An RNA Virus-Encoded Zinc-Finger Protein Acts as a PlantTranscription Factor and Induces a Regulator of Cell Size andProliferation in Two Tobacco Species C W

Nina I. Lukhovitskaya,a Anna D. Solovieva,b Santosh K. Boddeti,a Srinivas Thaduri,a Andrey G. Solovyev,c,d

and Eugene I. Savenkova,1

a Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural Sciences and LinneanCenter for Plant Biology, SE-75007 Uppsala, SwedenbDepartment of Virology, Biological Faculty, Moscow State University, Moscow 119992, Russiac A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119992 Moscow, Russiad Institute of Agricultural Biotechnology, Russian Academy of Agricultural Sciences, 127550 Moscow, Russia

Plant viruses cause a variety of diseases in susceptible hosts. The disease symptoms often include leaf malformations andother developmental abnormalities, suggesting that viruses can affect plant development. However, little is known about themechanisms underlying virus interference with plant morphogenesis. Here, we show that a C-4 type zinc-finger (ZF) protein,p12, encoded by a carlavirus (chrysanthemum virus B) can induce cell proliferation, which results in hyperplasia and severeleaf malformation. We demonstrate that the p12 protein activates expression of a regulator of cell size and proliferation,designated upp-L (upregulated by p12), which encodes a transcription factor of the basic/helix-loop-helix family sufficient tocause hyperplasia. The induction of upp-L requires translocation of the p12 protein into the nucleus and ZF-dependentspecific interaction with the conserved regulatory region in the upp-L promoter. Our results establish the role of the p12protein in modulation of host cell morphogenesis. It is likely that other members of the conserved C-4 type ZF family of viralproteins instigate reprogramming of plant development by mimicking eukaryotic transcriptional activators.

INTRODUCTION

Plant innate immunity has evolved as a multilayered defensesystem to recognize and resist invading pathogens (Jones andDangl, 2006). As a counterdefense strategy to overcome the firstlayer of defense, pathogens deliver various virulence determi-nants into the plant cell. These virulence factors have beentermed effectors (Bent and Mackey, 2007). Effectors hijackcellular machinery to suppress immune responses and enhancevirulence (Dodds and Rathjen, 2010). Gram-negative bacteriaemploy a type III secretion system to inject effectors into hostcells (Galán and Wolf-Watz, 2006). Plant pathogenic bacteria ofthe genus Xanthomonas deliver transcription activator-like ef-fector (TALe) proteins, which act as transcription factors (TFs)and activate host promoters through direct binding to UPT(upregulated by TALe) promoter boxes (Kay et al., 2007; Whiteet al., 2009; Römer et al., 2010). Thereby Xanthomonas TALes,depending on crop cultivar, can either promote disease throughhypertrophy and hyperplasia development or induce plant de-fense if the corresponding UPT box is located in the promoter of

a resistance gene (Römer et al., 2007). Interestingly, an AvrBs3TALe of Xanthomonas spp promotes tissue growth after in-filtration on leaves of Solanaceae species and, in pepper (Cap-sicum annuum), is known to induce a cell size regulator, basic/helix-loop-helix (bHLH)-type TF UPA20, sufficient to cause tis-sue growth (Kay et al., 2007).Whereas pathogen-induced expression of specific genes and

transcriptional reprogramming of host gene expression has beenlinked to hyperplasia and other morphological defects, especiallyfor some members of two genera of geminiviruses (Latham et al.,1997; Piroux et al., 2007; Ascencio-Ibáñez et al., 2008; Mills-Lujanand Deom, 2010), no TF-like proteins, which would directly bindto host promoters, have been reported for phytoviruses. The C4proteins of two monopartite members of the genus Begomovirusand Curtovirus induce hyperplasia probably due to interferencewith the brassinosteroid pathway and not as direct TFs (Lathamet al., 1997; Piroux et al., 2007). Although the C4 protein of beetcurly top virus (genus Curtovirus, family Geminiviridae) induceslocalized hyperplasia in infected phloem tissue and tumorogenicgrowth in transgenic plants, the C4 protein has not been linkedto activation of host promoters through direct binding to specificpromoter elements (Piroux et al., 2007; Mills-Lujan and Deom,2010). On the contrary, the C4 protein of beet curly top virus islocalized to the plasma membrane, interacts with plant SHAGGY-related protein kinases, and seems to act through the disruptionof brassinosteroid phytohormonal networks and signaling (Pirouxet al., 2007). For other geminiviruses, C4 and the homologous AC4/AL4 have been reported to be silencing suppressors (Vanitharaniet al., 2004).

1 Address correspondence to eugene.savenkov@slu.se.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Eugene I. Savenkov(eugene.savenkov@slu.se).C Some figures in this article are displayed in color online but in black andwhite in the print edition.W Online version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.112.106476

The Plant Cell, Vol. 25: 960–973, March 2013, www.plantcell.org ã 2013 American Society of Plant Biologists. All rights reserved.

Recently, we found that the p12 protein of chrysanthemumvirus B (CVB) is implicated in host gene induction as a patho-genicity and virulence determinant (Lukhovitskaya et al., 2009).CVB belongs to the genus Carlavirus, which comprises 35 de-finitive species and 29 tentative members with conserved ge-nome organization (Levay and Zavriev, 1991; Adams et al., 2004;Martelli et al., 2007). Their positive-stranded genomic RNAencodes six genes, of which the gene for p12 occupies theextreme 39-terminal position (see Supplemental Figure 1A on-line; Adams et al., 2004; Martelli et al., 2007). CVB p12 and itshomologs in other carlaviruses are Cys-rich proteins charac-terized by a central C-4 type zinc finger defined by itsRCxRCxRxxPx6-8CDxxxC motif and an adjacent nuclear locali-zation signal (NLS; see Supplemental Figure 1C online; Gramstatet al., 1990; Koonin et al., 1991; Lukhovitskaya et al., 2009; notethat the carlavirus C-4 superfamily should not be confused withthe C4 proteins of geminiviruses). The p12 protein binds bothRNA and DNA but exhibits a preference for DNA in the presenceof Zn2+ and its pathogenic effects depend on translocation intothe nucleus (Gramstat et al., 1990; Lukhovitskaya et al., 2009).Since carlaviruses are positive-stranded RNA viruses and repli-cate in the cytoplasm, the role of the p12 protein nuclear locali-zation and DNA binding in the virus–plant interactions has beenenigmatic. We hypothesized that the p12 might act as a tran-scriptional activator localized to the plant nucleus (Lukhovitskayaet al., 2009).

The p12-like protein of another carlavirus, potato virus M, hasa different function and acts in concert with the TGB1 (for TripleGene Block) protein as a silencing suppressor to enhance viralRNA accumulation, cell-to-cell movement, and systemic transport(Senshu et al., 2011). On the other hand, we demonstrated ex-plicitly that CVB p12 did not suppress RNA interference and,moreover, did not suppress microRNA-mediated mRNA cleavage(Lukhovitskaya et al., 2005, 2009). These results suggest thatthere are some fundamental differences even among homologousproteins encoded by the viruses of the same genus. Indeed, in thisarticle, characterization of the host responses to the p12 proteinexpression revealed additional unexpected levels of complexityduring virus–host interactions. We demonstrated that the p12protein acts as a eukaryotic TF to upregulate a bHLH-TF–encodinggene, which in turn stimulates ectopic cell proliferation and mod-ulates tissue growth in the mesophyll of infected leaves.

RESULTS AND DISCUSSION

p12 Expression Causes Severe Leaf Malformationand Hyperplasia

Chrysanthemum (Chrysanthemum morifolium) plants infectedwith CVB often show curling of the upper leaves. Cross sectionsand light microscopy revealed pronounced cell proliferation inCVB-infected chrysanthemum leaves compared with the mock-inoculated plants (Figure 1A; see Supplemental Figure 2A on-line). This aberrant tissue structure is referred to hereafter ashyperplasia (i.e., disorganized cell division). Interestingly, Agro-bacterium tumefaciens–launched CVB p12 expression resultedin hyperplasia (see Supplemental Figure 2B online), although it

was not as pronounced as during the virus infection, probablyowing to low levels of p12 protein accumulation upon agro-infiltration as was revealed by immunoblotting (see SupplementalFigure 2C online). Therefore, to achieve protein expression levelscomparable to those during CVB infection and to investigate thehost responses specifically to p12 in the absence of other car-laviral proteins, we expressed p12 from two unrelated virus vec-tors (to avoid vector-related bias), namely, the Tobacco mosaicvirus (TMV) vector (TMV-p12 construct) and the Potato virus X(PVX) vector (PVX-p12; see Supplemental Figure 1B online). Re-gardless of the vector used, we observed strong phenotypesmanifested as severe leaf malformation (Figure 1B; seeSupplemental Figures 2D and 2E online) in the susceptible Nico-tiana tabacum cv Samsun. Light microscopy revealed pronouncedcell proliferation (increase in cell number) in the p12-expressingleaf tissue, with palisade and spongy mesophyll cells beingstrongly reduced in size but more abundant in number com-pared with those in tissue of the control (see SupplementalFigure 2F online). Thus, p12 converted typical mosaic symptomsof TMV and PVX into leaf malformation because of hyperplasiaof the plant tissue.Severe leaf malformation and hyperplasia were observed in

Nicotiana occidentalis ssp hesperis plants, commonly known asnative tobacco, infected with CVB (Figures 1C and 1D), dem-onstrating that our findings reflect actual events during carlavi-rus infection not only in chrysanthemum but also in one of thetobacco species, which is commonly used as indicator host forCVB. Additional phenotypes associated with CVB infection innative tobacco included hypersensitive response (HR) inductionat later stages of virus infection (see Supplemental Figure 2Gonline) consistent with the previously reported role of p12 in HRinduction (Lukhovitskaya et al., 2009).The p12-induced hyperplasia might account for the leaf mal-

formation symptoms observed in infections caused by someother carlaviruses (Hakkaart and Maat, 1974; Dijkstra et al.,1985; Larsen et al., 2009; Tang et al., 2010). Although leafmalformation is a quite common symptom induced by manycarlaviruses, to the best of our knowledge, none of the host-carlavirus pathosystems other than CVB have been investigatedfor hyperplasia induction and this awaits characterization.

p12 Induces Expression of a bHLH TF

Since tobacco species represent a more genetically tractablemodel than chrysanthemum, and both react with hyperplasia top12 expression, all subsequent experiments were performed intobacco. The phenotypes caused by p12 resembled leaf malfor-mation induced by Ca UPA20 TF after infiltration on tobaccoleaves (Kay et al., 2007). Therefore, we wanted to analyze whethera tobacco ortholog of the pepper UPA20 is upregulated in thep12-expressing tissues. Since the gene sequence was not avail-able, we performed tobacco EST database searches and identifiedan EST encoding a polypeptide highly similar to the UPA20 proteinfrom pepper. Real-time RT-PCR experiments performed with pri-mers designed based on the EST sequence revealed upregulationof the tobacco gene in both TMV-p12 and PVX-p12–infectedplants (Figure 1E). Therefore, the upregulated tobacco gene wastermed upp (for upregulated by p12).

RNA Virus-Encoded Transcription Factor 961

We isolated the full-length cDNA of the upp genes from N.tabacum and N. occidentalis ssp hesperis by rapid amplificationof cDNA ends (RACE) and determined the corresponding ge-nomic sequence from clones generated by PCR and genomewalking. It appeared that the tobacco genome encodes two uppparalogs, designated Nt upp-L and Nt upp-S (Figure 1F),

whereas the genome of native tobacco encodes only the longergene (Nh upp-L). Compared with the upp-L genes, Nt upp-S hasa two nucleotide deletion in the beginning of the coding regionthat prevents initiation on the first AUG codon (Figure 1G) andresults in a shorter protein. Nt upp-L and Nt upp-S genes bothcomprise eight exons and seven introns (the 6th intron is longer

Figure 1. CVB Induces Hyperplasia in Chrysanthemum and Tobacco Leaves, and p12 Upregulates Expression of a bHLH TF.

(A) Cross sections and light microscopy of chrysanthemum upper leaves from mock-inoculated plants and from plants systemically infected with CVB.(B) Appearance of the symptoms induced by empty TMV vector and TMV-p12 in tobacco.(C) Leaf malformation symptoms induced by CVB in systemically infected leaves of N. occidentalis ssp hesperis.(D) Cross sections and light microscopy of N. occidentalis ssp hesperis leaves from mock-inoculated plants and from plants systemically infected withCVB.(E) Expression levels of upp were determined by quantitative RT-PCR. Values on the y axis represent normalized fold expression (n = 6); the error barsshow SD; *P < 0.05, **P < 0.01, and ***P < 0.001.(F) Structure of upp-L and upp-S in tobacco. Boxes and lines indicate exons and introns, respectively.(G) Alignment of N-terminal sequences of Upp/Upa20 and bHLH137 from Arabidopsis. The translation initiation codon for Nt upp-S is underlined.(H) Phylodendrogram of Upp/Upa-like TFs from 18 plant species. Arabidopsis At1g59640 BIGPRTALp bHLH TF was used as an outgroup. For plantspecies abbreviations and other details, see Supplemental Table 1 online. Numbers represent GenBank accession numbers for the correspondinggenes. The sequence alignment used for this analysis is available as Supplemental Data Set 1 online.(I) Expression levels of upp-L and upp-S were determined by quantitative RT-PCR. The mean 22DDCT values 6 SD from three independent experimentsare shown (n = 9); **P < 0.01 and ***P < 0.001. cDNA inputs were normalized to mRNA of three reference genes (Nt EF-1a, Nt b-actin, and Nt cdc2).(J) N. occidentalis ssp hesperis upp-L is upregulated during CVB infection as analyzed by RT-PCR.Bars in (A) and (D) = 100 µm.[See online article for color version of this figure.]

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Figure 2. Inducibility of the Nt upp-L and Nt upp-S Promoters and Promoter Fragments.

(A) to (C) Analysis of GFP fluorescence 3 DAI shows p12 inducibility of the Nt upp-L (A) and Nt upp-S (B) promoters compared with the constitutiveactin2 promoter (C). The Nt upp-L, Nt upp-S, and actin2 promoters were cloned upstream of GFP and tested by codelivery with p12 (right half of the leafin [A] to [C]) or empty T-DNA (left half of the leaves).(D) The GFP protein levels upon GFP codelivery with p12 and empty T-DNA. Coomassie blue staining of the membrane is presented as a loadingcontrol.(E) Fragments of the Nt upp-L and Nt upp-S promoters tested for p12 inducibility. Position of the UPA box is shown as a gray rectangle.(F) Results of GFP fluorometric analysis of the protein extracts from Agrobacterium-infiltrated N. benthamiana leaves. The promoter fragments shown in(E) were cloned upstream of GFP and assayed as before (see above). The baseline represents the level of fluorescence of the protein extract from theleaf expressing an empty T-DNA. Statistical analysis was performed in three independent experiments by t test, P < 0.05. Error bars denote SE of themeasurements.

RNA Virus-Encoded Transcription Factor 963

in Nt upp-L, 438 nucleotides versus 156 nucleotides in Nt upp-S;Figure 1F) and encode putative 362– and 260–amino acidbHLH-type TFs with predicted molecular masses of 42.2 and29.0 kD, respectively. As expected, BLAST analyses demon-strated that Nt upp-L, Nh upp-L, and Nt upp-S are related toUPA20 from pepper. The only homolog of Nt upp-L in Arabi-dopsis thaliana is an uncharacterized bHLH137 TF (At5g50915;54% identity and 65% similarity to Nt upp-L). The central part ofthe UPA/upp-L–related proteins is well conserved and com-prises the bHLH motif, whereas the N termini of the proteinsshow very little similarity between the UPA/upp-L proteins andAt bHLH137 (Figure 1G).

A phylogenetic analysis of the UPA/upp-L–related proteinsshowed that they formed four distinct clades in the phyloden-drogram with a high statistical probability (bootstrap values>70%), and these clades were named groups 1 to 4, respectively(Figure 1H; see Supplemental Table 1 and Supplemental Data Set1 online). Notably, group 1 includes the bHLH proteins fromSolanaceae species, group 3 comprises proteins from mono-cotyledons, and At bHLH137 falls into group 4 and is distantlyrelated to group 1 (Figure 1H).

Real-time RT-PCR experiments employing sets of gene-specificprimers, which are able to discriminate between Nt upp-L and Ntupp-S, revealed upregulation of both Nt upp-L and Nt upp-S byp12 (Figure 1I). Similarly, CVB infection activated the Nh upp-Lgene, the N. occidentalis ssp hesperis ortholog of tobacco upp-L(Figure 1J).

Both upp-L and upp-S Promoters Are p12 Responsive

Activation of the upp genes by p12 might be either direct (i.e.,involving interaction of p12 with the upp promoters and/or thepromoter-interacting TFs) or indirect via signal transduction path-ways where p12 interacts with a cellular receptor and initiatesa sequence of events resulting in the upregulation of the uppgenes. To elucidate the mechanism of upp activation, we studiedthe inducibility of the upp promoters by p12. We isolated the Ntupp-L and Nt upp-S promoters by genome walking. Sequencecomparisons of the Nt upp-L, Nt upp-S, and Ca UPA20 promotersrevealed the presence of a previously identified conserved motifwith the consensus sequence TATATAAACCN2-3CC that wastermed the UPA box (Kay et al., 2007, 2009; see SupplementalFigure 3A online). A TATA-box is located within the UPA box 33 bpupstream of the transcription start site.

To analyze the p12 inducibility of the upp promoters, we usedthe upp-Lpro:GFP (for green fluorescent protein) and upp-Spro:GFPcassettes as reporters and actinpro:GFP as a constitutive promotercontrol in N. benthamiana after Agrobacterium-mediated trans-formation. GFP fluorescence and immunoblots revealed that bothupp promoters are p12 responsive (Figure 2; see SupplementalFigures 3C to 3E online). Engineered promoter deletions from

both ends narrowed the p12-responsive region to a 103-bp se-quence 94% identical in both promoters (six variable positionsper 103 bp) (Figures 2E to 2G). Notably, the 103-bp fragments forboth promoters possessed the UPA box (see SupplementalFigure 3B online). Compared with upp/UPA, the At bHLH137promoter also has an UPA box, though slightly deviating from theconsensus (TATATAAACCN9CC), and could be activated by p12(Figure 2G, right panel). Nevertheless, Agrobacterium-mediatedAt bHLH137 overexpression did not induce any visible tissue orcellular changes. Concordant with these observations, transientexpression of Xanthomonas AvrBs3 TALe in Arabidopsis sppdoes not cause any cellular changes (Marois et al., 2002), furtherreinforcing the idea that the TFs of group 1 (Figure 1H) might differfrom those of group 4 in their role in plant morphogenesis.In addition, the p12 mutants altered in the NLS or zinc finger

(Lukhovitskaya et al., 2009) failed to activate the upp promoters(see Supplemental Figure 4 online). These data further sup-ported our hypothesis of direct promoter activation by p12, asour previous study revealed that the p12 ZF domain is involvedDNA binding and the NLS is required for p12 translocation intothe nucleus (Lukhovitskaya et al., 2009).

p12 Associates with upp Promoters

We performed a chromatin immunoprecipitation (ChIP) assay witha hemagglutinin (HA) epitope-specific antibody to determinewhether the HA epitope–tagged p12 associates with the upp pro-moters in vivo. Chromatin was isolated from plants infected withPVX or PVX-p12HA. Precipitated DNAs were extracted and am-plified by PCR using primers flanking the 103-bp p12-responsiveelement. p12 precipitated with the upp promoters containing theUPA box, but not with a sequence located 2.5 kb downstream ofthe TATA-box (Figures 3A and 3B; see Supplemental Figure 5Aonline). This demonstrates that the interaction of p12 with DNA invivo is specific. Cloning and sequencing of the 103-nucleotidefragments that precipitated with p12 revealed the presence of boththe Nt upp-L and Nt upp-S promoter sequences.The ChIP data were substantiated by subcellular fractionation

and anti-HA immunoblots. The p12HA protein was detected inthe nuclear P10 fraction and in the precipitated chromatin (Fig-ures 3C and 3D). Additionally, we found that p12 directly boundto the 32P-labeled upp promoter DNA in electrophoretic mobilityshift assays (EMSAs) (Figure 3E). Competition experiments in-volving two DNA substrates, a DNA fragment of the Nt upp-Lpromoter comprising the UPA box and the same fragment witha randomized UPA box sequence, revealed that the unlabeledUPA box freed more of the labeled UPA box from the complexthan the randomized UPA box sequence, suggesting higheraffinity of p12 to the UPA box–containing DNA (Figure 3F). Acontrol competition binding with p12 lacking the zinc-fingerdomain revealed no preference for the UPA box–containing DNA

Figure 2. (continued).

(G) The GFP protein levels upon GFP codelivery with p12 and empty T-DNA. The samples run in the gel included L0U0 (1179 bp upp-L promoterconstruct), L1U2 (103 bp upp-L promoter construct), S1U2 (103 bp upp-S promoter construct), and At bHLH137 promoter construct. Coomassie bluestaining of the membrane is presented as a loading control.

964 The Plant Cell

in the absence of the zinc finger (see Supplemental Figure 5Bonline). Collectively, these results demonstrate that p12 specif-ically binds to the UPA box–containing DNA.The important characteristic feature of a TF, and especially an

activator, is the presence of a trans-activating domain. To in-vestigate whether p12 possesses trans-activating activity, baittransactivation tests were performed in a yeast heterologoustranscription system. To this end, the yeast GAL4 DNA bindingdomain (BD) was fused either to full-length p12 or to p12 lackingthe zinc finger (p12ZF) as a result of point mutations in the p12gene (Lukhovitskaya et al., 2009). A yeast strain that has fourintegrated reporter genes under the control of three distinctunrelated Gal4-responsive promoters was employed. Aftertransformation, yeast colonies were tested on three separateplates for activation of the reporters, which included MEL1 en-coding a-galactosidase, AUR1-C conferring strong resistanceto the highly toxic drug Aureobasidin, and HIS3 enabling yeastgrowth on media that lack His. Both p12 and p12ZF appeared toactivate all three reporter genes tested (Figure 3G) comparedwith a negative control construct lacking autoactivation activity.Expression of the BD-p12 and BD-p12ZF fusion proteins inyeast cells was confirmed by immunoblotting (Figure 3H). Thesedata demonstrated that p12 possesses trans-activating activity.

Figure 3. Analysis of p12 Association with upp Promoters.

(A) Fragments in the Nt upp-L region analyzed by ChIP.(B) ChIP analysis was conducted with HA-specific antibodies on extractsfrom plants infected with PVX and PVX-p12HA. Conventional PCR with

34, 37, 40, and 42 cycles was conducted before immunoprecipitation(input) or on immunoprecipitated material (ChIP). Fragments 1 and 2indicated in (A) were amplified.(C) Presence of p12 in the nuclear P10 and cytoplasmic S30 fractions.The fractions were analyzed by immunoblotting using antisera to the HA-tag (top panel) and to Histone H3 as a nuclear marker (bottom panel).(D) Detection of p12HA in the precipitated chromatin. Immunoblottingwas conducted with samples collected before immunoprecipitation (in-put) and with immunoprecipitated material (ChIP) using antisera to theHA-tag (top panel) and to Histone H3 (bottom panel).(E) EMSA of the ability of p12 to interact with the Nt upp-L promoter(proupp). A 103-bp proupp fragment containing the UPA box was in-cubated with increasing amounts of p12 at the indicated molar protein:DNA ratios prior to electrophoresis.(F) EMSA of preformed complexes of labeled 40-bp proupp fragmentcontaining the UPA box (UPAbox) and p12 taken at the protein:DNAmolar ratio 4:1 (the amount required for the complete binding of thisparticular DNA substrate) incubated with the increasing amounts of un-labeled competitor DNA, either UPAbox or UPAbox-rand (the samefragment as the UPA box but with randomized UPA box sequence),added at the indicated molar ratios to the labeled UPA box.(G) Activation of the reporters by p12 and p12ZF in yeast. p12 and p12ZFfused to GAL4 DNA binding domain (BD) were transformed into the yeaststrain Y2HGold and plated on SD/-Trp/X-a-Gal medium to test for activa-tion ofMEL1 encoding a-galactosidase, plated on SD/-Trp/Aureobasidin totest for activation of the AUR1-C reporter, or plated on SD/-Trp/-His to testfor induction of the HIS3 reporter. The plates were incubated for 48 h at28°C. Yeast transformed with a Gateway vector containing an ORF, whichdoes not activate the reporters, fused to GAL4 BD was used as a negativecontrol (BD-control).(H) Detection of the BD-protein fusions in yeast extracts. Whole proteinextracts were analyzed by immunoblotting using antisera to the c-Mycepitope tag. Asterisks indicate nonspecific binding of the antibodies tounknown yeast protein. Molecular weights of protein marker are shownon the left.

RNA Virus-Encoded Transcription Factor 965

Overexpression of upp-L, but not upp-S, Resultsin Hyperplasia

To determine whether upregulation of Nt upp-L and/or Nt upp-Sexpression is involved in hyperplasia induction or, alternatively,just accompanies the changes in tissue morphology, Nt upp-Land Nt upp-S were individually expressed in tobacco and Ni-cotiana benthamiana leaves by Agrobacterium infiltration (Figure4A; see Supplemental Figure 6A online). The RT-PCRs con-firmed overexpression of Nt upp-L and Nt upp-S (Figure 4B; seeSupplemental Figure 6B online) in leaf patches infiltrated witheither upp-L or upp-S constructs, respectively, compared withthe wild-type plants or to the patches infiltrated with empty T-DNA. In both plant species, the Agrobacterium-mediated Ntupp-L overexpression induced hyperplasia (Figure 4C; seeSupplemental Figure 6C online) that was phenotypically similarto that in the tissues expressing p12 or observed during carla-virus infection. As before, palisade and spongy parenchymacells were smaller but more abundant in number compared withthe control infiltration. In contrast with Nt upp-L, overexpression

of Nt upp-S did not cause leaf malformation and hyperplasia(Figure 4C; see Supplemental Figure 6C online), demonstratingthat the truncated TF is dysfunctional in this respect. It should benoted that overexpression of Nt upp-L did not result in the alter-ation of Nt upp-S expression and vice versa (Figure 4B), indicatingthat the observed Nt upp-L overexpression phenotypes (hyper-plasia) were specific for Nt upp-L. Notably, Nt upp-L–mediatedhyperplasia caused by the Nt upp-L TF in N. benthamiana was notas pronounced as in N. tabacum and petunia (Petunia hybrida)(see Supplemental Figures 3A, 3C, and 7 online), suggesting thespecies-specific effect of Nt upp-L.

Overexpression of upp-L, but Not upp-S, Affects the Activityof E2F Genes in the Cell Cycle (CYCD/Rb/E2F) Pathway

Since factors that determine coordination between cell division,mitotic cell cycle, and differentiation play important roles in cellproliferation and often, when unregulated, induce hyperplasia,we analyzed whether abundance of the mRNAs of the genesinvolved in the CYCD/Rb/E2F pathway of the cell cycle and early

Figure 4. Overexpression of Nt upp-L but not Nt upp-S Causes Hyperplasia in Leaves.

(A) Transient expression by Agrobacterium infiltration of Nt upp-L (1) but not Nt upp-S (2) causes hyperplasia 4 DAI relative to empty transferred DNA(T-DNA) (3) in tobacco leaves.(B) RT-PCR on cDNA of tobacco leaves Agrobacterium infiltrated for delivery of the upp-L and upp-S constructs. The constitutively expressed gene forEF1a served as a normalization control. EP, empty T-DNA; wt, healthy plant not infiltrated with Agrobacterium.(C) Cross sections and light microscopy of tobacco leaves 4 DAI with Agrobacterium delivering Nt upp-L, Nt upp-S, or empty T-DNA. Bars = 100 µm.(D) Expression analysis of the core cell cycle genes and some leaf patterning genes in Nt upp-L Agrobacterium-infiltrated tobacco leaf discs. The dataare from two independent experiments (n = 8); the error bars denote SE; *P < 0.05, **P < 0.01, and ***P < 0.001; ns, not significant (P > 0.05).(E) Ploidy distribution of nuclei from Agrobacterium-infiltrated tobacco leaf halves. The average percentage of 2C and 4C from two independentexperiments is indicated.(F)Model for p12-mediated interference with mitotic cell cycle and endocycle. CVB p12 protein ectopically induces expression of the upp-L TF, which in turnupregulates E2FB and downregulates E2FC and CYCB1;1 expression, leading to stimulation of the mitotic cell cycle and inhibition of the endocycle. Geneswith elevated mRNA accumulation are shown in red, genes with reduced transcript are in blue, while genes with unaltered expression are in black.

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leaf development is affected by Nt upp-L and Nt upp-S over-expression.Tobacco mRNA sequences are available for Cyclin B1;1 (Nt

CYCB1;1), Cyclin D3;1 (Nt CYCD3;1), retinoblastoma (Nt RBR),one of the E2F genes (Nt E2FB1), AINTEGUMENTA (Nt ANTL),and PHAVOLUTA-like HD-ZIPIII (Nt PHUVL; see SupplementalTable 2 online). Additionally we performed tobacco EST data-base searches and identified ESTs encoding polypeptideshighly similar to the Arabidopsis E2FC and an additional ESTsimilar to At E2FB. Based on the available EST sequences, weisolated the full-length cDNA of Nt E2FC and Nt E2FB2 by RACEand determined the corresponding mRNA sequences (seeSupplemental Table 2 online) .At PHAVOLUTA is involved in the early stages of leaf development

(Ichihashi et al., 2010). The mRNA levels of the tobacco orthologof this gene were unchanged despite the cytological and mor-phological changes in the Nt upp-L–overexpressing tissue (seeSupplemental Figures 6D and 6E online).Nt ANTL is required for control of cell proliferation, expressed

during formation of leaf primordia, and regulates the number ofcells within the developing leaves (Mizukami and Fischer, 2000;Mudunkothge and Krizek, 2012). Using real-time RT-PCR, wefound that the mRNA levels of Nt ANTLwere reduced by Nt upp-L,but not by Nt upp-S overexpression (Figure 4D; see SupplementalFigures 6D and 6E online). The downregulation of the Nt ANTLgene is consistent with observed uncontrolled cell number duringhyperplasia development (Figure 4C). On the other hand, At ANTLoverexpression is associated with increased At CYCD3;1 levelsand promotes additional rounds of cell division during leaf de-velopment, suggesting that ANTL acts upstream of CYCD3;1(Mizukami and Fischer, 2000; Menges et al., 2006). Consistent withthis idea and the observed downregulation of Nt ANTL, we foundthat the mRNA levels of Nt CYCD3;1 were unaffected by either Ntupp-L or Nt upp-S overexpression (see Supplemental Figures 6Cand 6D online) despite pronounced hyperplasia and greatly in-creased number of cells within the Nt upp-L–overexpressing areasof a leaf. Similarly, no changes in Nt RBR and Nt E2FB1 levelswere observed upon Nt upp-L overexpression (Figure 4D; seeSupplemental Figures 6D and 6E online). On the other hand, Ntupp-L overexpression correlated with elevated levels of Nt E2FB2mRNAs and reduced levels of Nt E2FC and Nt CYB1;1 transcripts(Figure 4D). In agreement with these observations, At E2FCknockdown RNA interference lines undergo hyperplasia and showreduced ploidy, but Arabidopsis plants that overexpress E2FChave increased DNA content and a higher ratio of polyploid nuclei(del Pozo et al., 2006). Furthermore, tobacco mesophyll cells with

Figure 5. Phenotypes of Tobacco Plants Silenced for upp-L and N.occidentalis ssp. hesperis Infected with CVB.

(A) Nt upp-L knockdown suppresses hyperplasia development in TMV-p12 infected plants. Tobacco plants were challenged either with TRV:upp-L to initiate onset of VIGS or TRV:00 (empty vector control) followedby inoculation with TMV-p12 1 week later.(B) RT-PCR analysis of the Nt upp-L mRNA accumulation in cDNA of thesilenced and control plants. The constitutively expressed gene EF1aserved as a normalization control.(C) Cross sections and light microscopy of the leaves of nonsilenced andNt upp-L-silenced plants. Identity of the inoculated constructs is shownin each image. Bars = 100 µm.

(D) Images of tobacco plants silenced for Nt upp-L (TRV:upp-L) versusa control (TRV:00).(E) Images of N. occidentalis ssp hesperis plants infected with CVBversus mock-inoculated plants.(F) Growth response of tobacco plants to Nt upp-L silencing. The errorbars denote SE; n = 18.(G) Growth response of N. occidentalis ssp hesperis plants to CVB in-fection. The error bars show SE; n = 9.[See online article for color version of this figure.]

RNA Virus-Encoded Transcription Factor 967

higher 4C DNA content also contain high levels of Nt CYCB1;1transcripts (Qin et al., 1996). In Arabidopsis, E2FB promotes a mi-totic cell cycle, while E2FC can facilitate an endocycle (del Pozoet al., 2006). In tobacco and Arabidopsis, CYCB1;1 participates inthe regulation of the G2/M transition and elevated levels of CYCB1;1expression correlate with naturally occurring G2-arrested cellpopulations in leaves with higher ploidy (Qin et al., 1996; Ascencio-Ibáñez et al., 2008).

Collectively, these data suggest that Nt upp-L overexpressionmight facilitate the mitotic cell cycle through Nt E2FB2 activationand inhibit DNA endoreduplication and the endocycle throughdownregulation of Nt E2FC and Nt CYCB1;1 expression.Therefore, next, we examined the ploidy distribution in fully ex-panded leaves agroinfiltrated with the Nt upp-L and T-DNAconstructs. The leaves were at the same developmental stageand tobacco plants were grown under the same conditions asthose used for the gene expression experiments. DNA flow cy-tometry analysis and CyView profiles revealed ;1.5-fold totwofold reduction of the amount of 4C nuclei upon Nt upp-Loverexpression compared with the empty plasmid control in-filtrated onto the other half of the same leaf (Figure 4E). Notably,the most pronounced effect on the ploidy distribution was ob-served at 3 d after inoculation (DAI) and coincided with Nt E2FB2upregulation and Nt E2FC and Nt CYCB1;1 downregulation(Figures 2E and 4D). The asymmetric expression pattern ob-served for core cell cycle genes suggest that, indeed, Nt upp-Lexpression inhibits the capacity of the leaf cells to undergoendocycling and facilitates a mitotic cell cycle (Figure 4F).

The observed changes in the Nt E2FB and Nt E2FC levels ofexpression indicate that overexpression of Nt upp-L causes celldivision without induction of additional rounds of DNA synthesis(Figure 4F). This is the opposite of the case with geminiviruses,where DNA synthesis is induced but cell division is blocked(Ascencio-Ibáñez et al., 2008; Kittelmann et al., 2009). At thecellular level, ectopic Nt upp-L overexpression pushed palisadeand spongy mesophyll cells from the G1 phase to the mitoticcycle, promoting cell divisions in fully developed leaves in whichcell division is normally limited.

To explore the spatial expression pattern of Nt upp-L, wegenerated transgenic tobacco plants harboring the upp-Lpro:GUS(for b-glucuronidase) construct, which contains the promoter ofNt upp-L fused to the uidA gene. When we analyzed the GUSstaining pattern in the upp-Lpro:GUS lines, we observed that thestaining was confined to the shoot apical meristem (SAM; seeSupplemental Figure 6F online), where active cell division occurs.Collectively, the data suggest that Nt upp-L is mostly expressedin the SAM, but when ectopically induced by p12 in leaves, itpromotes cell division of the differentiated mesophyll. Overall,these findings support a direct role for Nt upp-L in promoting cellproliferation.

Knockdown of upp-L Expression Results in Suppression ofHyperplasia Development

To further confirm the role of Nt upp-L in the induction of hyper-plasia, we performed virus-induced gene silencing (VIGS) in to-bacco using a tobacco rattle virus vector carrying part of the Ntupp-L gene. An empty vector construct was used as a control. The

tobacco plants inoculated with the empty VIGS vector and theplants silenced for the Nt upp-L gene were challenged with TMV-p12 (Figure 5A). VIGS of Nt upp-L reduced the Nt upp-L transcriptlevels (Figure 5B) and the p12-dependent leaf malformation andhyperplasia compared with control inoculation with the emptyVIGS vector plus TMV-p12 (Figures 5A and 5C). Similarly, silencingof Nt upp-L in the absence of p12 expression had no effect on theplant tissue architecture with typical palisade and spongy meso-phyll cells layers (Figure 5C), reinforcing the idea that Nt upp-L isnormally expressed in the SAM and might act in pathways otherthan those that regulate leaf development. Indeed, tobacco plantssilenced for the Nt upp-L gene were characterized by shorterstems and shorter internodes (Figures 5D and 5F). The shorterstem phenotype was especially pronounced at earlier stages ofplant development (Figure 5F). Concordant with these ob-servations, N. occidentalis ssp hesperis plants infected with CVBdeveloped much longer stems; this phenotype was especiallyprominent at 14 to 21 DAI and earlier stages of plant development(Figures 5E and 5G). These lines of evidence suggest that the Ntupp-L TF might be directly or indirectly involved in early stemelongation. The upp-L–silenced plants were also delayed in settingflowers and seeds (see Supplemental Figure 8 online), and four outof 18 plants in two independent experiments did not undergovegetative to floral transition.Collectively, these results suggest that the Nt upp-L TF is a reg-

ulator that stimulates cell proliferation when ectopically expressed inleaves. Our results establish that the visible consequence of p12virulence activity, the induction of hyperplasia and leaf malformation,is elicited by p12 ectopically activating upp-L, which is normallyexpressed in the SAM.

Conclusions

Our data establish p12 as a virus-encoded TF that is translocatedto the nucleus and activates expression of the regulator of cellproliferation upp-L, which results in severe leaf malformation.Similar to numerous plant and animal TFs, which localize to thenucleus and directly bind to DNA via a zinc-finger domain (Klug2010), the function of the p12 protein as a transcriptional activatorrequires the Arg-rich NLS and Cys residues within the C-4 typezinc finger shared with cysteine-rich proteins (CRPs) of othercarlaviruses. This establishes CVB p12 as a TF encoded bya positive-stranded RNA virus. This finding is rather surprisingsince the life cycle of viruses, such as CVB, takes place in thecytoplasm and has been previously assumed to be unrelated tonuclear functions.The most obvious parallels in terms of hyperplasia induction can

be drawn between CVB and geminiviruses. Geminiviruses havesmall, single-stranded DNA genomes encoding a limited numberof genes and rely entirely on host machinery for replication in thenucleus, whereas CVB and other carlaviruses have a genomecomprising a single segment of single-stranded, positive-senseRNA, encode their own RNA polymerase and replicate in thecytoplasm. Nevertheless, both CVB and geminiviruses appearto interfere with cell cycle regulation and induce hyperplasia.Whereas CVB causes mostly proliferation of the leaf mesophylltissue, the majority of geminiviruses are phloem limited and inducecell division in the vascular tissue, which results in enations. For

968 The Plant Cell

example, cabbage leaf curl virus infection in Arabidopsis altersexpression of cell cycle genes, favorably inhibiting genes typicallyexpressed during G1-to-M phase progression (e.g., downregulationof CYCD3 and the division-promoting E2FB) and activatingthose normally expressed during S and G2 phases of the cellcycle (e.g., upregulation of E2FA/DPA and the division-inhibitingE2FC; Ascencio-Ibáñez et al., 2008). Conversely, in tobacco, theupp-L TF, ectopically induced by p12 in leaves, appeared toupregulate the division-promoting E2FB and downregulate thedivision-inhibiting E2FC. These differences suggest that while atleast some geminiviruses promote the endocycle and S phase ofthe cell cycle, which is beneficial for the viral DNA replication, CVBp12 seems to facilitate a mitotic cell cycle through induction ofupp-L, which is normally expressed in the SAM. Moreover, it hasbeen shown that overexpression of E2FB, which promotes themitotic cell cycle, strongly compromises cabbage leaf curl virusaccumulation (Ascencio-Ibáñez et al., 2008). Therefore, it seemsthat CVB and geminiviruses induce hyperplasia through differentpathways; while geminiviruses have been suggested to do thisthrough disturbing phytohormonal signaling (Piroux et al., 2007;Ascencio-Ibáñez et al., 2008; Mills-Lujan and Deom, 2010), CVBdoes it by encoding a TF, which in turn ectopically activates ex-pression of a SAM-specific TF.

Currently, the exact role of the p12 protein (as well as the C4proteins of geminiviruses) in virus infection is not known, al-though it might be required for efficient replication and accu-mulation in natural hosts and/or efficient transmission of thevirus by its insect vector through, for example, induction of stemelongation as we observed in this study. The infected plants withlonger stems might be more accessible to aphids comparedwith noninfected plants with shorter stems. It also is tempting tospeculate that p12, and p12-like proteins of other carlaviruses,may be considered as a latency factor (a latent protein) of thisgroup of viruses and the viral-programmed induction of hyper-plasia might be a way to delay the onset of HR (a type of pro-grammed cell death) and to maintain the latent state of the virusinfection (e.g., cell division without virus replication), as it hasrecently been shown that overexpression of Nt E2FB1 reducesprogrammed cell death induction in tobacco Bright Yellow-2cells (Smetana et al., 2012). Moreover, it has been well docu-mented that in animal/human viruses, some latent stages inducetumorogenesis (Poreba et al., 2011).

Despite the distinct evolutionary origin of bacteria and viruses,they can manipulate cell size regulators to trigger either hyper-trophy (Kay et al., 2007) or hyperplasia (this study) through acti-vation of similar pathways. Furthermore, our results show thatdespite the lack of obvious sequence similarity between p12 andthe Xanthomonas AvrBs3 TALe, both proteins activate UPA box–containing promoters and induce expression of orthologous genesencoding regulators of cell size and proliferation.

This article demonstrates that interaction of plant RNA viruses withtheir hosts are considerably more complex than previously thoughtand can involve virus manipulation of host transcription directly byuse of virally encoded TFs, such as p12. As an antiviral protectionstrategy, host defense genes could have evolved to contain the viralTF–responsive elements in their promoter regions to be upregulatedin the presence of the viral TF, as we previously found for a setof p12-induced defense-related genes (Lukhovitskaya et al., 2009).

Unraveling the mechanism of the p12-dependent HR induction inanother tobacco cultivar (Lukhovitskaya et al., 2009), which may besimilar to the transcriptional activation of a resistance gene by theAvrBs3 TALe in pepper (Römer et al., 2007), might shed further lighton this process.

METHODS

Plant Material and Growth Conditions

Chrysanthemum (Chrysanthemummorifolium), tobacco (Nicotiana tabacumcv Samsun), native tobacco (Nicotiana occidentalis ssp hesperis),Nicotianabenthamiana, Arabidopsis thaliana, and petunia (Petunia hybrida) plantswere grown under standard conditions (16 h light and 60% humidity).

Phylogenetic and Sequence Analysis

Theprotein sequencesencodedby tobaccoandnative tobaccowereanalyzedwith published sequence frompepper (Capsicumannum) andArabidopsis andseveral homologs from 17 other plant species (see Supplemental Table 1online). The sequences were aligned using ClustalW algorithm of theMegalignmodule of the Lasergene software package (DNASTAR), and the trees wereprepared using Phylip package. Bootstrap values were calculated from 1000replicates of the tree. GenBank accession numbers are indicated on thephylodendrograms. The sequences for some Solanaceae species were re-trieved from Sol genomics network database (http://solgenomics.net/tools/blast/index.pl).

Isolation of the upp Sequences and Promoters

The complete cDNAs of upp mRNAs were obtained by 59- and 39-RACEwith the SMART RACE cDNA amplification kit (Clontech) and the Ad-vantage 2 PCR kit (Clontech). Amplified PCR products were cloned intopJet1.2 (MBI Fermentas) and sequenced (by Macrogen). The genomicsequences of the upp genes and promoters were isolated from tobaccoSamsun genomic DNA with the GenomeWalker Universal kit (Clontech)and Advantage 2 PCR kit (Clontech).

Plant Expression Constructs and Promoter Studies

Standard recombinant DNA procedures were performed by use of a com-bination of PCRs, site-directed mutagenesis, and swapping of restrictionfragments. PVX-p12 construct was described previously (Lukhovitskayaet al., 2005). To construct TMV-p12, the p12 open reading frame (ORF) wasamplified with primers P12Age-FW and P12Xho-REV (see SupplementalTable 3 online), introducingAgeI and XhoI restriction sites. The resulting PCRproduct was cloned into the TMV30B vector (Shivprasad et al., 1999) as anAgeI-XhoI fragment.

TheplasmidpLH7000 (HausmannandTöpfer, 1999)wasused to generate allthebinary constructsdescribed in this study.CVBp12,CaUPA20,Ntupp-L, andNt upp-S were expressed in plants under control of the constitutive cauliflowermosaic virus 35S promoter. pro35S:p12 and its mutants for zinc finger (pro35S:p12ZF) and (pro35S:p12NLS) were described previously (Lukhovitskaya et al.,2009). To construct pro35S:CaUPA20, pro35S:Ntupp-L, and pro35S:Ntupp-S,the corresponding ORFs were amplified with the following primer combi-nations (see Supplemental Table 3 online): Ca-UPA20-Nco-P/UPA20-Xba-M, Nt-UppLS-NcoI-FW/Nt-UppL-Xba-gene-REV, Nt-UppLS-NcoI-FW/Nt-UppS-Xba-gene-REV digested with NcoI-XbaI and ligated into simi-larly digested pro35S:p12 to replace the p12 ORF.

GFP was expressed under control of either constitutive actin2 promoter,Ca UPA20, At bHLH137, or Nt upp promoters. proCaUPA20:GFP, proupp-L:GFP, proupp-S:GFP, probHLH137:GFP, L0U1, L0U2, L1U2, S0U1, S0U2,and S1U2 were engineered by ligating EcoRI-XhoI–digested PCR product

RNA Virus-Encoded Transcription Factor 969

generated with appropriate primers (see Supplemental Table 3 online) intoEcoRI-XhoI–digested pro35S:GFP (Lukhovitskaya et al., 2009) to replace the35S promoter. proACT:GFP and proCaUPA20:GFP were engineered by li-gating MunI-digested PCR products generated with ACT2-prom-Mun-Pand ACT2-prom-Mun-M primers and with Ca-F0R0-Mun-P and Ca-F0R0-Mun-M primers into EcoRI-digested pro35S:GFP to replace the 35S pro-moter. The correct orientation of the inserts was determined by restrictionanalysis and by sequencing. L0U0, L2U3, S0U0, and S2U3 were en-gineered by ligating EcoRI-NcoI–digested PCR product generated withappropriate primers (see Supplemental Table 3 online) into EcoRI-NcoI–digested pro35S:GFP (Lukhovitskaya et al., 2005) to replace the 35Spromoter.

To obtain TRV:upp-L silencing construct, XhoI-EcoRI–digested PCRproduct generated with Nt-Upp-XhoI-FW and Nt-Upp-EcoRI-R primers(see Supplemental Table 3 online) was ligated into TRV:00 digested withXhoI-EcoRI.

The binary constructs were used to transform Agrobacterium tume-faciens strain C58C1 pGV2260. Agrobacterium transformed with emptypLH7000 plasmid was used as a control in the promoter induction and CaUpa20, bHLH137, upp-L, and upp-S overexpression experiments.

Yeast Manipulations

For expressing p12 and p12ZF constructs in yeast, the p12 and p12ZFORFswere cloned to the destination vector pBD-Gate2 (Maier et al., 2008)using Gateway technology (Invitrogen). All the constructs were verified bysequencing. The Y2HGold yeast strain (Clontech) was transformed withthe plasmids using small-scale LiAc yeast transformation procedure asdescribed in Clontech’s Yeast Protocols Handbook. Transformants wereselected on2Trp plates. To test forHIS3 reporter self-activation, colonieswere incubated in 50 mL sterile distilled water for 36 h to deplete yeastfrom the internalized His. Prior to immunoblotting, yeast whole proteinwas extracted using the LiAc/NaOH extraction method as described(Zhang et al., 2011).

Protein Analysis, SDS-PAGE, and Immunoblotting

Protein extracts from tobacco and N. benthamiana leaves were preparedfrom leaf samples (20 mg, fresh weight). Leaf discs were ground andhomogenized in an ice-cold mortar in 20 mL 250 mM Tris-HCl, pH 7.8,mixed with 20 mL extraction buffer (75 mM Tris-HCl, pH 6.8, 9 M urea,4.3% SDS, and 7.5% b-mercaptoethanol), heated at 100°C for 5 min, andcentrifuged (5min at 10,000g) to remove insoluble material. Aliquots of thesupernatant (1 to 10 µL) were separated by SDS-PAGE on 20%gels. Afterelectrophoresis, proteins were transferred to a Hybond-C membrane (GEHealthcare Bio-Sciences) and subjected to immunoblot analysis with GFPantibody (Abcam), HA IgG (Santa Cruz Biotechnology), and anti-acetil-Histone H3 IgG, and the ECL protein gel blotting kit as recommended bythe supplier (GE Healthcare Bio-Sciences).

Agrobacterium-Mediated Transient Expression

Agrobacterium was grown overnight at 28°C in the presence of strep-tomycin, spectinomycin, carbenicillin, and rifampicin in Luria-Bertanibroth supplemented with MES (10 mM final concentration) and aceto-syringone (20 mM final concentration). Upon reaching OD600 ;0.5, cellswere pelleted by centrifugation at 3500g for 5 min, resuspended in in-filtration buffer (10 mMMES, 10 mMMgCl2, and 150 mM acetosyringone),and incubated for 2 h at room temperature with constant rotation. Six- toeight-week-old N. benthamiana plants were infiltrated using a needlelesssyringe. For promoter induction studies, two Agrobacterium strains de-livering a promoter construct and pro35S:p12 (or an empty pLH7000plasmid) were adjusted to OD600 = 0.6 each.

Microscopy

Sample preparation and light microscopy were performed as describedpreviously (Savenkov and Valkonen, 2001). Fluorescence microscopywas conducted as described (Zamyatnin et al., 2006).

RT-PCR and Real-Time RT-PCR

RNA was isolated from leaf discs using the Spectrum Plant total RNA kit(Sigma-Aldrich) and On-column DNaseI Digest Set (Sigma-Aldrich)according to the manufacturer’s instructions. One-microgram aliquots oftotal RNA samples were used for oligo(dT) primed cDNA synthesis withthe RevertAid H Minus First-Strand cDNA synthesis kit (MBI Fermentas).Obtained cDNAs were used as templates for PCR with DreamTaq DNAPolymerase (MBI Fermentas). Primers amplifying the tobacco elongationfactor 1a (EF-1a) were used as control for equal cDNA amounts (seeSupplemental Table 3 online). Suitable PCR cycle numbers specific toeach primer pair were determined by testing a range of cycle numbers. Aregime with exponential DNA amplification was chosen for each primercombination (see Supplemental Table 3 online). For real-time RT-PCR, 1mg of total RNA preparations was reverse transcribed into cDNAs usingqScript (Quanta). The cDNA was quantified with the DyNAmo Flash SYBRGreen qPCR kit (Finnzymes) and gene-specific primers (see SupplementalTable 3 online) using a Bio-Rad IQ5 icycler apparatus according to themanufacturer’s recommendations. PCR was run in 96-well optical re-action plates (Bio-Rad). The PCR conditions were as follows: 95°C for 10min to activate hot start TaqDNA polymerase, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. At the end of the PCR, dissociation kineticsanalysis was performed to check the specificity of annealing. mRNAquantifications were performed with specific primer combinations andwere normalized with three reference genes (Nt EF-1a, Nt b-actin, and Ntcdc2; see Supplemental Table 3 online). Four (or three) biological repli-cates were used, and three technical replicates were performed for eachbiological replicate. Three PCR reactions were performed for each samplein three independent experiments.

DNA Flow Cytometry Analyses

After themain ribwas removed, infiltrated tobacco leaf halves were choppedwith a razor blade in CyStain UV Precise P nuclei extraction buffer (Partec).The extracted nuclei were filtered through CellTrics 30 µm disposable filterunits (Partec), stained with CyStain UV Precise P staining buffer (Partec),and analyzed with a CyFlow Ploidy analyzer (Partec) and CyView 2 softwareaccording to manufacturer’s recommendations. A total of 7000 nuclei wereanalyzed per sample.

GFP Imaging and Fluorometric Analysis

Detection of GFP fluorescence in detached tobacco and N. benthamianaleaves was performed using a hand-held long-wave UV lamp UVL-56 (UVProducts). Plants and leaves were photographed with a digital camera(Canon G6). For promoter inducibility studies, fluorometric measurementsof GFP were performed. Leaf discs (50 mg fresh weight) were collectedfrom Agrobacterium-infiltrated halves of the leaves on fourth day afterinoculation. The samples were ground in an ice-cold mortar in 50 mLsample buffer (250 mM Tris-HCl and 0.1% [w/v] CHAPS, pH 7.8) sup-plemented with EDTA-free complete protease inhibitor cocktail (Roche),and centrifuged (5 min at 10,000g) to remove insoluble material. Theprotein concentrations were estimated by Bradford reagent (Bio-Rad) andstandardized prior to assays in 150 mL final volume. Fluorometric analysisof samples was performed against a total protein extract from the leafinfiltrated with Agrobacterium delivering an empty pLH700 using a Versa-Fluor Fluorometer (Bio-Rad) at the excitation wavelength of 490/10 nmand the emission wavelengths 520/10 nm.

970 The Plant Cell

VIGS and Systemic Viral Infection

Construction of the TRV:upp-L silencing plasmid is described above. The600-bp upp-L fragmentwas cloned in an antisense orientation into thepTRV:GFP vector by swapping GFP. The resulting construct TRV:upp-L wastransformed intoAgrobacterium and in combinationwith TRV-RNA1used forinduction of the Nt upp-L gene silencing. Seven days after TRV infiltration,tobacco leaves were challenged with TMV-p12. The uppermost leaves wereassayed for TMV-p12 infection and Nt upp-L silencing by RT-PCR.

Subcellular Fractionation of Plant Extracts

Leaf discs (100mg)were ground in liquid nitrogen to finepowder, whichwasresuspended in 2mLbuffer A (400mMSuc, 100mMTris/HCl, 10mMKCl, 5mMMgCl2, 10mMb-mercaptoethanol, and1mMPMSF, pH7.5). The slurrywas filtered through one layer of Miracloth, and the flow-through was splitinto two equal parts. One part was centrifuged at 30,000g for 30min to yielda pellet consisting of MEM fraction enriched in organelles and mis-cellaneous membranes and S30 (soluble protein fraction). Another part ofthe extractwas centrifuged for 10min at 1000g to yield a pellet fraction (P10)enriched in nuclei. P10 and S30 fractions were resuspended in Laemmlisample buffer containing 9 M urea and used for immunoblotting.

ChIP

To perform ChIP, 3 g of tobacco leaf material was collected from sys-temically infected leaves 14 DAI with PVX and PVX-p12HA. ChIP wasperformed as described (Nelson et al., 2006) with some minor mod-ifications. The ChIP kit (Millipore/Upstate) was used. EDTA-free completeprotease inhibitor cocktail (Roche) was used as protease inhibitor. Thechromatin was sonicated six times for 20 s at power 4 with Soniprep 150MSE sonicator to achieve an average DNA size of 0.5 kb. Aliquots ofcleared chromatin solution were saved as input controls and as samplesfor subsequent immunoblots with anti-HA antiserum. The recovered DNAwas analyzed by PCR and different number of cycles were tested.

EMSA

For expression of a C-terminal hexahistidine (His6)-tagged p12 in Escherichiacoli, we used pQE60 (Qiagen) plasmid carrying p12 (Lukhovitskaya et al.,2009). A similarly constructed vector was used for expression of p12ZFHis6(p12 zinc-finger mutant; Lukhovitskaya et al., 2009). Expression and purifi-cation of p12His6 andp12ZFHis6wasperformedasdescribed (Lukhovitskayaet al., 2009). In EMSA, PCR product corresponding to a UPA box–containingregion of the Nt upp-L promoter of 103 bp was incubated with increasingamounts of p12His6 in binding buffer (10 mM Tris/HCl, pH 8.0, and 50 mMNaCl) containing 3 mM ZnSO4 on ice for 30 min. Samples were analyzed ina 1.5% agarose gel containing ethidium bromide. In competition experi-ments, [a-32P]-labeled UPA box–containing PCR product corresponding to40 bp in the Ntupp-L promoter (UPAbox) was first incubated with eitherp12His6 or p12ZFHis6 at protein:DNA molar ratio of 4:1 in the binding bufferon ice for 30 min. The resulting complexes were incubated with increasingamounts of either unlabeled UPAbox or UPAbox-rand (same DNA fragmentas UPAbox but with randomized UPA box sequence) on ice for 30 min.Samples were analyzed in native 6% polyacrylamide gels (acrylamide:N,N9-methylenebisacrylamide ratio, 38.5:1) in TB buffer (89 mM Tris/HCl, pH 8.0,and 89 mM boric acid) and visualized by autoradiography.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL datalibraries under the following accession numbers: HE653922,NtE2FCmRNA;HE653923, Nt E2FB2 mRNA; HE653924, Nt upp-L mRNA; HE653925, Ntupp-SmRNA; HE6539226, Nh upp-LmRNA; HE653927, Nt upp-S gene andpromoter; and HE653928, Nt upp-L gene and promoter.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Position of p12 in the Virus Genome andSimilarity of the p12 Protein to Other Viral Zinc-Finger Proteins.

Supplemental Figure 2. The p12-Mediated Hyperplasia Induced inThree Plant Species.

Supplemental Figure 3. Analysis of the upp Promoter Sequences andupp Promoter Inducibility.

Supplemental Figure 4. p12 Mutants with an Altered NLS or ZincFinger Fail to Activate Both the Nt upp-L and Nt upp-S Promoters.

Supplemental Figure 5. Analysis of p12 Association with the uppPromoters.

Supplemental Figure 6. Ectopic Expression of Nt upp-L but Not Ntupp-S Causes Hyperplasia in Leaves, While Nt upp-L Is NormallyExpressed in the SAM.

Supplemental Figure 7. Hyperplasia Induction in Four Plant Speciesby Nt upp-L and Ca UPA20.

Supplemental Figure 8. Delay in Vegetative to Floral Transition ofTobacco Plants Silenced for upp-L.

Supplemental Table 1. Upp/UPA-Like Genes Used in the Phyloge-netic Analysis.

Supplemental Table 2. N. tabacum Genes Involved in Early LeafDevelopment and the CYCD/Rb/E2F Pathway of the Cell Cycle Usedin Our Analysis.

Supplemental Table 3. List of PCR Primers.

Supplemental Data Set 1. Text File of the Alignment Used for thePhylogenetic Analysis Shown in Figure 1H.

ACKNOWLEDGMENTS

We thank Richard H. Maier (University of Salzburg, Austria) for the pBD-Gate2 vector, Panagiotis N. Moschou (Swedish University of AgriculturalSciences, Sweden) for a negative control for the autoactivation test andhelpful discussions, Lesley Torrance (The James Hutton Institute, Dundee,UK) for critical reading of the article, and David Baulcombe (University ofCambridge, Cambridge, UK) for providing the cDNA clone of PVX.Financial support from the Swedish Research Council Formas (Grants2007-256, 2008-1047, and 2009-1979 to E.I.S.), the Carl TryggersFoundation (E.I.S. and N.I.L.) and the Swedish Institute (postdoctoralfellowship to N.I.L.) is gratefully acknowledged.

AUTHOR CONTRIBUTIONS

E.I.S. designed research. N.I.L., A.D.S., S.K.B., S.T., and E.I.S. performedresearch. N.I.L., A.D.S., A.G.S., and E.I.S. analyzed data. A.G.S. and E.I.S.wrote the article, and all authors commented on the article before submission.

Received October 19, 2012; revised January 29, 2013; accepted February 19,2013; published March 12, 2013.

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RNA Virus-Encoded Transcription Factor 973

DOI 10.1105/tpc.112.106476; originally published online March 12, 2013; 2013;25;960-973Plant Cell

and Eugene I. SavenkovNina I. Lukhovitskaya, Anna D. Solovieva, Santosh K. Boddeti, Srinivas Thaduri, Andrey G. Solovyev

Regulator of Cell Size and Proliferation in Two Tobacco SpeciesAn RNA Virus-Encoded Zinc-Finger Protein Acts as a Plant Transcription Factor and Induces a

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