The Plant Cell, Vol. 10, 495–510, April 1998 © 1998 American Society of Plant Physiologists

RESEARCH ARTICLE

Cell-to-Cell and Phloem-Mediated Transport of Potato Virus X: The Role of Virions

Simon Santa Cruz,

1

Alison G. Roberts, Denton A. M. Prior, Sean Chapman,

2

and Karl J. Oparka

Unit of Cell Biology, Department of Virology, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA,United Kingdom

Movement-deficient potato virus X (PVX) mutants tagged with the green fluorescent protein were used to investigatethe role of the coat protein (CP) and triple gene block (TGB) proteins in virus movement. Mutants lacking either a func-tional CP or TGB were restricted to single epidermal cells. Microinjection of dextran probes into cells infected with themutants showed that an increase in the plasmodesmal size exclusion limit was dependent on one or more of the TGBproteins and was independent of CP. Fluorescently labeled CP that was injected into epidermal cells was confined tothe injected cells, showing that the CP lacks an intrinsic transport function. In additional experiments, transgenic plantsexpressing the PVX CP were used as rootstocks and grafted with nontransformed scions. Inoculation of the PVX CPmutants to the transgenic rootstocks resulted in cell-to-cell and systemic movement within the transgenic tissue.Translocation of the CP mutants into sink leaves of the nontransgenic scions was also observed, but infection was re-stricted to cells close to major veins. These results indicate that the PVX CP is transported through the phloem,unloads into the vascular tissue, and subsequently is transported between cells during the course of infection. Evi-dence is presented that PVX uses a novel strategy for cell-to-cell movement involving the transport of filamentousvirions through plasmodesmata.

INTRODUCTION

Accumulating evidence demonstrates that both cellular andviral proteins can move between cells via plasmodesmata.To date, most of the experimental evidence demonstratingintercellular protein trafficking has been obtained from stud-ies using recombinant viral movement proteins (MPs) micro-injected into plant cells (Fujiwara et al., 1993; Noueiry et al.,1994; Waigmann et al., 1994; B. Ding et al., 1995). However,the finding that two maize proteins, the transcription factorKNOTTED (Lucas et al., 1995) and the pathogenesis-relatedprotein PRms (Murillo et al., 1997), can also move betweencells indicates that intercellular protein trafficking may be ofmore general relevance in plant biology and has rekindledthe debate about whether plant virus movement functionsrepresent stolen cellular functions (reviewed in Maule, 1994;Mezitt and Lucas, 1996).

A further example of intercellular transport of macromole-cules is the supply of protein from companion cells to theenucleate conducting cells, the sieve elements, which pos-sess no protein-synthesizing capability (Fisher et al., 1992;

Nakamura et al., 1993; Sjölund, 1997). The intimate relation-ship between sieve elements and companion cells is centralto the transportation of photoassimilates and other macro-molecules, including viruses, throughout the plant. The plas-modesmata connecting sieve elements to companion cellsare structurally distinct from other plasmodesmata (Leisnerand Turgeon, 1993; Van Bel and Kempers, 1997) and have asize exclusion limit (SEL) (Kempers et al., 1993; Kempersand Van Bel, 1997) substantially greater than the SEL ofplasmodesmata between either epidermal (Derrick et al.,1990) or mesophyll (Wolf et al., 1989) cells. In terms of sus-ceptibility to virus infection, plasmodesmata at the compan-ion cell–sieve element interface are particularly importantbecause they provide the last line of defense against sys-temic virus movement.

For a plant virus to establish a systemic infection, a seriesof productive interactions between the pathogen and hostmust occur. The most basic requirement is that the plantcan support the replication of the viral genome. Second, thevirus must interact with the host to move to and through plas-modesmata into adjacent cells. For mechanically transmittedviruses, such as potato virus X (PVX), infection initiates in epi-dermal cells and spreads by cell-to-cell movement via meso-phyll and bundle sheath cells to the phloem parenchyma

1

To whom correspondence should be addressed. E-mail ssanta@scri.sari.ac.uk; fax 44-1382-562426.

2

Current address: Axis Genetics PLC, Babraham, Cambridge CB24AZ, UK.

496 The Plant Cell

and companion cells. For long-distance movement to occur,the virus must then pass into the conducting sieve elements.Once in the phloem, virus translocation is entirely passiveand is determined by the pathway of photoassimilate trans-location through the plant (Leisner et al., 1992; Leisner andTurgeon, 1993; Roberts et al., 1997). The steps involved invirus entry into the sieve elements, and subsequent exit, arevery poorly understood; however, the availability of mutants,most notably coat protein (CP) mutants, that can move fromcell to cell but not systemically suggests that phloem-dependent transport is likely to involve specific interactionsseparate from those involved in cell-to-cell movement (Dawsonet al., 1988; Xiong et al., 1993; Dolja et al., 1994, 1995; Dinget al., 1996).

That cell-to-cell movement requires the participation of vi-rus-coded proteins has been known for some time (Nishiguchiet al., 1978, 1980). More recent research has begun to un-ravel the complex functions of the tobacco mosaic virus(TMV) MP in targeting plasmodesmata (Tomenius et al.,1987), modifying the plasmodesmal SEL (Wolf et al., 1989),binding single-stranded nucleic acids (Citovsky et al., 1990,1992), interacting with the cytoskeleton (Heinlein et al.,1995), and trafficking nucleic acid between cells (Waigmannet al., 1994). In contrast to the well-characterized role of theTMV MP, far less is known about the PVX cell-to-cell move-ment process. PVX is the type member of the potexvirusesand falls into a larger grouping of viruses that require theproducts of three overlapping open reading frames, the tri-ple gene block (TGB), for cell-to-cell movement (Petty andJackson, 1990; Beck et al., 1991; Jupin et al., 1991). Despitetheir central role in cell-to-cell movement, no clear evidenceexists for a role of the PVX TGB proteins in two key func-tions that have been ascribed to the TMV MP. First, neitherthe PVX 25-kD (Davies et al., 1993) nor the PVX 8-kD(Hefferon et al., 1997) TGB protein shows plasmodesmal lo-calization. Second, the binding of RNA by the PVX 25-kDprotein is extremely weak and, unlike the interaction be-tween the TMV MP and RNA, can be disrupted by very lowsalt concentrations (Citovsky et al., 1990; Kalinina et al.,1996). One feature of TMV infections that is also seen withPVX is an increase in the plasmodesmal SEL of infectedcells (Waigmann et al., 1994; Oparka et al., 1996, 1997). ForPVX, the ability to increase the plasmodesmal SEL is attrib-uted to the 25-kD TGB protein, although other viral proteinsmay also be involved (Angell et al., 1996). In addition to theTGB proteins, PVX also has an absolute requirement for theCP for intercellular movement (Chapman et al., 1992a, 1992b;Baulcombe et al., 1995). In fact, the only potexvirus protein todate that has been shown to localize to plasmodesmata is theviral CP (Rouleau et al., 1995; Oparka et al., 1996).

Although the precise role of the CP in cell-to-cell move-ment of PVX remains unknown, the presence of a fibrillarmaterial in plasmodesmata of infected cells has suggestedthe possibility that intercellular movement of PVX requiresvirions (Allison and Shalla, 1974; Oparka et al., 1996). Insupport of this suggestion, an analysis of PVX CP mutants

showed that mutations preventing particle assembly corre-lated with a failure of virus to accumulate in inoculatedleaves (Chapman et al., 1992a).

In this study, we investigated the potential role(s) of thePVX CP in plasmodesmal modification and virus transport.Microinjection of fluorescently labeled dextrans into epider-mal cells infected with movement-deficient PVX mutantswas used to probe the plasmodesmal SEL. Short- and long-distance movement of the CP was investigated by usingboth microinjection of fluorescently labeled CP and CPtransgenic plants. Data are presented on immunogold local-ization of PVX virions in plasmodesmata at different cell in-terfaces, and the possibility that cell-to-cell movement ofPVX differs from previously described movement mecha-nisms is discussed.

RESULTS

TGB Proteins but Not the CP InducePlasmodesmal Gating

To determine the role of the PVX CP and TGB proteins in themodification of the plasmodesmal SEL and cell-to-celltransport of PVX, three modified viruses, illustrated in Figure1, were inoculated onto leaves of

Nicotiana edwardsonii.

The mutant PVX.GFP

D

CP carries the green fluorescent pro-tein (

gfp

) gene in place of the

cp

gene and is unable to movefrom cell to cell (Baulcombe et al., 1995). PVX.GFP–CP

mut2A

expresses a GFP–CP fusion protein, fails to form virions,and also is unable to move from cell to cell (Santa Cruz etal., 1996). After manual inoculation, both PVX.GFP

D

CP andPVX.GFP–CP

mut2A

(Figure 2E) give rise to infections that arerestricted to single epidermal cells (Baulcombe et al., 1995;Santa Cruz et al., 1996). The mutant PVX.GFP

D

TGB, whichcarries a deletion between nucleotides 4945 to 5500 thatdisrupts all three TGB open reading frames (Figure 1), is alsorestricted to single infected cells (Figure 2G). In experimentsusing GFP-tagged mutants, the movement-competent virusPVX.GFP–CP (Santa Cruz et al., 1996) was used as a control(Figure 2A).

To assess the effect of the mutations on plasmodesmalmodification, inoculated leaves were detached from plants

z

40 hr after inoculation and imaged on a confocal laserscanning microscope for cells expressing GFP. Epidermalcells infected with each of the movement-deficient PVX mu-tants were microinjected with a Texas Red–labeled 10-kDdextran (Oparka et al., 1996, 1997) and monitored for es-cape of the fluorescent probe into adjacent cells. The re-sults, summarized in Table 1, demonstrate that in uninfectedcells and cells infected with the TGB deletion mutantPVX.GFP

D

TGB, dye was restricted to the injected cells (Fig-ures 2C and 2H). In contrast, cells infected with either of themovement-deficient mutants PVX.GFP–CP

mut2A

(Figure 2F)and PVX.GFP

D

CP (data not shown) or the movement-com-

Transport of PVX 497

petent control PVX.GFP–CP (Figures 2B and 2D) allowedrapid movement of the dye into adjacent cells.

Injected Viral CP Does Not Traffic between Cells

The injection of fluorescently labeled dextran into infectedcells showed that modification of the plasmodesmal SELoccurred in the absence of the PVX CP; however, a role ofthe viral CP in trafficking between cells could not be ruledout. To address this possibility, CP was prepared from dis-associated virions, labeled in vitro with the fluorescent dyeTexas Red, and microinjected into

N. edwardsonii

leaf epi-dermal cells. In 13 injections, the labeled CP was alwayscontained within the injected cell and failed to move into ad-jacent cells over the 10-min examination period (Figure 2I).

Localization of the GFP–CP Fusion Proteinto Plasmodesmata

Immunoelectron microscopy of PVX-infected tissue thatwas probed with antiserum raised against the viral CP hadshown the consistent association of antibody labeling with

plasmodesmata (Oparka et al., 1996). In our study, we alsofound GFP localized to punctate sites on cell walls in tissueinfected with PVX.GFP–CP (Figures 2J and 2K). The wall-associated GFP colocalized precisely with callose, as de-tected by probing PVX.GFP–CP—infected tissue with antibodyraised against

b

-1,3-glucan (Figure 2K), indicating the pres-ence of the GFP–CP fusion protein either in or close to theplasmodesmal pore.

Ultrastructure of Plasmodesmata in PVX-Infected Tissue

Previous studies had shown that the plasmodesmata ofPVX-infected cells frequently contained a fibrillar materialthat was of a size similar to that of the virus particles, sug-gesting that cell-to-cell transport of PVX may involve virions(Allison and Shalla, 1974; Oparka et al., 1996). In this study,we looked in more detail at the plasmodesmata connectingPVX-infected cells. Representative plasmodesmata fromcells infected with wild-type PVX are shown in Figure 3. InFigure 3A, which shows an unlabeled section, fibrillar mate-rial is clearly visible both within and extending from a plas-modesma. Plasmodesmata labeled with antiserum raisedagainst the PVX CP are shown in Figures 3B to 3D. Gold la-bel was consistently associated with the fibrillar material. InFigure 3C, both longitudinal and transverse sections of thefibrils are apparent.

Preparation of a Virion-Specific Antiserum

To confirm that the fibrils seen in plasmodesmata of PVX-infected cells were virus particles, we prepared antiserumthat specifically recognized virions. The polyclonal antiserumraised against PVX, which recognizes both disassociated CPsubunits and assembled virus particles, was cross-absorbedagainst CP subunits to generate an antiserum recognizingvirion-specific epitopes. We conducted several tests todemonstrate that the cross-absorbed antiserum retained theability to recognize virions but lacked any affinity for disas-sociated CP subunits. The affinity of the cross-absorbedantiserum for virions was demonstrated by immunogold la-beling of aggregated PVX.GFP–CP particles in the cyto-plasm of infected cells (Figure 4A). To determine whetherthe cross-absorbed antiserum retained any affinity for thedisassociated CP, an ELISA-based test and immunoblotting(protein gel blotting and slot blotting) were used. The ELISA-based test showed that the antiserum retained no detect-able affinity for the disassociated CP subunits, whereasaffinity for intact virions was reduced to approximately one-half of the level seen with the unfractionated antiserum (datanot shown).

Figure 4B shows a protein gel blot of purified PVX CPprobed with either the total polyclonal antiserum or thecross-absorbed antiserum. Whereas 25 ng of PVX CP wasdetected by the polyclonal antiserum at a dilution of

Figure 1. Organization of the Wild-Type PVX Genome and ModifiedPVX Genomes.

Shown are schematic representations of viral genomes. Untrans-lated regions are shown as lines, and open reading frames areboxes that include either the size of the predicted translation prod-uct in kilodaltons or the name of the encoded gene. PVX repre-sents the genome of wild-type PVX. PVX.GFP–CP, PVX.GFPDCP,PVX.GFP–CPmut2A, and PVX.GFPDTGB represent the modified formsof PVX used in this analysis. Arrowheads show the positions of thefoot and mouth disease virus 2A peptide in the translational fusion ofGFP to CP. Closed arrowheads indicate the functional 2A sequencethat cleaves cotranslationally to generate free GFP-2A and CP; theopen arrowhead indicates the mutant 2A sequence, which translatesto give a GFP–2A–CP fusion protein.

498 The Plant Cell

Figure 2.

Confocal Scanning Laser Microscopy of Fluorescence in Cells Infected with GFP-Tagged PVX and Cells Injected with Texas Red–Labeled Probes.

(A)

to

(D)

Injection of Texas Red–labeled 10-kD dextran into noninfected and PVX.GFP–CP—infected epidermal cells.

(A)

shows the GFP signalfrom infected cells at 3 days after inoculation, with the position of injected epidermal cells marked with asterisks. In

(B)

, the same field of view

Transport of PVX 499

1:25,000, the cross-absorbed antiserum at a dilution of1:250 failed to detect 250 ng of PVX CP. Slot blot analysisof the CP and virion immobilized on a nitrocellulose mem-brane and probed with either the total antiserum or thecross-absorbed antiserum also demonstrated that the cross-absorbed antiserum preparation was unable to bind disas-sociated CP subunits (data not shown).

Localization of PVX Virions to Plasmodesmata

The cross-absorbed antiserum was used to probe ultrathinsections of

N. benthamiana

tissue infected with wild-typePVX. Figures 5A to 5C show plasmodesmata labeled withthe virion-specific antiserum in cells at or very close to theinfection front, as determined by the presence of virus parti-cles and/or virus-induced laminate inclusions. Although theoverall level of labeling was greatly reduced (cf. Figures 5Ato 5C with Figures 5E to 5G), the fractionated antiserum washighly specific for aggregates of virus in the cytoplasm andplasmodesmata and did not give any labeling in uninfectedtissue (data not shown). Another feature of the plasmodes-mata of infected cells was their distended appearance (e.g.,Figure 5A) in both the neck region and central cavity whencompared with plasmodesmata from uninfected tissues(data not shown). In addition, the desmotubule of PVX-infected plasmodesmata was seldom discernible in either lon-gitudinal or transverse sections of infected plasmodesmata.

The CP Does Not Localize to Plasmodesmata in Cells within the Minor Vein Bundle Sheath

To determine whether plasmodesmal localization of the CPoccurs at all cell boundaries in the movement pathway fromepidermis to phloem, inoculated leaves of

N. benthamiana

infected with PVX.GFP–CP were processed for electron mi-croscopy and immunogold labeling with antiserum raisedagainst the PVX CP. Figure 5D shows a typical class V veininfected with PVX.GFP–CP; Figures 5E to 5H illustrate plas-

modesmata at different cellular interfaces. Gold labeling wasseen at all cell interfaces along the pathway from epidermisto the minor vein bundle (data not shown and Figure 5H).Within the minor vein bundle, gold label was seen in plas-modesmata between bundle sheath cells and companioncells and between bundle sheath cells and phloem paren-chyma (Figures 5E and 5F). The only plasmodesmata inwhich the CP antigen was not detected were those at the in-terface between companion cells and sieve elements (Figure5G) and those between phloem parenchyma and compan-ion cells (data not shown).

The CP Is Translocated in and Unloaded fromthe Phloem

Because the PVX CP is an absolute requirement for cell-to-cell movement, establishing whether the CP is essential for

Figure 2.

(continued).

as shown in

(A)

shows Texas Red fluorescence. In

(C)

, the noninfected control cell (lower left in

[B]

) shows that dextran is confined to the in-jected cell, whereas in

(D)

, an infected cell (upper right in

[B]

) shows that the dextran probe has moved to adjacent cells. Bar in

(A)

5

250

m

m for

(A)

and

(B)

; bar in

(C)

5

100

m

m for

(C)

and

(D)

.

(E)

and

(F)

Injection of PVX.GFP–CP

mut2A

—infected cell with Texas Red–labeled 10-kD dextran.

(E)

shows the GFP signal from an infected cell.In

(F)

, the injected dextran probe has moved out of the injected cell into surrounding cells. Bar in

(E)

5

100

m

m for

(E)

and

(F)

.

(G)

and

(H)

PVX.GFP

D

TGB-infected cell

(G)

injected with the 10-kD dextran probe.

(H)

shows Texas Red–labeled dextran confined to the in-jected cell. Bar in

(G)

5

100

m

m for

(G)

and

(H)

.

(I)

Uninfected epidermal cell injected with Texas Red–labeled CP. No movement of the CP was detected 10 min after injection. Bar

5

100

m

m.

(J)

and

(K)

GFP fluorescence in cells infected with PVX.GFP–CP.

(J)

shows epidermal cells with punctate, wall-localized fluorescence (arrowheads);an amorphous inclusion body, which is characteristic of PVX infection, is indicated with an arrow.

(K)

shows punctate fluorescence at the pit fieldsbetween mesophyll cells (arrowheads). The inset shows colocalization of GFP and callose (red fluorescence) in the pit field. Bars in

(J)

and

(K)

5

25

m

m; bar in the inset to

(K)

5

5

m

m.

Table 1.

Ability of PVX Mutants to Modify Plasmodesmal SELs and Move Cell to Cell

Inoculum

Movement of10-kD Dextran

a

Cell-to-Cell Movement of Virus

b

Non-transformed

CP Trans-genicInfected Healthy

PVX.GFP–CP 21 (21) 1 (16)

1 1

PVX.GFP

D

TGB 6 (20) 4 (17)

2 2

PVX.GFP

D

CP 13 (15) 1 (13)

2 1

PVX.GFP–CP

mut2A

4 (5) 1 (6)

2 1

a

PVX-infected leaf epidermal cells were injected with a 10-kD dex-tran–Texas Red conjugate. Numbers indicate the number of injec-tions showing movement of a fluorescent probe to adjacent cells;numbers within parentheses indicate the total number of injections.As a control, healthy cells on the same leaf were injected and scoredfor movement of the probe.

b

Movement or non-movement of the different viruses, on either non-transformed or PVX CP transgenic

N. benthamiana

, is indicated by(

1

) or (

2

), respectively.

500 The Plant Cell

phloem-mediated transport was not possible. As an alternative,we investigated whether the CP was able to move throughthe phloem using grafts between transgenic

N. benthamiana

rootstocks expressing the PVX CP and nontransformed

N.benthamiana

scions. Previous experiments had shown thattransgenic

N. tabacum

plants expressing the viral CP wereable to rescue the movement-deficient phenotypes of bothPVX.GFP

D

CP and PVX.GFP–CP

mut2A

(Oparka et al., 1996;Santa Cruz et al., 1996). After inoculation of PVX.GFP–CP,PVX.GFP

D

CP, or PVX.GFP–CP

mut2A

onto leaves of the trans-genic

N. benthamiana

rootstocks, cell-to-cell movement ofPVX.GFP–CP and both of the mutants was observed. Cell-to-cell movement of all three viruses in leaves of transgenicplants occurred at approximately two-thirds the rate atwhich PVX.GFP–CP moved on nontransformed

N. bentha-miana

, as determined by the expansion of fluorescent infec-

tion foci (data not shown). In contrast, inoculation of the CPtransgenic plants with PVX.GFP

D

TGB resulted in single-cellinfections (data not shown). Systemic movement of bothPVX.GFP

D

CP and PVX.GFP–CP

mut2A

was first detected assmall fluorescent flecks, which were associated with theclass III veins of noninoculated leaves on transgenic root-stock plants, from 12 days after inoculation. Subsequentcell-to-cell movement away from the vascular tissue led to apattern of infection that, although slightly delayed, resem-bled the normal progress of systemic infection by PVX(Figures 6A and 6B) (Baulcombe et al., 1995; Roberts et al.,1997).

The appearance of virus in the nontransgenic scion tissuewas delayed in comparison to systemic movement intotransgenic rootstock leaves; however, from 15 days after in-oculation, GFP was detected in leaves of the nontransgenic

Figure 3. Ultrastructure of Plasmodesmata from PVX-Infected Tissue.

Shown is electron microscopy of plasmodesmata between PVX-infected cells.(A) Longitudinal section of plasmodesma showing fibrils both within and extending from the pore (open arrows).(B) to (D) Plasmodesmata showing labeling with antiserum raised against the viral CP. In (B) and (D), fibrils are clearly visible within the plas-modesmal pores (open arrows) and are closely associated with gold labeling. In (C), fibrils are visible in the transverse section (arrow).Bars in (A) to (D) 5 100 mm.

Transport of PVX 501

scions. Unloading of virus from the phloem of the nontrans-genic scion tissue was seen in four of four plants inoculatedwith PVX.GFP–CP

mut2A

and four of six plants inoculated withPVX.GFPDCP. The most significant difference between theprogress of the infection in systemically infected leavesabove and below the graft junction was that after virus un-loading from the phloem, virus infection of nontransgenicscion tissue failed to progress significantly into the meso-phyll, and the infection remained restricted to cells adjacentto the class III veins (cf. Figures 6B and 6C). Microscopic ex-amination of systemically infected nontransgenic scionleaves infected with either PVX.GFPDCP or PVX.GFP–CPmut2A (Figures 6D to 6F) illustrates more clearly the extentto which virus had escaped from the vascular tissue. In gen-eral, the distribution of PVX.GFPDCP was more restricted tocells close to the phloem than was PVX.GFP–CPmut2A (cf.Figures 6D and 6E). However, both PVX.GFP–CPmut2A (Fig-ure 6F) and PVX.GFPDCP (data not shown) were detected inmesophyll and epidermal cells. To determine whether thenontransgenic tissue was infected with the inoculated mu-tants and to rule out the presence of recombinant viral ge-nomes, infected tissue was used to prepare inoculum toback-inoculate nontransformed plants. In all cases, back-inoculations resulted in single-cell infections, as determinedby GFP fluorescence, and the inoculated plants did notshow any visible symptoms of infection in noninoculatedleaves (data not shown).

Virus Distribution around Class III Veins of Systemically Infected Leaves

To examine in greater detail the pattern of virus distributionin systemically infected leaves, tissue showing the presenceof GFP was taken from both below and above the graftunion and prepared for electron microscopy. Figure 7 showselectron microscopic analysis of sections probed with anti-serum raised against either the CP or the 25-kD TGB pro-tein. Figures 7A and 7B show representative cross-sectionsillustrating the distribution of the two viral proteins in the vi-cinity of class III veins of nontransgenic scion tissue infectedwith PVX.GFPDCP and PVX.GFP–CPmut2A, respectively. De-spite the obvious presence of virus-induced laminate inclu-sions that are characteristic of PVX infection (Shalla andShepard, 1972) and heavy labeling with the antiserum raisedagainst the 25-kD protein in PVX.GFPDCP-infected sciontissue (Figure 7C), no CP antigen was detected in infectedcells (Figure 7D). This lack of CP labeling in scion tissuecontrasts with the labeling seen in rootstock tissue systemi-cally infected with PVX.GFPDCP in which immunogold la-beling of CP is clearly visible (cf. Figures 7D and 7E). Thedistribution of viral antigens in PVX.GFP–CPmut2A—infectedscion tissue is illustrated in Figure 7B. In cells close to thephloem, viral CP was detected in PVX.GFP–CP mut2A—infected tissue (Figure 7F), whereas farther from the site of

phloem unloading, no labeling of the CP was observed,even though the virus-induced laminate inclusions wereclearly visible (Figure 7G).

DISCUSSION

Two distinct mechanisms for viral cell-to-cell movementhave been described (reviewed in Maule, 1991; Carringtonet al., 1996). One strategy is typified by TMV. It is indepen-dent of the CP (Siegel et al., 1962; Takamatsu et al., 1987)and requires a single MP that can traffic between cells(Waigmann et al., 1994). A second well-described move-ment strategy used by the comoviruses and nepoviruses isCP dependent and involves the transport of virions throughvirus-induced tubules that span the cell walls of adjacentcells (Wellink and Van Kammen, 1989; Van Lent et al., 1990;Wieczoreck and Sanfaçon, 1993). Other plant viruses, in-cluding cucumber mosaic virus (Suzuki et al., 1991), to-bacco etch virus (Dolja et al., 1994, 1995), and severalpotexviruses (Chapman et al., 1992a; Forster et al., 1992; Sitand AbouHaidir, 1993), are also known to depend on the CPfor cell-to-cell movement; however, the exact role of the CPin the movement processes of these viruses has not yetbeen determined. The CPs of potexviruses and potyviruses,

Figure 4. Analysis of the Cross-Absorbed Antiserum by Immu-nogold Labeling of Virions and Protein Gel Blotting of the CP.

(A) An infected cell with an aggregate of PVX.GFP–CP virions heavilylabeled with gold after probing ultrathin sections of infected leaf tis-sue with the cross-absorbed antiserum. Bar 5 500 nm.(B) Protein gel blot of PVX CP (lanes 1 and 4, 25 ng; lanes 2 and 5,100 ng; lanes 3 and 6, 250 ng) probed with either polyclonal antise-rum raised against PVX (lanes 1 to 3; antiserum diluted 1:25,000) orthe same antiserum after cross-absorption against disassociatedCP subunits (lanes 4 to 6; antiserum diluted 1:250). Numbers at leftindicate relative molecular mass standards (31000).

502 The Plant Cell

Figure 5. Electron Microscopy and Immunogold Localization of the PVX CP in Plasmodesmata.

(A) to (C) Virion-specific antiserum resulting in gold labeling of mesophyll plasmodesmata in cells infected with wild-type PVX. (A) shows plas-modesmata in longitudinal section; (B) and (C) show transverse sections. Note the distended appearance of the neck region of the plasmodes-mata shown in the longitudinal section in (A). Bars 5 250 nm.

Transport of PVX 503

both of which encapsidate viral RNA as flexuous rods, havebeen shown to be structurally related (Dolja et al., 1991). Therecent demonstration that for the potyviruses, tobacco veinmottling virus (Rodriguez-Cerezo et al., 1997), and peaseed-borne mosaic virus (A. Maule, personal communica-tion), the CP localizes to plasmodesmata raises the possibil-ity that the potexviruses and potyviruses may share someaspects of their movement processes.

The results presented here demonstrate that the increasein the plasmodesmal SEL seen in PVX-infected tissue re-quires one or more of the TGB proteins and is completely in-dependent of the CP. The failure of the CP Texas Redconjugate to move from injected epidermal cells is consis-tent with the observation that in the absence of infection, theCP in transgenic plants shows no affinity for plasmodes-mata (Oparka et al., 1996) and indicates that the PVX CPlacks the intrinsic trafficking ability associated with charac-terized movement proteins (Fujiwara et al., 1993; Noueiry etal., 1994; Waigmann et al., 1994; B. Ding et al., 1995). Theseresults show that for PVX, plasmodesmal modification aloneis insufficient to permit cell-to-cell movement of virus andthat the PVX CP does not play an active role in the modifica-tion of plasmodesmata.

This localization of the CP to the plasmodesmal pores im-plies an association of CP and viral RNA during the cell-to-cell transport process, either as virions or some other formof ribonucleoprotein complex. The possibility that virions arepresent in plasmodesmata is consistent with the observa-tion that flexuous fibers are frequently seen within the pore.To determine whether virus particles were present in plas-modesmata, the polyclonal antiserum raised against PVXvirions was cross-absorbed against intact and denaturedCP subunits in an attempt to generate a virion-specific anti-serum. Several assays confirmed that the cross-absorbedantiserum retained an affinity for virions but was unable tobind disassociated CP subunits. Using this antiserum, wewere able to demonstrate that PVX virions are present in theplasmodesmata connecting infected cells. Moreover, anti-body labeling was seen in plasmodesmata of cells at theleading edge of the infection, suggesting that this localiza-tion is biologically significant and does not reflect cytologi-cal changes at a late stage in the infection process. The

localization of virions in plasmodesmata would also explainthe bright GFP signal detected from plasmodesmata in cellsinfected with the fluorescent virus PVX.GFP–CP (see Figures2J and 2K).

All cell interfaces between the epidermis and bundlesheath showed the CP localized to plasmodesmata. Withinthe vascular bundle of the minor veins, the CP was localizedat the bundle sheath–phloem parenchyma and the bundlesheath–companion cell interfaces but was not detected inplasmodesmata connecting either phloem parenchyma andcompanion cell or companion cell and sieve element. Thelack of labeling at the phloem parenchyma–companion cellboundary is unlikely to be of great significance because inmembers of the genus Nicotiana, the bundle sheath cellswithin the minor veins contact companion cells directly (X.S.Ding et al., 1995). More important was the complete ab-sence of detectable CP, and also fibrillar material, in thespecialized plasmodesmata connecting companion cells tosieve elements. It is these plasmodesmata through whichthe virus must pass to gain access to the long-distancetransport pathway. The lack of CP labeling at this key gate-way to systemic movement could be due to the fact that thevirus crosses these plasmodesmata in a form different fromthat used at other cell boundaries (i.e., in non-virion form).Alternatively, the large SEL of the plasmodesmata at thecompanion cell–sieve element interface (Kempers et al.,1993; Kempers and Van Bel, 1997) may permit the relativelyfree passage of intact virus particles and lack the tendencyto block seen at other plasmodesmata.

That the PVX CP can enter, move through, and exit thephloem is clearly demonstrated by the results obtained fromthe grafting experiments in which cell-to-cell movementwithin the nontransgenic scion tissue of the mutantsPVX.GFPDCP and PVX.GFP–CPmut2A was dependent on CPsynthesized in the transgenic rootstock. Movement of thePVX CP outward from the phloem is shown by the progressof the infection in the scion tissue into mesophyll and epi-dermal cells. This ability of the CP to move between cells isnot an intrinsic property of the CP itself but a consequenceof the infection process. The “freezing” of the progress of in-fection to cells within and immediately adjacent to the classIII veins, as shown in Figure 6C, emphasizes the conclusion

(D) to (G) Sections probed with unfractionated polyclonal antiserum showing gold labeling of plasmodesmata within a minor vein. In (D), thestructure of a class V vein is shown. Arrows numbered 1, 2, and 3 indicate the cell interfaces shown in (E), (F), and (G), respectively. Bar in (D) 55 mm; bars in (E) and (F) 5 250 nm; bar in (G) 5 200 nm.(H) Interface between two mesophyll cells. Bar 5 500 nm.The arrows in (E) and (H) show gold-labeled plasmodesmata. In (G), there is no gold labeling at the interface between the companion cell andsieve element. b, bundle sheath; c, companion cell; m, mesophyll; p, phloem parenchyma; s, sieve element; x, xylem.

Figure 5. (continued).

504 The Plant Cell

Figure 6. Long-Distance Movement and Phloem Unloading of Viral Mutants on Transgenic N. benthamiana Expressing the PVX CP.

(A) A grafted plant showing GFP fluorescence in a transgenic rootstock systemically infected with PVX.GFP–CPmut2A. The nontransformed scionis outlined, and the graft union is shown by a white rectangle.

Transport of PVX 505

of a previous study that the phloem unloading of PVX in N.benthamiana occurs predominantly from this branchedveinal system (Roberts et al., 1997).

That the infection stops abruptly after phloem unloadingindicates that movement of the mutant viruses seen in thenontransgenic tissue did not reflect the presence of recom-binant viral genomes that had acquired a functional CP. Thispoint was also confirmed by the back-inoculation experi-ments that showed only single-cell infections, with no evi-dence of the recombinant virus lacking the gfp gene butexpressing the functional CP. The restriction of the infectionto within a few cells of the phloem presumably reflects thefact that the virus simply runs out of sufficient CP for particleassembly to occur. The low amount of CP that is unloadedfrom the phloem in nontransgenic scion tissue infected withPVX.GFPDCP is evident from the failure to detect PVX CPby immunoelectron microscopy. Even in scion tissue in-fected with PVX.GFP–CPmut2A, the CP was only detected incells within the phloem bundle. The inability of the antiserumraised against the CP to detect the assembly-deficient GFP–CP fusion protein in infected cells most probably reflects thelow affinity of the antiserum for the unassembled CP (S.Santa Cruz, unpublished data). Also, the immunodominantdomain at the N terminus of the CP (Baratova et al., 1992) ismasked by the fusion with GFP. However, the presence ofinfected mesophyll and epidermal cells indicates that in thecourse of infection, the CP had been transported through anumber of plasmodesmata. One unresolved question relat-ing to the cell-to-cell transport of the CP is whether the CPfrom disassembled virions is recycled to encapsidate prog-eny virus or whether virus entering an already infected cellcan be passed straight into the transport pathway andmoved to an adjacent cell without entering the cycle of dis-assembly, replication, and encapsidation.

The data presented here suggest that the dependence onCP for intercellular movement of PVX reflects a requirementfor encapsidation of the viral RNA before cell-to-cell trans-port and that it is the intact virion that is transported throughplasmodesmata. This is consistent with a previous study ofPVX (Allison and Shalla, 1974) and also with reports of otherfilamentous viruses localizing to the plasmodesmal pore(Esau et al., 1967; Weintraub et al., 1976; discussed in Lucas et

al., 1990). Intercellular movement of the filamentous PVX vi-rion implies a mechanism distinct from the CP-independenttransport process used by TMV. In addition, the absenceof virus-induced tubules in the plasmodesmata of PVX-infected cells indicates that the PVX movement process differsfrom the movement via tubules of nepovirus and comovirusvirions.

The necessary plasmodesmal modification required tosupport transport of PVX virions (diameter of 13 nm; Koenigand Leseman, 1989) would be greater than the modificationneeded for transport of the TMV RNA–MP complex (diame-ter of 2 nm; Citovsky et al., 1992) but less than the dilationrequired to allow passage of icosohedral viruses, such asthe comovirus cowpea mosaic virus (diameter of 20 to 24nm; Van Kammen and de Jager, 1978). The apparent lack ofa desmotubule and the distorted appearance of plasmodes-mata in infected cells suggest that the PVX movement pro-cess may involve removal from the plasmodesmal pore ofinternal membranes and proteins. This could allow theopening of a passage that even in the constricted neckregion would have a diameter of .20 nm (Olesen andRobards, 1990) between the inner leaflets of the plasmamembrane lining the plasmodesmal pore. The challengenow is to understand how the proteins encoded by the TGBfunction in the movement process, both in the modificationof the plasmodesmal SEL and in their possible role in thetransport of PVX virions to and/or through plasmodesmata.

METHODS

Virus, Viral cDNA Clones, and Plant Inoculation

Wild-type potato virus X (PVX) strain UK3 was propagated on Nicoti-ana benthamiana. Sap from infected plants, diluted 1:100 (w/v) in 50mM sodium borate, pH 8.2, was used to inoculate aluminium oxide–dusted leaves. Plasmids pTXS.GFP–CP, pTXS.GFP–CPmut2A, andpTXS.GFPDCP (Figure 1) have been described previously (Baulcombeet al., 1995; Santa Cruz et al., 1996). The plasmid pTXS.GFPDTGBwas created by deleting the sequence in pTXS.GFP–CP between anApaI restriction site at nucleotide 4945 and an EcoNI restriction siteat nucleotide 5550 (Figure 1). All plasmids were digested with SpeI

Figure 6. (continued).

(B) The leaf of a transgenic rootstock systemically infected with PVX.GFP–CPmut2A showing virus distributed across the leaf lamina.(C) The leaf of a nontransformed scion systemically infected with PVX.GFP–CPmut2A. Note that the virus is closely associated with the major veinnetwork.(D) to (F) Magnified images of nontransgenic leaves systemically infected with PVX.GFPDCP (D) and PVX.GFP–CPmut2A ([E] and [F]) showing therestriction of the virus to the vicinity of class III veins. In (F), the virus has unloaded from phloem and entered mesophyll and epidermal cells. In(D) to (F), xylem has been allowed to import Texas Red to aid vizualization of the vascular tissue. Bar in (D) 5 250 mm; bar in (E) 5 500 mm; barin (F) 5 200 mm.

506 The Plant Cell

Figure 7. Immunogold Labeling of PVX-Infected Cells after Phloem Unloading.

(A) and (B) Sections viewed under a light microscope showing the distribution of viral antigens in infected cells (as determined from immuno-electron microscopy of serial sections) around infected class III veins. (A) was infected with PVX.GFPDCP; (B) was infected with PVX.GFP–CPmut2A.

Transport of PVX 507

before in vitro transcription by using an mMessage mMachine T7RNA polymerase kit (Ambion, Austin, TX), according to the manufac-turer’s instructions, and a template concentration of 0.2 mg/mL. Thetranscription reactions were inoculated directly onto aluminium ox-ide–dusted leaves by using 5 mL of reaction product per leaf. Infec-tions established with transcripts from the plasmids described aboveare referred to by replacing the prefix pTXS with the prefix PVX.

Preparation of Fluorescently Labeled Probes

The Texas Red–labeled 10-kD dextran probe was purchased fromMolecular Probes (Eugene, OR). Because of the significant levels oflow molecular weight contaminants and unconjugated dye in thedextran preparation, it was necessary to filter the dye by using a Mi-crocon-10 membrane exclusion filter (Amicon, Beverly, MA) with anominal 10-kD cutoff. The absence of unconjugated dye was con-firmed by thin-layer chromatography, as described previously(Oparka et al., 1996, 1997). Aliquots were stored at 2208C until re-quired for microinjection. Freeze–thaw cycles were avoided to pre-vent accumulation of breakdown products. Labeling of the coatprotein (CP) with Texas Red was performed with disassociated CPsubunits prepared according to the method of Goodman (1975). Theabsence of either intact or fragmented virions in the CP preparationwas confirmed by electron microscopic analysis of poly-L-lysine–treated carbon support films that had been incubated with the CPpreparation, as described by Roberts (1986). Disassociated CP sub-units were labeled using a Texas Red protein conjugation kit, accordingto the manufacturer’s instructions (Molecular Probes). Unconjugateddye was removed by centrifugation of the sample through a MicroBio-Spin 6 column (Bio-Rad), and the labeled CP was dialyzedagainst 10 mM Sörensen’s phosphate buffer (Na2HPO4/KH2PO4, pH7.2). The concentration of Texas Red–labeled CP was checked byPAGE alongside known quantities of PVX CP, and samples were ad-justed to a concentration of 0.25 mg/mL. Labeled CP was stored at48C and used within 2 days of preparation.

Microinjection

Microinjection of the Texas Red–labeled dextran and CP was per-formed as described previously (Oparka et al., 1996), using a modi-fied pressure probe to prevent vacuolar rupture during impalement(Oparka et al., 1990). Injected cells were observed for 10 min after in-

jection and scored for the presence or absence of fluorescent label incells adjacent to the injected cell. The injections of 10-kD dextran toepidermal cells infected with the movement-deficient mutants were,as far as was possible, performed blind (i.e., without the operatorknowing which mutant was infecting the target cells).

Labeling of Xylem with Texas Red

In some experiments, to allow the vascular tissue to be seen easilyunder the confocal laser scanning microscope, the petioles of de-tached leaves were immersed in a solution containing 0.1 mg/mL of3-kD Texas Red dextran (Molecular Probes), as described by Robertset al. (1997).

Immunolocalization of b-1,3-Glucan (Callose)

Callose was localized in epidermal and mesophyll tissues, essentiallyas described in Oparka et al. (1997). Rabbit antibody raised againstb-1,3-glucan (Genosys Biotechnologies, Cambridge, UK) was usedto probe sections prepared from PVX.GFP–CP—infected leaves.Callose was detected by labeling the primary antibody, using ananti–rabbit antibody conjugated to CY3 (Sigma Immunochemicals,Poole, UK).

Detection of Green Fluorescent Protein in Leaves andWhole Plants

Plants were illuminated with long-wavelength UV light (365 nm) byusing a Blak-Ray hand-held lamp (UV Products, Upland, CA). Photo-graphs were taken on Kodachrome PKL 200 daylight film with agreen 31 filter (Hoya, Japan), as described previously (Oparka et al.,1996; Roberts et al., 1997).

Detection of CY3, the Green Fluorescent Protein, and Texas Red by Confocal Laser Scanning Microscopy

Detached leaves were viewed with an MRC 1000 confocal laserscanning microscope (Bio-Rad) to image the green fluorescent pro-tein (GFP), CY3, and Texas Red fluorescence. The imaging proce-dures used have been described in detail previously (Baulcombe etal., 1995; Oparka et al., 1997; Roberts et al., 1997).

Figure 7. (continued).

Uninfected cells are unshaded (1), cells labeled with the antiserum raised against the PVX 25-kD protein are lightly shaded (2), and cells labeledwith the antiserum raised against the CP are heavily shaded (3). x, xylem vessels. (C) to (G) Electron microscopy of ultrathin sections prepared from systemically infected tissues. (C) to (E) show cells systemically infected withPVX.GFPDCP. (C) shows labeling in nontransgenic scion tissue probed with the antiserum raised against the 25-kD protein. Gold label is seenon the laminate inclusion structures typical of PVX infections. (D) shows an infected nontransgenic cell probed with the antiserum raised againstthe CP. No label is seen despite the presence of laminate inclusion structures. In (E), an infected transgenic cell shows clear labeling, with theantiserum raised against the CP around a laminate inclusion. In (F), in cells close to the phloem, nontransgenic cells infected with PVX.GFP–CPmut2A show clear labeling, with the antiserum raised against the CP. Farther from the phloem, no CP labeling is seen, despite the obvious pres-ence of PVX-induced inclusions (G). c, companion cell; s, sieve element. Bars in (C) to (G) 5 1 mm.

508 The Plant Cell

Plant Material and Grafting

Plants (N. benthamiana and N. edwardsonii) were grown from seed ina heated greenhouse and used for experiments when they were be-tween 20 and 30 days old. Grafting experiments were performed us-ing transgenic N. benthamiana expressing the PVX CP (the generousgift of T. Lough and R. Forster, Horticulture and Food Research Insti-tute of New Zealand, Auckland, New Zealand). Grafts were per-formed using a standard wedge graft technique, as described byGarner (1979). N. edwardsonii was used for microinjection experi-ments because the leaf lamina is flat and epidermal cells can be eas-ily injected.

Preparation of the Antiserum Raised against the 25-kD Protein

Recombinant 25-kD protein was expressed in Escherichia coliBL21(DE3) pLysS as a fusion to thioredoxin from the expression vec-tor pET32a (Novagen, Madison, WI) and purified according to thesupplier’s instructions. Purified protein was diluted to a concentra-tion of 1 mg/mL in 50 mM sodium phosphate, pH 7.0, and mixed withan equal volume of Freund’s incomplete adjuvant. Intramuscular in-jection of a New Zealand White rabbit was performed at 2-week in-tervals using 1 mL of antigen. Serum was prepared from samplebleeds collected 6 weeks after the first immunization and used forimmunogold labeling, as described below.

Preparation and Analysis of Virion-Specific Antiserum

Polyclonal antiserum raised against PVX particles (generously pro-vided by I. Barker, Central Science Laboratories, York, UK) waspassed through a HiTrap NHS-activated Sepharose column (Phar-macia, Uppsala, Sweden) to which PVX CP had been coupled. ThePVX CP subunits, prepared as described above, were dialyzedagainst 50 mM Sörensen’s buffer, pH 7.2, and coupled to the col-umn, according to the manufacturer’s instructions. The column flow-through was purified to give an IgG fraction by using an E-Z-Sep kit,according to the manufacturer’s instructions (Pharmacia), and equil-ibrated in 50 mM Sörensen’s buffer, pH 7.2. The affinity of the cross-absorbed antiserum for virions was tested by immunoelectronmicroscopy of PVX.GFP–CP—infected tissue, as described below.The affinity of the cross-absorbed antiserum for disassociated CPsubunits was tested by using SDS-PAGE of purified PVX CP, fol-lowed by protein gel blotting and probing of blots with either the totalpolyclonal antiserum or the cross-absorbed antiserum. An ELISA-based test, using microtiter plates coated with either 1 mg of CP or 1mg of virion, was also used to determine the affinity of serial dilutionsof the total polyclonal antiserum and the fractionated antiserum forboth CP and virion.

Immunoelectron Microscopy

Leaf tissues were fixed and embedded in epoxy resin for immu-nogold labeling, as described by Fasseas et al. (1989). Ultrathin sec-tions on nickel grids were labeled using antiserum to either the PVXCP or PVX 25-kD proteins, followed by goat anti–rabbit gold conju-gate (15 nm; GAR-G; Amersham). To examine the cellular distributionof PVX after its unloading from the phloem, leaf veins were examinedunder the confocal laser scanning microscope to detect the first ap-

pearance of the GFP-tagged virus. Areas of leaf with veins showingGFP were then excised with a razor blade and processed for electronmicroscopy, as described above.

ACKNOWLEDGMENTS

We are grateful to Tony Lough and Richard Forster for providingtransgenic N. benthamiana plants expressing the PVX CP. We thankPeter Nettleton and Tracy Fitzgerald for preparing the antiserumraised against the 25-kD protein and Ian Barker for providing antise-rum raised against PVX. We also acknowledge George Duncan forassistance with the electron microscopy and Paul Donaghy for anti-body fractionation. We thank Peter Palukaitis for helpful discussions.The Scottish Crop Research Institute is grant-aided from the ScottishOffice Agriculture Environment and Fisheries Department. A.G.R.was the recipient of a postgraduate research award from DundeeUniversity.

Received November 19, 1997; accepted January 26, 1998.

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DOI 10.1105/tpc.10.4.495 1998;10;495-510Plant Cell

Simon Santa Cruz, Alison G. Roberts, Denton A. M. Prior, Sean Chapman and Karl J. OparkaCell-to-Cell and Phloem-Mediated Transport of Potato Virus X: The Role of Virions

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