Invasion of minor veins of tobacco leaves inoculated with tobacco ...

Proc. Natl. Acad. Sci. USA Vol. 93, pp. 11155-11160, October 1996

Microbiology

Invasion of minor veins of tobacco leaves inoculated with tobacco mosaic virus mutants defective in phloem-dependent movement (coat protein/companion cells/tobamoviruses/vascular parenchyma cells/viral genetics)

XINSHUN DING, MICHAEL H. SHINTAKU*, SHELLY A. CARTER, AND RICHARD S. NELSONt The Samuel Roberts Noble Foundation, Plant Biology Division, P.O. Box 2180, Ardmore, OK 73402

Communicated by George Bruening, University of California, Davis, CA, July 9, 1996 (received for review March 1, 1996)

either in an encapsidated form (i.e., as a virion) or as a viral ribonucleoprotein (14, 15). Both forms require functional CP for the phloem-dependent accumulation of virus. Many other plant viruses require their CP for phloem-dependent accumulation (e.g., refs. 16-21). Recently, Dolja et al. (20, 22) determined that the N-terminal 29 residues and the C-terminal 18 residues of the tobacco etch potyvirus CP were necessary for phloem-dependent accumulation. Also, it has been suggested that the capsid of red clover necrotic mosaic dianthovirus, and not some other CP-containing form, is necessary for the long distance movement of the virus (23). Results from all these studies indicate the necessity of the CP for phloemdependent accumulation, but in no instance has the accumulation of a CP mutant within the vasculature of the inoculated leaves been studied. Invasion of minor veins by mutant viruses is only now coming under study. Cronin et al. (24) have recently determined that the helper component-proteinase of tobacco etch potyvirus functions within the vein to allow phloemdependent accumulation. Phloem-dependent accumulation of virus requires that the virus invade the vein cells from the mesophyll cells of the inoculated leaf. Minor veins (class IV and V; ref. 25) account for approximately 90% of the veins in sections from mature tobacco leaves (ref. 26; X.S.D. and R.S.N., unpublished data) and are likely the first veins spreading virus would contact from a site of inoculation. These veins are generally sheathed by long bundle sheath (BS) cells and contain various cell types including xylem tracheary elements, phloem or vascular parenchyma (VP) cells, companion (C) cells, and sieve elements (SE) (5, 8, 26). Hilf and Dawson (27) have shown that host factors different from those required for cell-to-cell movement affect phloem-dependent accumulation of tobamoviruses. The location of these host factors within the veins has not been determined. In regard to virally encoded movement factors, cell-to-cell movement of TMV is facilitated by at least one specific viral protein; the 30-kDa or movement protein (28, 29). The movement protein interacts with the plasmalemma and the plasmodesmata (30, 31), and can cause a significant increase in the molecular size exclusion limits of plasmodesmata (30). However, the movement protein expressed by transgenic tobacco was unable to alter the size exclusion limits of plasmodesmata between BS cells and VP cells (32). Based on this observation and the evidence that the CP of TMV is necessary for phloem-dependent accumulation of this virus, it was suggested that the CP may be the additional viral factor required for TMV to move from BS to VP cells (32, 33). We have addressed this hypothesis using TMV CP mutants unable to accumulate via the phloem due to an altered or

To fully understand vascular transport of ABSTRACT plant viruses, the viral and host proteins, their structures and functions, and the specific vascular cells in which these factors function must be determined. We report here on the ability of various cDNA-derived coat protein (CP) mutants of tobacco mosaic virus (TMV) to invade vascular cells in minor veins of Nicotiana tabacum L. cv. Xanthi nn. The mutant viruses we studied, TMV CP-O, UlmCP15-17, and SNCO15, respectively, encode a CP from a different tobamovirus (i.e., from odontoglossum ringspot virus) resulting in the formation of nonnative capsids, a mutant CP that accumulates in aggregates but does not encapsidate the viral RNA, or no CP. TMV CP-O is impaired in phloem-dependent movement, whereas UlmCP15-17 and SNCO15 do not accumulate by phloemdependent movement. In developmentally-defined studies using immunocytochemical analyses we determined that all of these mutants invaded vascular parenchyma cells within minor veins in inoculated leaves. In addition, we determined that the CPs of TMV CP-O and UlmCP15-17 were present in companion (C) cells of minor veins in inoculated leaves, although more rarely than CP of wild-type virus. These results indicate that the movement of TMV into minor veins does not require the CP, and an encapsidation-competent CP is not required for, but may increase the efficiency of, movement into the conducting complex of the phloem (i.e., the C cell/sieve element complex). Also, a host factor(s) functions at or beyond the C cell/sieve element interface with other cells to allow efficient phloem-dependent accumulation of TMV CP-O.

The movement of plant viruses through vascular tissue is essential to allow their maximum accumulation and symptom development in the host plant (1). In crops infected with viruses, the greatest yield reductions are due to vasculardependent movement (2). Although a considerable amount of information is known about the cell-to-cell movement of plant viruses (3-5), much less is known about vascular-dependent movement (5-8). The majority of plant viruses, including tobacco mosaic virus (TMV), move through the phloem (1). The coat protein (CP) of TMV is critical for phloemdependent accumulation of this virus. Mutant strains of TMV that produce CP, but are defective in virion assembly, spread as labile infectious entities from cell-to-cell, but do not accumulate in tissue accessed by phloem-dependent movement (9). More recently, it has been possible to delete or modify the CP open reading frame of cDNA clones of TMV, and infectious transcripts from these clones yielded virus that moved cell-tocell but did not accumulate in upper uninoculated leaves unless complemented by CP expressed in transgenic plants (10-13). Saito et al. (14) mutated the nucleotide sequence within the origin of capsid assembly and observed that inefficient assembly led to inefficient accumulation of the virus in upper leaves. Results from this and other work indicate that the virus moves

Abbreviations: BS, bundle sheath; C, companion; CP, coat protein; cpbs, coat protein bodies; DPI, days post-inoculation; ORSV, odontoglossum ringspot virus; SE, sieve element; TMV, tobacco mosaic virus; VP, vascular parenchyma; PB, phosphate buffer. *Present address: College of Agriculture, University of Hawaii at Hilo, Hilo, HI 96720. tTo whom reprint requests should be addressed. e-mail: RSNelson@

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

noble.org. 11155

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absent CP. We also determined the location where a host factor(s) must function to allow efficient phloem-dependent accumulation of a TMV mutant encapsidated with a non-native CP. The accumulation of these mutants in vein cells was monitored through immunocytochemical studies during periods when rapid phloem-dependent movement would be expected.

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

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MATERIALS AND METHODS Viruses, Plants, and Virus Inoculation of Plants. U1-TMV was obtained and purified as described (34). UlmCP15-17, a CP mutant of U1-TMV, was made from an infectious cDNA clone of U1-TMV (p35SU1R; refs. 13 and 35) using a slightly modified protocol described in the Altered Sites mutagenesis kit (Promega; ref. 35). The mutagenic primer used was UBstSac (5'-CCGGCCACCGCGGTGGCAAGAACACGAACTGAG-3'), which introduces unique BstXI and SacII sites in the vicinity of nucleotide 5756 of the TMV genome. The CP of UlmCP15-17 was thus altered at positions 15-17 (i.e., from Ser-Ser-Ala to Pro-Pro-Arg) in the CP sequence. SNCO15, a CP mutant of U1-TMV containing the CP coding sequence but lacking an initiation codon (12), was produced by in vitro transcription of a cDNA clone behind a T7 promoter as described (35). TMV CP-O, was produced by exchanging the Ul strain CP gene with the CP gene from odontoglossum ringspot tobamovirus (ORSV; ref. 27). Nicotiana tabacum L. cv. Xanthi nn, a systemic host for TMV, was used in this study. The plants were grown, both prior to and after inoculation with virus, as described (26). At one month after planting, the 5th leaf of each plant was mechanically inoculated with purified TMV CP-O virus (20 ,tg/ml, 20 ,ul/leaf) or with total RNA (-4 ,ug/leaf) isolated from leaves of Nicotiana benthamiana infected with mutant viruses or from leaves of N. tabacum L. cv. Xanthi nn infected with the Ul strain of TMV (U1-TMV). Total RNA was isolated by phenol extraction and ethanol precipitation. Plants were inoculated with viral RNA as described (35) or with TMV CP-O as described for purified virus (35). Antisera. Antiserum against TMV was prepared by injecting a rabbit with purified TMV and harvesting blood after the first booster injection. Antiserum against the 126-kDa protein of TMV was obtained from Hal S. Padgett and Roger N. Beachy (Scripps Research Institute, La Jolla, CA). Antiserum against ORSV was obtained from William 0. Dawson (University of Florida, Lake Alfred). All antisera were used without further purification. Reverse Transcription-PCRs. At 10 or 30 days postinoculation (DPI), the inoculated leaf and stem nodes of plants inoculated with UlmCP15-17 or U1-TMV were harvested individually and stored at -70°C before extraction of RNA. Three plants were harvested for each virus analyzed. Two plants inoculated with 0.1 M sodium phosphate buffer (PB) (pH 7.0) were harvested as controls for each experiment. Total RNA was isolated from individual samples via phenol extraction and ethanol precipitation. First strand cDNAs were synthesized with Superscript reverse transcriptase (Life Technologies, Gaithersburg, MD) using Primer B (5'-ATGCATCTTGACTACCTCCAGTTGCAGGACCA-3') complementary to nucleotides 6176-6207 of the TMV genome and the cDNAs then amplified by PCR using the B primer and Joint primer (5'-CGATGATGATTCGGAGGCTACTGT-3', which corresponds to nucleotides 5664-5687 of the TMV genome) as described (35). The resulting PCR products were electrophoresed in 1% agarose gels and stained with EtBr. Tissue Fixation and Embedding. Individual chlorotic lesions were randomly sampled from Xanthi nn leaves inoculated with UlmCP15-17 or SNC015 at 3 and 6 DPI, respectively. Xanthi nn leaves inoculated with U1-TMV in PB or PB alone were sampled at the same time as those for the mutant viruses. Sampled tissues were fixed for 3 h as described (36) except

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Inoculated leaf FIG. 1. Accumulation of a virion-defective mutant of TMV (UlmCP15-17) in inoculated and systemically infected tissue of N. tabacum cv. Xanthi nn at 10 DPI. Three plants were inoculated with 4 ,ug/ml total RNA containing UlmCP15-17 or U1-TMV. Tissue from the inoculated plants was harvested at 10 DPI and analyzed for accumulation of UlmCP15-17 or U1-TMV by reverse transcriptionPCRs with primers identical or complementary to the TMV sequence. The expected fragment size was 543 bases. Reaction products in lanes 1-3 were obtained using extracts from three plants inoculated with UlmCP15-17 and lanes 4-6 were obtained using extracts from three plants inoculated with U1-TMV. Lane M, molecular size markers of 1.02 kbp and unresolved doublet of 507 and 517 bp. Lane H, reaction product obtained using extracts from mock-inoculated control. Node 0, stem at node adjacent to the petiole of the inoculated leaf. Node 1, stem at node one above the inoculated leaf. Node 6+ shoot apex, stem at node 6 above the inoculated leaf and the shoot apex above this node.

2.5% (wt/vol) sucrose was added to the wash buffer. Procedures for dehydration, LR White resin embedding and sectioning were as described (26). Tissues from leaves inoculated with TMV CP-0 were harvested at 4 DPI, fixed in a microwave oven, and embedded in LR White resin as described (26). Immunocytochemistry. Semi-thin sections (2 p,m) prepared from tissues infected with TMV CP-0 or UlmCP15-17 were analyzed using antiserum against ORSV or TMV, respectively, as described (36). Sections were examined and photographed as brightfield or polarized light images with a Nikon Microphot-FX microscope equipped with a Nikon camera. For electron microscopy, thin sections (-90 nm) were mounted on slot (2 x 1 mm) nickel grids precoated with 0.3% Formvar/ carbon film. The sections were incubated in a 0.03 M glycine solution for 15 min at room temperature. After three washes (2 min per wash) in distilled water, the sections were incubated in 2% (wt/vol) bovine serum albumin (BSA) in PB (BSA-PB) for 30 min at room temperature and then probed using antiserum against TMV (1:15,000 dilution, vol/vol) or TMV 126-kDa protein (1:2,000 dilution, vol/vol) in the BSA-PB solution. The sections were washed five times (5 min per wash) in 0.02 M Tris buffer containing 0.5 M NaCl and 0.01% Tween 20 (pH 7.5). The sections were then incubated in the BSA-PB solution for 30 min followed by a 1-h incubation in a goat anti-rabbit IgG gold (20 nm) conjugate (BioCell Research Laboratories, Cardiff, U.K) diluted 1:200 in the BSA-PB solution at room temperature. Post-staining of the sections and their analysis by electron microscopy were done as described (26).

RESULTS Symptom Induction by UlmCP15-17, SNCO15, TMV CP-O, and U1-TMV. UlmCP15-17 induced chlorotic lesions on in-

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oculated Xanthi nn leaves by 3 DPI. On occasion, chlorotic i 10> Xc j;t * !lesions adjacent to major veins expanded slowly along the veins

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in^similar to that observed for TMV CP mutants a fashion ?-$ffi~?ii.; studied previously (i.e., TMV mutants PM1 and PM2; ref. 9). SNC015 induced the formation of chlorotic lesions on inoculated leaves of Xanthi nn by 4 DPI. No systemic symptoms were observed in plants inoculated.with UlmCP15-17 or SNCO15. TMV CP-O induced chlorotic lesions on inoculated Xanthi nn leaves by 3 DPI. At approximately 7 DPI, expanding leaves at ;t . T ! top of the plant began to show mild vein-clearing symptoms ............s, C1 I

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lated with the presence of a virus-dependent PCR product. Necrotic lesions were observed on leaves only after inoculation with extract that had yielded a viral-specific PCR product (data not shown). These results indicated that UlmCP15-17 spreads through plant tissue in a manner consistent with cell-to-cell movement and not phloem-dependent movement. Immunocytochemical Detection of UlmCP15-17 and TMV CP-O in Inoculated Leaves. UlmCP15-17 produces a CP, but vis defective in virion assembly as determined by Western blot analysis and immunosorbent electron microscopy (data not shown). Thin sections of tissue prepared from chlorotic lesions induced by UlmCP15-17 on inoculated leaves revealed numerous dark-staining bodies within cells similar to the coat protein bodies (cpbs) observed for another CP mutant of TMV ....(i.e., CP 10; ref. 37). As observed for CP 10 in that study, the bodies we observed often were associated with -.xtI dark-staining X-bodies; cytoplasmic structures containing TMV-encoded 126 kDa- and 183 kDa-proteins known to be associated with viral replication (38-43). Sections of mesophyll cells probed

with antiserum against TMV showed that the dark-staining

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FIG. 2. Immunodetection of cpbs in infected cells of N. tabacum cv. Xanthi nn leaves inoculated with a virion assembly defective TMV mutant, UlmCP15-17. Sections prepared from chlorotic lesions were labeled using an antiserum against TMV followed with a goat antirabbit IgG gold (20 nm) conjugate. All panel insets are magnifications of the boxed areas within the veins to the left of the insets. (A) Immunogold-labeled cpbs detected in the cytoplasm of an infected

(x 18,000; bar = 0.5,um.) (B) Immunogold-labeled cpbs detected in a VP and a C cell in a class V vein of an inoculated leaf. (C) Immunogold-labeled cpbs detected in a VP cell in a second class V vein. The SEs of low magnification images in B and C are the two small cells above the C cell and to the left of the VP cell, respectively. (D) Immunogold-labeled U1-TMV virions detected in a VP cell in a class V vein from tissue infected with U1-TMV. (E) Vein from a healthy control-probed as for the other sections. (Veins X900; bars = 2 gm/Insets x 16,000; bars = 0.6 ,um.)

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pared from UlmCP15-17-infected leaf tissues were subjected to double-sided labeling immunocytochemistry using antiserum against TMV and then examined under a light microscope, black spots, representative of cpbs, were seen in the cytoplasm of infected mesophyll cells (data not shown). When 14 infected typical class V veins (26) in 8 sections from 8 different chlorotic lesions were examined under the light microscope, 40% of the VP and 5% of the C cells were found to have immunogold-labeled cpbs. Similar results were obtained when thin sections containing typical class V veins were immunolabeled and examined under the electron microscope (Fig. 2 B and C, Table 1). No immunogold-labeled cpbs were seen in SEs in typical class V veins (Fig. 2 B and C and data not shown). Also no cpbs and no signal were observed in sections from plants inoculated with U1-TMV or PB, respectively (Fig. 2 D and E). TMV CP-O accumulated in VP cells within class V veins of inoculated leaves (Fig. 3). Immunocytochemical analyses of 8 semi-thin sections containing 12 class V veins showed that by 4 DPI, TMV CP-O had accumulated in 94% of the VP cells and 6% of the C cells. Cytological and Immunocytochemical Detection of SNCO15 in Inoculated Leaves. Thin sections prepared from chlorotic lesions induced by SNCO15 and U1-TMV at 6 DPI were analyzed by immunocytochemistry using antiserum against the TMV 126-kDa protein. Many X-bodies were observed in the cytoplasm of mesophyll cells infected with U1-TMV, and these X-bodies were specifically labeled with gold particles (Fig. 4A). X-bodies in VP cells from tissue infected with SNCO15 or U1-TMV were also labeled (Fig. 4 B and C). Examination of typical class V veins infected with SNCO15 showed that the virus accumulated in VP cells, but not in C cells unlike U1-TMV (Table 1). No SEs or xylem tracheary elements contained X-bodies or were labeled (Fig. 4 B and C; data not shown). When sections from SNCO15-infected plants were incubated with antiserum against TMV, no labeling was detected (data not shown).

DISCUSSION Ding et al. (32) found that TMV movement protein could significantly increase the size exclusion limits of plasmodesmata between mesophyll cells and mesophyll and BS cells in mature tobacco leaves, but was unable to modify the size exclusion limits of plasmodesmata between BS and VP cells. This observation, and the fact that TMV CP is required for phloem-dependent accumulation in tobacco, led to a suggestion that TMV CP may be required to allow the transport of TMV across this boundary (5, 32, 33). In this study we have Table 1. Percentage of cells infected for three cell types around and within typical class V veins of tobacco leaves inoculated with CP mutants of U1-TMV or U1-TMV Percentage of cells infected No. of veins Virus C VP Exp. analyzed BS 1 35 (18)* UlmCP15-17 78 1 40 U1-TMV 2 (2) 100 100 17 SNCO15 2 17 (9) 27 16 0 U1-TMV 6 (4) 100 88 17 Xanthi nn plants were inoculated in separate experiments with total RNA (4 ,ug/ml) containing UlmCP15-17, SNCO15, or U1-TMV. At 3 DPI (UlmCP15-17, experiment 1) or 6 DPI (SNCO15, experiment 2), tissue containing chlorotic lesions were randomly sampled and analyzed, respectively, for TMV CP or 126-kDa protein accumulation by immunocytochemistry and electron microscopy. For each experiment, sections from Xanthi nn plants inoculated with U1-TMV were analyzed for comparison. BS, Bundle sheath cell; VP, vascular parenchyma cell; C, companion cell. *Numbers in parentheses equal total number of lesions analyzed.


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FIG. 3. Immunodetection of TMV CP-O in VP cells of N. tabacum cv. Xanthi nn leaves at 4 DPI. Sections prepared from inoculated leaves were labeled using an antiserum against ORSV followed by a goat anti-rabbit IgG gold (5 nm) conjugate and silver enhancement. Cells were stained with toluidine blue. Signal appears as silver speckles within the cell interior in the polarized images. (A) Class V vein from leaf inoculated with TMV CP-O. (B) Class V vein from leaf inoculated with PB. (C) Schematic identifying cell types within vein shown in A. C, companion cell; VP, vascular parenchyma cell; BS, bundle sheath cell; XT, xylem tracheary element; unlabeled small cells, SEs. (X250; bar = 43 ,um.)

determined that, like U1-TMV, a virion defective mutant (UlmCP15-17) and a coat proteinless mutant of TMV (SNCO15) could accumulate in VP cells in inoculated tobacco leaves (Figs. 2 B and C and 4B, Table 1). Therefore, neither the virion nor the CP is required for TMV to enter VP cells, the potential first step in the process of phloem-dependent movement. We have recently suggested that a viral factor other than the CP potentiates TMV movement into VP cells (26). We determined that a strain of TMV (MIc-TMV), whose attenuated phenotype maps to the 126-kDa protein open reading frame (34, 35, 44), was deficient in its ability to accumulate in VP and C cells within the inoculated leaf. The CP of MIc-TMV cannot be involved in the lack of phloem-dependent accumulation because the CP sequence of this virus and its accumulation in inoculated leaves were identical to that of U1-TMV (34, 44). Other researchers (45-47) have also determined that, for several plants viruses, sequences analogous to the TMV 126kDa protein open reading frame have an influence on cellto-cell and phloem-dependent accumulation. de Zoeten and Gaard (48), studying pea enation mosaic virus, found that a vesicular material, containing RNA-dependent RNA polymerase and virus-specific double-stranded RNA, is likely transported from VP or C cells to SEs. Therefore, the involvement of virally encoded proteins other than the CP in phloemdependent movement has support and requires further study. Host factors also have a role in the phloem-dependent movement of viruses (reviewed in refs. 5 and 7). Several studies have demonstrated that although some TMV CP mutants could produce virion-like particles they were still defective in phloem-dependent accumulation in plants (11, 49). Hilf and Dawson (27) replaced the native CP of U1-TMV with the CP from ORSV, and found that the chimeric virus (TMV CP-O) produced nuclease-resistant particles, but the virus moved very poorly from leaf to leaf in tobacco. They suggested that the virion produced by the chimeric virus was unable to interact with host factors to allow efficient phloem-dependent accumulation. In our study, using the same chimeric virus, we determined-that the virus could accumulate in the VP cells of minor veins in inoculated leaves. Therefore, if the hypothesis of Hilf and Dawson (27) is correct, the host factor(s) required for this chimeric virus to efficiently accumulate systemically function at the C cell interface with other cells, within the C cells, or beyond. Numerous studies have shown that the transport of photosynthate in plants encounters a specialized barrier between the VP cells and the C cell/SE complexes, and this barrier allows the maintenance of extremely different osmotic and electrical potentials in the VP cell and the complex (for review see ref.



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meric virus are required for invasion of C cells. However, the ratios of VP to C cells infected with UlmCP15-17 and TMV CP-O (8 to 40:1 and 16:1) were greater than those observed in this study and previously for U1-TMV at a similar stage of infection (4 to 5:1, ref. 26 and Table 1). Also, we have not observed any C cells infected with the CP-less mutant, SNCO15, although in theory we should have if the ratio of VP to C cell infection for this virus is similar to that of U1-TMV (Table 1). The results suggest that a native encapsidationcompetent CP of TMV may aid in the efficient transport of the virus into the C cell/SE complexes, although it is not essential. The results also suggest that a CP is necessary to allow virus movement into the C cell/SE complex; however, the lack of signal in C cells of tissues infected by SNCO15 may be due to an insufficient number of samples analyzed or a lack of sensitivity in the detection method employed. Both UlmCP15-17 and SNCO15 accumulated in fewer BS or vascular bundle cells within chlorotic lesions than the parental U1-TMV (Table 1). Free RNA viruses are known to accumulate to only low steady-state levels and this trait has been attributed to the instability of the unprotected RNA (10, 13, 51, 52). It has been suggested that a general impairment of cell-to-cell movement and accumulation of virus may disproportionately inhibit phloem-dependent accumulation (7). Mise and Ahlquist (53) found that a movement protein mutant of cowpea chlorotic mottle bromovirus capable of cell-to-cell spread stops moving and accumulating before it reaches a novel cell boundary, i.e., its cell-to-cell movement is confined to epidermal cells through which the corresponding wild-type virus moves unimpeded. Also, as noted, MIc-TMV accumulates in fewer vascular cells than U1-TMV and this characteristic correlates with the impaired systemic accumulation of this virus (26, 34). Whether the general decrease in infection observed for UlmCP15-17 and SNCO15 contributes to their lack of phloem-dependent accumulation remains to be determined. We thank Drs. W. 0. Dawson, R. A. Dixon, and R. Hajimorad for critical comments on the manuscript; Dr. Tim Clark, Stacy Hefner, and Blake Hartwell for growth and maintenance of plants; and Dr. Robert Gonzales, Ann Harris, and Valerie Graves for synthesizing PCR primers and sequencing PCR products. We also thank Ty Meyers, Darla Boydston, and Cuc Ly for the production of glossy prints and Allyson Wilkins for typing and help in proofing the manuscript.

FIG. 4. Immunodetection of 126/183-kDa protein in vein cells of N. tabacum cv. Xanthi nn leaves inoculated with a coat proteinless TMV mutant, SNCO15, or the parental U1-TMV. Sections prepared from chlorotic lesions harvested at 6 DPI were labeled using an anti-serum against the TMV 126-kDa protein followed with a goat anti-rabbit IgG gold (20 nm) conjugate. (A) An immunogold-labeled X-body adjacent to a virus aggregate in an infected mesophyll cell. X, X-body; R, virus aggregate. (X30,000; bar = 0.3 ,um.) (B) X-bodies (denoted by arrowheads) containing 126/183-kDa protein detected in two VP cells of a class V vein from a section infected with SNCO15. Inset (indicated by arrow) is a magnification of the boxed area within the vein and shows the deposition of gold particles over an X-body. C, companion cell; VP, vascular parenchyma cell, XT, xylem tracheary element. (C) An X-body containing 126/183-kDa protein detected in a VP cell within a class V vein from a section infected with U1-TMV. Inset (indicated by arrow) is a magnification of the boxed area within the vein and shows the deposition of gold particles over an X-body. (Veins, x1,300; bars = 4 ,um; Insets, x25,000; bars = 0.4 ,im.)

50). We have shown that transport of TMV between these cell types is also controlled, because U1-TMV and MIC-TMV accumulate in more VP cells than C cells in inoculated tobacco leaves both early and late after infection (Table 1; ref. 26). In this study we have shown that neither a virion nor host factors essential for phloem-dependent movement of a specific chi-

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