Proc. Natl. Acad. Sci. USAVol. 93, pp. 6286-6290, June 1996Plant Biology

Assembly and movement of a plant virus carrying a greenfluorescent protein overcoatSIMON SANTA CRUZ*, SEAN CHAPMAN, ALISON G. ROBERTS, IAN M. ROBERTS, DENTON A. M. PRIOR,AND KARL J. OPARKAScottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, United Kingdom

Communicated by Robert J. Shepherd, University of Kentucky, Lexington, KY March 11, 1996 (received for review October 30, 1995)

ABSTRACT Potato virus X (PVX) is a filamentous plantvirus infecting many members of the family Solanaceae. Amodified form of PVX, PVX.GFP-CP which expressed achimeric gene encoding a fusion between the 27-kDa Aequoreavictoria green fluorescent protein and the amino terminus ofthe 25-kDa PVX coat protein, assembled into virions andmoved both locally and systemically. The PVX.GFP-CP yri-ons were over twice the diameter of wild-type PVX virions.Assembly of PVX.GFP-CP virions required the presence offree coat protein subunits in addition to the fusion proteinsubunits. PVX.GFP-CP virions accumulated as paracrystal-line arrays in infected cells similar to those seen in cellsinfected with wild-type PVX. The formation ofvirions carryinglarge superficial fusions illustrates a novel approach forproduction of high levels of foreign proteins in plants. Aggre-gates of PVX.GFP-CP particles were fluorescent, emittinggreen light when excited with ultraviolet light and could beimaged using confocal laser scanning microscopy. The detec-tion of virus particles in infected tissue demonstrates thepotential of fusions between the green fluorescent protein andvirus coat protein for the non-invasive study of virus multi-plication and spread.

Plant virus-based vectors have been exploited for severaldifferent purposes, most commonly viral genomes modified toexpress marker proteins have been used to investigate viralpathology (1-3). Tagged viral genomes allow the localizationof virus-infected cells and have been used to study the phe-notypes of mutant viral genomes (4, 5). Another potential useof virus-based vectors is for the production of valuable pep-tides and proteins in plants. The high levels to which manyviruses and virus-encoded proteins accumulate, the avoidanceof costly fermentation systems and the flexibility in the scaleof production afforded by plants are possible advantages ofvirus-mediated protein production in plants (6).

Attempts to exploit viral vectors for overproduction offoreign proteins in plants have relied either on free proteinproduction or the production of fusions between the foreignprotein and the viral coat protein (CP). The former approach,exemplified by work on tobacco mosaic virus (TMV), is basedon the insertion of an additional open reading frame under thetranscriptional control of a viral subgenomic RNA promoter(7, 8). For rod-shaped viruses, such as potato virus X (PVX)and TMV, no apriori constraint on the size of the inserted geneis imposed by packaging. However, where the protein encodedby the inserted gene is required in a purified state, it must beextracted from infected tissue and isolated from other cellularmaterials. The second approach, using fusions to the viral CP,has been developed for both TMV (9, 10) and the icosahedralvirus cowpea mosaic virus (11, 12). An advantage of thisapproach is the relative ease with which modified viral particlescan be isolated from infected tissues and the fact that the

recombinant protein is stabilized by attachment to a macro-molecular carrier.The structures ofTMV and cowpea mosaic virus have been

determined to atomic resolution (13, 14) allowing the designof fusion proteins that carry modifications to either internal orterminal sequences of the CP that are predicted to lie at thesurface of the assembled virion. TMV CP-fusion proteinscarrying either a 7-amino acid extension to the carboxy ter-minus or a 12-amino acid insertion in a surface loop of the CPhave been shown to assemble into virions (9, 15). Othermodifications to the carboxy terminus of theTMV CP howeverhave proved to be assembly defective. This limitation has beenovercome, for peptides of up to 21 amino acids, by exploitingthe TMV 130-kDa protein read-through motif that determinesthe suppression of an amber termination codon in the TMVpolymerase reading frame (16). Insertion of a suppressiblestop codon between the end of the CP open reading frame anda carboxyl-terminal extension allows the generation of a mixedpool of free and fused CP subunits that can assemble intovirions comprising heterologous subunits (15, 17). The limitedsize of peptides that can be fused to the CPs of TMV andcowpea mosaic virus, while retaining the ability to assembleinto virions, has restricted these systems to expression ofpeptide immunogens (11, 15, 17) and a peptide hormone (10).The 27-kDa Aequorea victoria green fluorescent protein

(GFP) (18), expressed as a free protein, has been describedpreviously as a reporter for PVX infections (5). Here we reporton a genetically modified PVX expressing a fusion between theGFP and the 25-kDa CP that assembles into virus particles andmoves systemically in infected plants.

MATERIALS AND METHODSPlasmid Constructions. pTXS.GFP (5) a modified form of

the PVX cDNA clone pTXS (19) carries the GFP cDNA underthe transcriptional control of a duplicated subgenomic RNApromoter. pTXS.GFP was used as template to produce thegfp-2a-cp fusion gene by overlap extension PCR (20) usingflanking oligonucleotides complementary to the PVX genomeand mutagenic oligonucleotides to incorporate the foot-and-mouth disease virus (FMDV) 2A oligopeptide coding se-quence. The GFP termination codon, the intergenic regionand first nine nucleotides of the PVX CP in pTXS.GFP werereplaced with an in-frame sequence encoding 16 amino acidsof the 2A oligopeptide (NFDLLKLAGDVESNPG) (21). Am-plified product was subcloned into pTXS.GFP, as a 1.5-kbpfragment, using the unique restriction sites EagI and XhoI togive pTXS.GFP-CP. A derivative of pTXS.GFP-CP carryinga 3-amino acid substitution within the 2A oligopeptide codingsequence, pTXS.GFP-CPmut2A, was prepared by PCR ampli-fication of pTXS.GFP-CP by using a mutagenic oligonucleo-

Abbreviations: CP, coat protein; FMDV, foot-and-mouth diseasevirus; GFP, green fluorescent protein; PVX, potato virus X; TMV,tobacco mosaic virus.*To whom reprint requests should be addressed.

6286

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

Dow

nloa

ded

by g

uest

on

Oct

ober

1, 2

020

Proc. Natl. Acad. Sci. USA 93 (1996) 6287

tide and a downstream oligonucleotide complementary to thePVX 3'-untranslated region. The amplified product was di-gested with Aflll and XhoI and used to replace the corre-sponding sequence in pTXS.GFP-CP. The mutant 2A oli-gopeptide encoded by pTXS.GFP-CPmut2A was NFDLLK-LGAAVESNPG, with the substituted residues indicated inboldface type.

In Vitro Transcription and Plant Inoculation. Plasmids werelinearized with SpeI before in vitro transcription reactions.Transcripts were synthesized using bacteriophage T7 RNApolymerase as described previously (19). In vitro transcriptswere inoculated directly to young leaves of either Nicotianabenthamiana or Nicotiana tabacum cv Petite Havana by rub-bing aluminium oxide dusted leaves. Two leaves were inocu-lated per plant, and each leafwas inoculated with the transcriptproducts derived from 0.2 ,g plasmid template.

Detection of Fluorescence. Leaves were viewed under UVillumination (365 nm) generated from a Blak Ray B100-APlamp (Ultraviolet Products, San Gabriel, CA) and photo-graphed by using a Wratten 58 filter to eliminate chlorophyllautofluorescence. For confocal imaging, leaves were excisedfrom the plant and sectioned transversely into 200-,um slices byusing a vibrotome. The sections were immediately mounted inwater and viewed under a Bio-Rad MRC 1000 confocal laserscanning microscope as described (5). The conditions used forconfocal microscopy, excitation at 488 nm using a krypton-argon laser and a 522 nm emission filter, allowed detection ofGFP-mediated fluorescence with no significant autofluores-cence.

Transgenic Plants Expressing the PVX Coat Protein. Trans-genic tobacco lines expressing the PVX CP were prepared byleaf disc transformation of N. tabacum cv Petite Havana. PCRamplification of PVX CP cDNA from pTXS template wasperformed by using an upstream mutagenic oligonucleotidedesigned to insert an NcoI recognition site at the CP initiationcodon and a downstream oligonucleotide 3' to the viral cDNAsequence. The amplified product was digested with NcoI andXbaI and used to replace the f3-glucuronidase coding sequencein the shuttle vector pSLJ4D4 (22). The resultant plasmid,p4D4UK3, carried the PVX CP coding sequence and 3'-untranslated region positioned between the cauliflower mosaicvirus 35S promoter and the octopine synthase 3' terminator.The promoter-CP-terminator cassette in p4D4UK3 was iso-lated by digestion with EcoRI and HindIII and cloned into thebinary vector pSLJ4654A (22) to give p35UK3. Agrobacteriumtumefaciens strain LBA4404 was transformed with p35UK3 byelectroporation (23). Transformation of tobacco leaf discs andregeneration of transgenic plants by cocultivation with A.tumefaciens and selection on media containing kanamycin wasas described by Horsch et al. (24). The transgenic plants usedfor viral inoculations were the F1 progeny of a CP expressingline; individual plants were checked for the presence of PVXCP by probing Western blots of plant protein extracts withantiserum to the PVX CP.

Preparation of Anti-GFP Antiserum. Recombinant GFP,prepared from yeast, was diluted to a concentration of 1 mg/mlin Tris*HCl (pH 8.0) and mixed with an equal volume ofFreund's incomplete adjuvant. Intramuscular injection of aNew Zealand White rabbit was performed at two weeklyintervals by using 1 ml of immunogen. Serum was preparedfrom sample bleeds collected 4 and 5 weeks after the firstimmunization and used for immunoblot and immunogoldanalyses as described below.Immunoblot Analysis. Protein extracts were prepared by

grinding leaf tissue in two volumes (wt/vol) protein extractionbuffer (25) to which an equal volume of 2x SDS load bufferwas added, and the extracts were boiled for 2 min beforesamples were loaded on SDS/polyacrylamide gels. Proteinswere electrophoresed, blotted to nitrocellulose, and probed

with rabbit polyclonal antiserum, raised against either the PVXCP or the GFP, as described by Davies et al. (25).Sample Preparation for Electron Microscopy. Leaf tissues

were fixed and embedded in Araldite resin for immunogoldlabeling (26). Ultrathin sections on nickel grids were labeled byusing polyclonal rabbit antiserum to either the PVX CP or theGFP followed by goat anti-rabbit gold conjugate (GAR-15 nm;Amersham). For negative staining, virus particles weretrapped from virus-infected sap extracts by immunosorbentelectron microscopy (27) by using anti-PVX CP antiserum, andstained with 2% sodium phosphotungstate (pH 7).

RESULTS AND DISCUSSIONTwo factors influenced the design of a PVX-based CP fusionvector that would assemble and move. First, although there isno crystallographic data available for PVX, structural (28) andserological (29) evidence suggest that the surface of the virionis formed from the amino terminus of the CP. Therefore afusion was made between the carboxy terminus of the GFP andamino terminus of the PVX CP. Second, because the GFP andPVX CP are of similar sizes, having molecular weights of 26.9kDa (18) and 25.1 kDa (30), respectively, it was expected thatin a homogeneous population of fusion protein steric effectswould prevent virion formation. Assembly of fusion proteininto virions might be facilitated by the presence of a pool offree CP as has been demonstrated for carboxy-terminallymodified forms of the TMV CP (10).To generate free and fused forms of the PVX CP, we

developed a strategy based on the FMDV 2A peptide. TheFMDV 2A peptide mediates a processing event between the2A and 2B regions of the FMDV polyprotein cleaving betweenthe carboxy-terminal glycine residue of the 2A peptide and theamino terminal proline residue of the 2B protein (21). The16-amino acid 2A sequence has previously been demonstratedto result in the processing of a synthetic polyprotein both invitro and in vivo (31). 2A-mediated processing requires aproline residue following the 2A sequence (M. Ryan, personalcommunication); thus in the plasmid pTXS.GFP-CP (Fig. 1A)the 2A sequence was fused between the last codon of the GFPgene and the fourth codon of the CP gene, which encodes thefirst proline in the PVX CP.

Processing of the FMDV polyprotein at the 2A/2B junctionis highly efficient; however, processing of a synthetic ,3-gluc-uronidase-2A-chloramphenicol acetyltransferase polyproteinwas incomplete resulting in the production of unprocessedpolyprotein in addition to the processed products, ,B-glucuron-idase-2A and chloramphenicol acetyltransferase (31). Al-though the mechanism by which 2A-mediated processingoccurs is not known the available evidence indicates thatprocessing is entirely cotranslational as unprocessed polypro-tein products do not undergo subsequent cleavage (31). Hencetranslation of the gfp-2a-cp gene was expected to result in theaccumulation of a GFP-2A-CP fusion protein, a GFP-2Afusion protein and PVX CP lacking the first 3 amino acids.

In vitro run-off transcripts, synthesized from pTXS.GFP-CP, were infectious when inoculated to plants; virus derivedfrom transcript inoculation is subsequently indicated by sub-stitution of the prefix PVX for the prefix pTXS in the name ofthe progenitor plasmid. Following inoculation of either N.benthamiana or N. tabacum, PVX.GFP-CP caused the devel-opment of green fluorescent lesions which were first detect-able by eye under UV illumination between 2 and 3 dayspostinoculation. Subsequent long-distance movement of thevirus to developing leaves led to the appearance of greenfluorescence in systemically infected leaves (Fig. 2A). The rateat which fluorescent lesions spread on inoculated leaves wasslower in PVX.GFP-CP-infected plants than plants infectedwith the previously described GFP expression vectorPVX.GFP (5), and the appearance of fluorescence in system-

Plant Biology: Santa Cruz et aL

Dow

nloa

ded

by g

uest

on

Oct

ober

1, 2

020

6288 Plant Biology: Santa Cruz et al.

A

TIS

TIs WY

237a.

1~~~~~~~~~I Is" --la | m roI.ila 237

I~~~~~~~~~~~r- --tIr--RA:::--,

* ~ ~ ~ 2"13M*3

B CWe

66.0 -10

kDa

21.5 -+1 2 3 4 5

FIG. 1. Construction and analysis of gfp-2a-cp fusion gene. (A)Schematic representation of viral cDNAs used to synthesize infectiousrun-off transcripts. Boxes represent coding sequences. The predictedMr values of the four viral proteins common to all constructs areindicated (K = kDa). The polypeptide chain lengths of the CP, theGFP and the 2A oligopeptide (2A) encoded by the constructs are

shown. Bars indicate the position of the subgenomic promoter for theCP. TXS, wild-type PVX; TXS.GFP, PVX modified to express freeGFP from a duplicated subgenomic promoter; TXS.GFP-CP, PVXmodified to express the GFP-2A-CP fusion protein. (B and C)Immunoblot analysis of extracts from systemically infected N.benthamiana leaves probed with either anti-CP antiserum (B) or

anti-GFP antiserum (C). Protein was prepared from mock-inoculatedcontrol plants (lane 2) or from plants inoculated with in vitro tran-scripts synthesized from plasmid DNAs (TXS, lane 3; TXS.GFP, lane4; TXS.GFP-CP, lane 5). Lane 1 contains 80 ng of either PVX CP (B)or histidine-tagged GFP (C). Mr values of native CP and GFP are 25.1kDa and 26.9 kDa, respectively. The Mr of the GFP-2A-CP fusion is53.2 kDa, and the predicted Mr values of the GFP-2A and CP releasedfollowing 2A-mediated processing are 28.4 kDa and 24.8 kDa, respec-tively. The Mr values of standards (X 10-3) are shown on the left.

ically infected leaves was retarded in plants infected withPVX.GFP-CP- compared with PVX.GFP-infected plants. Thereasons for the slower spread of PVX.GFP-CP compared withPVX.GFP are not known, however a requirement for CP incell-to-cell movement ofPVX has been reported previously (1,5, 32). The impaired movement may result from interferenceby the fusion protein either in virion assembly or in some directrole of the CP in cell-to-cell movement.

In systemically infected leaves, PVX.GFP-CP moved fromthe phloem into surrounding bundle sheath and mesophyllcells and eventually into the epidermis. Under the confocalmicroscope transverse sections of the systemically infectedleaves showed that, in PVX.GFP-CP-infected cells, greenfluorescence was detected predominantly in large, fibrillaraggregates within infected cells (Fig. 2B). By contrast, inPVX.GFP-infected cells, the green fluorescence is associatedwith nuclei and shows a relatively uniform distributionthroughout the cytoplasm (5).Western blotting of protein extracts from inoculated N.

benthamiana leaves was carried out to demonstrate that thefusion protein was produced and processed. Probing withCP-specific antiserum showed that most of the immunoreac-

FIG. 2. Detection of green fluorescence in tissues and cells infectedwith modified forms of PVX that express the GFP. (A) Leaf ofsystemically infected N. benthamiana plant 14 days postinoculationwith PVX.GFP-CP showing green fluorescence associated predomi-nantly with the leaf veins. (B) Confocal image of palisade cell from aleaf systemically infected with PVX.GFP-CP showing the GFP-containing virus particles assembled into fibrillar structures. (Bar = 10,um.) (C) Confocal image of inoculated N. tabacum leaf infected withPVX.GFP-CPmut2A 7 days postinoculation, green fluorescence isrestricted to a single epidermal cell, no autofluorescence is seen in thesurrounding uninfected epidermal cells. (Bar = 50 ,im.) (D) Confocalimage of inoculated leaf of PVX CP transgenic N. tabacum 7 dayspostinoculation showing cell-cell movement of PVX.GFP-CPmut2Ahas occurred. (Bar = 100 jim.)

tive protein in PVX.GFP-CP infected plants comprised thefusion protein (Fig. 1B). A lower level of a smaller immuno-reactive protein was also detected. This protein, which mi-grated slightly faster than wild-type PVX CP, represents theprocessed form ofPVX CP that lacks the three amino-terminalamino acids. A similar pattern was observed on blots probedwith GFP-specific antiserum (Fig. 1C). The smaller proteinreacting with the GFP antiserum in PVX.GFP-CP-infectedtissue migrated more slowly than the wild-type GFP producedin PVX.GFP infected plants due to the presence of the16-amino acid 2A peptide at the carboxy terminus of the GFP.A similar ratio of free protein to fusion protein was observedin all other samples analyzed, and reverse transcription-PCRanalysis, using the same tissue samples used for proteinanalysis, showed no evidence of deleted forms of the viralgenome (data not shown).The distribution of fluorescence, observed under the con-

focal laser scanning microscope, suggested that the majority ofGFP produced in PVX.GFP-CP-infected plants was still fusedto the CP and that these fusion proteins were assembling intovirions, which subsequently aggregated into the fibrillar struc-tures observed in infected cells (Fig. 2B). To confirm that viruswas formed and that the fluorescent structures observed underthe confocal microscope were composed of virus particles,rather than nonspecific aggregates containing fluorescentprotein, ultrathin sections of inoculated leaves were preparedfor immunogold labeling and probed with polyclonal antibodyto either the PVX CP or the GFP. Dense gold labeling, insections of PVX.GFP-CP-infected tissue, probed with eitherthe PVX CP or GFP antisera, was predominantly associatedwith virus particles aligned side-by-side in bundles (Fig. 3A andB). When viewed at lower magnification, the bundles ofPVX.GFP-CP particles were seen to be connected end-to-end,forming elongated structures (data not shown). These struc-tures are believed to correspond to the fibrillar aggregates seenunder the confocal microscope.Comparison of negatively stained virus particles under the

electron microscope revealed that PVX.GFP-CP virions hada different morphology to wild-type PVX virions. This differ-

66.0 -*

45.0 -*

31.0 -_ am

w4.0 -

31.0 -* * 4'

21.5 -.1 2 3 4 5

Proc. Natl. Acad. Sci. USA 93 (1996)

Dow

nloa

ded

by g

uest

on

Oct

ober

1, 2

020

Proc. Natl. Acad. Sci. USA 93 (1996) 6289

FIG. 3. (A and B) Electron microscope images of mesophyll cells infected with PVX.GFP-CP. (Bar = 200 nm.) The sections were probed withpolyclonal antibodies to either the PVX CP (A) or the GFP (B) and show the aggregates of the filamentous virions. (C and D) Negatively stainedvirus particles isolated from leaf tissue systemically infected with virus. (Bar = 50 nm.) (C) PVX.GFP virions. (D) PVX.GFP-CP virions. Virionsproduced in PVX.GFP-CP-infected plants were over twice the diameter of virions assembled from free CP. This difference in diameter is seenmost clearly where virions are aligned side-by-side (arrowheads indicate the outer edges of adjacent virus particles).

ence is seen as a diffuse staining at the edges of PVX.GFP-CPvirions (Fig. 3D) which masks the discrete edges seen in thePVX.GFP particles (Fig. 3C). The PVX.GFP-CP virions hada mean diameter of 29.7 nm, more than twice the diameter ofPVX.GFP virions (12.6 nm). This difference in diameter ismost obvious when particles aligned side-by-side are compared(Fig. 3 C and D; arrowheads).To test whether the presence of free CP subunits was

essential for assembly of virions containing fusion protein amodified form of PVX.GFP-CP was constructed with threeamino acid substitutions within the FMDV 2A peptide. Thismutant, termed PVX.GFP-CPmut2A, was designed to be de-fective in processing of the polyprotein and produce only theGFP-2A-CP fusion protein. Infections of N. benthamiana andN. tabacum with this mutant were restricted to single epider-mal cells (Fig. 2C). Green fluorescence in PVX.GFP-CPmut2A_infected cells was distributed throughout the cytoplasm andalso localized to cytoplasmic aggregates. When viewed at highmagnification these aggregates showed no obvious substruc-ture and lacked the fibrillar appearance of the fluorescentaggregates induced by PVX.GFP-CP (Fig. 2B). It is believedthat the fluorescent aggregates seen in PVX.GFP-CPmut2A-infected cells represent localization of the fluorescent proteinto the virus induced inclusion bodies which are found in PVXinfected cells (25). The absence of the fibrillar aggregates ofvirus particles in PVX.GFP-CPmut2A-infected cells suggestedthat the presence of free CP was essential for either initiationor elongation of virions. To confirm this hypothesis

PVX.GFP-CPmut2A was inoculated to transgenic N. tabacumthat expressed the PVX CP. On the transgenic tobacco plantsthe movement deficient phenotype of the mutant was rescued(Fig. 2D). The presence of assembled virus in PVX.GFP-CPmut2A-infected transgenic tobacco was confirmed by immu-nogold labeling of sections from infected tissue probed withanti-PVX CP antibodies (data not shown). Thus assembly ofthe fusion protein encoded by PVX.GFP-CP into virionsappears to depend on the presence of free CP subunits. Theresults demonstrate that both of the strategies employed togenerate a heterologous CP subunit population, partial co-translational processing of a polyprotein and transgenic CPcomplementation, are able to generate sufficient free CP forvirus assembly to occur.The ability of a plant RNA virus to assemble into virions

coated with a foreign protein offers the possibility of usingplant RNA viruses as vectors for the production of a widerange of proteins. For some applications, for example the useof virus decorated with an immunogenic protein as a vaccine,purified virus could be used without further treatment. How-ever, where the attached protein is required free from the viruscarrier, it will be necessary to detach the foreign protein fromthe fusion protein. Separation of the foreign protein frompurified particles could be achieved by engineering an endo-protease target sequence upstream of the 2A peptide to enableproteolytic release of the foreign protein. Previous descrip-tions of assembly competent plant RNA viruses carrying CPextensions have involved small oligopeptide fusions (12, 15,

Plant Biology: Santa Cruz et aL

T. 4,

0. ,

Dow

nloa

ded

by g

uest

on

Oct

ober

1, 2

020

6290 Plant Biology: Santa Cruz et al.

17). The data presented here suggest that the system describedcould be used for the production of proteins that are at leastas large as the PVX CP. The high level of accumulation andease of virion purification make PVX ideally suited for thispurpose. The general utility of the approach described here,for assembly of fusion protein-containing virions, requires thatproteins other than the GFP can be fused to the PVX CP andretain the ability to assemble into virions and move systemi-cally. Fusions between the PVX CP and neomycin phospho-transferase II (31 kDa), chloramphenicol acetyltransferase(25.6 kDa), and 13-galactosidase (8.5 kDa) have all resulted inassembly and movement competent viruses (S.C. and S.S.C.,unpublished data), indicating that assembly of PVX.GFP-CPvirions does not reflect a unique attribute of the GFP-CPsubunit.

In addition, the noninvasive detection of virus particleswithin individual living cells under the confocal microscopewill be of practical benefit in studying the local and longdistance movement of PVX.

We are grateful to Michael Wilson for advice and guidance, DavidBaulcombe for providing the plasmids pTXS and pTXS.GFP and toJonathan Jones for the plasmids pSUJ4D4 and pSLJ4654A. The giftsof recombinant GFP, provided by Jim Haseloff, and anti-PVX anti-serum, provided by Lesley Torrance, are gratefully acknowledged. Wethank Kate Harrison for assistance with tobacco transformation andGraham Cowan for preparing the anti-GFP antiserum. This work wassupported by the Scottish Office Agriculture Environment and Fish-eries Department. A.G.R. is in receipt of a studentship from DundeeUniversity.

1. Chapman, S. N., Kavanagh, T. & Baulcombe, D. C. (1992) PlantJ. 2, 549-557.

2. Dolja, V. V., McBride, H. J. & Carrington, J. C. (1992) Proc.Natl. Acad. Sci. USA 89, 10208-10212.

3. Scholthof, H. B., Morris, T. J. & Jackson, A. 0. (1993) Mol.Plant-Microbe Interact. 6, 309-322.

4. Dolja, V. V., Haldeman, R., Robertson, N. L., Dougherty, W. G.& Carrington, J. C. (1994) EMBO J. 13, 1482-1491.

5. Baulcombe, D. C., Chapman, S. N. & Santa Cruz, S. (1995) PlantJ. 7, 1045-1053.

6. Lomonossoff, G. P. (1995) Agro Food Ind. Hi Tech. 6, 7-11.7. Donson, J., Kearney, C. M., Hilf, M. E. & Dawson, W. 0. (1991)

Proc. Natl. Acad. Sci. USA 88, 7204-7208.8. Kumagai, M. H., Turpen, T. H., Weinzettl, N., della-Cioppa, G.,

Turpen, A. M., Donson, J., Hilf, M. E., Grantham, G. L., Daw-son, W. O., Chow, T. P., Piatak, M. & Grill, L. K. (1993) Proc.Natl. Acad. Sci. USA 90, 427-430.

9. Takamatsu, N., Watanabe, Y., Yanagi, H., Tadayoshi, S. &Okada, Y. (1990) FEBS Lett. 269, 73-76.

10. Hamamoto, H., Sugiyama, Y., Nakagawa, N., Hashida, E., Mat-sunaga, Y., Takemoto, S., Watanabe, Y. & Okada, Y. (1993)Bio-Technology 11, 930-932.

11. Usha, R., Rohll, J. B., Spall, V. E., Shanks, M., Maule, A. J.,Johnson, J. E. & Lomonossoff, G. P. (1993) Virology 197, 366-374.

12. Porta, C., Spall, V. E., Loveland, J., Johnson, J. E., Barker, P. J.& Lomonossoff, G. P. (1994) Virology 202, 949-955.

13. Namba, K., Pattanayek, R. & Stubbs, G. (1989) J. Mol. Biol. 208,307-325.

14. Lomonossoff, G. P. & Johnson, J. E. (1991) Prog. Biophys. Mol.Biol. 55, 107-137.

15. Turpen, T. H., Reinl, S. J., Charoenvit, Y., Hoffman, S. L.,Fallarme, V. & Grill, L. K. (1995) Bio-Technology 13, 53-57.

16. Skuzeski, J. M., Nichols, L. M., Gesteland, R. F. & Atkins, F.(1991) J. Mol. Biol. 218, 365-373.

17. Sugiyama, Y., Hamamoto, H., Takemoto, S., Watanabe, Y. &Okada, Y. (1995) FEBS Lett. 359, 247-250.

18. Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast,F. G. & Cormier, M. J. (1992) Gene 111, 229-233.

19. Kavanagh, T., Goulden, M., Santa Cruz, S., Chapman, S., Barker,I. & Baulcombe, D. (1992) Virology 189, 609-617.

20. Higuchi, R., Krummel, B. & Saiki, R. K. (1988) NucleicAcids Res.16, 7351-7367.

21. Ryan, M. D., King, A. M. Q. & Thomas, G. P. (1991) J. Gen.Virol. 72, 2727-2732.

22. Jones, J. D. G., Shulmukov, L., Carland, F., English, J., Scofield,S. R., Bishop, G. J. & Harrison, K. (1992) Transgenic Res. 1,285-297.

23. Wen-jun, S. & Forde, B. G. (1989) Nucleic Acids Res. 17, 8385.24. Horsch, R. B., Fry, J. E., Hoffmann, N. L., Eichholtz, D., Rogers,

S. G. & Fraley, R. T. (1985) Science 227, 1229-1231.25. Davies, C., Hills, G. & Baulcombe, D. C. (1993) Virology 197,

166-175.26. Fasseas, C., Roberts, I. M. & Murant, A. F. (1989) J. Gen. Virol.

70, 2741-2749.27. Roberts, I. M. (1986) in Electron Microscopy of Proteins, eds.

Harris, J. R. & Horne, R. W. (Academic, New York), Vol. 5, pp.293-357.

28. Baratova, L. A., Grebenshchikov, N. I., Dobrov, E. N., Gedrov-ich, A. V., Kashirin, I. A., Shishkov, A. V., Efimov, A. V.,Jarvekiilg, L., Radavsky, Yu, L. & Saarma, M. (1992) Virology188, 175-180.

29. Koenig, R. & Torrance, L. (1986) J. Gen. Virol. 67, 2145-2151.30. Huisman, M. J., Linthorst, H. J. M., Bol, J. F. & Cornelissen, B. J.

C. (1988) J. Gen. Virol. 69, 1789-1798.31. Ryan, M. D. & Drew, J. (1994) EMBO J. 13, 928-933.32. Chapman, S. N., Hills, G., Watts, J. & Baulcombe, D. C. (1992)

Virology 191, 223-230.

Proc. Natl. Acad. Sci. USA 93 (1996)

Dow

nloa

ded

by g

uest

on

Oct

ober

1, 2

020