The Plant Cell, Vol. 7, 2101-21 14, December 1995 O 1995 American Society of Plant Physiologists Tobacco Mosaic Virus Movement Protein Associates with the Cytoskeleton in Tobacco Cells 8. Gail McLean, John Zupan, and Patricia C. Zambryskil Department of Plant Biology, University of California-Berkeley, Berkeley, California 94720-3102 Tobacco mosaic virus movement protein P30 complexes with genomic viral RNA for transport through plasmodesmata, the plant intercellular connections. Although most research with P30 focuses on its targeting to and gating of plasmodes- mata, the mechanisms of P30 intracellularmovement to plasmodesmata have not been defined. To examine P30 intracellular localization, we used tobacco protoplasts, which lack plasmodesmata, for transfection with plasmids carrying P30 cod- ing sequences under a constitutive promoter and for infection with tobacco mosaic virus particles. In both systems, P30 appears as filaments that colocalize primarily with microtubules. To a lesser extent, P30 filaments colocalize with actin filaments, and in vitro experiments suggested that P30 can bind directly to actin and tubulin. This association of P30 with cytoskeletal elements may play a critical role in intracellular transport of the P30-vira1 RNA complex through the cytoplasm to and possibly through plasmodesmata. INTRODUCTION To establish a systemic infection, plant viruses must move from that the cytoskeleton acts as a trafficking system for intracel- the infection site to the rest of the plant. For many plant viruses, lular transport, translocating vesicles, organelles, protein, and a virus-encoded product, the movement protein, actively poten- even mRNA to specific cellular locations (Williamson, 1986; tiates viral cell-to-cell spread through plasmodesmata, the Vale, 1987; Dingwall, 1992; Singer, 1992; Wilhelm and Vale, cytoplasmic bridges that function as intercellular connections 1993; Bassell et al., 1994; Hesketh, 1994). An intriguing pos- (Gibbs, 1976; reviewed in Deom et al., 1992; Citovsky and sibility is that directional movement of the P30-RNA complex Zambryski, 1993; McLean et al., 1993; Lucas and Gilbertson, through the cytoplasm occurs on cytoskeletal components. 1994). One of the most extensively studied viral movement pro- In a somewhat analogous situation, many animal viruses teins is the P30 protein of tobacco mosaic virus (TMV), a spread through the host cell by interacting with host cell relatively simple, positive sense, single-stranded RNA virus. cytoskeletal components. Specifically, the microtubule network P30 (also termed TMV MP) is proposed to form a complex with appears to play an important role in viral protein trafficking the genomic TMV RNA, to target this protein-nucleic acid com- and distribution in animal cells (Pasick et al., 1994). For ex- plex to plasmodesmata, and to transport it through the now- ample, the viral matrix protein of vesicular stomatitus virus enlarged, open plasmodesmata (Citovsky and Zambryski, associates with tubulin in vitro and in vivo; the in vitro data 1991). Virtually all research on P30-mediated cell-cell move- show that this viral protein can bind to both polymerized and ment focuses on the interaction, that is, targeting and gating, unpolymerized tubulin (Melki et al., 1994). In sensory neurons, with plasmodesmata. Yet, synthesis of P30, replication of TMV the transport of herpes simplex virus occurs in a plus-minus RNA, and presumably, formation of the P3O-vira1 RNA complex direction on microtubules, suggesting that directional trans- all occur in the host cell cytoplasm. Therefore, the P3O-RNA port of herpes simplex virus is mediated by a minus end- complex must move through the cytoplasm to the plasmo- directed motor, such as cytoplasmic dynein (Topp et al., 1994). desmata. Like the animal cytoskeleton,the plant cytoskeleton is com- The mechanism by which P30 moves intracellularly to reach posed of filamentous networks of actin, tubulin (microtubules), the plasmodesmata is not known. Since the protein content and intermediate filaments. Although the plant cytoskeleton and organized nature of the cytoplasm probably restrict diffu- and its components are not as well characterized as those in sion of large molecular complexes, such as protein-nucleic animals, experimental data and evolutionary conservation of acid complexes (Luby-Phelps, 1993, 1994), movement of the the cytoskeletal proteins suggest that both the general mech- P30-RNA complex to plasmodesmata most likely is not by pas- anisms and the functions of the cytoskeleton are conserved sive diffusion. Along these lines, numerous studies suggest between animals and plants (reviewed in Lloyd, 1982; Staiger and Lloyd, 1991; Shibaoka and Nagai, 1994). Thus, both plants To whom correspondence should be addressed and animals may use cytoskeletal filaments and motor proteins 2102 The Plant Cell Figur . Filamentoue1 s Appearanc f Transientleo y Expressed Wild-Typ Tobaccn i 0 eP3 o Protoplasts P30 was detected by affinity-purified P30 polyclonal antibody and fluorescein-conjugated goat anti-rabbit secondary antibody. Arrows in (B), ) denot (E aggregates 0 d (D)eP3 an , . (A) to (C) P30 in aldehyde-fixed protoplasts. (D) and (E) P30 in a detergent-permeabilized unfixed protoplasts. (F) P30 sb-6 mutant (Citovsky et al., 1993) in an aldehyde-fixed protoplast. (F)o t .) (A urr 0 1 nfo = ) (F Ba n i r to move macromolecular complexes, such as ribonucleopro- RESULTS tein particles additionn I . , evolutionary studies suggest that viruses act as scavengers, exploiting host cellular genes and processes and adapting them for the viruses' life cycle (Haseloff Expressio Protoplastn i 0 P3 f no s et al., 1984; Zimmern, 1988; Morozov et al., 1989; Citovsky, 1993; Kooni Doljad nan , 1993). Hypothetically virae ,th l P30- To examine expressio tobaccn i TMf no 0 VP3 o protoplastse ,th RNA complexes may mimic ribonucleoprotein particles and P30 coding region was cloned into pRTL2, a plant expression use the cytoskeleton as a highway through the cytoplasm to vector containin cauliflowee gth r mosaic virus (CaMVS )35 the plasmodesmata. promoter and the untranslated leader of tobacco etch virus To begin characterizing the process of P30 movement within (Restrep t al.oe , 1990). Consequently alon0 P3 ,produce es i d througd an plane hth t cell cytoplasm examinee ,w intrae dth - in the cell, thus separating P30 expression from other TMV cellular localization of P30 in tobacco cells. P30 must be proteins. Protoplasts were prepared from tobacco cell suspen- confine singla o dt e celsucr fo l h studies preveno .T t transport sion cultures, electroporated with plasmid DNA, and incubated celle th ,f o plasmodesmat t ou 0 oP3 f a absenneee b o dt r o t 18 to 20 hr to allow the expression of the introduced DNA. P30 nonfunctional (Deo t mal.e , 1987; Mesh t al.e i , 1987; Moser was detected by affinity-purified anti-P30 polyclonal antibod- et al., 1988). Consequently, tobacco protoplasts were used. ies, followed by fluorescein-conjugated secondary antibody. Protoplast idean a e l ssystear studyinr mfo g intracellula- lo r Fluorescenc t detecteno s ewa untransfecten d i d protoplasts calizatio proteinsf no protoplase Th . t itsel singla s i f e celld ,an or with preimmune serum. importantly procese ,th removinf so cele gth l wall severs plas- As shown in Figure 1, when expressed alone in protoplasts, modesmata. Thus, P30 is restricted to a single cell, allowing appear0 P3 filamentoua s a s s network reminiscene th f o t cytoplase th n analysii 0 mP3 f beforso e targetin plasmodesgo t - cytoskeleton in both fixed (Figures 1A to 1C) and unfixed mata. Here, we show that P30 forms a filamentous network (Figures 1D and 1E) protoplasts. The appearance of P30 var- that is coincident with the cytoskeleton in tobacco protoplasts. ied from very dense, fine filaments (Figures 1A and 1C) to less Cytoskeletal Associatio f TMno 0 VP3 2103 dense, thicker filaments (Figures 1B, 1D, and 1E), depending To confirm that the localization of P30 in the transient assay particulaoe nth r protoplast being examined. Protoplasts per- reflected localizatio virus-encodef no d P30, virus-infected pro- meabilized or fixed without DMSO also showed both fine and toplasts were examined. Protoplasts, prepared from tobacco thick filaments, indicating that the presence of DMSO did not suspension cells, were inoculated with TMV in the presence cause bundling of the P30 filaments (data not shown). In some of polyethylene glycol (Maul t al.ee , 1980 incubated )an r dfo protoplasts, small aggregate coul0 seee P3 d b f nso associated 10 halloo rt w expressio P30f no . Previous studies have shown with the P30 filaments. To confirm that the filamentous net- that, in both inoculated tobacco protoplasts and infected cells work results from a specific activity of P30 and not simply from of intact tobacco leaves, P30 accumulates to maximal amounts its overexpression substitutioa , n mutan f P30to , sb-6 alss o,wa durin earle gth y infectiostageV TM f so n (Kibersti al.t se , 1983; transfecte expressed dan d in tobacco protoplasts (Citovskt ye Watanab t al.ee , 1984; Blu t mal.e , 1989; Leht t al.oe , 1990). al., 1993). Although wild-type P30, a tenacious single-stranded In an electron microscopy study, Meshi et al. (1992) found P30 nucleic acid binding protein, bind singled an s A bot- hRN nea nucleue th r TMV-infecten si d protoplasts; however, dur- stranded DNA (Citovsky et al., 1990), sb-6 binds RNA but no ing later stages of virus infection (15 to 24 hr after inoculation), longer binds single-stranded DNA; sb- phose 6b alsn - oca observeP3s 0wa confinet da d cytoplasmareae th r n si ou n I . phorylate cele th l y wall-associatedb d kinase describen di studies, fluorescence microscopy has been used because it Citovsk . (1993)al t ye shows A . Figurn i , sb-e1F 6 doet sno permits gentler and potentially less disruptive fixation of P30- form the filamentous network typical of wild-type P30 in pro- expressing cells.