:RFP) together with the ER stress-inducing marker GFP: HDEL. Upon transient expression of CDC48BQQ: RFP, in which both ATPase domains are disrupted, the ER retracts to the vertices and forms aggregates. Single optical sections are shown. Bars = 10 mm.

2096

Plant Physiol. Vol. 160, 2012

Plant CDC48 Controls Viral Movement Protein Fate

Interestingly, differences in ER structure were observed when GFP:HDEL was expressed together with specific CDC48B mutants (Ye et al., 2003; Müller et al., 2005) in which either the minor ATPase domain (CDC48BE308Q, also referred to as CDC48QE:RFP), the major ATPase domain (CDC48BE581Q, also referred to as CDC48EQ:RFP), or both ATPase domains (CDC48QQ: RFP) were mutated. Whereas expression of CDC48BQE: RFP had no effect on the GFP:HDEL-labeled ER network (Fig. 2C), expression of CDC48BEQ:RFP or of CDC48BQQ: RFP resulted in an extensively modified ER network (Fig. 2, D and E; Supplemental Movie S2). Similar results were obtained when we used RFP:HDEL as ER marker or when N-terminal fluorescent protein fusions to wild-type or mutant CDC48B were used (data not shown). Interestingly, the dominant effects of CDC48EQ:RFP and CDC48BQQ:RFP on ER structure were seen only when the ER marker GFP:HDEL was transiently expressed. When we used the GFP:HDEL transgenic line 16c (Ruiz et al., 1998), this effect was not observed (Supplemental Fig. S3). This suggests a critical role of CDC48B function in the maintenance of the ER network upon sudden ER stress, such as caused by strong, transient overexpression of the ER marker. If the altered ER phenotype observed in the presence of CDC48BQQ:RFP was due to transient overaccumulation of misfolded protein in the ER, the effect should be

alleviated by coexpression of ER chaperones that maintain ER proteins in the quality control cycle (Liu and Howell, 2010). Indeed, transient expression of CALNEXIN (CNX-2:GFP; At5g07340) instead of GFP: HDEL as ER marker revealed an intact ER network upon coexpression with CDC48BQQ:RFP (Supplemental Fig. S3). Moreover, expression of CNX-2:GFP weakened the effect of CDC48BQQ:RFP on the ER structure in the presence of the transiently expressed ER marker RFP:HDEL (Fig. 3). The accumulation of misfolded proteins in the ER upon transient expression of GFP:HDEL was confirmed by quantitative reverse transcription (qRT)PCR experiments, which revealed a strong induction of genes encoding the N. benthamiana ER luminal stress marker proteins BLP4 (the ER-luminal BINDING PROTEIN [BIP]; Ye et al., 2011) and CRT (Fig. 4). Compared with transient expression of GFP as control, these genes were also induced upon ectopic expression of the dominant-negative CDC48BQQ:GFP protein. Importantly, the ER stress markers were not induced upon expression of CDC48B:GFP or CNX-2:GFP compared with the GFP-expressing control. As expected, the markers were also not significantly induced in transgenic GFP:HDEL-expressing plants (16c) compared with the wild type (data not shown). We also tested whether ER stress causes induction of CDC48 in N. benthamiana. Transient expression of GFP: Figure 3. Expression of CNX-2:GFP dampens the disruptive effect of CDC48BQQ:RFP on ER structure in the presence of ER stress. N. benthamiana epidermal cells transiently expressing Arabidopsis CDC48B QQ :RFP, RFP:HDEL, and CNX-2:GFP have an intact ER network. A, A 13.5-mm thick z-projection of 30 optical confocal sections. B, Single optical section taken from the z-stack in A showing the cortical ER network. C, Single optical section in central view taken from the z-stack in A. Red fluorescence in the nucleoplasm confirms that CDC48QQ:RFP is expressed in this cell. Bars = 10 mm.

Plant Physiol. Vol. 160, 2012

2097

Niehl et al.

Figure 4. ER stress markers and CDC48 are induced upon ectopic expression of ER stress-inducing proteins in N. benthamiana. qRT-PCR analysis of the luminal ER stress markers BLP4 and CRT and of N. benthamiana CDC48 in N. benthamiana leaves transiently expressing Arabidopsis CDC48B:GFP, CDC48BQQ:GFP, GFP:HDEL, or CNX-2: GFP. The figure shows the relative fold changes of the normalized expression levels compared with those in control plants transiently expressing 35S::GFP. Induction of BLP4, CRT, and NbCDC48 expression upon expression of CDC48BQQ:GFP and GFP:HDEL is statistically significant (P # 0.02, Student’s t test). Data represent mean relative fold changes in three biological replicates. Error bars show SD.

HDEL and Arabidopsis CDC48BQQ:GFP in N. benthamiana leaves induced expression of NbCDC48 compared with the GFP-expressing control, while transiently expressed CNX-2:GFP and Arabidopsis CDC48B: GFP did not (Fig. 4). NbCDC48 was also not significantly induced in 16c plants compared with the wild type (data not shown). Collectively, these findings indicate that plant CDC48 proteins function in alleviating ER stress and maintaining a native ER structure in the presence of accumulated, misfolded ER proteins. CDC48B Interacts with MP in ER-Associated Inclusions

To alleviate ER stress, the misfolded proteins are usually removed from the ER via ERAD (Tsai et al., 2002; Meusser et al., 2005; Müller et al., 2005; Marshall et al., 2008; Liu and Howell, 2010). Thus, CDC48B could be induced during virus infection to support the retrotranslocation of ER-associated, misfolded viral proteins to the cytoplasm. As mentioned above, TMV MP accumulates in ER-associated, proteinaceous inclusions during late infection (Heinlein et al., 1998) as well as when ectopically expressed (Reichel and Beachy, 1998; Supplemental Fig. S1). Since recombinant MPHis-6 interacted with a putative tobacco CDC48 in vitro (Supplemental Table S1), we surmised that the ER-accumulated MP may represent a substrate for CDC48-mediated retrotranslocation. To test a potential role of CDC48 in relation to MP in ER inclusions, we expressed CDC48B:GFP in N. benthamiana leaves 2098

carrying infection sites of a TMV derivative expressing a functional MPTMV:RFP fusion protein (TMV-MP: RFP). Interestingly, CDC48B:GFP colocalized with MP TMV:RFP in ER-associated inclusions but not with MP:RFP localized at PD or along microtubules (Fig. 5A). Colocalization of CDC48B:GFP to MPTMV:RFP at inclusions was also observed when MPTMV:RFP was expressed in the absence of virus infection (Fig. 5B). To test whether CDC48B and MP undergo direct protein-protein interactions, we used fluorescence lifetime imaging microscopy (FLIM) to investigate the efficiency of fluorescence resonance energy transfer (FRET) between the GFP and RFP moieties of the CDC48B:GFP and MPTMV:RFP proteins ectopically expressed in N. benthamiana and colocalizing in MP inclusions. Compared with CDC48B:GFP when expressed alone and localizing to the cytosol or nucleus (Fig. 6A), the protein exhibited a reduced GFP fluorescence lifetime when expressed with MPTMV:RFP and colocalizing with MPTMV:RFP in the ER inclusions (Fig. 6B, arrow). The observed reduction in fluorescence lifetime represents a FRET efficiency of 14.4%, which is strongly indicative for molecular proteinprotein interactions (Padilla-Parra and Tramier, 2012; Table I). A comparable reduction in GFP fluorescence lifetime and representing a FRET efficiency of 11.7% was observed when we expressed CDC48B:GFP together with MPORMV:RFP (Table I; Fig. 6, A and C). Thus, CDC48B appears to interact with the MPs of both TMV and ORMV, although the two proteins share only 63% amino acid similarity. Interestingly, no significant FRET values were obtained when amino acids at positions 2 to 5 or 2 to 30 were deleted from MPTMV (MPTMVDN5:RFP and MPTMVDN30:RFP, respectively; Table I; Fig. 6, D and E), suggesting that the interaction requires the N terminus of MPTMV. No significant change in GFP fluorescence lifetime and, thus, no significant FRET was observed when MPTMV: RFP was coexpressed with CDC48B carrying the fusion to RFP at its N terminus rather than at its C terminus (Fig. 6, F and G; Table I), indicating that interaction might require a free CDC48B N terminus. To further confirm the interaction between MP and CDC48B as indicated by FLIM-FRET, we performed coimmunoprecipitation experiments with protein extracts of N. benthamiana leaf samples in which we expressed MPTMV:RFP alone or together with CDC48B:GFP. As is shown in Figure 6H, MPTMV:RFP precipitated with CDC48B:GFP using anti-GFP antibodies (Fig. 6H, lane 7) but did not precipitate with anti-GFP antibodies in the absence of CDC48B:GFP (lane 3). MP:RFP did also not precipitate when expressed together with CDC48B:GFP, but normal rabbit serum was used for immunoprecipitation (lane 4). However, anti-MP antibodies precipitated MP under these conditions (lane 6). Using RFP-Trap, we further confirmed that the interaction between MP: RFP and CDC48B:GFP is specific and depends on the MP N terminus and on a free CDC48B N terminus (Supplemental Fig. S4). Plant Physiol. Vol. 160, 2012

Plant CDC48 Controls Viral Movement Protein Fate

Figure 5. CDC48B:GFP colocalizes with MPTMV in ER-associated inclusions. A, Subcellular localization of CDC48B:GFP and MPTMV:RFP in TMV-MP:RFP infection sites in N. benthamiana leaves. MPTMV:RFP localizes to microtubules (top row, arrowheads), PD (bottom row, arrowheads), and ER-localized inclusions (arrows). Cytosolic CDC48B:GFP colocalizes with MPTMV:RFP in ERlocalized inclusions (arrows). B, Subcellular localization of ectopically coexpressed CDC48B: GFP and MPTMV:RFP in N. benthamiana. Colocalization of CDC48B:GFP with MPTMV:RFP is seen in inclusions (arrows) but not at microtubules (top row, arrowheads) or PD (bottom row, arrowheads). Single optical sections are shown. Bars = 10 mm.

MPTMV:RFP Degradation Depends on CDC48 Enzyme Activity

The interaction between CDC48B and MP in ERassociated inclusions suggests that CDC48B may be involved in ER-associated degradation of MP. Since the MP accumulates in ER inclusions during late infection stages, this process may lead to removal of misfolded, accumulated MP, recovery of a native ER structure, and reduction of ER stress. To investigate whether interaction of MP with CDC48 results in degradation of MP, we analyzed the effect of CDC48B: GFP or CDC48BQQ:GFP on the levels of MPTMV:RFP in N. benthamiana leaves in the presence of the protein synthesis inhibitor cycloheximide (CHX). Interestingly, whereas MPTMV:RFP levels decreased over time in cells expressing GFP or CDC48B:GFP, MPTMV:RFP levels did not decrease, but rather increased in cells Plant Physiol. Vol. 160, 2012

expressing CDC48BQQ:RFP (Fig. 7, A and B). This finding suggests that MP degradation is dependent on CDC48 enzyme activity. The increase of MPTMV:RFP protein levels in the presence of CDC48BQQ:RFP may result from incomplete inhibition of protein synthesis by the inhibitor. Stability of GFP, CDC48B:GFP, and CDC48BQQ:GFP in the CHX-treated tissues was verified by western blot (Supplemental Fig. S5). Interestingly, MPTMVDN5:RFP levels did not decrease upon CHX treatment irrespective of whether GFP, CDC48B: GFP, or CDC48BQQ:GFP was coexpressed (Fig. 7, C and D). This observation may indicate that the interaction between MPTMV and CDC48 is a prerequisite for MP degradation. Evidence for the hypothesis that CDC48-mediated degradation of MPTMV may indeed reduce ER stress induced by MPTMV is provided by western-blot experiments, in which we used a maize (Zea mays) CRT 2099

Niehl et al.

Figure 6. CDC48B interacts with tobamovirus MP. A to G, FRET-FLIM analysis. Left images show fluorescence intensity, and right images show the color-coded GFP fluorescence lifetime. The color code referring to fluorescent lifetimes from 1 to 3 ns is shown below D. A, CDC48B:GFP localized to the cytosol (arrowhead) and nucleus (N) has a specific fluorescence lifetime of 2.6 ns as indicated by a yellowish color. Asterisk indicates a fluorescence lifetime signal of the chloroplast autofluorescence. B, In the presence of MPTMV:RFP, CDC48B:GFP localizes to MP inclusions (arrow). In these inclusions, CDC48B:GFP shows a decreased fluorescence lifetime of 2.2 ns (green color), which is indicative of FRET with MPTMV:RFP. CDC48B:GFP present in the cytosol of the same cells shows a yellowish signal (arrowhead), thus indicating the absence of interactions with MPTMV:RFP at this location. C, CDC48B:GFP localizes to MP inclusions also in the presence of MPORMV:RFP (arrow). In these inclusions, CDC48B:GFP fluorescence lifetime is reduced to 2.3 ns (green color) indicative of FRET with MPORMV:RFP. D and E, Mutations in the N terminus of MP abolish FRET with CDC48B:GFP. D, In the presence of MPTMVDN5:RFP, CDC48B:GFP localizes to MP inclusions (arrow) but does not exhibit a significant decrease in fluorescence lifetime (yellow color, similar as if expressed alone [A]), thus indicating the absence of FRET. E, In the presence of MPTMVDN30:RFP, CDC48B:localizes to MP inclusions (arrow) but does not exhibit a significant decrease in fluorescence lifetime (yellow color, similar as if expressed alone [A]), thus indicating the absence of FRET. F and G, GFP:CDC48B (GFP fused to the N terminus of CDC48B) does not show FRET with MPTMV:RFP. F, GFP:CDC48B is localized to the cytosol (arrowhead) and nucleoplasm and has a specific lifetime of 2.6 ns (yellows color) when expressed alone. G, In the presence of MPTMV:RFP, GFP:CDC48B localizes to MP inclusions (arrow) but does not exhibit a significant decrease in fluorescence lifetime (yellow color, similar as if expressed alone [F]), thus indicating the absence of FRET. H, Co-IP of MPTMV:RFP and CDC48B:GFP. Protein extracts of N. benthamiana leaves expressing MPTMV: RFP or MPTMV:RFP together with CDC48B:GFP were used. CDC48B:GFP (120 kD) and MP:RFP (57 kD) are detected with specific GFP and MP antibodies, respectively. These antibodies were also used for immunoprecipitation as indicated below the lanes. Lane 1, presence of CDC48B:GFP and MPTMV:RFP in the input sample. Lane 3, absence of MPTMV:RFP after precipitation with GFP antibody in the absence of CDC48B:GFP. Lane 4, absence of MPTMV:RFP after precipitation with normal rabbit serum (NRS) in the presence of CDC48B:GFP. Lane 6, presence of MPTMV:RFP after precipitation with MP antibody in the presence of CDC48B:GFP. Lane 7, presence of MPTMV:RFP after precipitation with GFP antibody in the presence of CDC48B:GFP (Co-IP). Lanes 2 and 5 were used for protein size markers. 2100

Plant Physiol. Vol. 160, 2012

Plant CDC48 Controls Viral Movement Protein Fate

Table I. CDC48B interacts with tobamovirus MPs in vivo FRET between transiently expressed proteins was determined by FLIM. Lifetimes are mean values of independent measurements in different cells 6 SD. FRET percentage was calculated according to the formula: 1 2 t (donor + acceptor)/t(donor). t, Fluorescence lifetime; n, number of measurements. n

Protein

Average t ns

14 25 35 50 32 14 21 33 38 20

CDC48B:GFP CDC48B:GFP CDC48B:GFP CDC48B:GFP CDC48B:GFP GFP:CDC48B GFP:CDC48B GFP:CDC48B GFP:CDC48B GFP:CDC48B

+ + + +

MPTMV:RFP MPTMVDN5:RFP MPTMVDN30:RFP MPORMV:RFP

+ + + +

MPTMV:RFP MPTMVDN5:RFP MPTMVDN30:RFP MPORMV:RFP

2.59 2.21 2.49 2.44 2.28 2.62 2.52 2.55 2.55 2.52

SD

FRET %

0.02 – 0.05 14.0 0.04 3.7 0.04 5.8 0.05 11.7 0.03 – 0.04 3.8 0.05 2.8 0.06 2.6 0.04 3.6

antibody (Baluška et al., 1999) to analyze endogenous CRT levels in N. benthamiana leaves ectopically coexpressing MPTMV:RFP or MPTMVDN5:RFP together with tagged or untagged CDC48B or CDC48BQQ (Fig. 7E). These experiments revealed higher levels of the ER stress-induced chaperone CRT in the presence of the MPTMVDN5:RFP mutant, which, unlike MPTMV:RFP, is unable to interact with CDC48B. Increased amounts of CRT observed upon expression of tagged or untagged ATPase-deficient CDC48BQQ compared with functional CDC48B validate the induction of ER-resident chaperones in the presence of the dominant-negative CDC48QQ protein already seen at the transcript level (Fig. 4) and confirm that the GFP fusion to CDC48B does not interfere with the functionality of the protein. CDC48B Activity Modulates MPTMV Subcellular Localization

Deletion mutations in the N terminus of MPTMV, such as DN5, interfere with protein function in virus movement and with the localization of the protein along microtubules and at PD (Supplemental Fig. S6; Gafny et al., 1992; Lapidot et al., 1993; Cooper et al., 1995; Kahn et al., 1998; Boyko et al., 2000c; Kotlizky et al., 2001). Since the N terminus of the protein is important for interaction with CDC48B, we wondered whether CDC48B could play a role in directing the MP away from ER inclusions for association with microtubules and PD. As a test for this hypothesis, we analyzed whether overexpression of CDC48B:GFP would trigger a redistribution of MPTMV:RFP from inclusions to microtubules and PD. Because MPTMV shows extensive association with both inclusions and microtubules, it was difficult to visualize and quantify a gradual change in the tendency of accumulation to these alternative localizations using the wild-type protein. Therefore, for this test, we used MPR3, a functional MPTMV variant that carries a mutation in one of the putative transmembrane domains and accumulates Plant Physiol. Vol. 160, 2012

predominantly in ER-associated inclusions and less along microtubules and shows a more homogeneous subcellular localization throughout infection sites (Gillespie et al., 2002). Expression of CDC48B:RFP in TMV-MPR3:GFP infection sites on N. benthamiana leaves indeed resulted in an increased accumulation of MPR3:GFP along microtubules (Fig. 8A). Quantification of this phenomenon revealed that in cells expressing CDC48B:RFP, the number of cells with MPR3:GFP fluorescence associated with microtubules increased to 71% compared with 19% upon expression of RFP. In contrast, expression of CDC48BQQ:RFP decreased the number of cells showing microtubule labeling to 11% of the cells (Fig. 8B). These results suggest the involvement of CDC48 activity in the relocalization of MP from ER-associated inclusions to microtubules. Expression of CDC48B Ahead of Infection Reduces the Size of Viral Infection Sites

To further test the role of CDC48B in directing MP away from the ER, we wondered whether CDC48 overexpression would influence the cell-to-cell spread of TMV infection. According to the current model, TMV movement occurs in form of MPTMV-containing viral ribonucleoprotein/replication complexes that are transported in association with the ER to the PD for intercellular spread (Niehl and Heinlein, 2011; Peña and Heinlein, 2012). Since MP is essential for this process, we expected that TMV movement should be inhibited if CDC48 activity removes MP from the ER upon introduction into cells at the leading front of infection. In order to test the effect of CDC48 activity on virus movement, we expressed free RFP in one half and CDC48B:RFP or CDC48BQQ:RFP in the other half of N. benthamiana leaves carrying TMV-MPR3:GFP infection sites and measured the size of fluorescent infection sites 4 d later. The sizes of infection sites in leaf halves expressing wild-type CDC48B:RFP were approximately 30% reduced compared with infection sites in leaf halves expressing free RFP (Fig. 7C). No such difference was observed between infection sites in leaf halves expressing CDC48BQQ:RFP and RFP, respectively. These observations are consistent with a role of CDC48B in virus movement by removing MP from the ER-associated transport pathway. Moreover, since CDC48B interacts with MP in ER inclusions, CDC48-mediated removal of accumulated MP from the ER may primarily occur as part of an ER stressinduced response that allows ER recovery during late stages of infection. DISCUSSION

Previous studies demonstrated a requirement of CDC48 homologs in yeast (Cdc48p) and mammals (p97/VCP) for the extraction of misfolded or unassembled substrates from the ER for delivery to proteasomes 2101

Niehl et al.

Figure 7. Degradation of MPTMV:RFP depends on CDC48B activity. A, Degradation of MPTMV:RFP in N. benthamiana leaves in the presence of coexpressed GFP, CDC48B:GFP, or CDC48BQQ:GFP and incubated in 100 mm CHX solution for 0 min (T0), 30 min (T1), 60 min (T2), and 120 min (T3). B, Quantification of the MPTMV:RFP signal in A. Numbers represent percentage amount of the total MP amount (100%) detected at all time points. The presence of ATPase-deficient CDC48BQQ:RFP interferes with degradation of MPTMV:RFP. C, Degradation of MPTMVDN5:RFP in leaves in the presence of coexpressed GFP, CDC48B:GFP, or CDC48BQQ:GFP and incubated in 100 mm CHX solution for 0 min (T0), 30 min (T1), 60 min (T2), and 120 min (T3). D, Quantification of the MPTMVDN5:RFP signal in C. Numbers represent percentage amount of the total MP amount (100%) detected at all time points. The protein is stable irrespective of coexpression of either GFP, CDC48B:GFP, or CDC48BQQ:GFP. E, CRT protein levels are induced upon MPTMVDN5:RFP and CDC48BQQ expression. A western blot probed with maize anti-CRT antibody and peroxidase-labeled secondary antibody is shown. The blot shows the approximately 53-kD band of CRT in protein extracts of N. benthamiana leaf samples coexpressing MPTMV:RFP or MPTMVDN5:RFP together with GFP-tagged or nontagged CDC48B (CDC:G, CDC) or CDC48BQQ (QQ:G, QQ). Endogenous CRT levels are induced by both GFP-tagged or nontagged CDC48BQQ and further increased in samples expressing MPTMVDN5:RFP compared to samples expressing MPTMV:RFP.

during ERAD (Meusser et al., 2005; Römisch, 2005). In plants, only little is known about cellular CDC48 functions and substrates. Nevertheless, Arabidopsis CDC48A was shown to play a role in the quality control of the powdery mildew (Blumeria graminis) resistance protein MLO and of the catalytic A subunit of ricin, suggesting that CDC48 functions in ERAD-like processes also in plants (Di Cola et al., 2001; Müller et al., 2005; Marshall et al., 2008). Our findings demonstrate that plant CDC48 targets tobamoviral MP, 2102

extracts it from the ER where it is produced, and thus determines its subsequent fate and function during infection. The tobamoviral MPs investigated here are targets of Arabidopsis CDC48B, which is highly induced upon virus infection and other stresses. We show that overexpression of ATPase-deficient CDC48BQQ interferes with ER membrane structure upon ER stress conditions, indicating a requirement of CDC48 activity for ER maintenance. The observed ER phenotype upon Plant Physiol. Vol. 160, 2012

Plant CDC48 Controls Viral Movement Protein Fate

Figure 8. Expression of CDC48B:RFP during TMV-MPR3:GFP infection causes increased accumulation of MPR3:GFP along microtubules and smaller fluorescent infection sites. A, Subcellular localization of virusencoded MPR3:GFP in the presence of RFP, CDC48B:RFP, or CDC48BQQ:RFP. MPR3:GFP fluorescence is mainly associated with inclusions in the presence of RFP or CDC48BQQ:RFP but more microtubule associated in the presence of CDC48B:RFP. Bars = 10 mm. Single optical sections are shown. B, Quantification of microtubule labeling by virus-encoded MPR3:GFP in N. benthamiana leaves expressing either RFP, CDC48B:RFP, or CDC48BQQ:RFP. The number of cells showing MPR3:GFP along microtubules is increased in the presence of CDC48B:RFP. Infection sites obtained in two independent experiments were analyzed. C, Fold size difference of TMV-MPR3:GFP infection sites developed in N. benthamiana leaves expressing RFP, CDC48B:RFP, or CDC48BQQ:RFP, 4 d after agroinfiltration. Expression of RFP fluorescence was verified by fluorescence microscopy. The calculated relative mean fold size refers to the difference in size of infection sites in tissues expressing the indicated proteins in comparison to RFP. CDC48B:RFP or CDC48BQQ:RFP were expressed in leaf Plant Physiol. Vol. 160, 2012

overexpression of ATPase-deficient CDC48BQQ is consistent with an ERAD function of Arabidopsis CDC48. In mammalian and yeast systems, CDC48 function in different cellular processes is governed through binding of specific cofactors (Schuberth and Buchberger, 2008; Yeung et al., 2008; Buchberger et al., 2010; Stolz et al., 2011). Given that Arabidopsis encodes three CDC48 isoforms and that the expression of especially CDC48B is substantially increased upon tobamovirus infection, we suggest that the different isoforms have different cofactor preferences. As CDC48A is expressed at generally higher levels compared with CDC48B (Feiler et al., 1995; Rancour et al., 2002), CDC48A appears to maintain cellular housekeeping functions while CDC48B is induced upon ER stress conditions. During infection, MPTMV accumulates in the ER, which leads to strong deformations of the native ER structure and to the formation of proteinaceous ER inclusions (i.e. viral factories) during late stages of infection. Subsequently, the MP is removed from these inclusions and degraded, which may involve an intermitting accumulation of the protein along microtubules. Here, we provide strong indications for a role of CDC48B in these processes by showing that CDC48B physically interacts with MP in ER-associated inclusions and by presenting evidence that the interaction between the two proteins as well as CDC48 activity are involved in the degradation of MPTMV. Moreover, CDC48B overexpression shifts the localization of MP TMV from ER-associated inclusions to microtubules dependent on CDC48B ATPase activity. When CDC48B is overexpressed in cells ahead of infection, MPTMV function is perturbed, leading to reduced sizes of infection sites. Taken together, our data suggest that MP accumulating during the course of infection is subject to retrotranslocation from ER and ER-associated proteinaceous inclusions (i.e. viral factories) by CDC48 in an ERAD-like mechanism. Consistent with a role of CDC48 in MP degradation, an N-terminal deletion mutant of MP, which does not interact with CDC48B, is stable after CHX treatment. A role of the N terminus of MPTMV in MPTMV degradation during late stages of TMV infection is consistent with the previous observation that TMV-MP:GFP mutants with N-terminal deletions in MP form disk-shaped fluorescent infection sites in MPTMV transgenic tobacco plants as opposed to the typical ring-shaped infection sites produced

halves with RFP expressed in the other half of the same leaf as control. Mean values were obtained from n = 31 (leaves infiltrated with RFP on one half and CDC48B:RFP on the other half) and n = 13 (leaves infiltrated with RFP on one half and CDC48BQQ:RFP on the other half) leaves. Error bars show SD. Per leaf half, between three and 15 individual infection sites were analyzed. Whereas expression of CDC48BQQ:RFP did not influence the size of infection sites, the sites were significantly smaller (P # 0.01) in leaves expressing CDC48B: RFP. 2103

Niehl et al.

by wild-type TMV-MP:GFP, which is indicative of MP: GFP degradation behind the infection front (Boyko et al., 2000c). As the TMV sequence contains several Lys residues close to its N terminus (Lys-6, Lys-8, Lys-19, and Lys-22), it would be interesting to investigate whether these residues are involved in MP ubiquitylation. Recent studies demonstrate that ERAD substrates are deubiquitylated for dislocation from the ER (Ernst et al., 2011; Tsai and Weissman, 2011). Indeed, CDC48 occurs in association with ubiquitin ligase and deubiquitylating activities (Crosas et al., 2006; Rumpf and Jentsch, 2006). This may be in agreement with our previous finding that microtubule-aligned MPTMV is not ubiquitylated (Ashby et al., 2006), although MPTMV is ubiquitylated in crude cell extracts (Reichel and Beachy, 2000). Given that MPTMV associates with microtubules after its production in association with the ER, and the predicted membranespanning domains of MPTMV coincide with the domains responsible for microtubule binding (Boyko et al., 2000b; Brill et al., 2004; Peña and Heinlein, 2012), ER-produced MP may be first deubiquitylated and then extracted from the ER by CDC48 before it accumulates along microtubules and is finally degraded. Since CDC48 acts also in response to other proteins expressed in the ER, we propose that CDC48 functions as part of an ER stress-induced response that removes specific proteins from the ER. As MP accumulates and participates in the formation of ER-associated viral inclusions during late stages of infection (Heinlein et al., 1998; Reichel and Beachy, 1998), retrotranslocation of MP from ER-associated inclusions may be important for the cell to restore a native ER structure. Extracted MP, which is likely not ubiquitylated and may have alternative folds, may at this stage be either reubiquitylated for subsequent degradation or remain nonubiquitylated to accumulate along and stabilize microtubules (Ashby et al., 2006) in a process correlated with the inhibition of further virus movement behind the spreading infection front (Gillespie et al., 2002; Kragler et al., 2003; Boyko et al., 2007; Peña and Heinlein, 2012). The finding that transient CDC48B expression indeed interferes with virus movement supports this model. Although the interaction between MP and CDC48 may primarily cause the removal of MP from the ER for degradation and for controlling viral RNA transport along the ER, this interaction may also have a role in sequestering CDC48 away from other cellular substrates and thus to interfere with the fate of other proteins in the ER. ERAD-like protein quality control has recently been shown to control environmental stress signaling in plants by targeting receptor-like kinases (Liu et al., 2011; Su et al., 2011; Cui et al., 2012; Su et al., 2012). It is compelling that Arabidopsis CDC48A interacts with the Leu-rich repeat receptorlike kinase SERK1 (for SOMATIC EMBROGENESIS RECEPTOR-LIKE KINASE1), which belongs to a family of Leu-rich repeat receptor-like kinases involved in diverse signaling pathways (Aker et al., 2006, 2007; Albrecht et al., 2008). CDC48A appears to be involved in the ERAD-like quality control of the ER2104

synthesized receptor (Aker and de Vries, 2008). Sequestration of CDC48 by MP may thus modulate stress signal transduction by interfering with proper folding and targeting of receptor proteins. MATERIALS AND METHODS Plant Growth and Virus Inoculation Arabidopsis (Arabidopsis thaliana) Columbia-0 plants were grown from seeds on soil with 12-h/12-h light/dark cycles at 21°C/18°C for 5 weeks. Plants were infected by rub-inoculating two leaves per plant with 150 ng purified ORMV virions in 10 mM sodium-phosphate buffer, pH 7.4, or mock (10 mM sodium-phosphate buffer, pH 7.4). Upper infected leaves were harvested for RNA extraction and qRT-PCR at 7 and 14 dpi. Wild-type or 16c (Ruiz et al., 1998) Nicotiana benthamiana plants were grown from seeds on soil under 16-h/8-h light/dark cycles at 22°C/18°C. Expanding leaves of 4- to 5-week-old plants were rub-inoculated with infectious RNA of TMV-MPR3:GFP (Toth et al., 2002) or TMV-MP:RFP (Hofmann et al., 2009) in vitro transcribed (RiboMAX; Promega) from infectious complementary DNA (cDNA) clones.

Virion Preparation Virions were prepared from ORMV-infected N. benthamiana leaves. Leaves were homogenized to fine powder in liquid N2. After addition of 1 mL 0.5 M sodium-phosphate buffer, pH 7.4, and 0.1% 2-mercaptoethanol per g leaf material, virions were extracted with 1 volume butanol/chloroform (1/1 [v/v]) and the phases separated by centrifugation (23 15 min at 12,000g). Virions in the upper aqueous phase were precipitated with 4% polyethylene glycol 8000 at 20,000g. The pellet was resuspended in 10 mM sodium-phosphate buffer, pH 7.4, and cleared by centrifugation at 5,000g for 10 min. The supernatant was precipitated again with 4% polyethylene glycol 8000 and 1% NaCl and resuspended in 10 mM sodium-phosphate buffer, pH 7.4. Virion concentration was estimated from absorbance values at 260 nm.

DNA Constructs Binary vectors pB7-MP:GFP (MPTMV:GFP), pB7-MP:RFP (MPTMV:RFP), pK7-MPDC55:GFP (MPTMVDC55:GFP), and pH7-MPDC55:RFP (MPTMVDC55: RFP) are described elsewhere (Brandner et al., 2008; Boutant et al., 2010). Binary GFP:HDEL constructs were previously described (Ruiz et al., 1998). Binary vectors for expression of N-terminal MPTMV deletion mutants, MPORMV, CDC48B (At3g53230), and CNX-2 (At5g07340) fused to fluorescent proteins and for expression of nonfused CDC48B were created by Gateway cloning. To construct N-terminal deletion mutants of MPTMV, the MPTMV open reading frame lacking the first 15 or 90 nucleotides, respectively, but retaining an ATG start codon was PCR amplified from TMV-U1 (NC_001367) cDNA. The open reading frame for full-length MPORMV was PCR amplified from ORMV (U30944.1) cDNA. Full-length coding sequences for CDC48B and CNX-2 were PCR amplified from Arabidopsis leaf cDNA. PCR products were recombined into pDONR/Zeo (Invitrogen). Following DNA sequence confirmation, entry clones were used for recombination with the destination vectors pMDC32, pK7FWG2, pK7WGF2, pH7WGR2, and pH7RWG2 (Karimi et al., 2002). Amino acid exchange mutants CDC48B E308Q , CDC48B E581Q , and CDC48BE308QE581Q (CDC48BQQ) were created by site-directed mutagenesis. PCR reactions contained 5 ng CDC48B entry vector as template DNA, 23 Phusion high-fidelity Taq master mix (Finnzymes), and 0.5 mM primer. PCR was performed according to the Phusion Taq protocol. Following mutagenesis, the PCR products were purified using the Nucleospin extract II kit (Macharey Nagel), DpnI digested, and transformed into Escherichia coli. After DNA sequence confirmation, the resulting entry vectors were used for recombination with pK7FWG2, pK7WGF2, pH7WGR2, or pH7RWG2 destination vectors (Karimi et al., 2002). Primer sequences to generate DNA constructs are listed in Supplemental Table S3.

Gene Transcript Analysis Analysis of publicly available gene expression data was performed using the Genevestigator analysis tool (https://www.genevestigator.com/gv/) and Plant Physiol. Vol. 160, 2012

Plant CDC48 Controls Viral Movement Protein Fate

the AtGenExpress visualization tool (http://jsp.weigelworld.org/expviz/ expviz.jsp). Microarray profiling and analysis are described elsewhere (Hu et al., 2011). For qRT-PCR, upper leaves of untreated, mock-inoculated, and virus-infected Arabidopsis plants were homogenized in liquid nitrogen and RNA extracted using the RNeasy Plant mini kit (Qiagen). Following treatment with TURBO-DNA-free DNAse (Ambion), 1 mg of the total RNA was reverse transcribed into cDNA using oligo(dT) primers and SuperScript III (Invitrogen). qRT-PCR with primers for CDC48A, CDC48B, CDC48C, CRT (At1g09210), and BIP-3 (At1g09080) as well as for reference genes (GAPDH, At1g13440; EXPRESSED PROTEIN, At3g01150; Czechowski et al., 2005) was performed with cDNA of at least three biological replicates each. Each biological replicate represented a pool of four plants. For qRT-PCR analysis of agroinfiltrated N. benthamiana leaf tissue, RNA was isolated using the trireagent (MRC), DNAse treated, and reverse transcribed as above. qRTPCR with primers for N. benthamiana BLP4 (FJ463755) and CRT (TIGR EST: TC18084) and with EF1a (AY206004) as reference gene, as well as with primers for tobacco (Nicotiana tabacum) CDC48 (NCBI accession 98962497), was performed with three biological replicates of cDNA of plants transiently expressing GFP, AtCDC48B:GFP, AtCDC48BQQ:GFP, or GFP:HDEL for 2 d or with cDNA of wild-type or GFP:HDEL transgenic 16c plants kept under the same conditions. Ten-microliter PCR reactions contained 0.5 mM primer, 0.05 mM cDNA, and 5 mL SYBR-green master mix (Roche). PCR was conducted in a Roche 480II light cycler. PCR conditions were 5 min denaturation at 95°C and 40 cycles with 10 s at 95°C, 15 s at 60°C, and 15 s at 72° C. N. benthamiana BLP4 and Arabidopsis CRT-2 primers are described elsewhere (Ye et al., 2011). All other primer sequences used for qRT-PCR are listed in Supplemental Table S3. Relative quantification of gene expression was made by normalization of the obtained cycle threshold (CT) values for the tested genes to those for the endogenous reference genes Arabidopsis GAPDH, Arabidopsis EXPRESSED PROTEIN, and N. benthamiana EF1a, respectively, yielding DCT values. Relative fold expression changes were calculated by forming the ratio between the 22DCT values for treated and control conditions. Coefficients of variation for relative expression values (22DCT) were derived by calculating the variation coefficients between biological replicates of both test and control conditions according to the formula cv = = (cv12 + cv22). SDs of relative fold expression changes were then calculated by multiplying the coefficient of variation with the ratio mean value.

Transient Protein Expression Binary vectors designed to express fluorescent protein fusions were transformed into Agrobacterium tumefaciens strain LBA4404 or GV3101. The bacteria were syringe infiltrated into N. benthamiana leaf tissues with a final optical density at 600 nm of 0.5. For coexpression experiments, agrobacteria suspensions were mixed (1:1) before infiltration. Expression of the agroinfiltrated constructs was observed at 30 to 48 h after infiltration by fluorescence microscopy.

Confocal Laser Scanning Microscopy Leaf samples were mounted on a microscope slide and vacuum infiltrated with water and epidermal cells imaged using a Zeiss LSM510 laser scanning confocal microscope with a C-Apo-chromat (633 1.2 W Korr) water objective lens in multitrack mode. Excitation/emission wavelengths were 488 nm/505 to 545 nm for GFP and 543/585 to 615 nm for RFP. Images were acquired using LSM510 version 2.8 software (Zeiss) and processed with Adobe Photoshop and Image J software.

CHX Treatment Leaf discs of N. benthamiana leaves transiently coexpressing MPTMV:RFP or MPTMVDN5:RFP together with GFP, CDC48B:GFP, or CDC48QQ:GFP for 40 h were vacuum infiltrated with a 100 mM CHX solution for 20 min and incubated in the solution for the duration of the experiment. Four leaf disks were withdrawn from the solution as pooled sample after 30 min (T1), 60 min (T2), and 120 min (T3). For the T0 time point, leaf disks were taken immediately before infiltration. Crude protein extracts were prepared by processing equal amounts of leaf tissue in sample buffer (10% glycerol, 5% 2-mercaptoethanol, and 2% SDS in 75 mM Tris, pH 6.8). For immunoblot analysis, samples were separated by 12.5% SDS-PAGE and blotted onto polyvinylidene difluoride membrane (Millipore). Immunoblot labeling was performed with anti-MP antibody reactive against MP amino acid residues 209 to 222 or with anti-GFP antibody (Invitrogen) and peroxidase-labeled secondary antibodies (Invitrogen) for luminescence detection (Roche). Luminescent signal was quantified with the ImageJ (http://rsbweb.nih.gov/ij/) gel analysis tool.

Coimmunoprecipitation Coimmunoprecipitation experiments were conducted with extracts of N. benthamiana leaf material expressing MPTMV:RFP alone or together with CDC48B:GFP harvested at 1.5 d after agroinfiltration. Leaves were homogenized in coimmunoprecipitation (Co-IP) buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 1 mM dithiothreitol, 4 mM EGTA, 2 mM MgCl2, 10% Suc, 0.3% Nonidet P-40, and 13 protease inhibitor cocktail) and cleared by centrifugation and the supernatant incubated with Protein-A Sepharose for 10 min to remove unspecific binding. Subsequently, the supernatant was incubated with Protein-A Sepharose and 10 mL anti-GFP antibody (Invitrogen), anti-MP antibody, or preimmune serum, respectively, overnight at 4°C with gentle end-over mixing. After incubation, the beads were washed three times in Co-IP buffer, eluted with Gly, pH 2.5, and neutralized with Tris base. Recovered proteins were analyzed by western blot with anti-MP antibody or anti-GFP antibody (Invitrogen) and peroxidase-labeled secondary antibody (Invitrogen) for luminescence detection (Roche). Co-IP using RFP-Trap M beads (ChromoTek) was conducted with N. benthamiana leaf material ectopically expressing CDC48B:GFP or GFP:CDC48B together with MPTMV:RFP or MPTMVDN5:RFP, respectively, for 2 d. As control, leaf samples expressing MPTMV:RFP and GFP were included. Samples were ground to fine powder in liquid N2 and vortexed with 1 volume (v/v) of lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% Nonidet P40, and 13 Protease inhibitor cocktail [Roche]). Samples were subsequently incubated in lysis buffer on ice for 30 min and cleared by filtration through Miracloth. After filtration, an aliquot of the protein extract was withdrawn as input control. RFP-Trap M beads were equilibrated according to the manufacturer’s instructions and 500 mL protein extract added to 20 mL beads in 500 mL dilution buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% Nonidet P-40, and 13 Protease inhibitor cocktail). The protein extract was incubated with the beads for 2 h at 4°C and gentle end-over mixing. Washing was performed by magnetically separating the beads five times and resuspension in wash buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% Nonidet P-40, and 13 Protease inhibitor cocktail). Remaining proteins were eluted by boiling the beads for 10 min in 23 sample loading buffer (126 mM Tris-HCl, pH 6.8, 20% glycerol, 4% SDS, and 0.02% bromophenol blue), magnetic separation, and recovery of the supernatant containing the trap sample. The input and trap samples were analyzed by western blot using antiGFP (Invitrogen) and anti-RFP (Clontech) antibodies and peroxidase-labeled secondary antibody (Invitrogen) for luminescence detection (Roche).

FRET-FLIM Analysis

Affinity Chromatography

Time-correlated single-photon-counting FLIM measurements were performed as previously described using a home-built two-photon system (Brandner et al., 2008; Boutant et al., 2010). Typically, samples were scanned for approximately 60 s to achieve appropriate photon statistics for the fluorescence decays. Data were analyzed using SPCImage version 2.8 software (Becker and Hickl), which uses an iterative deconvolution method to recover the lifetimes from the fluorescence decays. FRET efficiency reflecting the distance between donor and acceptor chromophores was calculated according to: E = (R06/(R06 +R6)) = 1 2 (t fret/t free), where R0 is the Förster radius, R the distance between donor and acceptor molecules, t fret the donor lifetime in presence of the acceptor, and t free the donor lifetime in the absence of the acceptor.

Recombinant His-tagged MPTMVHis6 expressed in E. coli was regenerated on nickel-nitrilotriacetic acid agarose (Ni-NTA) agarose (Qiagen) and used for affinity chromatography as described previously (Brandner et al., 2008). Soluble proteins of tobacco suspension cell line BY2 were extracted essentially as described (Brandner et al., 2008). After preincubation for 1 h with Ni-NTA agarose, the proteins were incubated with MPTMVHis6-complexed Ni-NTA agarose beads in PDB-I (50 mM HEPES, pH 7, 25 mM imidazole, 250 mM NaCl, 2 mM dithiothreitol, 2 mM MgCl2, 10% glycerol, 0.5% Triton X-100, 2 mM phenylmethylsulfonyl fluoride, and 13 protease inhibitor cocktail [Roche]) for 2h at 4°C with gentle end-over shaking. Following elution by incubation in elution buffer (500 mM NaCl, 50 mM HEPES, and 500 mM imidazole, pH 7.5),

Plant Physiol. Vol. 160, 2012

2105

Niehl et al.

recovered proteins were analyzed by SDS-PAGE. Bands with differential intensity in MPTMV:His6-containing samples compared with empty control samples were excised, in-gel trypsin digested, and submitted to matrixassisted laser-desorption ionization time of flight analysis for identification. Mascot searches were performed in the NCBI 20070324 database to identify proteins corresponding to the measured peptide masses.

Measurement of Fluorescent Infection Sites Photographs of TMV-MPR3:GFP fluorescent infection sites in N. benthamiana leaves transiently expressing RFP in one leaf half and Arabidopsis CDC48B:RFP or CDC48BQQ:RFP in the other leaf half were taken under UV light, and areas of fluorescent infection sites on each leaf half were measured using ImageJ software. Mean values of areas of three to 15 fluorescent infection sites per leaf half were calculated. Mean values of infection site areas on leaf halves expressing CDC48B:RFP or CDC48BQQ:RFP were normalized to mean values of fluorescent infection site areas for the RFP-expressing control leaf half, respectively. Data were acquired in four independent experiments with a total number of 31 and 13 leaves expressing CDC48B:RFP or CDC48BQQ:RFP, respectively.

Supplemental Data The following materials are available in the online version of this article. Supplemental Figure S1. Subcellular localizations of MPTMV:RFP are similar during infection and upon transient expression. Supplemental Figure S2. Expression pattern of the different Arabidopsis CDC48 isoforms. Supplemental Figure S3. Expression of ATP hydrolysis-deficient CDC48B: RFP does not interfere with ER maintenance in the absence of ER stress. Supplemental Figure S4. Coimmunoprecipitation of CDC48B:GFP with MPTMV:RFP using RFP-Trap. Supplemental Figure S5. GFP, CDC48B:GFP, and CDC48BQQ:GFP are expressed during cycloheximide treatment. Supplemental Figure S6. Localization of MPTMV to PD and microtubules depends on the N terminus. Supplemental Table S1. MP-binding proteins in tobacco BY2 cells identified by affinity chromatography using recombinant MPTMV:His6. Supplemental Table S2. Relative expression levels of CDC48 isoforms and ER protein quality control genes in ORMV-infected compared with mock-inoculated Arabidopsis plants revealed by Affymetrix RNA profiling. Supplemental Table S3. Primer sequences. Supplemental Movie S1. N. benthamiana epidermal cell expressing CDC48B:RFP and GFP:HDEL. Supplemental Movie S2. N. benthamiana epidermal cell expressing CDC48BQQ:RFP and GFP:HDEL.

ACKNOWLEDGMENTS We thank Ulrike Eilers for assistance in generating the CDC48B entry clone. We thank Quanan Hu for provision of microarray data, Inmaculada Ferriol for help in establishment of quantitative PCR protocols, and Pascal Didier for assistance with FRET-FLIM measurements. We also thank Eduardo Peña and Camilla Kørner for discussion and Karl Oparka for providing TMVMPR3:GFP plasmid. Received September 15, 2012; accepted September 27, 2012; published October 1, 2012.

LITERATURE CITED Aguilar I, Sánchez F, Martin Martin A, Martinez-Herrera D, Ponz F (1996) Nucleotide sequence of Chinese rape mosaic virus (Oilseed rape mosaic virus), a crucifer tobamovirus infectious on Arabidopsis thaliana. Plant Mol Biol 30: 191–197 2106

Aker J, Borst JW, Karlova R, de Vries S (2006) The Arabidopsis thaliana AAA protein CDC48A interacts in vivo with the somatic embryogenesis receptor-like kinase 1 receptor at the plasma membrane. J Struct Biol 156: 62–71 Aker J, de Vries SC (2008) Plasma membrane receptor complexes. Plant Physiol 147: 1560–1564 Aker J, Hesselink R, Engel R, Karlova R, Borst JW, Visser AJWG, de Vries SC (2007) In vivo hexamerization and characterization of the Arabidopsis AAA ATPase CDC48A complex using forster resonance energy transfer-fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy. Plant Physiol 145: 339–350 Albrecht C, Russinova E, Kemmerling B, Kwaaitaal M, de Vries SC (2008) Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR KINASE proteins serve brassinosteroid-dependent and -independent signaling pathways. Plant Physiol 148: 611–619 Ashby J, Boutant E, Seemanpillai M, Groner A, Sambade A, Ritzenthaler C, Heinlein M (2006) Tobacco mosaic virus movement protein functions as a structural microtubule-associated protein. J Virol 80: 8329–8344 Baluška F, Šamaj J, Napier R, Volkmann D (1999) Maize calreticulin localizes preferentially to plasmodesmata in root apex. Plant J 19: 481–488 Bawden FC, Sheffield FML (1939) The intracellular inclusions of some plant virus diseases. Ann Appl Biol 26: 102–115 Boutant E, Didier P, Niehl A, Mély Y, Ritzenthaler C, Heinlein M (2010) Fluorescent protein recruitment assay for demonstration and analysis of in vivo protein interactions in plant cells and its application to Tobacco mosaic virus movement protein. Plant J 62: 171–177 Boyko V, Ashby JA, Suslova E, Ferralli J, Sterthaus O, Deom CM, Heinlein M (2002) Intramolecular complementing mutations in Tobacco mosaic virus movement protein confirm a role for microtubule association in viral RNA transport. J Virol 76: 3974–3980 Boyko V, Ferralli J, Ashby J, Schellenbaum P, Heinlein M (2000b) Function of microtubules in intercellular transport of plant virus RNA. Nat Cell Biol 2: 826–832 Boyko V, Ferralli J, Heinlein M (2000a) Cell-to-cell movement of TMV RNA is temperature-dependent and corresponds to the association of movement protein with microtubules. Plant J 22: 315–325 Boyko V, Hu Q, Seemanpillai M, Ashby J, Heinlein M (2007) Validation of microtubule-associated Tobacco mosaic virus RNA movement and involvement of microtubule-aligned particle trafficking. Plant J 51: 589–603 Boyko V, van der Laak J, Ferralli J, Suslova E, Kwon MO, Heinlein M (2000c) Cellular targets of functional and dysfunctional mutants of Tobacco mosaic virus movement protein fused to green fluorescent protein. J Virol 74: 11339–11346 Brandner K, Sambade A, Boutant E, Didier P, Mély Y, Ritzenthaler C, Heinlein M (2008) Tobacco mosaic virus movement protein interacts with green fluorescent protein-tagged microtubule end-binding protein 1. Plant Physiol 147: 611–623 Brill LM, Dechongkit S, DeLaBarre B, Stroebel J, Beachy RN, Yeager M (2004) Dimerization of recombinant Tobacco mosaic virus movement protein. J Virol 78: 3372–3377 Brill LM, Nunn RS, Kahn TW, Yeager M, Beachy RN (2000) Recombinant tobacco mosaic virus movement protein is an RNA-binding, a-helical membrane protein. Proc Natl Acad Sci USA 97: 7112–7117 Buchberger A, Bukau B, Sommer T (2010) Protein quality control in the cytosol and the endoplasmic reticulum: brothers in arms. Mol Cell 40: 238–252 Citovsky V, Knorr D, Schuster G, Zambryski P (1990) The P30 movement protein of Tobacco mosaic virus is a single-strand nucleic acid binding protein. Cell 60: 637–647 Cooper B, Lapidot M, Heick JA, Dodds JA, Beachy RN (1995) A defective movement protein of TMV in transgenic plants confers resistance to multiple viruses whereas the functional analog increases susceptibility. Virology 206: 307–313 Crosas B, Hanna J, Kirkpatrick DS, Zhang DP, Tone Y, Hathaway NA, Buecker C, Leggett DS, Schmidt M, King RW, et al (2006) Ubiquitin chains are remodeled at the proteasome by opposing ubiquitin ligase and deubiquitinating activities. Cell 127: 1401–1413 Cui F, Liu L, Zhao Q, Zhang Z, Li Q, Lin B, Wu Y, Tang S, Xie Q (2012) Arabidopsis ubiquitin conjugase UBC32 is an ERAD component that functions in brassinosteroid-mediated salt stress tolerance. Plant Cell 24: 233–244 Curin M, Ojangu E-L, Trutnyeva K, Ilau B, Truve E, Waigmann E (2007) MPB2C, a microtubule-associated plant factor, is required for microtubular Plant Physiol. Vol. 160, 2012

Plant CDC48 Controls Viral Movement Protein Fate

accumulation of Tobacco mosaic virus movement protein in plants. Plant Physiol 143: 801–811 Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible W-R (2005) Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol 139: 5–17 Deom CM, Oliver MJ, Beachy RN (1987) The 30-kilodalton gene product of Tobacco mosaic virus potentiates virus movement. Science 237: 389–394 Di Cola A, Frigerio L, Lord JM, Ceriotti A, Roberts LM (2001) Ricin A chain without its partner B chain is degraded after retrotranslocation from the endoplasmic reticulum to the cytosol in plant cells. Proc Natl Acad Sci USA 98: 14726–14731 Ding B, Haudenshield JS, Hull RJ, Wolf S, Beachy RN, Lucas WJ (1992) Secondary plasmodesmata are specific sites of localization of the Tobacco mosaic virus movement protein in transgenic tobacco plants. Plant Cell 4: 915–928 Eisele F, Schäfer A, Wolf DH (2010) Ubiquitylation in the ERAD pathway. In M Groettrup, ed, Conjugation and Deconjugation of Ubiquitin Family Modifiers. Vol 54. Springer, New York, pp 136–148 Ernst R, Claessen JHL, Mueller B, Sanyal S, Spooner E, van der Veen AG, Kirak O, Schlieker CD, Weihofen WA, Ploegh HL (2011) Enzymatic blockade of the ubiquitin-proteasome pathway. PLoS Biol 8: e1000605 Esau K, Cronshaw J (1967) Relation of Tobacco mosaic virus to the host cells. J Cell Biol 33: 665–678 Feiler HS, Desprez T, Santoni V, Kronenberger J, Caboche M, Traas J (1995) The higher plant Arabidopsis thaliana encodes a functional CDC48 homologue which is highly expressed in dividing and expanding cells. EMBO J 14: 5626–5637 Fujiki M, Kawakami S, Kim RW, Beachy RN (2006) Domains of Tobacco mosaic virus movement protein essential for its membrane association. J Gen Virol 87: 2699–2707 Gafny R, Lapidot M, Berna A, Holt CA, Deom CM, Beachy RN (1992) Effects of terminal deletion mutations on function of the movement protein of Tobacco mosaic virus. Virology 187: 499–507 Gillespie T, Boevink P, Haupt S, Roberts AG, Toth R, Valentine T, Chapman S, Oparka KJ (2002) Functional analysis of a DNA-shuffled movement protein reveals that microtubules are dispensable for the cellto-cell movement of Tobacco mosaic virus. Plant Cell 14: 1207–1222 Harries P, Ding B (2011) Cellular factors in plant virus movement: at the leading edge of macromolecular trafficking in plants. Virology 411: 237–243 He B (2006) Viruses, endoplasmic reticulum stress, and interferon responses. Cell Death Differ 13: 393–403 Heinlein M, Epel BL, Padgett HS, Beachy RN (1995) Interaction of tobamovirus movement proteins with the plant cytoskeleton. Science 270: 1983–1985 Heinlein M, Padgett HS, Gens JS, Pickard BG, Casper SJ, Epel BL, Beachy RN (1998) Changing patterns of localization of the Tobacco mosaic virus movement protein and replicase to the endoplasmic reticulum and microtubules during infection. Plant Cell 10: 1107–1120 Hills GJ, Plaskitt KA, Young ND, Dunigan DD, Watts JW, Wilson TMA, Zaitlin M (1987) Immunogold localization of the intracellular sites of structural and nonstructural Tobacco mosaic virus proteins. Virology 161: 488–496 Hofmann C, Niehl A, Sambade A, Steinmetz A, Heinlein M (2009) Inhibition of Tobacco mosaic virus movement by expression of an actinbinding protein. Plant Physiol 149: 1810–1823 Hong Z, Jin H, Tzfira T, Li J (2008) Multiple mechanism-mediated retention of a defective brassinosteroid receptor in the endoplasmic reticulum of Arabidopsis. Plant Cell 20: 3418–3429 Hu Q, Hollunder J, Niehl A, Kørner CJ, Gereige D, Windels D, Arnold A, Kuiper M, Vazquez F, Pooggin M, et al (2011) Specific impact of tobamovirus infection on the Arabidopsis small RNA profile. PLoS ONE 6: e19549 Kahn TW, Lapidot M, Heinlein M, Reichel C, Cooper B, Gafny R, Beachy RN (1998) Domains of the TMV movement protein involved in subcellular localization. Plant J 15: 15–25 Karimi M, Inzé D, Depicker A (2002) GATEWAY vectors for Agrobacteriummediated plant transformation. Trends Plant Sci 7: 193–195 Kotlizky G, Katz A, van der Laak J, Boyko V, Lapidot M, Beachy RN, Heinlein M, Epel BL (2001) A dysfunctional movement protein of Tobacco mosaic virus interferes with targeting of wild-type movement protein to microtubules. Mol Plant Microbe Interact 14: 895–904 Plant Physiol. Vol. 160, 2012

Kragler F, Curin M, Trutnyeva K, Gansch A, Waigmann E (2003) MPB2C, a microtubule-associated plant protein binds to and interferes with cellto-cell transport of Tobacco mosaic virus movement protein. Plant Physiol 132: 1870–1883 Laliberté J-F, Sanfaçon H (2010) Cellular remodeling during plant virus infection. Annu Rev Phytopathol 48: 69–91 Lapidot M, Gafny R, Ding B, Wolf S, Lucas WJ, Beachy RN (1993) A dysfunctional movement protein of Tobacco mosaic virus that partially modifies the plasmodesmata and limits virus spread in transgenic plants. Plant J 4: 959–970 Liu J-X, Howell SH (2010) Endoplasmic reticulum protein quality control and its relationship to environmental stress responses in plants. Plant Cell 22: 2930–2942 Liu L, Cui F, Li Q, Yin B, Zhang H, Lin B, Wu Y, Xia R, Tang S, Xie Q (2011) The endoplasmic reticulum-associated degradation is necessary for plant salt tolerance. Cell Res 21: 957–969 Lucas WJ (2006) Plant viral movement proteins: agents for cell-to-cell trafficking of viral genomes. Virology 344: 169–184 Mansilla C, Sánchez F, Padgett HS, Pogue GP, Ponz F (2009) Chimeras between Oilseed rape mosaic virus and Tobacco mosaic virus highlight the relevant role of the tobamoviral RdRp as pathogenicity determinant in several hosts. Mol Plant Pathol 10: 59–68 Marshall RS, Jolliffe NA, Ceriotti A, Snowden CJ, Lord JM, Frigerio L, Roberts LM (2008) The role of CDC48 in the retro-translocation of nonubiquitinated toxin substrates in plant cells. J Biol Chem 283: 15869– 15877 Más P, Beachy RN (1999) Replication of Tobacco mosaic virus on endoplasmic reticulum and role of the cytoskeleton and virus movement protein in intracellular distribution of viral RNA. J Cell Biol 147: 945–958 Meusser B, Hirsch C, Jarosch E, Sommer T (2005) ERAD: the long road to destruction. Nat Cell Biol 7: 766–772 Meyer H, Bug M, Bremer S (2012) Emerging functions of the VCP/p97 AAA-ATPase in the ubiquitin system. Nat Cell Biol 14: 117–123 Müller J, Piffanelli P, Devoto A, Miklis M, Elliott C, Ortmann B, SchulzeLefert P, Panstruga R (2005) Conserved ERAD-like quality control of a plant polytopic membrane protein. Plant Cell 17: 149–163 Niehl A, Heinlein M (2011) Cellular pathways for viral transport through plasmodesmata. Protoplasma 248: 75–99 Ouko MO, Sambade A, Brandner K, Niehl A, Peña E, Ahad A, Heinlein M, Nick P (2010) Tobacco mutants with reduced microtubule dynamics are less susceptible to TMV. Plant J 62: 829–839 Padgett HS, Epel BL, Kahn TW, Heinlein M, Watanabe Y, Beachy RN (1996) Distribution of tobamovirus movement protein in infected cells and implications for cell-to-cell spread of infection. Plant J 10: 1079–1088 Padilla-Parra S, Tramier M (2012) FRET microscopy in the living cell: different approaches, strengths and weaknesses. Bioessays 34: 369–376 Peña EJ, Heinlein M (2012) RNA transport during TMV cell-to-cell movement. Front Plant Sci 3: 193 Rancour DM, Dickey CE, Park S, Bednarek SY (2002) Characterization of AtCDC48. Evidence for multiple membrane fusion mechanisms at the plane of cell division in plants. Plant Physiol 130: 1241–1253 Reichel C, Beachy RN (1998) Tobacco mosaic virus infection induces severe morphological changes of the endoplasmic reticulum. Proc Natl Acad Sci USA 95: 11169–11174 Reichel C, Beachy RN (2000) Degradation of Tobacco mosaic virus movement protein by the 26S proteasome. J Virol 74: 3330–3337 Römisch K (2005) Endoplasmic reticulum-associated degradation. Annu Rev Cell Dev Biol 21: 435–456 Ruiz MT, Voinnet O, Baulcombe DC (1998) Initiation and maintenance of virus-induced gene silencing. Plant Cell 10: 937–946 Rumpf S, Jentsch S (2006) Functional division of substrate processing cofactors of the ubiquitin-selective Cdc48 chaperone. Mol Cell 21: 261–269 Sambade A, Brandner K, Hofmann C, Seemanpillai M, Mutterer J, Heinlein M (2008) Transport of TMV movement protein particles associated with the targeting of RNA to plasmodesmata. Traffic 9: 2073– 2088 Schrader EK, Harstad KG, Matouschek A (2009) Targeting proteins for degradation. Nat Chem Biol 5: 815–822 Schuberth C, Buchberger A (2008) UBX domain proteins: major regulators of the AAA ATPase Cdc48/p97. Cell Mol Life Sci 65: 2360–2371 Stolz A, Hilt W, Buchberger A, Wolf DH (2011) Cdc48: a power machine in protein degradation. Trends Biochem Sci 36: 515–523 2107

Niehl et al.

Su W, Liu Y, Xia Y, Hong Z, Li J (2011) Conserved endoplasmic reticulumassociated degradation system to eliminate mutated receptor-like kinases in Arabidopsis. Proc Natl Acad Sci USA 108: 870–875 Su W, Liu Y, Xia Y, Hong Z, Li J (2012) The Arabidopsis homolog of the mammalian OS-9 protein plays a key role in the endoplasmic reticulumassociated degradation of misfolded receptor-like kinases. Mol Plant 5: 929–940 Tilsner J, Linnik O, Wright KM, Bell K, Roberts AG, Lacomme C, Santa Cruz S, Oparka KJ (2012) The TGB1 movement protein of Potato virus X reorganizes actin and endomembranes into the X-body, a viral replication factory. Plant Physiol 158: 1359–1370 Toth RL, Pogue GP, Chapman S (2002) Improvement of the movement and host range properties of a plant virus vector through DNA shuffling. Plant J 30: 593–600 Tsai B, Ye Y, Rapoport TA (2002) Retro-translocation of proteins from the endoplasmic reticulum into the cytosol. Nat Rev Mol Cell Biol 3: 246–255 Tsai YC, Weissman AM (2011) Ubiquitylation in ERAD: reversing to go forward? PLoS Biol 9: e1001038 Waigmann E, Curin M, Heinlein M (2007) Tobacco mosaic virus: a model for macromolecular cell-to-cell spread. In E Waigmann, M Heinlein, eds, Viral Transport in Plants, Vol 7. Springer, Berlin-Heidelberg, pp 29–62

2108

Wolf S, Deom CM, Beachy RN, Lucas WJ (1989) Movement protein of Tobacco mosaic virus modifies plasmodesmatal size exclusion limit. Science 246: 377–379 Wright KM, Wood NT, Roberts AG, Chapman S, Boevink P, Mackenzie KM, Oparka KJ (2007) Targeting of TMV movement protein to plasmodesmata requires the actin/ER network: evidence from FRAP. Traffic 8: 21–31 Yamanaka K, Sasagawa Y, Ogura T (2012) Recent advances in p97/VCP/ Cdc48 cellular functions. Biochim Biophys Acta 1823: 130–137 Ye C, Dickman MB, Whitham SA, Payton M, Verchot J (2011) The unfolded protein response is triggered by a plant viral movement protein. Plant Physiol 156: 741–755 Ye Y, Meyer HH, Rapoport TA (2001) The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol. Nature 414: 652–656 Ye Y, Meyer HH, Rapoport TA (2003) Function of the p97-Ufd1-Npl4 complex in retrotranslocation from the ER to the cytosol: dual recognition of nonubiquitinated polypeptide segments and polyubiquitin chains. J Cell Biol 162: 71–84 Yeung HO, Kloppsteck P, Niwa H, Isaacson RL, Matthews S, Zhang X, Freemont PS (2008) Insights into adaptor binding to the AAA protein p97. Biochem Soc Trans 36: 62–67

Plant Physiol. Vol. 160, 2012