Inhibition of tobacco mosaic virus replication in lateral roots is ...

Protoplasma (2002) 219: 184–196

PROTOPLASMA © Springer-Verlag 2002 Printed in Austria

Inhibition of tobacco mosaic virus replication in lateral roots is dependent on an activated meristem-derived signal T. A. Valentine, I. M. Roberts, and K. J. Oparka* Unit of Cell Biology, Scottish Crop Research Institute, Dundee Received July 23, 2001 Accepted October 11, 2001

Summary. Viral invasion of the root system of Nicotiana benthamiana was studied noninvasively with a tobacco mosaic virus (TMV) vector expressing the green-fluorescent protein (GFP). Lateral root primordia, which developed from the pericycle of primary roots, became heavily infected as they emerged from the root cortex. However, following emergence, a progressive wave of viral inhibition occurred that originated in the lateral-root meristem and progressed towards its base. Excision of source and sink tissues suggested that the inhibition of virus replication was brought about by the basipetal movement of a root meristem signal. When infected plants were inoculated with tobacco rattle virus (TRV) expressing the red-fluorescent protein, DsRed, TRV entered the lateral roots and suppressed the host response, leading to a reestablishment of TMV infection in lateral roots. By infecting GFP-expressing transgenic plants with TMV carrying the complementary GFP sequence it was possible to silence the host GFP, leading to the complete loss of fluorescence in lateral roots. The data suggest that viral inhibition in lateral roots occurs by a gene-silencing-like mechanism that is dependent on the activation of a lateral-root meristem. Keywords: Tobacco mosaic virus; Nicotiana benthamiana; Root; Meristem; Viral suppression; Green-fluorescent protein. Abbreviations: GFP green-fluorescent protein; PTGS posttranscriptional gene silencing; TMV tobacco mosaic virus; TRV tobacco rattle virus.

Introduction The cell-to-cell and long-distance movement of plant viruses has been the subject of intense recent investigation (for reviews, see Carrington et al. 1996, Lucas and Wolf 1999, Oparka et al. 1996). In the case of

* Correspondence and reprints: Unit of Cell Biology, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, United Kingdom.

phloem-mobile viruses, the distribution patterns of virus in developing sink tissues are strongly influenced by source–sink relationships (A. Roberts et al. 1997). It appears that once an infectious viral complex enters the translocation stream, its final destination is largely dependent on the flow of assimilates within the plant (reviewed by Santa Cruz 2000). If systemic viruses enter the phloem at a stage of development during which the root system is a dominant sink, it might be expected that such viruses would also be delivered to developing roots. However, almost nothing is known of the patterns of virus invasion in roots, despite the fact that several viruses invade the root systems of many commercially important crop species (Matthews 1991). It appears that in both roots and shoots the apical meristem is largely devoid of virus infection (Faccioli and Colombarini 1996, Hsu et al. 2000, Matthews 1991, Walkey et al. 1969). The inability of viruses to infect apical meristems has been exploited commercially and forms the basis of meristem tip culture, a method by which virus-free clones can be obtained by growing excised shoot tips in tissue culture (Matthews 1991, Walkey et al. 1987). In general, a zone of variable length (but usually within 100 mm of the root or shoot tip) is free of replicating virus. At present, the reasons why meristems fail to support virus replication are unknown. It is possible that a barrier to meristem invasion is enforced by cells some distance from the meristem (see Matthews 1991), although such a putative barrier has not been experimentally demonstrated. While many viruses are thought to be unable to invade meristems, virus particles from several unrelated

T. A. Valentine et al.: Inhibition of tobacco mosaic virus replication in developing roots

groups have been detected in apical meristems by electron microscopy (Appiano and Pennazio 1972, D. Roberts et al. 1970, Walkey and Webb 1968). An attractive hypothesis for the ability of meristems to escape virus infection is that a mechanism for viral gene silencing occurs in meristematic cells that targets the viral RNA for degradation, allowing newly differentiated tissues to escape viral infection (Baulcombe 1999, Ratcliff et al. 1997, van Kammen 1997). There is now substantial evidence for a parallel between the phenomenon of posttranscriptional gene silencing (PTGS), in which transgenes are silenced by a sequence-specific RNA degradation process (Carthew 2001, Davies et al. 1997, Fire 1999, Grant 1999, Lee et al. 1997, Lindbo et al. 2001, Matzke et al. 2001, Metzlaff et al. 1997, Vaucheret and Fagard 2001), and the RNA-mediated defence that occurs against plant viruses (Covey et al. 1997; Kooter et al. 1999; Marano and Baulcombe 1998; Ratcliff et al. 1997, 1999). It appears that there may be a surveillance system in plants that can target viral nucleic acids and give sequence specificity to RNA-mediated defence (Baulcombe 1999, Waterhouse et al. 2001). The recent discovery that some viruses possess genes that act as suppressors of antiviral defence in the host (Carrington and Whitham 1998, Kasschau and Carrington 1998, Li and Ding 2001, Voinnet et al. 1999) lends support to the view that these suppressors of gene silencing act as part of a viral counter-defence system that allows viruses to accumulate after activation of the host defence mechanism. With many viruses, the final steady state of virus accumulation appears to be determined by the effectiveness of the suppressors (Baulcombe 1999, Voinnet et al. 1999). While the analysis of molecular trafficking in the shoot meristem is hindered by the inaccessibility of the shoot apex, and usually involves the surgical removal of several leaves and lateral primordia to expose the meristem (e.g., Gisel et al. 1999), the developing root apical meristem is amenable to noninvasive studies (Malamy and Benfey 1997a, b; Dolan et al. 1994; Wright and Oparka 1996). In particular, the formation of lateral roots from the pericycle provides a convenient model system in which to examine the developmental events that give rise to the root meristem (Malamy and Benfey 1997a, b). To date, however, there have been no studies of viral movement in root meristems. In the present work we used confocal microscopy to study the movement of a tobacco mosaic virus (TMV)

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vector tagged with the green-fluorescent protein (GFP) in roots of the host plant Nicotiana benthamiana, using intact plantlets grown on nutrient agar (see Oparka et al. 1995, Wright and Oparka 1996). We show that the emerging lateral-root primordium becomes heavily infected with virus. However, with continued development, the growing root escapes infection due to a wave of viral inhibition that progresses in a basipetal pattern away from the root tip. We demonstrate that the mobile signal for inhibition originates within the root apex, rather than in source tissues, and that its propagation is dependent on the formation of an active lateral-root meristem.

Material and methods Plant material Nicotiana benthamiana plants were used in all experiments. For viral tracking in roots, seeds were surface sterilised (10% bleach for 20 min, followed by several washes in distilled water), incubated in 0.3 mM gibberellic acid for 4 h, and sown directly onto Murashigeand-Skoog medium (Melford Laboratories, Ipswich, U.K.) containing 2% sucrose and 0.8% agar (Difco, Detroit, Mich., U.S.A.) in square 10 cm petri dishes. Petri dishes were sealed with Nescofilm (Bando Chemical Industries, Kobe, Japan) and vented with micropore tape (3M Healthcare, St. Paul, Minn., U.S.A.) before being placed at a near vertical position at 21 °C with a cycle of 18 h daylight and 6 h darkness. Removal of meristems and cutting of primary roots was achieved with sterile razor blade fragments attached to wooden cocktail sticks. After cuts had been made, all petri dishes were resealed as above.

Inoculation of plants with TMV.GFP A modified form of a previously described construct of TMV expressing GFP (Shivprasad et al. 1999), carrying the cycle 3 enhanced GFP (Crameri et al. 1996), was kindly supplied by G. Pogue (Large Scale Biology Corporation, Calif., U.S.A.). Sap inoculum was produced by inoculating 4- to 6-week-old greenhousegrown N. benthamiana with in vitro transcripts of TMV.GFP. Systemically infected fluorescent leaves were harvested approximately 9 days post inoculation and ground in Sörensen’s phosphate buffer (1 g/ml). For all root experiments, 21- to 25-day-old N. benthamiana seedlings grown in tissue culture were inoculated by rubbing a 1-in-5 dilution of infectious sap plus carborundum onto the first true leaf using a cotton bud.

Superinfection of roots with tobacco rattle virus In one experiment, plants were inoculated with TMV.GFP as described above. Following the inhibition of TMV.GFP replication in lateral roots the plants were superinfected with a tobacco rattle virus (TRV) vector expressing the red-fluorescent protein, DsRed (from plasmid pDsRed; Clonetech, Palo Alto, Calif., U.S.A.; for details, see Vassilakos et al. 2001). The subsequent progression of TMV.GFP and TRV.DsRed infections was monitored in situ with a Coolview digital camera (Photonic Science) attached to a Leica fluorescence stereomicroscope (MZFLIII).

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Virus-induced gene silencing To study the potential for virus-induced gene silencing within the root system, transgenic N. benthamiana plants constitutively expressing GFP within the endoplasmic reticulum (Voinnet et al. 1998) were infected with TMV.GFP, exactly as described above. As a control, transgenic plants were infected with TMV lacking the GFP sequence.

within the pericycle of infected roots, were highly fluorescent and visible as bright plaques of cells within the root stele. As the lateral primordium emerged, the cells within it remained highly fluorescent due to the presence of replicating virus (Fig. 1 B, C). In all cases, the lateral primordia were more highly fluorescent than the primary root from which they were derived.

Confocal laser scanning microscopy GFP expression was tracked with a Bio-Rad MRC 1000 confocal laser scanning microscope (Bio-Rad, Hemel Hempstead, U.K.). Plants were observed in situ through the bottom of the petri dish with either a ¥4 or a ¥10 long-working distance lens (see Oparka et al. 1995) and 488 nm blue laser excitation. Whole-root pictures were mapped in sections and montaged by Photoshop software (Adobe, Mountain View, Calif., U.S.A.). Immuno-electron microscopy Lateral-root tips were excised in 5% glutaraldehyde in piperazineN,N-bis(2-ethanesulfonic acid) buffer, pH 8.0, fixed for further 18–24 h in the same fixative, and then dehydrated in alcohol before embedding in Araldite resin (Agar Scientific Ltd., Stansted, U.K.), essentially as described by Ryabov et al. (1999). Alternate groups of 0.5 mm and 0.1 mm sections were prepared for light microscopy, and electron microscopy, respectively. Light microscope sections were stained with 1% Toluidine Blue, while those for electron microscopy were poststained with uranyl acetate and lead citrate. Immunogold labelling was optimised and performed as described previously (Ryabov et al. 1999). For mapping of the position of infected cells, median sections of roots were photographed at low magnifications and reference prints made for several sections. All cells in a single section were subsequently examined at higher magnification to determine the presence or absence of immunogold labelling for TMV. Microinjection The fluorescent, membrane-impermeant probe HPTS (Molecular Probes, Cambridge, U.K.) was microinjected into single lateral-root primordia with a modified pressure-microinjection system (Oparka et al. 1990). Following injection, the petri dish lid was replaced and dye distribution within the root monitored with a confocal laser scanning microscope (see above).

Inhibition of TMV replication in lateral roots We found that as lateral roots began to elongate they gradually lost fluorescence, despite the maintenance of unaltered levels of fluorescence in the primary root. We therefore imaged noninvasively the developmental sequence of lateral-root formation from over 30 infected primary roots. Two such sequences are shown in Fig. 2. Following their emergence from the parent root the lateral roots remained fluorescent for approximately 20 h, by which time they had reached an average length of 150 mm. When the lateral roots had reached an approximate length of 200 mm (about 30–40 h after emergence), a distinct zone lacking GFP was observed close to the lateral-root meristem (Fig. 3). With time this “cone” of inhibition spread basipetally along the lateral root to its point of connection with the infected primary root. In comparison with the differentiating cells behind the root meristem, cells of the root cap remained highly fluorescent and could be distinguished clearly from the underlying inhibited tissues (Fig. 2). With time, however, root cap cells also lost GFP fluorescence (Fig. 2). In all virus-inhibited roots, a sharp demarcation was observed between the base of the lateral root and its connection with the primary root (Fig. 2). Primary roots did not diminish in fluorescence, indicating that the signal for viral inhibition did not spread into mature infected cells of the primary root.

Results Production of lateral roots from infected primary roots Plants of N. benthamiana infected with TMV.GFP showed fluorescence within the primary root system within 5–10 days after inoculation of the upper leaves. Virus presence was first observed towards the root apex as intermittent “streaks” arising from the root vascular cylinder (Fig. 1). With time these streaks expanded longitudinally along the vasculature and also outwards into the root cortex and epidermis. Eventually, root hairs also became infected with the virus (Fig. 1 A). Lateral-root primordia, which formed

Virus particles are present in inhibited lateral roots In one experiment, lateral roots showing viral inhibition were imaged under the confocal microscope and subsequently prepared for immuno-electron microscopy using polyclonal antibodies to the TMV coat protein. Median longitudinal serial sections of the root were mapped at low magnification and the positions of infected cells recorded (Fig. 4).Although these roots had lost fluorescence, labelled virus particles were detected in cells at the base of the lateral root, within the root meristem, and within cells of the root cap (Fig. 4).


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Fig. 1. Progression of infection of TMV.GFP on the root system of Nicotiana benthamiana. Bar: 5 mm. At 6 days post inoculation (d.p.i) virus is absent from the root system, which displays only background autofluorescence. The small arrow indicates the primary root tip. At 9 d.p.i intermittent streaks of replicating virus (arrowheads) have escaped from the vascular system. At 12 d.p.i the root is heavily infected. Note the presence of a virus-free zone extending behind the primary- and lateral-root meristems. Insets Detail of infected root hairs (A), details of infected lateral-root primordia (B and C). Bars: 100 mm

Infection of mature lateral roots In one experiment, a set of plants was infected by TMV.GFP when extensive lateral roots had already formed from the primary root. The pattern of infection of these lateral roots was identical to that observed in the primary-root system; the virus escaped from the

phloem and invaded the cortex and epidermis by cellto-cell movement (data not shown). When established lateral roots became infected, they did not undergo the complete loss of fluorescence observed with newly forming lateral primordia but remained fluorescent for several days. However, a consistent feature in mature lateral roots was a zone that lacked replicating virus

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Fig. 2. Suppression of TMV.GFP in developing lateral roots. The emerging lateral-root primordium is heavily infected with virus. At approximately 20–30 h after emergence a dark zone is apparent behind the apical meristem (for detail, see Fig. 3). 48 h after emergence the suppression of viral replication has extended to the junction of the lateral root with the primary root, although some root cap cells (arrows) remain fluorescent. By 60 h after emergence, the lateral roots have lost fluorescence. Note that the primary-root system does not diminish in fluorescence. Numbers in right-hand corners represent hours post emergence of the lateral root. Bars: 100 mm

basipetal to the apical meristem (e.g., Fig. 5 A). This apparent barrier to infection of the meristem extended for approximately 500 mm behind the root tip, within the zone of root elongation (Fig. 5 A). As the infected lateral root continued to grow, this “exclusion zone” was maintained at a constant distance behind the root meristem.

Source and sink removal experiments In order to determine whether the initial signal for viral inhibition originated in source leaves or in the root meristem, we examined the effect of removing source and sink tissues on infected seedlings. To isolate the source leaves, the primary root was completely severed above the first detectable lateral-root initials (Fig. 5 B, C). The lateral roots were then observed for a further 3–4 days. During this time period the infected


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Fig. 3. Detail of lateral root at 35 h post emergence. Note the dark cone of cells lacking fluorescence (asterisk) immediately behind the root cap. Bar: 100 mm

lateral roots continued to emerge and underwent a progression of viral suppression identical to that observed in intact infected plants (Fig. 5 B, C). These results suggest that the signal for viral inhibition did not arise in source tissues. As the phloem is not fully connected in lateral roots until some time after their emergence (Wright and Oparka 1996), we concluded that a phloem-mobile signal from the shoot was unlikely to have been the trigger for viral inhibition in the emerging lateral root. In sink removal experiments, we surgically removed the apical regions of infected roots, including the meristem, and monitored these roots for further 4 days. During this time, replicating virus moved into the nonfluorescent cells of the exclusion zone, up to the point of meristem excision (Fig. 5 A). However, no virus infection was established in cells apical to the excision point (Fig. 5 A). These data suggest that viral inhibition in the subapical region of the root was brought about by a meristem-derived signal that moved basipetally. Formation of symplastic domains In order to examine symplastic domains in developing root primordia, we injected the membrane-impermeant fluorescent probe HPTS into infected and noninfected developing lateral roots. When HPTS was injected into emerging lateral roots, the dye spread rapidly within the lateral primordium but failed to

Fig. 4 A, B. Detection of virus particles in virus-inhibited lateral roots. A Map of an infected lateral root that had lost GFP fluorescence. Dark regions depict cells that contained TMV particles as detected by immunogold labelling. B Electron micrograph of an infected cell showing gold-labelled TMV particles found in the cell marked by an asterisk in A. Bar: 0.1 mm

move into the primary root, indicating the formation of a symplastic barrier at the base of the enlarging primordium (Fig. 6 A, B). In another experiment, we infected the primary root with TMV.GFP after

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lateral-root primordia had already formed. In cases where the primordium was established before the arrival of replicating virus in the stele, the virus was unable to invade the cells of the primordium by cell-to-cell movement, and fluorescence remained restricted to stelar tissues of the primary root (Fig. 6 C–H). These data indicate that during lateral-root formation a symplastic barrier is established at the

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base of the developing primordium that is retained during lateral-root development. Superinfection with TRV suppresses host inhibition mechanism and releases TMV infection If inhibition of TMV replication in lateral roots was brought about by a host resistance mechanism based

Fig. 6 A–H. Symplastic domains in developing lateral roots. A and B Following microinjection of the membrane-impermeant probe HPTS into a developing lateral root, the dye was restricted to the primordium and did not enter the primary root. The arrow indicates the point of injection. Panel A, fluorescence; panel B, bright field. Bars: 100 mm. C–H Progression of infection of TMV.GFP subsequent to the initiation of an uninfected lateral root. The virus escaped from the stele (C) and continued to invade the primary root cortex (D and E). However, it did not enter the developing lateral root. Panels C, D, and E, fluorescence; panels F, G, and H, bright field. Bars: 100 mm

Fig. 5 A–C. Source and sink removal experiments. A Removal of the apical meristem from an infected root system (time zero; dashed line). Virus continued to spread along the lateral root and by 120 h had reached the point of excision (see also enlargement to right). Note that the region apical to the excision continued to grow but remained uninfected. Bar: 500 mm. B and C The primary root was cut above the point of insertion of developing, infected lateral roots. The lateral roots continued to grow and underwent a progressive loss of GFP fluorescence. In sequence in B, the lateral root was completely devoid of fluorescence at 120 h following emergence. Bar: 100 mm

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on PTGS, we hypothesised that suppression of this signal would be achieved by superinfection with a second virus known to suppress host gene silencing. We selected TRV, a virus with a known tropism for root systems (MacFarlane and Popovich 2000), and which possesses an effective suppressor of PTGS in N. benthamiana (Voinnet et al. 1999). We constructed a TRV vector that expressed the red-fluorescent protein, DsRed (TRV.DsRed; Vassilakos et al. 2001), in order to distinguish TRV and TMV infection sites and to ensure that the viruses did not contain homologous GFP sequences which might lead to silencing of TRV infection. TMV.GFP-infected plants were monitored for the inhibition of viral replication in lateral

roots as described above. The plants were then infected with TRV.DsRed and monitored for further 10 days. The results are shown in Fig. 7. In several plants, TRV was able to infect lateral roots in which TMV replication had been inhibited. Figure 7 B shows the establishment of TRV infection (red) within a lateral root in which TMV infection had been almost completely eliminated (cf. Fig. 7 A). Subsequently, TMV infection (green; Fig. 7 C) was restored at the apex of the same root. Higher-magnification images of the root tip showed that both TMV.GFP (Fig. 7 E) and TRV.DsRed (Fig. 7 F) were present in this region. Identical results were observed with virus vectors expressing the reciprocal fluorescent proteins (i.e.,

Fig. 7 A–H. Superinfection of lateral roots with TRV restores TMV replication. Following inhibition of TMV replication in lateral roots (A; arrow indicates root tip), the plants were inoculated with TRV.DsRed. TRV entered the TMVinhibited lateral roots and replicated (B). 3 days later, TMV replication was restored at the root apex (C, arrow) and in more basal parts of the lateral root. TRV.DsRed replication was also evident in the elongating lateral root, including the apex (D, arrow). E and F Same root tip at higher magnification showing the presence of both TMV.GFP and TRV.DsRed in the root apex. In a reciprocal experiment, roots were first infected with TMV.DsRed. Following the characteristic inhibition of TMV replication, the plants were superinfected with TRV.GFP, which entered the lateral roots and commenced replication (G, arrows). 3 days later, TMV.DsRed replication had resumed throughout most of the lateral root (H)


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TRV.GFP was able to release TMV.DsRed infection in lateral roots; Fig. 7 G, H). When TMV.GFP-silenced lateral roots were superinfected with TMV.DsRed, no infection was established in the lateral roots, consistent with a gene-silencing mechanism directed specifically against the TMV RNA (data not shown). TMV induces gene silencing of integrated GFP in lateral-root meristems The replication of TMV.GFP in the lateral-root primordium, and its subsequent suppression following meristem activation, prompted us to explore the possibility of silencing endogenous meristem-expressed genes with a TMV-based vector. It is now well established that plant viruses carrying elements of plant host genes produce symptoms that are phenocopies of mutations in the corresponding host genes (Baulcombe 1999, Lindbo et al. 2001, Matzke et al. 2001). To study the potential for such virus-induced gene silencing in lateral-root meristems, we utilised transgenic N. benthamiana plants that expressed integrated GFP (intGFP) in the endoplasmic reticulum (Voinnet et al. 1998). In these plants the intGFP was distributed throughout cells of the root system and was particularly conspicuous in both primary and lateralroot meristems (Fig. 8 A). We then inoculated these plants with TMV.GFP and examined them for subsequent loss of intGFP fluorescence. At 20 days post inoculation, fluorescence was conspicuously absent in more than 95% of emerging lateral roots, indicating that silencing of the endogenous intGFP gene had occurred in parallel with suppression of TMV (Fig. 8 B). Uninfected root systems, or root systems infected with TMV lacking the GFP gene, failed to show intGFP silencing (data not shown). As shown previously for infected, nontransgenic roots, the silencing signal spread along the lateral root to its connection with the primary root but did not enter the connecting primary root (compare Fig. 2 with Fig. 8 B). These results confirmed the presence of a barrier to the mobile gene-silencing signal at this interface. Discussion It is well established that plant apical meristems are able to escape infection by several viruses (see Matthews 1991), although the mechanism by which this occurs is poorly understood. The root system of infected plants provides an amenable system for exam-

Fig. 8 A, B. Virus-induced gene silencing in lateral roots. A Transgenic N. benthamiana plant expressing GFP within the root system. Note the intense fluorescence from the lateral-root meristem. B Silencing of host intGFP 20 days after infection of the leaves with TMV.GFP. Note that the primary root remains unsilenced. Bars: 100 mm

ining the ability of plant viruses to infect apical meristems. Using a noninvasive imaging system and a GFPtagged TMV, we were able to follow the progression of viral infection in primary- and developing lateralroot systems in the host plant N. benthamiana. Lateral roots are initiated by anticlinal cell divisions in the pericycle, a layer of cells found immediately within the root endodermis (Casero et al. 1993; Malamy and Benfey 1997a, b). One of the first events in lateral-root formation is the onset of anticlinal cell divisions of individual pericycle cells as these undergo reentry into the cell cycle (Malamy and Benfey 1997b). Our observations of infected primary-root systems revealed that the pericycle initials that gave rise to lateral primordia became heavily infected with virus during these early stages in the formation of the lateral-root primordium. As enlargement of the primordium proceeded these cells remained heavily infected. Malamy and Benfey (1997a, b) have sepa-

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rated the development of Arabidopsis lateral roots into a number of distinct stages. During the preemergence and emergence phases the number of cells in the primordium epidermis and root cap remain constant, suggesting that growth during these phases is achieved largely through cell expansion within the primordium. After emergence, the number of cells begins to increase, and new cells appear near the apex. The root then starts to grow via new cell divisions at the apex, concomitant with the formation of a functional lateralroot meristem. Malamy and Benfey (1997a, b) have referred to this stage as meristem activation, which in Arabidopsis thaliana occurs when the expanding lateral root has reached a length of approximately 200 mm. The onset of viral inhibition seen in lateral roots of N. benthamiana appears to correspond well with the stage of meristem activation. Loss of GFP fluorescence was first observed in cells near the lateral-root apex when the primordium had reached a length of 150– 200 mm, with a progressive loss of fluorescence in cells basipetal to this region. Removal of source tissues failed to prevent the progression of viral inhibition in the emerging lateral roots, suggesting that the signal was initiated in the newly formed lateral-root meristem. Prior to this developmental stage the cells that formed the primordium were unable to inhibit viral replication. Once the inhibition signal was initiated, it progressed basipetally along the lateral root to its junction with the primary root. Microinjection experiments demonstrated that a symplastic barrier was present at this interface, providing evidence that the mobile viral inhibition signal moves through plasmodesmata. Following the formation of the symplastic barrier, it is likely that the phloem connecting the primary- and lateral-root systems provides the predominant means of molecular exchange between the two root systems (see Oparka et al. 1996). The prolonged fluorescence seen in root cap cells is most likely due to the progressive symplastic isolation of the root cap from the underlying root epidermal cells (Zhu et al. 1998). Initially the root cap cells are formed from the division of an epidermal and lateralroot cap initial that undergoes a periclinal division to generate both epidermal cells and cells of the lateralroot cap (Malamy and Benfey 1997b). As all the cells of the root primordium are initially infected with virus, following meristem activation virus would be divided between the newly forming tissues. Electron microscopy of TMV-inhibited lateral roots confirmed that

virus particles were present in both root cap cells and meristem cells of the primordium. The eventual loss of fluorescence from the entire lateral root, including the root cap, is possibly due to the continued turnover of new root cap cells as increasing numbers of the originally infected cells are sloughed off from the root surface (Hawes et al. 2000). The presence of nonreplicating virus particles in meristematic cells of inhibited roots may explain the several reports, on the basis of ultrastructural studies, that virus particles of different viral groups may be present in meristems (see Matthews 1991). Our observations would indicate that such virus particles entered the meristem prior to the initiation of viral inhibition and may have triggered the host response. Although many of our observations point to a PTGS-like mechanism of viral inhibition, we have not shown, unequivocally, that viral inhibition results directly from a mechanism in which viral RNA was detected and degraded specifically by a host surveillance system (Baulcombe 1999). It remains possible that viral replication was impeded by more indirect mechanisms, such as the degradation of one or more viral proteins, or competition of viral RNA with host RNA for the translational machinery in meristematic cells (Matthews 1991). However, the events we observed bear a striking resemblance to virus-induced gene silencing phenomena reported in other plant tissues (Baulcombe 1999, Marano and Baulcombe 1998, Kooter et al. 1999, Ratcliff et al. 1997, Waterhouse et al. 1999). The ability of TRV, a virus known to express a suppressor of PTGS (Voinnet et al. 1999), to infect TMV-silenced lateral roots and suppress the host response is consistent with a model in which TMV replication was held in check by a PTGS-like mechanism. Also, the ability of TMV to resume replication following TRV infection is consistent with the observed presence of TMV particles in these roots and indicates that the TMV vector had not lost its GFP insert through genetic instability. Finally, the ability of TMV.GFP to silence transgenically expressed intGFP lends support to the view that a PTGS-based mechanism was initiated in root tips during, or immediately after, meristem activation. In the present study, two distinct infection “phenotypes” were observed. In the case of infected lateralroot primordia, replicating virus was present in the meristem initials, but viral replication was inhibited following meristem activation. In the case of mature lateral roots, differentiated root cells became infected


during post-phloem transport, but the subapical region remained uninfected. It is possible that these infection patterns arise from the same defence mechanism, operating to varying degrees of intensity. We have found that the extent of viral gene silencing is in many cases proportional to the amount of viral RNA encountered by initially infected host cells (T. A. Valentine unpubl. data). We hypothesise that, in the case of infected lateral primordia, the high viral RNA titre detected during meristem activation gives rise to a strong, mobile RNA silencing signal that is transmitted back along the emerging lateral root. In contrast, in already emerged laterals, the lateral-root meristem is devoid of viral RNA at the time of infection of the primary-root system. In this case, the first virus particles that enter meristematic cells following phloem unloading may give rise to a weaker gene silencing response that spreads a limited distance back from the meristem. In both the above scenarios a functional meristem is required to maintain the inhibition of viral replication. Our present study points to a relationship between meristem activation and viral gene silencing. In the future it will be interesting to study the behaviour of a range of viruses that infect Arabidopsis roots. Several Arabidopsis mutants have been created that have specific mutations in root meristem development. For example, in the rml1 and rml2 (root meristemless) mutants, growth of the primary root is arrested soon after germination (Cheng et al. 1995). Lateral roots emerge from the short parent root, and then the growth of the lateral roots is arrested. [3H]thymidine labelling studies showed that there is little or no cell proliferation at the tips of the lateral roots, and so it appears that the rml mutants are unable to activate a root meristem (Cheng et al. 1995). Similarly, Arabidopsis mutants have been identified that are unable to initiate gene silencing mechanisms (Elmayan et al. 1998) and would make useful tools to determine the factors that influence the ability of viruses to infect meristems. Further studies exploiting viral vectors on plant root systems are likely to give new insights into the complex interplay between plant meristems and viral pathogens.

Acknowledgments We are grateful to Large Scale Biology Corporation (LSBC) for financial support and for providing the modified TMV.GFP vector used in these studies. We also thank Stuart MacFarlane for con-

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structing TRV.DsRed and David Baulcombe for the kind gift of ERGFP transgenic N. benthamiana plants. The Scottish Crop Research Institute is grant aided by the Scottish Executive Rural Affairs Department (SERAD).

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