Research ArticleResearch Article

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

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

Published March 2013. DOI: https://doi.org/10.1105/tpc.112.106476

Abstract

Plant viruses cause a variety of diseases in susceptible hosts. The disease symptoms often include leaf malformations and other developmental abnormalities, suggesting that viruses can affect plant development. However, little is known about the mechanisms 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 severe leaf 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 to cause hyperplasia. The induction of upp-L requires translocation of the p12 protein into the nucleus and ZF-dependent specific interaction with the conserved regulatory region in the upp-L promoter. Our results establish the role of the p12 protein in modulation of host cell morphogenesis. It is likely that other members of the conserved C-4 type ZF family of viral proteins instigate reprogramming of plant development by mimicking eukaryotic transcriptional activators.

INTRODUCTION

Plant innate immunity has evolved as a multilayered defense system to recognize and resist invading pathogens (Jones and Dangl, 2006). As a counterdefense strategy to overcome the first layer of defense, pathogens deliver various virulence determinants into the plant cell. These virulence factors have been termed effectors (Bent and Mackey, 2007). Effectors hijack cellular machinery to suppress immune responses and enhance virulence (Dodds and Rathjen, 2010). Gram-negative bacteria employ a type III secretion system to inject effectors into host cells (Galán and Wolf-Watz, 2006). Plant pathogenic bacteria of the genus Xanthomonas deliver transcription activator-like effector (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; White et al., 2009; Römer et al., 2010). Thereby Xanthomonas TALes, depending on crop cultivar, can either promote disease through hypertrophy and hyperplasia development or induce plant defense if the corresponding UPT box is located in the promoter of a resistance gene (Römer et al., 2007). Interestingly, an AvrBs3 TALe of Xanthomonas spp promotes tissue growth after infiltration on leaves of Solanaceae species and, in pepper (Capsicum annuum), is known to induce a cell size regulator, basic/helix-loop-helix (bHLH)-type TF UPA20, sufficient to cause tissue growth (Kay et al., 2007).

Whereas pathogen-induced expression of specific genes and transcriptional reprogramming of host gene expression has been linked to hyperplasia and other morphological defects, especially for some members of two genera of geminiviruses (Latham et al., 1997; Piroux et al., 2007; Ascencio-Ibáñez et al., 2008; Mills-Lujan and Deom, 2010), no TF-like proteins, which would directly bind to host promoters, have been reported for phytoviruses. The C4 proteins of two monopartite members of the genus Begomovirus and Curtovirus induce hyperplasia probably due to interference with the brassinosteroid pathway and not as direct TFs (Latham et al., 1997; Piroux et al., 2007). Although the C4 protein of beet curly top virus (genus Curtovirus, family Geminiviridae) induces localized hyperplasia in infected phloem tissue and tumorogenic growth in transgenic plants, the C4 protein has not been linked to activation of host promoters through direct binding to specific promoter elements (Piroux et al., 2007; Mills-Lujan and Deom, 2010). On the contrary, the C4 protein of beet curly top virus is localized to the plasma membrane, interacts with plant SHAGGY-related protein kinases, and seems to act through the disruption of brassinosteroid phytohormonal networks and signaling (Piroux et al., 2007). For other geminiviruses, C4 and the homologous AC4/AL4 have been reported to be silencing suppressors (Vanitharani et al., 2004).

Recently, we found that the p12 protein of chrysanthemum virus B (CVB) is implicated in host gene induction as a pathogenicity and virulence determinant (Lukhovitskaya et al., 2009). CVB belongs to the genus Carlavirus, which comprises 35 definitive species and 29 tentative members with conserved genome organization (Levay and Zavriev, 1991; Adams et al., 2004; Martelli et al., 2007). Their positive-stranded genomic RNA encodes six genes, of which the gene for p12 occupies the extreme 3′-terminal position (see Supplemental Figure 1A online; Adams et al., 2004; Martelli et al., 2007). CVB p12 and its homologs in other carlaviruses are Cys-rich proteins characterized by a central C-4 type zinc finger defined by its RCxRCxRxxPx6-8CDxxxC motif and an adjacent nuclear localization signal (NLS; see Supplemental Figure 1C online; Gramstat et al., 1990; Koonin et al., 1991; Lukhovitskaya et al., 2009; note that the carlavirus C-4 superfamily should not be confused with the C4 proteins of geminiviruses). The p12 protein binds both RNA and DNA but exhibits a preference for DNA in the presence of Zn2+ and its pathogenic effects depend on translocation into the nucleus (Gramstat et al., 1990; Lukhovitskaya et al., 2009). Since carlaviruses are positive-stranded RNA viruses and replicate in the cytoplasm, the role of the p12 protein nuclear localization and DNA binding in the virus–plant interactions has been enigmatic. We hypothesized that the p12 might act as a transcriptional activator localized to the plant nucleus (Lukhovitskaya et al., 2009).

The p12-like protein of another carlavirus, potato virus M, has a different function and acts in concert with the TGB1 (for Triple Gene Block) protein as a silencing suppressor to enhance viral RNA accumulation, cell-to-cell movement, and systemic transport (Senshu et al., 2011). On the other hand, we demonstrated explicitly 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 that there are some fundamental differences even among homologous proteins encoded by the viruses of the same genus. Indeed, in this article, characterization of the host responses to the p12 protein expression revealed additional unexpected levels of complexity during virus–host interactions. We demonstrated that the p12 protein acts as a eukaryotic TF to upregulate a bHLH-TF–encoding gene, which in turn stimulates ectopic cell proliferation and modulates tissue growth in the mesophyll of infected leaves.

RESULTS AND DISCUSSION

p12 Expression Causes Severe Leaf Malformation and Hyperplasia

Chrysanthemum (Chrysanthemum morifolium) plants infected with CVB often show curling of the upper leaves. Cross sections and light microscopy revealed pronounced cell proliferation in CVB-infected chrysanthemum leaves compared with the mock-inoculated plants (Figure 1A; see Supplemental Figure 2A online). This aberrant tissue structure is referred to hereafter as hyperplasia (i.e., disorganized cell division). Interestingly, Agrobacterium tumefaciens–launched CVB p12 expression resulted in hyperplasia (see Supplemental Figure 2B online), although it was not as pronounced as during the virus infection, probably owing to low levels of p12 protein accumulation upon agroinfiltration as was revealed by immunoblotting (see Supplemental Figure 2C online). Therefore, to achieve protein expression levels comparable to those during CVB infection and to investigate the host responses specifically to p12 in the absence of other carlaviral proteins, we expressed p12 from two unrelated virus vectors (to avoid vector-related bias), namely, the Tobacco mosaic virus (TMV) vector (TMV-p12 construct) and the Potato virus X (PVX) vector (PVX-p12; see Supplemental Figure 1B online). Regardless of the vector used, we observed strong phenotypes manifested as severe leaf malformation (Figure 1B; see Supplemental Figures 2D and 2E online) in the susceptible Nicotiana tabacum cv Samsun. Light microscopy revealed pronounced cell proliferation (increase in cell number) in the p12-expressing leaf tissue, with palisade and spongy mesophyll cells being strongly reduced in size but more abundant in number compared with those in tissue of the control (see Supplemental Figure 2F online). Thus, p12 converted typical mosaic symptoms of TMV and PVX into leaf malformation because of hyperplasia of the plant tissue.

Figure 1.
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 with CVB.

(E) Expression levels of upp were determined by quantitative RT-PCR. Values on the y axis represent normalized fold expression (n = 6); the error bars show 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 plant species abbreviations and other details, see Supplemental Table 1 online. Numbers represent GenBank accession numbers for the corresponding genes. 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 2−ΔΔCT values ± sd from three independent experiments are shown (n = 9); **P < 0.01 and ***P < 0.001. cDNA inputs were normalized to mRNA of three reference genes (Nt EF-1α, Nt β-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.]

Severe leaf malformation and hyperplasia were observed in Nicotiana occidentalis ssp hesperis plants, commonly known as native tobacco, infected with CVB (Figures 1C and 1D), demonstrating that our findings reflect actual events during carlavirus infection not only in chrysanthemum but also in one of the tobacco species, which is commonly used as indicator host for CVB. Additional phenotypes associated with CVB infection in native tobacco included hypersensitive response (HR) induction at later stages of virus infection (see Supplemental Figure 2G online) consistent with the previously reported role of p12 in HR induction (Lukhovitskaya et al., 2009).

The p12-induced hyperplasia might account for the leaf malformation symptoms observed in infections caused by some other carlaviruses (Hakkaart and Maat, 1974; Dijkstra et al., 1985; Larsen et al., 2009; Tang et al., 2010). Although leaf malformation is a quite common symptom induced by many carlaviruses, to the best of our knowledge, none of the host-carlavirus pathosystems other than CVB have been investigated for hyperplasia induction and this awaits characterization.

p12 Induces Expression of a bHLHTF

Since tobacco species represent a more genetically tractable model than chrysanthemum, and both react with hyperplasia to p12 expression, all subsequent experiments were performed in tobacco. The phenotypes caused by p12 resembled leaf malformation induced by Ca UPA20 TF after infiltration on tobacco leaves (Kay et al., 2007). Therefore, we wanted to analyze whether a tobacco ortholog of the pepper UPA20 is upregulated in the p12-expressing tissues. Since the gene sequence was not available, we performed tobacco EST database searches and identified an EST encoding a polypeptide highly similar to the UPA20 protein from pepper. Real-time RT-PCR experiments performed with primers designed based on the EST sequence revealed upregulation of the tobacco gene in both TMV-p12 and PVX-p12–infected plants (Figure 1E). Therefore, the upregulated tobacco gene was termed upp (for upregulated by p12).

We isolated the full-length cDNA of the upp genes from N. tabacum and N. occidentalis ssp hesperis by rapid amplification of cDNA ends (RACE) and determined the corresponding genomic sequence from clones generated by PCR and genome walking. It appeared that the tobacco genome encodes two upp paralogs, designated Nt upp-L and Nt upp-S (Figure 1F), whereas the genome of native tobacco encodes only the longer gene (Nh upp-L). Compared with the upp-L genes, Nt upp-S has a two nucleotide deletion in the beginning of the coding region that prevents initiation on the first AUG codon (Figure 1G) and results in a shorter protein. Nt upp-L and Nt upp-S genes both comprise eight exons and seven introns (the 6th intron is longer in Nt upp-L, 438 nucleotides versus 156 nucleotides in Nt upp-S; Figure 1F) and encode putative 362– and 260–amino acid bHLH-type TFs with predicted molecular masses of 42.2 and 29.0 kD, respectively. As expected, BLAST analyses demonstrated that Nt upp-L, Nh upp-L, and Nt upp-S are related to UPA20 from pepper. The only homolog of Nt upp-L in Arabidopsis thaliana is an uncharacterized bHLH137 TF (At5g50915; 54% identity and 65% similarity to Nt upp-L). The central part of the UPA/upp-L–related proteins is well conserved and comprises the bHLH motif, whereas the N termini of the proteins show very little similarity between the UPA/upp-L proteins and At bHLH137 (Figure 1G).

A phylogenetic analysis of the UPA/upp-L–related proteins showed that they formed four distinct clades in the phylodendrogram 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 Set 1 online). Notably, group 1 includes the bHLH proteins from Solanaceae species, group 3 comprises proteins from monocotyledons, and At bHLH137 falls into group 4 and is distantly related to group 1 (Figure 1H).

Real-time RT-PCR experiments employing sets of gene-specific primers, which are able to discriminate between Nt upp-L and Nt upp-S, revealed upregulation of both Nt upp-L and Nt upp-S by p12 (Figure 1I). Similarly, CVB infection activated the Nh upp-L gene, 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 the promoter-interacting TFs) or indirect via signal transduction pathways where p12 interacts with a cellular receptor and initiates a sequence of events resulting in the upregulation of the upp genes. To elucidate the mechanism of upp activation, we studied the inducibility of the upp promoters by p12. We isolated the Nt upp-L and Nt upp-S promoters by genome walking. Sequence comparisons of the Nt upp-L, Nt upp-S, and Ca UPA20 promoters revealed the presence of a previously identified conserved motif with the consensus sequence TATATAAACCN2-3CC that was termed the UPA box (Kay et al., 2007, 2009; see Supplemental Figure 3A online). A TATA-box is located within the UPA box 33 bp upstream of the transcription start site.

To analyze the p12 inducibility of the upp promoters, we used the upp-Lpro:GFP (for green fluorescent protein) and upp-Spro:GFP cassettes as reporters and actinpro:GFP as a constitutive promoter control in N. benthamiana after Agrobacterium-mediated transformation. GFP fluorescence and immunoblots revealed that both upp promoters are p12 responsive (Figure 2; see Supplemental Figures 3C to 3E online). Engineered promoter deletions from both ends narrowed the p12-responsive region to a 103-bp sequence 94% identical in both promoters (six variable positions per 103 bp) (Figures 2E to 2G). Notably, the 103-bp fragments for both promoters possessed the UPA box (see Supplemental Figure 3B online). Compared with upp/UPA, the At bHLH137 promoter also has an UPA box, though slightly deviating from the consensus (TATATAAACCN9CC), and could be activated by p12 (Figure 2G, right panel). Nevertheless, Agrobacterium-mediated At bHLH137 overexpression did not induce any visible tissue or cellular changes. Concordant with these observations, transient expression of Xanthomonas AvrBs3 TALe in Arabidopsis spp does not cause any cellular changes (Marois et al., 2002), further reinforcing the idea that the TFs of group 1 (Figure 1H) might differ from those of group 4 in their role in plant morphogenesis.

Figure 2.
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 constitutive actin2 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 leaf in [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 loading control.

(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 the leaf expressing an empty T-DNA. Statistical analysis was performed in three independent experiments by t test, P < 0.05. Error bars denote se of the measurements.

(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 promoter construct), L1U2 (103 bp upp-L promoter construct), S1U2 (103 bp upp-S promoter construct), and At bHLH137 promoter construct. Coomassie blue staining of the membrane is presented as a loading control.

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 supported our hypothesis of direct promoter activation by p12, as our previous study revealed that the p12 ZF domain is involved DNA binding and the NLS is required for p12 translocation into the nucleus (Lukhovitskaya et al., 2009).

p12 Associates with upp Promoters

We performed a chromatin immunoprecipitation (ChIP) assay with a hemagglutinin (HA) epitope-specific antibody to determine whether the HA epitope–tagged p12 associates with the upp promoters in vivo. Chromatin was isolated from plants infected with PVX or PVX-p12HA. Precipitated DNAs were extracted and amplified by PCR using primers flanking the 103-bp p12-responsive element. p12 precipitated with the upp promoters containing the UPA box, but not with a sequence located 2.5 kb downstream of the TATA-box (Figures 3A and 3B; see Supplemental Figure 5A online). This demonstrates that the interaction of p12 with DNA in vivo is specific. Cloning and sequencing of the 103-nucleotide fragments that precipitated with p12 revealed the presence of both the Nt upp-L and Nt upp-S promoter sequences.

Figure 3.
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 extracts from 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 2 indicated 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. Immunoblotting was conducted with samples collected before immunoprecipitation (input) and with immunoprecipitated material (ChIP) using antisera to the HA-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 incubated 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 fragment containing the UPA box (UPAbox) and p12 taken at the protein:DNA molar ratio 4:1 (the amount required for the complete binding of this particular DNA substrate) incubated with the increasing amounts of unlabeled competitor DNA, either UPAbox or UPAbox-rand (the same fragment 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 p12ZF fused to GAL4 DNA binding domain (BD) were transformed into the yeast strain Y2HGold and plated on SD/-Trp/X-α-Gal medium to test for activation of MEL1 encoding α-galactosidase, plated on SD/-Trp/Aureobasidin to test for activation of the AUR1-C reporter, or plated on SD/-Trp/-His to test for induction of the HIS3 reporter. The plates were incubated for 48 h at 28°C. Yeast transformed with a Gateway vector containing an ORF, which does not activate the reporters, fused to GAL4 BD was used as a negative control (BD-control).

(H) Detection of the BD-protein fusions in yeast extracts. Whole protein extracts were analyzed by immunoblotting using antisera to the c-Myc epitope tag. Asterisks indicate nonspecific binding of the antibodies to unknown yeast protein. Molecular weights of protein marker are shown on the left.

The ChIP data were substantiated by subcellular fractionation and anti-HA immunoblots. The p12HA protein was detected in the nuclear P10 fraction and in the precipitated chromatin (Figures 3C and 3D). Additionally, we found that p12 directly bound to the 32P-labeled upp promoter DNA in electrophoretic mobility shift assays (EMSAs) (Figure 3E). Competition experiments involving two DNA substrates, a DNA fragment of the Nt upp-L promoter comprising the UPA box and the same fragment with a randomized UPA box sequence, revealed that the unlabeled UPA box freed more of the labeled UPA box from the complex than the randomized UPA box sequence, suggesting higher affinity of p12 to the UPA box–containing DNA (Figure 3F). A control competition binding with p12 lacking the zinc-finger domain revealed no preference for the UPA box–containing DNA in the absence of the zinc finger (see Supplemental Figure 5B online). Collectively, these results demonstrate that p12 specifically 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 investigate whether p12 possesses trans-activating activity, bait transactivation tests were performed in a yeast heterologous transcription system. To this end, the yeast GAL4 DNA binding domain (BD) was fused either to full-length p12 or to p12 lacking the zinc finger (p12ZF) as a result of point mutations in the p12 gene (Lukhovitskaya et al., 2009). A yeast strain that has four integrated reporter genes under the control of three distinct unrelated Gal4-responsive promoters was employed. After transformation, yeast colonies were tested on three separate plates for activation of the reporters, which included MEL1 encoding α-galactosidase, AUR1-C conferring strong resistance to the highly toxic drug Aureobasidin, and HIS3 enabling yeast growth on media that lack His. Both p12 and p12ZF appeared to activate all three reporter genes tested (Figure 3G) compared with a negative control construct lacking autoactivation activity. Expression of the BD-p12 and BD-p12ZF fusion proteins in yeast cells was confirmed by immunoblotting (Figure 3H). These data demonstrated that p12 possesses trans-activating activity.

Overexpression of upp-L, but not upp-S, Results in Hyperplasia

To determine whether upregulation of Nt upp-L and/or Nt upp-S expression is involved in hyperplasia induction or, alternatively, just accompanies the changes in tissue morphology, Nt upp-L and Nt upp-S were individually expressed in tobacco and Nicotiana benthamiana leaves by Agrobacterium infiltration (Figure 4A; see Supplemental Figure 6A online). The RT-PCRs confirmed overexpression of Nt upp-L and Nt upp-S (Figure 4B; see Supplemental Figure 6B online) in leaf patches infiltrated with either upp-L or upp-S constructs, respectively, compared with the wild-type plants or to the patches infiltrated with empty T-DNA. In both plant species, the Agrobacterium-mediated Nt upp-L overexpression induced hyperplasia (Figure 4C; see Supplemental Figure 6C online) that was phenotypically similar to that in the tissues expressing p12 or observed during carlavirus infection. As before, palisade and spongy parenchyma cells were smaller but more abundant in number compared with the 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), demonstrating that the truncated TF is dysfunctional in this respect. It should be noted that overexpression of Nt upp-L did not result in the alteration of Nt upp-S expression and vice versa (Figure 4B), indicating that the observed Nt upp-L overexpression phenotypes (hyperplasia) were specific for Nt upp-L. Notably, Nt upp-L–mediated hyperplasia caused by the Nt upp-L TF in N. benthamiana was not as pronounced as in N. tabacum and petunia (Petunia hybrida) (see Supplemental Figures 3A, 3C, and 7 online), suggesting the species-specific effect of Nt upp-L.

Figure 4.
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 for EF1α 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 data are 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 independent experiments 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 turn upregulates E2FB and downregulates E2FC and CYCB1;1 expression, leading to stimulation of the mitotic cell cycle and inhibition of the endocycle. Genes with elevated mRNA accumulation are shown in red, genes with reduced transcript are in blue, while genes with unaltered expression are in black.

Overexpression of upp-L, but Not upp-S, Affects the Activity of 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 cell proliferation and often, when unregulated, induce hyperplasia, we analyzed whether abundance of the mRNAs of the genes involved in the CYCD/Rb/E2F pathway of the cell cycle and early leaf development is affected by Nt upp-L and Nt upp-S overexpression.

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 Supplemental Table 2 online). Additionally we performed tobacco EST database searches and identified ESTs encoding polypeptides highly similar to the Arabidopsis E2FC and an additional EST similar to At E2FB. Based on the available EST sequences, we isolated the full-length cDNA of Nt E2FC and Nt E2FB2 by RACE and determined the corresponding mRNA sequences (see Supplemental Table 2 online) .

At PHAVOLUTA is involved in the early stages of leaf development (Ichihashi et al., 2010). The mRNA levels of the tobacco ortholog of this gene were unchanged despite the cytological and morphological changes in the Nt upp-L–overexpressing tissue (see Supplemental Figures 6D and 6E online).

Nt ANTL is required for control of cell proliferation, expressed during formation of leaf primordia, and regulates the number of cells within the developing leaves (Mizukami and Fischer, 2000; Mudunkothge and Krizek, 2012). Using real-time RT-PCR, we found that the mRNA levels of Nt ANTL were reduced by Nt upp-L, but not by Nt upp-S overexpression (Figure 4D; see Supplemental Figures 6D and 6E online). The downregulation of the Nt ANTL gene is consistent with observed uncontrolled cell number during hyperplasia development (Figure 4C). On the other hand, At ANTL overexpression is associated with increased At CYCD3;1 levels and promotes additional rounds of cell division during leaf development, suggesting that ANTL acts upstream of CYCD3;1 (Mizukami and Fischer, 2000; Menges et al., 2006). Consistent with this idea and the observed downregulation of Nt ANTL, we found that the mRNA levels of Nt CYCD3;1 were unaffected by either Nt upp-L or Nt upp-S overexpression (see Supplemental Figures 6C and 6D online) despite pronounced hyperplasia and greatly increased number of cells within the Nt upp-L–overexpressing areas of a leaf. Similarly, no changes in Nt RBR and Nt E2FB1 levels were observed upon Nt upp-L overexpression (Figure 4D; see Supplemental Figures 6D and 6E online). On the other hand, Nt upp-L overexpression correlated with elevated levels of Nt E2FB2 mRNAs and reduced levels of Nt E2FC and Nt CYB1;1 transcripts (Figure 4D). In agreement with these observations, At E2FC knockdown RNA interference lines undergo hyperplasia and show reduced ploidy, but Arabidopsis plants that overexpress E2FC have increased DNA content and a higher ratio of polyploid nuclei (del Pozo et al., 2006). Furthermore, tobacco mesophyll cells with higher 4C DNA content also contain high levels of Nt CYCB1;1 transcripts (Qin et al., 1996). In Arabidopsis, E2FB promotes a mitotic cell cycle, while E2FC can facilitate an endocycle (del Pozo et al., 2006). In tobacco and Arabidopsis, CYCB1;1 participates in the regulation of the G2/M transition and elevated levels of CYCB1;1 expression correlate with naturally occurring G2-arrested cell populations in leaves with higher ploidy (Qin et al., 1996; Ascencio-Ibáñez et al., 2008).

Collectively, these data suggest that Nt upp-L overexpression might facilitate the mitotic cell cycle through Nt E2FB2 activation and inhibit DNA endoreduplication and the endocycle through downregulation of Nt E2FC and Nt CYCB1;1 expression. Therefore, next, we examined the ploidy distribution in fully expanded leaves agroinfiltrated with the Nt upp-L and T-DNA constructs. The leaves were at the same developmental stage and tobacco plants were grown under the same conditions as those used for the gene expression experiments. DNA flow cytometry analysis and CyView profiles revealed ∼1.5-fold to twofold reduction of the amount of 4C nuclei upon Nt upp-L overexpression compared with the empty plasmid control infiltrated onto the other half of the same leaf (Figure 4E). Notably, the most pronounced effect on the ploidy distribution was observed at 3 d after inoculation (DAI) and coincided with Nt E2FB2 upregulation and Nt E2FC and Nt CYCB1;1 downregulation (Figures 2E and 4D). The asymmetric expression pattern observed for core cell cycle genes suggest that, indeed, Nt upp-L expression inhibits the capacity of the leaf cells to undergo endocycling and facilitates a mitotic cell cycle (Figure 4F).

The observed changes in the Nt E2FB and Nt E2FC levels of expression indicate that overexpression of Nt upp-L causes cell division 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 the cellular level, ectopic Nt upp-L overexpression pushed palisade and spongy mesophyll cells from the G1 phase to the mitotic cycle, promoting cell divisions in fully developed leaves in which cell division is normally limited.

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

Knockdown of upp-L Expression Results in Suppression of Hyperplasia Development

To further confirm the role of Nt upp-L in the induction of hyperplasia, we performed virus-induced gene silencing (VIGS) in tobacco using a tobacco rattle virus vector carrying part of the Nt upp-L gene. An empty vector construct was used as a control. The tobacco plants inoculated with the empty VIGS vector and the plants silenced for the Nt upp-L gene were challenged with TMV-p12 (Figure 5A). VIGS of Nt upp-L reduced the Nt upp-L transcript levels (Figure 5B) and the p12-dependent leaf malformation and hyperplasia compared with control inoculation with the empty VIGS vector plus TMV-p12 (Figures 5A and 5C). Similarly, silencing of Nt upp-L in the absence of p12 expression had no effect on the plant tissue architecture with typical palisade and spongy mesophyll cells layers (Figure 5C), reinforcing the idea that Nt upp-L is normally expressed in the SAM and might act in pathways other than those that regulate leaf development. Indeed, tobacco plants silenced for the Nt upp-L gene were characterized by shorter stems and shorter internodes (Figures 5D and 5F). The shorter stem phenotype was especially pronounced at earlier stages of plant development (Figure 5F). Concordant with these observations, N. occidentalis ssp hesperis plants infected with CVB developed much longer stems; this phenotype was especially prominent at 14 to 21 DAI and earlier stages of plant development (Figures 5E and 5G). These lines of evidence suggest that the Nt upp-L TF might be directly or indirectly involved in early stem elongation. The upp-L–silenced plants were also delayed in setting flowers and seeds (see Supplemental Figure 8 online), and four out of 18 plants in two independent experiments did not undergo vegetative to floral transition.

Figure 5.
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) followed by inoculation with TMV-p12 1 week later.

(B) RT-PCR analysis of the Nt upp-L mRNA accumulation in cDNA of the silenced and control plants. The constitutively expressed gene EF1α served as a normalization control.

(C) Cross sections and light microscopy of the leaves of nonsilenced and Nt upp-L-silenced plants. Identity of the inoculated constructs is shown in each image. Bars = 100 µm.

(D) Images of tobacco plants silenced for Nt upp-L (TRV:upp-L) versus a control (TRV:00).

(E) Images of N. occidentalis ssp hesperis plants infected with CVB versus mock-inoculated plants.

(F) Growth response of tobacco plants to Nt upp-L silencing. The error bars denote se; n = 18.

(G) Growth response of N. occidentalis ssp hesperis plants to CVB infection. The error bars show se; n = 9.

[See online article for color version of this figure.]

Collectively, these results suggest that the Nt upp-L TF is a regulator that stimulates cell proliferation when ectopically expressed in leaves. Our results establish that the visible consequence of p12 virulence activity, the induction of hyperplasia and leaf malformation, is elicited by p12 ectopically activating upp-L, which is normally expressed in the SAM.

Conclusions

Our data establish p12 as a virus-encoded TF that is translocated to the nucleus and activates expression of the regulator of cell proliferation upp-L, which results in severe leaf malformation. Similar to numerous plant and animal TFs, which localize to the nucleus and directly bind to DNA via a zinc-finger domain (Klug 2010), the function of the p12 protein as a transcriptional activator requires the Arg-rich NLS and Cys residues within the C-4 type zinc finger shared with cysteine-rich proteins (CRPs) of other carlaviruses. This establishes CVB p12 as a TF encoded by a positive-stranded RNA virus. This finding is rather surprising since the life cycle of viruses, such as CVB, takes place in the cytoplasm and has been previously assumed to be unrelated to nuclear functions.

The most obvious parallels in terms of hyperplasia induction can be drawn between CVB and geminiviruses. Geminiviruses have small, single-stranded DNA genomes encoding a limited number of genes and rely entirely on host machinery for replication in the nucleus, whereas CVB and other carlaviruses have a genome comprising a single segment of single-stranded, positive-sense RNA, encode their own RNA polymerase and replicate in the cytoplasm. Nevertheless, both CVB and geminiviruses appear to interfere with cell cycle regulation and induce hyperplasia. Whereas CVB causes mostly proliferation of the leaf mesophyll tissue, the majority of geminiviruses are phloem limited and induce cell division in the vascular tissue, which results in enations. For example, cabbage leaf curl virus infection in Arabidopsis alters expression of cell cycle genes, favorably inhibiting genes typically expressed during G1-to-M phase progression (e.g., downregulation of CYCD3 and the division-promoting E2FB) and activating those normally expressed during S and G2 phases of the cell cycle (e.g., upregulation of E2FA/DPA and the division-inhibiting E2FC; Ascencio-Ibáñez et al., 2008). Conversely, in tobacco, the upp-L TF, ectopically induced by p12 in leaves, appeared to upregulate the division-promoting E2FB and downregulate the division-inhibiting E2FC. These differences suggest that while at least some geminiviruses promote the endocycle and S phase of the cell cycle, which is beneficial for the viral DNA replication, CVB p12 seems to facilitate a mitotic cell cycle through induction of upp-L, which is normally expressed in the SAM. Moreover, it has been shown that overexpression of E2FB, which promotes the mitotic cell cycle, strongly compromises cabbage leaf curl virus accumulation (Ascencio-Ibáñez et al., 2008). Therefore, it seems that CVB and geminiviruses induce hyperplasia through different pathways; while geminiviruses have been suggested to do this through disturbing phytohormonal signaling (Piroux et al., 2007; Ascencio-Ibáñez et al., 2008; Mills-Lujan and Deom, 2010), CVB does it by encoding a TF, which in turn ectopically activates expression of a SAM-specific TF.

Currently, the exact role of the p12 protein (as well as the C4 proteins of geminiviruses) in virus infection is not known, although it might be required for efficient replication and accumulation in natural hosts and/or efficient transmission of the virus by its insect vector through, for example, induction of stem elongation as we observed in this study. The infected plants with longer stems might be more accessible to aphids compared with noninfected plants with shorter stems. It also is tempting to speculate that p12, and p12-like proteins of other carlaviruses, may be considered as a latency factor (a latent protein) of this group of viruses and the viral-programmed induction of hyperplasia might be a way to delay the onset of HR (a type of programmed cell death) and to maintain the latent state of the virus infection (e.g., cell division without virus replication), as it has recently been shown that overexpression of Nt E2FB1 reduces programmed cell death induction in tobacco Bright Yellow-2 cells (Smetana et al., 2012). Moreover, it has been well documented that in animal/human viruses, some latent stages induce tumorogenesis (Poreba et al., 2011).

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

This article demonstrates that interaction of plant RNA viruses with their hosts are considerably more complex than previously thought and can involve virus manipulation of host transcription directly by use of virally encoded TFs, such as p12. As an antiviral protection strategy, host defense genes could have evolved to contain the viral TF–responsive elements in their promoter regions to be upregulated in the presence of the viral TF, as we previously found for a set of p12-induced defense-related genes (Lukhovitskaya et al., 2009). Unraveling the mechanism of the p12-dependent HR induction in another tobacco cultivar (Lukhovitskaya et al., 2009), which may be similar to the transcriptional activation of a resistance gene by the AvrBs3 TALe in pepper (Römer et al., 2007), might shed further light on this process.

METHODS

Plant Material and Growth Conditions

Chrysanthemum (Chrysanthemum morifolium), tobacco (Nicotiana tabacum cv Samsun), native tobacco (Nicotiana occidentalis ssp hesperis), Nicotiana benthamiana, Arabidopsis thaliana, and petunia (Petunia hybrida) plants were grown under standard conditions (16 h light and 60% humidity).

Phylogenetic and Sequence Analysis

The protein sequences encoded by tobacco and native tobacco were analyzed with published sequence from pepper (Capsicum annum) and Arabidopsis and several homologs from 17 other plant species (see Supplemental Table 1 online). The sequences were aligned using ClustalW algorithm of the Megalign module of the Lasergene software package (DNASTAR), and the trees were prepared using Phylip package. Bootstrap values were calculated from 1000 replicates of the tree. GenBank accession numbers are indicated on the phylodendrograms. The sequences for some Solanaceae species were retrieved 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 5′- and 3′-RACE with the SMART RACE cDNA amplification kit (Clontech) and the Advantage 2 PCR kit (Clontech). Amplified PCR products were cloned into pJet1.2 (MBI Fermentas) and sequenced (by Macrogen). The genomic sequences of the upp genes and promoters were isolated from tobacco Samsun 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 combination of PCRs, site-directed mutagenesis, and swapping of restriction fragments. PVX-p12 construct was described previously (Lukhovitskaya et al., 2005). To construct TMV-p12, the p12 open reading frame (ORF) was amplified with primers P12Age-FW and P12Xho-REV (see Supplemental Table 3 online), introducing AgeI and XhoI restriction sites. The resulting PCR product was cloned into the TMV30B vector (Shivprasad et al., 1999) as an AgeI-XhoI fragment.

The plasmid pLH7000 (Hausmann and Töpfer, 1999) was used to generate all the binary constructs described in this study. CVB p12, Ca UPA20, Nt upp-L, and Nt upp-S were expressed in plants under control of the constitutive cauliflower mosaic 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 combinations (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 similarly 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 generated with appropriate primers (see Supplemental Table 3 online) into EcoRI-XhoI–digested pro35S:GFP (Lukhovitskaya et al., 2009) to replace the 35S promoter. proACT:GFP and proCaUPA20:GFP were engineered by ligating MunI-digested PCR products generated with ACT2-prom-Mun-P and 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 promoter. The correct orientation of the inserts was determined by restriction analysis and by sequencing. L0U0, L2U3, S0U0, and S2U3 were engineered by ligating EcoRI-NcoI–digested PCR product generated with appropriate primers (see Supplemental Table 3 online) into EcoRI-NcoI–digested pro35S:GFP (Lukhovitskaya et al., 2005) to replace the 35S promoter.

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

The binary constructs were used to transform Agrobacterium tumefaciens strain C58C1 pGV2260. Agrobacterium transformed with empty pLH7000 plasmid was used as a control in the promoter induction and Ca Upa20, bHLH137, upp-L, and upp-S overexpression experiments.

Yeast Manipulations

For expressing p12 and p12ZF constructs in yeast, the p12 and p12ZF ORFs were cloned to the destination vector pBD-Gate2 (Maier et al., 2008) using Gateway technology (Invitrogen). All the constructs were verified by sequencing. The Y2HGold yeast strain (Clontech) was transformed with the plasmids using small-scale LiAc yeast transformation procedure as described in Clontech’s Yeast Protocols Handbook. Transformants were selected on −Trp plates. To test for HIS3 reporter self-activation, colonies were incubated in 50 μL sterile distilled water for 36 h to deplete yeast from the internalized His. Prior to immunoblotting, yeast whole protein was 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 prepared from leaf samples (20 mg, fresh weight). Leaf discs were ground and homogenized in an ice-cold mortar in 20 μL 250 mM Tris-HCl, pH 7.8, mixed with 20 μL extraction buffer (75 mM Tris-HCl, pH 6.8, 9 M urea, 4.3% SDS, and 7.5% β-mercaptoethanol), heated at 100°C for 5 min, and centrifuged (5 min at 10,000g) to remove insoluble material. Aliquots of the supernatant (1 to 10 µL) were separated by SDS-PAGE on 20% gels. After electrophoresis, proteins were transferred to a Hybond-C membrane (GE Healthcare Bio-Sciences) and subjected to immunoblot analysis with GFP antibody (Abcam), HA IgG (Santa Cruz Biotechnology), and anti-acetil-Histone H3 IgG, and the ECL protein gel blotting kit as recommended by the supplier (GE Healthcare Bio-Sciences).

Agrobacterium-Mediated Transient Expression

Agrobacterium was grown overnight at 28°C in the presence of streptomycin, spectinomycin, carbenicillin, and rifampicin in Luria-Bertani broth supplemented with MES (10 mM final concentration) and acetosyringone (20 μM final concentration). Upon reaching OD600 ∼0.5, cells were pelleted by centrifugation at 3500g for 5 min, resuspended in infiltration buffer (10 mM MES, 10 mM MgCl2, and 150 μM acetosyringone), and incubated for 2 h at room temperature with constant rotation. Six- to eight-week-old N. benthamiana plants were infiltrated using a needleless syringe. For promoter induction studies, two Agrobacterium strains delivering a promoter construct and pro35S:p12 (or an empty pLH7000 plasmid) were adjusted to OD600 = 0.6 each.

Microscopy

Sample preparation and light microscopy were performed as described previously (Savenkov and Valkonen, 2001). Fluorescence microscopy was 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 of total RNA samples were used for oligo(dT) primed cDNA synthesis with the RevertAid H Minus First-Strand cDNA synthesis kit (MBI Fermentas). Obtained cDNAs were used as templates for PCR with DreamTaq DNA Polymerase (MBI Fermentas). Primers amplifying the tobacco elongation factor 1α (EF-1α) were used as control for equal cDNA amounts (see Supplemental Table 3 online). Suitable PCR cycle numbers specific to each primer pair were determined by testing a range of cycle numbers. A regime with exponential DNA amplification was chosen for each primer combination (see Supplemental Table 3 online). For real-time RT-PCR, 1 μg of total RNA preparations was reverse transcribed into cDNAs using qScript (Quanta). The cDNA was quantified with the DyNAmo Flash SYBR Green qPCR kit (Finnzymes) and gene-specific primers (see Supplemental Table 3 online) using a Bio-Rad IQ5 icycler apparatus according to the manufacturer’s recommendations. PCR was run in 96-well optical reaction plates (Bio-Rad). The PCR conditions were as follows: 95°C for 10 min 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 kinetics analysis was performed to check the specificity of annealing. mRNA quantifications were performed with specific primer combinations and were normalized with three reference genes (Nt EF-1α, Nt β-actin, and Nt cdc2; see Supplemental Table 3 online). Four (or three) biological replicates were used, and three technical replicates were performed for each biological replicate. Three PCR reactions were performed for each sample in three independent experiments.

DNA Flow Cytometry Analyses

After the main rib was removed, infiltrated tobacco leaf halves were chopped with a razor blade in CyStain UV Precise P nuclei extraction buffer (Partec). The extracted nuclei were filtered through CellTrics 30 µm disposable filter units (Partec), stained with CyStain UV Precise P staining buffer (Partec), and analyzed with a CyFlow Ploidy analyzer (Partec) and CyView 2 software according to manufacturer’s recommendations. A total of 7000 nuclei were analyzed per sample.

GFP Imaging and Fluorometric Analysis

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

VIGS and Systemic Viral Infection

Construction of the TRV:upp-L silencing plasmid is described above. The 600-bp upp-L fragment was cloned in an antisense orientation into the pTRV:GFP vector by swapping GFP. The resulting construct TRV:upp-L was transformed into Agrobacterium and in combination with TRV-RNA1 used for induction of the Nt upp-L gene silencing. Seven days after TRV infiltration, tobacco leaves were challenged with TMV-p12. The uppermost leaves were assayed for TMV-p12 infection and Nt upp-L silencing by RT-PCR.

Subcellular Fractionation of Plant Extracts

Leaf discs (100 mg) were ground in liquid nitrogen to fine powder, which was resuspended in 2 mL buffer A (400 mM Suc, 100 mM Tris/HCl, 10 mM KCl, 5 mM MgCl2, 10 mM β-mercaptoethanol, and 1 mM PMSF, pH 7.5). The slurry was filtered through one layer of Miracloth, and the flow-through was split into two equal parts. One part was centrifuged at 30,000g for 30 min to yield a pellet consisting of MEM fraction enriched in organelles and miscellaneous membranes and S30 (soluble protein fraction). Another part of the extract was centrifuged for 10 min at 1000g to yield a pellet fraction (P10) enriched in nuclei. P10 and S30 fractions were resuspended in Laemmli sample buffer containing 9 M urea and used for immunoblotting.

ChIP

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

EMSA

For expression of a C-terminal hexahistidine (His6)-tagged p12 in Escherichia coli, 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 purification of p12His6 and p12ZFHis6 was performed as described (Lukhovitskaya et al., 2009). In EMSA, PCR product corresponding to a UPA box–containing region of the Nt upp-L promoter of 103 bp was incubated with increasing amounts of p12His6 in binding buffer (10 mM Tris/HCl, pH 8.0, and 50 mM NaCl) containing 3 mM ZnSO4 on ice for 30 min. Samples were analyzed in a 1.5% agarose gel containing ethidium bromide. In competition experiments, [α-32P]-labeled UPA box–containing PCR product corresponding to 40 bp in the Ntupp-L promoter (UPAbox) was first incubated with either p12His6 or p12ZFHis6 at protein:DNA molar ratio of 4:1 in the binding buffer on ice for 30 min. The resulting complexes were incubated with increasing amounts of either unlabeled UPAbox or UPAbox-rand (same DNA fragment as 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 data libraries under the following accession numbers: HE653922, Nt E2FC mRNA; HE653923, Nt E2FB2 mRNA; HE653924, Nt upp-L mRNA; HE653925, Nt upp-S mRNA; HE6539226, Nh upp-L mRNA; HE653927, Nt upp-S gene and promoter; and HE653928, Nt upp-L gene and promoter.

Supplemental Data

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

Acknowledgments

We thank Richard H. Maier (University of Salzburg, Austria) for the pBD-Gate2 vector, Panagiotis N. Moschou (Swedish University of Agricultural Sciences, Sweden) for a negative control for the autoactivation test and helpful discussions, Lesley Torrance (The James Hutton Institute, Dundee, UK) for critical reading of the article, and David Baulcombe (University of Cambridge, Cambridge, UK) for providing the cDNA clone of PVX. Financial support from the Swedish Research Council Formas (Grants 2007-256, 2008-1047, and 2009-1979 to E.I.S.), the Carl Tryggers Foundation (E.I.S. and N.I.L.) and the Swedish Institute (postdoctoral fellowship 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. performed research. 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.

Footnotes

  • The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Eugene I. Savenkov (eugene.savenkov{at}slu.se).

  • www.plantcell.org/cgi/doi/10.1105/tpc.112.106476

  • ↵[C] Some figures in this article are displayed in color online but in black and white in the print edition.

  • ↵[W] Online version contains Web-only data.

Glossary

TALe
transcription activator-like effector
TF
transcription factor
bHLH
basic/helix-loop-helix
CVB
chrysanthemum virus B
NLS
nuclear localization signal
TMV
Tobacco mosaic virus
PVX
Potato virus X
HR
hypersensitive response
RACE
rapid amplification of cDNA ends
GFP
green fluorescent protein
ChIP
chromatin immunoprecipitation
HA
hemagglutinin
EMSA
electrophoretic mobility shift assay
DAI
days after inoculation
SAM
shoot apical meristem
VIGS
virus-induced gene silencing
ORF
open reading frame
  • Received October 19, 2012.
  • Revised January 29, 2013.
  • Accepted February 19, 2013.
  • Published March 12, 2013.

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An RNA Virus-Encoded Zinc-Finger Protein Acts as a Plant Transcription Factor and Induces a Regulator of Cell Size and Proliferation in Two Tobacco Species
Nina I. Lukhovitskaya, Anna D. Solovieva, Santosh K. Boddeti, Srinivas Thaduri, Andrey G. Solovyev, Eugene I. Savenkov
The Plant Cell Mar 2013, 25 (3) 960-973; DOI: 10.1105/tpc.112.106476
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