The Nanovirus-Encoded Clink Protein Affects Plant Cell Cycle Regulation through Interaction with the Retinoblastoma-Related Protein

Nanoviruses, multicomponent single-stranded DNA plant viruses,encode a unique cell cycle link protein, Clink, that interactswith retinoblastoma-related proteins (RBR). We have establishedtransgenic Arabidopsis thaliana lines that conditionally expressClink or a Clink variant deficient in RBR binding. By controlledinduction of Clink expression, we demonstrated the capacityof the Clink protein to alter RBR function in vivo. We showedthat transcription of both S-phase-specific and G₂/M-phase-specificgenes was up-regulated depending on the RBR-binding proficiencyof Clink. Concomitantly, ploidy levels increased in a substantialfraction of leaf cell nuclei. Also, leaf epidermis cells oftransgenic plants producing Clink were smaller and more numerous,indicating additional cell divisions in this tissue. Furthermore,cytogenetic analyses following induction of Clink expressionin mature leaves revealed the presence of metaphasic and anaphasicnuclei, clear evidence that Clink-mediated RBR inactivationis sufficient to induce quiescent cells to reenter cell cycleprogression and, for at least a fraction of them, to pass throughmitosis. Expression of Clink had no effect on genes transcribedby RNA polymerases I and III, suggesting that, in contrast toits mammalian homologue, A. thaliana RBR is not involved inthe repression of polymerase I and polymerase III transcription.The results of these in vivo analyses firmly establish Clinkas a member of the diverse class of multifunctional cell cyclemodulator proteins encoded by small DNA viruses.

INTRODUCTION

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Introduction

Due to their restricted genome size, small DNA viruses do notencode polymerases and other enzymes of the DNA synthesis machinery.Instead, they exploit host DNA replication to multiply theirgenomes (19). This is a general feature of mammalian tumor viruses,e.g., simian virus 40 (SV40) or the papillomaviruses, whichencode multifunctional regulatory proteins that cause the hostcell to enter S phase, thereby making the host's DNA synthesismachinery available for virus DNA replication. Key regulatorsof cell cycle progression are the members of the retinoblastomaprotein (RB) family, which sequester E2F/DP transcription factorsin inactive complexes, thereby preventing them from gene activation(13, 53). The RB-controlled block of cell cycle progressionis released in various ways, frequently by the binding of otherproteins to RB and the subsequent release of the previouslysequestered transcription factors. Various cellular or viralproteins bind to RB or otherwise prevent it—by hyperphosphorylation(48) or degradation (8)—from complexing S-phase relevanttranscription factors. Among the best-studied examples are theSV40 large T antigen (T-ag), human papillomavirus E7, and adenovirusE1A protein, all of which bind to the pocket domain of RB througha sequence containing the conserved amino acid motif LxCxE (11,16).

In mammals, RB also acts as a general repressor of transcriptionby RNA polymerase III (PolIII) and PolI, potentially to controlcell growth (reviewed in reference 29). Repression of PolI transcriptionis mediated through interaction between RB and the RNA PolItranscription factor UBF (10), while the different classes ofPolIII-transcribed genes appear to be down-regulated via distinctmechanisms involving the interaction of RB with different transcriptionfactors (21, 22, 47, 54). These effects require the pocket domainof RB, and LxCxE-containing viral proteins such as E1A, T-ag,and E7 are able to relieve the repressive effects of RB on PolIIIand PolI transcription (29, 54).

Plant DNA viruses include members of the families Geminiviridaeand Nanoviridae (43, 52). These viruses possess small single-strandedDNA (ssDNA) genomes, as opposed to the double-stranded genomesof the mammalian tumor viruses cited above, but still show strikingsimilarities with them in the way they induce host cells toenter S phase or trigger progress beyond the G₁/S checkpoint(20).

The multifunctional replication initiator protein Rep (or AL1)of geminiviruses has been shown to bind to the plant homologuesof RB, retinoblastoma-related proteins (RBR), and release theblock imposed by RBR on cell cycle progression (18, 26). Thebinding of geminivirus Rep proteins to RBR is not always mediatedby an LxCxE motif; expression of Rep proteins from viruses ofthe genus Begomovirus which lack this sequence systematicallyleads to the induction of cell cycle progression and S-phase-relevantgenes, such as the proliferating cell nuclear antigen gene (PCNA)(18, 35, 36).

Members of the second plant virus family with ssDNA genomes,the nanoviruses, encode a multitude of replication initiatorproteins, but only one of them, the master Rep (M-Rep), is essentialfor nanovirus genome multiplication (49). In addition, all nanovirusesencode a small (20-kDa) protein named Clink (for "cell cyclelink") that has been shown to bind human RB and plant RBR invitro through its LxCxE motif (5). Furthermore, Clink is anF-box protein that also interacts with Skp1 protein, a memberof the Skp1/cullin/F-box protein complex targeting proteinsfor ubiquitin-dependent degradation by the 26S proteasome. Clinkacts as a replication enhancer during infection by a nanovirus(5). The stimulating effect of Clink on DNA replication is notnanovirus specific; geminivirus DNA replication is equally enhanced(3). Although present in the genomes of all known nanoviruses,Clink is not essential for the replication of faba bean necroticyellows virus (FBNYV) DNA or for the infectivity and symptomexpression of this nanovirus (50).

The in vitro interaction of Clink with RBR and its functionas a replication enhancer in vivo suggested that Clink mightinteract with RBR in planta and trigger cell cycle progressionin a manner similar to that of geminivirus Rep proteins or,for instance, SV40 T-ag. Therefore, we studied here the influenceof Clink on cell cycle regulation in vivo by using transgenicArabidopsis thaliana plants carrying clink gene constructs,conditionally inducible by the glucocorticoid hormone dexamethasone(Dex) (2).

MATERIALS AND METHODS

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Materials and Methods

Recombinant DNA plasmids and plant transformation. The binary transformation vector pTA7002, containing the completetwo-component glucocorticoid-inducible system (2), was cleavedwith XhoI and SpeI. The DNA sequence coding either for wild-typeClink or for the mutated Clink^{C112R E114A} (Clink^mut) protein(5) was introduced into the pTA7002 vector as an XhoI-SpeI fragmentto create pTA7002-Clink and pTA7002-Clink^mut, respectively.The correctness of all constructs was verified by sequencingusing an automated capillary sequencer (CEQ2000) and the DTCSsequencing kit (both from Beckman Coulter).

A. thaliana Col4 plants were transformed with the pTA7002-Clinkor -Clink^mut construct as previously described (12). TransgenicT1 seedlings were selected after 2 to 3 weeks of growth underhygromycin selection (20 µg/ml), and several independenttransgenic plant lines were produced. A preliminary screeningof eight clink and six clink^mut independent lines was performedto confirm that the expression of the viral transgenes was dependenton the presence of Dex (see Fig. S1 in the supplemental material).E2F-controlled PCNA gene expression was also tested and foundto be up-regulated in response to Dex treatment for the eighttransgenic clink lines, while Dex treatment had no effect onPCNA expression in the six clink^mut lines (see Fig. S1 in thesupplemental material). Therefore, for each construct, one representativeline that segregated for a single T-DNA insertion locus accordingto the hygromycin resistance segregation pattern was selectedand established at the homozygous state. These two transgenicclink and clink^mut plant lines display roughly similar transgeneexpression under glucocorticoid induction and were used forthe whole set of experiments described.

Glucocorticoid treatments and plant growing conditions. Dex (Sigma) was dissolved in 98% ethanol at 30 mM Dex (stocksolution) and stored at –20°C in a light-tight vial.clink and clink^mut plants were grown in the greenhouse for 6to 7 weeks. For detached-leaf inductions, fully expanded matureleaves were cut from each rosette and incubated either in asolution of distilled water including Dex at a final concentrationof 30 µM in 0.1% ethanol or in 0.1% ethanol as a negative(mock) control. At 0, 2, 5, 24, 48, or 72 h after induction,the leaves were collected and used for total-RNA extraction.In addition, leaves were used for total-protein extraction after24 h of induction.

Plants were germinated in vitro on standard 0.5x Murashige andSkoog (MS) medium (M-5519; Sigma) supplemented with 1% sucroseand 0.8% Bacto agar at 21°C under growth conditions of 16h of light and 8 h of darkness. For inductions lasting 3 to4 weeks, 3-day-old seedlings were transferred either to fresh0.5x MS medium-1% sucrose plates containing 30 µM Dexor to control plates containing 0.1% ethanol and were maintainedunder the same growth conditions. After the respective inductionperiod, the third and fourth rosette leaves and the first caulineleaf were harvested for flow cytometry or scanning electronmicroscopy (SEM) analysis. Whole plants were harvested for proteinextraction and immunoblot analysis. Alternatively, fully expandedmature leaves from greenhouse plants were incubated in 0.1%ethanol in the presence or absence (mock control) of 30 µMDex and were analyzed by flow cytometry and immunoblotting after4 days of treatment.

Infection of plants by FBNYV. A. thaliana plants were either infected with FBNYV by viruliferousAphis craccivora aphids or mock treated with nonviruliferousaphids reared as described elsewhere (51). Two-week-old A. thalianaplants were exposed for an inoculation access period of 3 to5 days, and aphids were killed with 0.2% Dedevap (Bayer). Healthyand infected plants as well as nonviruliferous and viruliferousaphids were maintained at 25°C and 50% humidity with 16h of light in cages inside growth chambers in a restricted-accessS3 confinement facility.

Cytogenetics. Plant leaf material was obtained as described for detached-leafinductions (see above). Following fixation of the leaves inethanol-acetic acid (3:1), nuclei were spread on a slide accordingto reference 40. Nuclei were stained with 4',6'-diamidino-2-phenylindole(DAPI).

Flow cytometry. One to two square centimeters of fresh leaves was finely choppedin 600 µl of Galbraith buffer (17) supplemented with 0.1%Triton X-100 and 1% polyvinylpyrrolidone 10,000 with a sharprazor blade. The extract was filtered on 48-µm mesh, treatedwith 20 µg/ml of RNase A (Roche) for 10 min on ice, andstained with 20 µg/ml propidium iodide. The samples wereanalyzed on an EPICS Elite ESP cytometer (Coulter, Hialeah,FL). Excitation of the fluorochrome was performed by an air-cooledargon laser (Uniphase) at 488 nm and 20 mW.

Total protein extraction and immunoblot analysis. A. thaliana leaves or whole plants were harvested after Dexor ethanol treatment (see above). Total-protein extracts wereprepared as described elsewhere (32). Leaf tissue (fresh weight,100 mg) was ground to a fine powder using an MM301 mixer mill(Retsch) and resuspended at a 1:2 (wt/vol) ratio in 2x Laemmlibuffer. The extracts were centrifuged at 15,000 x g for 15 minat 4°C, and 1/20 (10 µl) of the total proteins wasfractionated by sodium dodecyl sulfate (SDS)-12% polyacrylamidegel electrophoresis and analyzed by immunoblotting using polyclonalanti-Clink antibodies (1:500 dilution) as primary antibodiesas described previously (4). Antigen was detected using an ECLdetection kit (Amersham).

SEM. The leaf epidermal surface was visualized by SEM with a HitachiS-3000N microscope under the environmental secondary electrondetector mode. Samples were slowly frozen at –18°Cunder a partial vacuum (90 Pa) on the Peltier stage prior toobservation. The adaxial lamina surfaces of the third and fourthexpanded leaves of 4-week-old plants grown in the presence ofDex or ethanol were visualized. Cell areas were measured usingImageJ 1.28u software.

Total-RNA isolation, Northern blot analysis, quantification, and cell cycle gene-specific probes. Total RNA was extracted as described elsewhere (31). For Northernblot analysis, RNA samples were denatured and separated on 1%agarose-formaldehyde gels (39). The RNAs were transferred toHybond-N⁺ membranes (Amersham-Pharmacia Biotech.) by capillaryblotting and fixed by UV cross-linking. Prehybridizations andhybridizations were carried out in 50% formamide-5x SSC (1xSSC is 0.15 M NaCl plus 0.015 M sodium citrate)-1% SDS-1x Denhardtsolution-100 µg/ml of sheared, denatured herring spermDNA-10% (wt/vol) dextran sulfate at 42°C. Membranes werewashed with 1x SSC-1% SDS for 15 min at 42°C and then for15 min at 65°C, followed by two washes with 0.1x SSC-1%SDS for 15 min at 65°C. Signals were visualized by autoradiographyor by using a phosphorimager (Molecular Imager FX; Bio-Rad)for quantification.

For the detection of cell cycle-regulated gene transcripts,probes corresponding to parts of the PCNA-2 (At2g29570), CDC6a(At2g29680), CDKB2;1 (At1g76540), CycB1;1 (At4g37490), and CycB1;4(At2g26760) cDNAs were PCR amplified (primers available on request).

RESULTS

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Results

Production of stably transformed A. thaliana plants with a glucocorticoid-inducible clink gene. In vitro and yeast two-hybrid assays both suggested that Clinkis able to interact with RB and potentially modulate the hostcell cycle (5). Because we failed to establish Arabidopsis linescontaining a clink transgene under the control of the strong,constitutive cauliflower mosaic virus 35S promoter, we tookadvantage of the tightly regulated glucocorticoid-induciblesystem (2). With this system, we established transgenic A. thalianalines with conditional expression of Clink or of a mutated versionof Clink (Clink^mut). The Clink^mut protein differs from wild-typeClink by 2 amino acids that convert the LxCxE motif to LxRxAand render it incompetent for binding to the A/B pocket domainof RBR (5). Based on a preliminary screening, representativehomozygous lines expressing Clink or Clink^mut were selectedfor further studies (see Materials and Methods; also Fig. S1in the supplemental material). Following Dex treatment for 2to 72 h, efficient expression of the respective clink transgeneswas obtained, leading to accumulation of comparable levels ofclink-specific transcripts (Fig. 1A). Transcript levels werecorrelated with the accumulation of Clink or Clink^mut proteinin leaf tissue as revealed by Western blot analysis using Clink-specificantibodies (Fig. 1B; see also Fig. 3D).

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FIG. 1. Expression of viral sequences in transgenic plants, showing the impact of Clink on cell cycle-regulated gene expression. Following Dex (D) or ethanol (E) treatment of detached, fully expanded leaves, total RNAs (panel A) or proteins (panel B) were extracted and analyzed. (A) Six micrograms of total RNAs extracted from the expanded rosette leaves was used for Northern blot analysis. The time (T) of the treatment and the identity of the transgenic plant line (C, Clink; C*, Clink^mut) are given above the panels. The two membranes were hybridized successively with probes specific for the classes of genes given on the left (see Materials and Methods). The transcript level of actin was used as a control for quantification. Six micrograms of total RNA extracted from cells from an actively growing A. thaliana T87 suspension culture (6) was also loaded and served as a positive control for hybridization with the cell cycle-specific probes. (B) Typical immunoblot result obtained with the anti-Clink antibodies. Molecular masses of protein markers (in kilodaltons) are given on the right. As previously reported (5), the Clink^mut protein migrates slightly faster than the Clink protein. Equivalent amounts of proteins were loaded in each lane, as confirmed by the similar intensities of the ${cong}$ 45-kDa bands resulting from cross-reactions to endogenous proteins.

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FIG. 3. Effect of Clink expression on cell size. (A) SEM of the leaf adaxial epidermis of Clink-expressing, Clink^mut-expressing, and nontransgenic Col4 plants. Bars, 200 µm. (B) Cell area distribution of Clink-expressing (open bars), Clink^mut-expressing (shaded bars), and nontransgenic (solid bars) Col4 plants. (C) Number of cells per square millimeter of leaf epidermis of Clink-expressing (open bar), Clink^mut -expressing (shaded bar), and nontransgenic (solid bar) Col4 plants. (D) Immunoblot analysis of total proteins from Col4 plants and from the two transgenic plant lines using Clink-specific antibodies. The identity of the plant line is given above the panel. As explained for Fig. 1B, the Clink^mut protein migrates faster than the Clink protein. Plants for which results are shown in panels A and D were grown and induced under the same conditions.

Clink-specific induction of cell cycle-regulated genes. To assess the cellular impact of Clink, we studied the expressionof two well-known E2F-regulated genes, encoding PCNA and thechromosomal DNA replication origin protein (CDC6), in relationto Clink induction. Expression of Clink, but not Clink^mut, indetached, expanded 6- to 7-week-old rosette leaves was clearlyassociated with the transcriptional activation of both genes(Fig. 1A). PCNA- and CDC6-specific transcripts were alreadydetectable 5 h after the beginning of Dex treatment, and a longerinduction time with Clink correlated consistently with PCNAand CDC6 gene expression. No impact on PCNA or CDC6 expressionwas observed for Dex-treated Clink^mut plants or ethanol-treatedClink or Clink^mut plants. To analyze whether Clink expressioncould impact the transcription of cell cycle genes other thanE2F-regulated S-phase genes, the same Northern blots were hybridizedwith probes specific for B2-type cyclin-dependent kinases (CDKB2)and the B1-type cyclins CycB1;1 and CycB1;4. In plants, CDKB2proteins are thought to control entry and progression throughthe M phase, and this class of genes (CDKB2;1 and CDKB2;2) isexpressed only during the G₂ and M phases (24, 27, 34). Similarly,accumulation of transcripts of plant mitotic cyclins of theB1 class is limited to the G₂ and M phases of dividing tissues(23, 37). For all three G₂/M markers, specific up-regulationwas detected only when wild-type Clink was expressed (Fig. 1A).In contrast to that of PCNA and CDC6, transcriptional expressionof the CDKB2, CycB1;1 and CycB1;4 genes became clearly detectableat 48 h after Clink induction (Fig. 1A). When whole plants weretreated with Dex, analysis of mature leaves showed results similarto those reported in Fig. 1 (data not shown). Therefore, forshort-term induction experiments, we decided to work on detachedmature leaves.

To further investigate the impact of Clink on the cell cycle,nuclei were isolated from detached, expanded rosette leavesof 6- to 7-week-old Clink and Clink^mut transgenic plants thathad been treated for 4 days with Dex or ethanol, spread, andstained with DAPI. Ethanol-treated tissues and Dex-treated Clink^mutplant tissues displayed similar distributions of only two typesof nuclei (Fig. 2A and B), a situation typical of mature A.thaliana leaves (41). About 95% of these nuclei showed a mostlyoval shape with irregular contours and exhibited about 10 intenselyDAPI stained regions termed chromocenters (CCs), which reflectthe classical organization of the centromeric and nucleolusorganizer heterochromatic regions (Fig. 2A). The other 5% ofnuclei (Fig. 2B) appeared larger, with a higher number of CCs,a characteristic associated with a high (usually 16C to 32C)ploidy level (41). In Clink-expressing tissues, these two typesof nuclei were also observed, but the proportion of the majorfraction was drastically reduced, from 95% to about 33%, whilethe proportion of the minor fraction was increased from 5% to10%, and new nucleus configurations became visible. Among these,a large fraction of nuclei clearly displayed some of the morphologicalchanges associated with young, dividing cells, such as nuclearrounding and smaller nuclei (Fig. 2C, D, E, and F). For 19%of the nuclei, this phenotype also correlated with the presenceof more than 10 CCs, suggesting increased levels of ploidy (Fig.2C and D). Seven percent of the nuclei displayed an overallintense DAPI staining indicative of highly condensed euchromatin(Fig. 2E). Moreover, 1% of the nuclei were unambiguously identifiedas metaphasic (not shown) or anaphasic (Fig. 2F), thus providingdirect evidence of mitotic activity in the Clink-expressingleaf tissue.

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FIG. 2. Representative DAPI-stained nuclei isolated after 4 days of Dex treatment on detached, expanded 6- to 7-week-old rosette leaves from Clink-transgenic plants. The relative proportions of these different types of nuclei in Clink-expressing tissues were as follows: 33% for oval nuclei with irregular contours and $~$ 10 CCs (A), 10% for larger nuclei with more CCs (B), 19% for smaller, rounded nuclei with >10 CCs (C and D), 7% for nuclei with highly condensed euchromatin (E), and 1% for dividing (e.g. anaphasic) nuclei (F). For Dex-treated Clink^mut plant leaves and ethanol-treated Clink or Clink^mut plant leaves, only nuclei of the types shown in panels A and B were observed, in proportions of about 95% and 5%, respectively. In each case, the relative proportions are based on the observation of 800 to 1,100 nuclei. Bars, 15 µm.

Phenotypic effects of Clink expression in A. thaliana plants. Three-day-old A. thaliana seedlings germinated in the absenceof Dex were transferred to new plates containing 30 µMDex or control plates containing 0.1% ethanol and kept at 21°Cwith a photoperiod of 16 h of light. Under these conditions,no visible differences in the growth or morphology of leavesor roots between A. thaliana plants expressing Clink or Clink^mutand wild-type Col4 plants were observed (data not shown). Theaccumulation of Clink or Clink^mut protein in leaves was confirmedby Western blot analysis using Clink-specific antibodies (Fig.3D). The adaxial lamina surfaces of the third and fourth expandedrosette leaves of 4-week-old plants grown in the presence ofDex or ethanol were analyzed by SEM. The wild-type A. thalianaleaf epidermis contains three cell types: pavement cells ina jigsaw puzzle-like pattern, stomatal cells, and trichomes.Rosette leaves of Clink-expressing plants showed clusters ofsmall pavement cells surrounded by almost normal puzzle-shapedcells (Fig. 3A). Ethanol-treated plants (data not shown) orDex-treated nontransgenic Col4 and Clink^mut plants (Fig. 3A)showed epidermal pavement cells of a normal puzzle shape andsize. Comparison of nontransgenic Col4 plants and Clink-expressingplants revealed a decrease in cell size in the Clink-expressingplants (Fig. 3A and B). Cell size was quantified using SEM imagesof the third rosette leaves of each genotype. The results showeda significant (P < 0.05 by Student's t test) decrease inthe mean cell surface area ( $~$ 65%) for Clink-expressing plants(1,633 µm² for 210 cells measured) compared to wild-typeCol4 plants (4,612 µm² for 180 cells measured). No significantchange (P < 0.1) in the mean cell surface area was observedfor Clink^mut-expressing plants (4,769 µm² for 210 cellsmeasured) compared to nontransgenic Col4 plants (4,612 µm²for 180 cells measured). The reduction in the size of pavementcells for Clink-expressing plants was accompanied by a 4.5-foldincrease in the number of pavement cells (Fig. 3C), while thenumbers of pavement cells in leaves of Clink^mut-expressing plantsand nontransgenic control plants were similar. In addition,the number of stomatal cells was 2.5-fold higher in Clink-expressingplants than in Clink^mut-expressing and nontransgenic controlplants (Fig. 3A). These results suggest that the arrest of celldivision of epidermal leaf cells in Clink-expressing plantswas less synchronous than that in nontransgenic or Clink^mutexpressing plants.

Effect of Clink expression on ploidy levels. To analyze the impact of Clink expression on endoreduplicationcycles, the ploidy levels of leaves were measured by flow cytometry.The third and fourth expanded rosette leaves from 4-week-oldplants of the three genotypes, grown on plates and subjectedto the same induction treatment as that for plants analyzedby SEM, were used (Fig. 4A and B). Six replicates were analyzedin three independent experiments. The ploidy level of ethanol-treatedplants (data not shown) and those of Dex-treated nontransgenicCol4 and Clink^mut-expressing plants did not show significantdifferences. For Clink-expressing plants, however, the fractionsof 2C (P < 0.001 by Student's t test) and 4C (P < 0.01)ploidy level nuclei were lower, and the fractions of 16C (P< 0.001) and 32C (P < 0.01) nuclei were drastically higher,than those for nontransgenic Col4 plants. Similar results wereobtained using detached leaves of 6-week-old greenhouse-grownplants when the leaves were induced for 4 days in liquid Dexmedium (Fig. 4C). Collectively, these results indicate thatleaf cells in Clink-expressing plants had undergone more endoreduplicationcycles than those in nontransgenic Col4 and Clink^mut-expressingplants. This induction of the endoreduplication cycles clearlydepended on an intact RBR-binding domain (LxCxE) of Clink; hence,interaction with RBR is required.

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FIG. 4. Effect of Clink expression on endoreduplication in rosette and cauline leaves. (A) Flow cytometry profiles of the DNA contents of nuclei from third and fourth expanded rosette leaves of 4-week-old Clink-expressing, Clink^mut-expressing, and nontransgenic Col4 plants grown on plates in the presence of Dex. (B) Comparison of the DNA contents in nuclei of rosette leaves derived from Clink-expressing (open bars), Clink^mut-expressing (shaded bars), and nontransgenic (solid bars) Col4 plants. (C) Comparison of the DNA contents in nuclei of detached cauline leaves following 4 days of Dex treatment. For panels B and C, three independent experiments with six replicates were performed; representative results for one replicate are shown in panel A.

Ploidy levels in FBNYV-infected tissue. Infection of several ecotypes of A. thaliana with FBNYV by meansof viruliferous Aphis craccivora has been established previously(J. Vega-Arreguin et al., unpublished data). Figure 5A showsthe symptoms of FBNYV infection in a Col4 plant 3 weeks afterinoculation by viruliferous aphids. Infected plants were stuntedcompared to plants that had been exposed to nonviruliferousaphids (Fig. 5A). FBNYV levels were measured by an enzyme-linkedimmunosorbent assay (kindly provided by H. J. Vetten). The virusaccumulated to high levels in newly developed tissues (datanot shown). As in the case of Clink expression, infection withFBNYV resulted in a significant increase in ploidy levels inthe leaf cells (Fig. 5B). Infected cauline leaves showed lowerlevels of 2C (P < 0.001) and 4C (P < 0.05) nuclei andhigher levels of 8C (P < 0.01), 16C (P < 0.05), and 32C(P < 0.01) nuclei than leaves exposed to nonviruliferousaphids. The shift of ploidy to higher levels in the nuclei ofFBNYV-infected cauline leaves suggests that the nuclei withelevated ploidy levels underwent virus-induced endoreduplicationthat paralleled viral DNA replication.

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FIG. 5. FBNYV infection of A. thaliana plants. (A) Symptoms of an infected plant (left) compared to a healthy plant (right). (B) Ploidy levels in cauline leaves of FBNYV-infected plants (shaded bars) or healthy control plants (solid bars) 29 days postinfection.

Clink expression does not influence expression of PolIII- and PolI-transcribed genes. In mammals, RB also acts as a general repressor of transcriptionby PolIII and PolI, potentially to control cell growth, andinteracting viral proteins such as T-ag, E7, and E1A are ableto neutralize this repression (29). To address the questionwhether the same may occur in plants following the Clink-RBRinteraction, we used the same RNA samples analyzed in Fig. 1Aand performed polyacrylamide gel electrophoresis and severalhybridizations using probes specific for (i) the PolI-transcribed5.8S rRNA gene, (ii) the PolIII-transcribed class-1 5S rRNAgene and class-2 tRNAala gene (controlled by different intragenicpromoter elements), and (iii) the PolIII-transcribed class-37SL, U6, and U3 genes (relying solely on extragenic promoterelements) (data not shown). For the 5.8S rRNA gene and all PolIII-transcribedgenes tested, induction of Clink did not correlate with a significantchange in the accumulation of the respective RNA. A similarresult was observed for the PolI-transcribed 18S rRNA gene (datanot shown). Therefore, these results indicate that expressionof Clink is not accompanied by a significant increase in genetranscription by PolIII or PolI, suggesting a difference inthe pathways of cell growth regulation by RB and RBR in mammalsand plants.

DISCUSSION

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Discussion

Having established that the nanovirus protein Clink binds thekey cell cycle regulator protein RBR and enhances the replicationof FBNYV DNA (5), we studied here the influence of Clink oncell cycle regulation. For that purpose, transgenic A. thalianalines were constructed in which the expression of Clink or theRBR-binding-deficient variant Clink^mut could be controlled byinduction with the glucocorticoid hormone Dex (2).

Depending on the expression of a functional Clink protein, transcriptionof the E2F/DP-controlled S-phase-specific genes encoding thePCNA and CDC6 proteins was readily induced, whereas expressionof Clink^mut, an RBR-binding-deficient Clink variant, did notcause any induction of PCNA- and CDC6-specific transcripts.This is consistent with the assumption that via interactionwith Arabidopsis RBR, Clink triggers a cell cycle progressionbeyond the RBR-controlled checkpoint. Similar effects on theS-phase transition were reported for another plant DNA virusRBR-binding protein, the tomato golden mosaic virus Rep (orAL1) protein (15, 26, 36). Clink expression also caused increasedtranscription of G₂-M-specific genes encoding the B2-type cyclin-dependentkinases and the B1-type cyclins CycB1;1 and CycB1;4 (Fig. 1A).This is suggestive evidence that not only DNA synthesis (duringS phase) but also, at least to some extent, mitoses and celldivisions might be triggered by the induced expression of Clink.These findings were further supported by in situ fluorescenceanalyses of DAPI-stained nuclei derived from Clink- and Clink^mut-transgenicplant leaves (Fig. 2). Following expression of Clink, but notClink^mut, the fraction of leaf nuclei displaying more than 10CCs increased from 5% to 29% (Fig. 2B, C, and D); moreover,about 1% of metaphasic or anaphasic nuclei (Fig. 2F) were clearlyidentified among Clink-expressing cells, a situation unexpectedin mature Arabidopsis leaves. These results provide clear evidenceof Clink-induced cell cycle reactivation in mature leaf tissuesassociated with enhanced endoreduplication and some mitoticactivity. Recently, the geminivirus RBR-binding RepA proteinwas also shown to affect the G₂-M transition in transgenic Arabidopsisplants (14). Following disruption of RBR function by RepA inlate-developing rosette leaves, a general increase in the ploidylevels of leaf nuclei was observed, and fully expanded pavementcells displayed the capacity to engage in a single divisionevent. In our case, these epidermal cells are also likely toaccount for at least a fraction of the mitotic events observedin response to Clink induction in fully differentiated rosetteleaves from 6- to 7-week-old seedlings.

In contrast to the small DNA viruses of mammals, which causeextended cell proliferation (tumors), such excessive cell proliferationhas never been observed to be associated with infection of plantsby ssDNA viruses. Only limited tissue proliferation or cellenlargement is caused by some geminivirus proteins (30, 44).It was therefore of interest to assess the effect of prolonged(3- to 4-week) expression of the cell cycle "deregulator" Clinkon plant tissue. Clink-expressing as well as Clink^mut-expressingplants that had been grown for 4 weeks in the presence of Dexshowed no obvious morphological differences. A closer inspectionof the adaxial epidermis by SEM, however, revealed a clearlydifferent phenotype for Clink-expressing plants than for Clink^mut-expressingplants or nontransgenic Col4 plants (Fig. 3). Clink-expressingplants had a significantly increased fraction of smaller epidermalcells (extrapolated from their smaller surface area), and thissize reduction was accompanied by increases in the numbers ofpavement and stomatal cells (Fig. 3). Similar phenotypes havebeen described for Arabidopsis plants overexpressing E2Fa-DPa(7), AtCDT1, or AtCDC6 (9) and for Arabidopsis plants subjectto geminivirus RepA-induced RBR inactivation (14). This is inline with the assumption that due to prolonged expression offunctional Clink, the RBR-imposed block of transcriptional activatorsof the E2F family remained suspended, and some mitotic cyclesand subsequent epidermal cell divisions occurred. Concomitantly,the ploidy levels of nuclei from the third and fourth expandedrosette leaves and cauline leaves from the same plants (4 weeksof Dex induction), assessed by flow cytometry, revealed a clearshift toward 16C and 32C DNA contents (Fig. 4A and B). The beginningof such a shift toward a higher ploidy level could already beobserved after 4 days of Clink induction (Fig. 4C). The Clink-inducedincrease in epidermal pavement cell and stomatal cell divisionand the concomitantly elevated level of ploidy of total nucleifrom the corresponding leaves suggest a differential effectof Clink on the nuclear cycle in epidermal cells and total tissuenuclei. These changes are the consequence of transcriptionalreprogramming suggested by the up-regulation of E2F target genes.In epidermal cells, no endoreduplication occurred beyond thealready existing level (not determined), because larger epidermalcells, expected in the case of a higher degree of ploidy (see,for instance, references 28 and 33), were not observed. At thesame time, in total cells from the same leaves, a fraction ofnuclei existed that had undergone more endocycles. Since Clinkclearly triggers transcription of S-phase-specific genes, e.g.,PCNA and CDC6, as well as the mitotic cyclin cycB1;1, a dichotomyin the effect of Clink on cell cycle regulation in the nucleiof cells of different tissues is easily conceivable. Such abalanced dichotomy in E2F/DP-dependent effects on the cell cycleand cell fate has also been proposed by others (7, 9, 14). Indeed,the authors concluded that the RBR/E2F pathway regulates thebalance between cell proliferation, endocycle program, and differentiation.They also concluded that the importance of this pathway dependson the cell type, the tissue, and the developmental stage.

A clear shift of the nuclear ploidy to elevated levels (8, 16,and 32C) was observed in cauline leaves of FBNYV-infected A.thaliana plants (Fig. 5). It is tempting to speculate that theendocycling nuclei are derived either from cells that are targetedby FBNYV for its replication or from cells that contain at leastsome FBNYV DNA components. Nanoviruses are assumed to be restrictedto the phloem (52), but the impact on the overall ploidy leveldetected for FBNYV-infected cauline leaves suggests that thisis not the case. No data are available on the multiplicationof FBNYV in epidermal cells, and whether nanoviruses invadethe mesophyll is not known. Attempts to determine the tropismof FBNYV in A. thaliana by immunolocalization with the availablepolyclonal antibodies against Clink, M-Rep, and FBNYV particleswere unsuccessful, due to cross-reaction of these antibodieswith host plant proteins. Nanoviruses (52), including FBNYV(B. C. Ramirez, unpublished results), code for two proteinsinvolved in cell-to-cell movement. DNA-N codes for a nuclearshuttle protein and DNA-M for a movement protein. The movementprotein and nuclear shuttle protein coordinate the movementof viral DNA as viral nucleoprotein complexes from the nucleusto the cytoplasm and from the cytoplasm to the neighboring cells.It is possible that nanoviruses have the capacity to invadenonphloem tissues, since this has been observed for some bipartitegeminiviruses such as the begomovirus bean dwarf mosaic virus(38). On the other hand, it is also possible that some FBNYVDNA components are not limited to the phloem, at least in A.thaliana. Recent studies (42) with the nanovirus milk vetchdwarf virus (MDV) indicate that the expression of the individualDNA components is differentially regulated: the promoter ofMDV-M is active not only in phloem and meristematic tissuesbut also in mesophyll and cortex cells. It therefore appearslogical to assume that nuclei with an elevated ploidy levelunderwent virus-induced (i.e., Clink-triggered) endoreduplicationthat paralleled viral DNA replication. A similar situation waspreviously reported for infection of Digitaria setigera withthe mastrevirus Digitaria streak virus, which was accompaniedby an increase in the fraction of S-phase nuclei (1).

Since the effects of Clink on the expression of cell cycle-relevantgenes involving interaction with RBR proteins are in many aspectscomparable to those of the cell cycle regulators of small DNAtumor viruses such as SV40 T-ag or E7 of human papillomaviruses,we were interested in seeing whether Clink might also influencethe transcription by PolI and PolIII of genes implicated incell growth regulation (for a review, see reference 29). Northernblot analyses of the PolI-transcribed 5.8S rRNA gene and offive PolIII-transcribed genes, the 5S rRNA, tRNAala, 7SL, U6,and U3 genes, revealed no significant differences in transcriptaccumulation upon Clink induction (data not shown). Therefore,these classes of genes involved in cell growth regulation arenot affected by Clink, in agreement with the observation thatno gross increase in cell size was observed in Clink-expressingtissue. Also, for the RBR-interacting cell cycle modulatorsof other ssDNA plant viruses, e.g., the mastrevirus RepA proteinand the begomovirus Rep (AL1) protein, no increase in cell sizehas been reported. The only protein known to cause an increasein cell size is the C4 protein of beet curly top virus (30).Hence, to regulate their cell growth (size), plants and animalshave obviously evolved diverging molecular strategies (for reviews,see references 25, 45, and 46).

In the present study, we have shown that the nanovirus cellcycle link protein Clink significantly interferes with the plantcell cycle in vivo, conceivably via interaction with cell RBR.An intriguing question still remains, however: what is the biologicalsignificance of Clink as an F-box protein, its binding to SKP1,and its connection to the SCF-regulated protein turnover pathway?A "transgenic" approach comparable to that described here, employingthe tightly regulated conditional expression of Clink or mutantClink, may be useful for shedding further light on the moleculardetails of other Clink functions and on nanovirus biology ingeneral.

ACKNOWLEDGMENTS

We thank Ingo Schubert and Olivier Mathieu for assistance inthe analysis of the cytogenetics data, Julien Douet for helpwith the cytogenetics experiments, Séverine Domenichinifor assistance with SEM, and Julio Vega-Arreguín forhelp with the aphid colonies. We thank the "Dynamique de lacompartimentation cellulaire" laboratory for their help andvaluable discussions with the immunolocalization and flow cytometrystudies. We thank Susanne Bolte and Marie-Noëlle Solerfor expert support with microscopy. We are grateful to DanielleClérot for her valuable help concerning the measurementsof the cell surfaces. We are also grateful to Nam-Hai Chua forthe generous gift of the PTA7002 vector, to Eva Kondorosi forproviding the CycB1;1 probe, and to H.-J. Vetten for the enzyme-linkedimmunosorbent assay and for antibodies to FBNYV. We thank Anne-LiseHaenni for valuable suggestions.

This work was partly supported by the CNRS (UMR 6547 BIOMOVEand GDR2157), by the Université Blaise Pascal, and bya grant from the European Commission as part of the RIBOREGEU FP6 project (LSHG-CT-2003503022). S. Lageix is the recipientof a French MNERT grant. The Imaging and Cell Biology facilityof the IFR87 (FR-W2251) "La plante et son environnement" issupported by "Action de Soutien à la Technologie et laRecherche en Essonne," Conseil Général de l'Essonne.

FOOTNOTES

* Corresponding author. Mailing address for Thierry Pélissier: CNRS UMR 6547 BIOMOVE, Université Blaise Pascal, 24 Avenue des Landais, 63177 Aubière Cedex, France. Phone: (33) 4 73 40 74 08. Fax: (33) 4 73 40 77 77. E-mail: thierry.pelissier{at}univ-bpclermont.fr. Present address for Bertha Cecilia Ramírez: Institut Pasteur, Unité de Régulation Enzymatique des Activités Cellulaires, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France. Phone: (33) 1 40 61 35 34. Fax: (33) 1 45 68 83 99. E-mail: cecilia{at}pasteur.fr

Published ahead of print on 31 January 2007.

Supplemental material for this article may be found at http://jvi.asm.org/.

T.P. and B.C.R. contributed equally to this work.

REFERENCES

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