Percentage distribution of up- and down-regulated genes in <i>VFP3</i> RNAi-1 plants as compared to the wild-type Col-0 plants.

<p>Annotation is based on MapMan categories. Categories with gene number less than 10 are not shown. Gray bars indicate up-regulated gene categories, and blue bars indicate down-regulated gene categories.</p

VFP3 interacts with VirF in the cell nucleus.

<p>(A-C) BiFC assay for the VFP3-VirF interaction in planta. Constructs encoding nYFP-VFP3 and cYFP-VirF were coexpressed in microbombarded <i>N</i>. <i>benthamiana</i> leaves. (A) Plastid autofluorescence. (B) YFP signal. (C) Merged plastid autofluorescence and YFP signals. (D-F) BiFC assay for the VFP3-VBF interaction in planta. Constructs encoding nYFP-VFP3 and cYFP-VBF were coexpressed in microbombarded <i>N</i>. <i>benthamiana</i> leaves. (D) Plastid autofluorescence. (E) YFP signal. (F) Merged plastid autofluorescence and YFP signals. (G-I) Subcellular localization of CFP-tagged VFP3 coexpressed with free DsRed2 in agroinfiltrated <i>N</i>. <i>benthamiana</i> leaves. (G) DsRed2 signal. (H) CFP signal. (I) Merged plastid autofluorescence, DsRed2 and CFP signals. Location of the cell nucleus is indicated by a white arrowhead. All images are projections of single confocal sections. Scale bars, 20 μm.</p

Amino acid sequence analysis of VFP3.

<p>(A) Sequence alignment of VFP3 and its homologs from Arabidopsis. The deduced amino acid sequence of VFP3 (At3g11100) was aligned with the sequences of proteins encoded by At5g05550 (VFP5) and At3g58630 of Arabidopsis and of tobacco NtSIP1 (GenBank accession number BAB83610.1) using ClustalX (ver. 2.1) (<a href="http://www.clustal.org/clustal2/" target="_blank">http://www.clustal.org/clustal2/</a>). Three α-helices of the trihelix domain, delineated with an open box, and the fourth C-terminal α-helical region were predicted using the Garnier-Robson-Osguthorpe (GOR) algorithm [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142128#pone.0142128.ref026" target="_blank">26</a>]. The MADF domain, predicted by InterPro (<a href="http://www.ebi.ac.uk/interpro" target="_blank">http://www.ebi.ac.uk/interpro</a>), is delineated with a gray box. Asterisks indicate the putative monopartite NLS predicted by cNLS Mapper (nls-mapper.iab.keio.ac.jp). Identical residues in the aligned sequences are highlighted in white letters on black/dark gray background and similar residues are shaded in gray. (B) Ribbon diagram of the trihelix domain VFP3 showing the three predicted helical structures was constructed using the Hhpred (<a href="http://toolkit.tuebingen.mpg.de/" target="_blank">http://toolkit.tuebingen.mpg.de/</a>) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142128#pone.0142128.ref059" target="_blank">59</a>] and UCSF Chimera tools [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142128#pone.0142128.ref060" target="_blank">60</a>]. (C) Phylogenetic tree of the members of the SIP1 clade of Arabidopsis trihelix transcription factors and tobacco NtSIP1. VFP3 (At3g11100) and its close homolog VFP5 (At5g05550) are highlighted by a shaded box and white letters. Known gene names are indicated in parenthesis next to their locus names. The evolutionary history was inferred using the Neighbor-Joining method [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142128#pone.0142128.ref061" target="_blank">61</a>]. The optimal tree with the sum of branch length of 6.60887794 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142128#pone.0142128.ref062" target="_blank">62</a>]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142128#pone.0142128.ref063" target="_blank">63</a>] and are in the units of the number of amino acid substitutions per site. All positions containing gaps and missing data were eliminated. There were a total of 199 positions in the final dataset. Evolutionary analyses were conducted using the Molecular Evolutionary Genetics Analysis tool (MEGA, version 6.0.5 for Mac OS) (<a href="http://www.megasoftware.net" target="_blank">http://www.megasoftware.net</a>) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142128#pone.0142128.ref064" target="_blank">64</a>], which also generated this description of the analysis. Bar, 0.2 amino acid substitutions per site.</p

Suppression of <i>VFP3</i> gene expression in <i>VFP3</i> RNAi-1 plants has no detectable effect on their genetic transformation by Agrobacterium.

<p>Root explants were infected with Agrobacterium cultures at the indicated optical densities. (A) Numbers of tumors and roots scored for each plant. (B) Tumorigenicity expressed as percent of roots showing tumors. Black bars, wild-type plants; gray bars, <i>VFP3</i> RNAi-1 plants. Standard deviations are indicated.</p

Reduction of <i>VFP3</i> gene expression in <i>VFP3</i> RNAi-1 plants.

<p>(A) Semi-quantitative RT-PCR analysis of the <i>VFP3</i> transcript levels in leaves of the wild-type Col-0 and <i>VFP3</i> RNAi-1 plants. <i>ACTIN2</i> was used as internal reference. (B) Quantification of <i>VFP3</i> transcript levels described in (A) normalized to the levels of the <i>ACTIN2</i> reference. (C) Semi-quantitative RT-PCR analysis of the <i>VFP3</i> transcript in roots of the wild-type Col-0 and <i>VFP3</i> RNAi-1 plants. <i>ACTIN2</i> was used as internal reference. (D) Quantification of <i>VFP3</i> transcript levels described in (C) normalized to the levels of the <i>ACTIN2</i> reference. The data represent average values of three independent experiments with indicated standard deviations.</p

Summary of functional categories of 507 up-regulated and 611 down-regulated DEGs in VFP3 RNAi plants.

<p>Numbers represent -log10(q-value).</p

Reduction of <i>VFP5</i> gene expression in <i>VFP3</i> RNAi-1 plants.

<p>(A) Semi-quantitative RT-PCR analysis of the <i>VFP5</i> transcript in leaves of the wild-type Col-0 and <i>VFP3</i> RNAi-1 plants. <i>ACTIN2</i> was used as internal reference. (B) Quantification of <i>VFP5</i> transcript levels described in (A) normalized to the levels of the <i>ACTIN2</i> reference. The data represent average values of three independent experiments with indicated standard deviations.</p

Metabolism overview of genes differentially expressed in <i>VFP3</i> RNAi-1 plants.

<p>The analysis employed the MapMan software. Values are log2 fold changes between the analyzed plants. Blue indicates up-regulation in gene expression, and red indicates down-regulation in gene expression.</p

VirF does not destabilize VFP3 or VFP5 in a cell-free degradation assay.

<p>(A) Western blot analysis of CFP-VFP3 following coexpression with Myc-VirF. (B) Quantification of CFP-VFP3 accumulation described in (A). (C) Western blot analysis of CFP-VFP5 following coexpression with Myc-VirF. (D) Quantification of CFP-VFP5 accumulation described in (C). (E) Western blot analysis of CFP-VFP3 following coexpression with Myc-VirF and free VirD5. (F) Quantification of CFP-VFP3 accumulation described in (E). The tested proteins were coexpressed in <i>N</i>. <i>benthamiana</i> leaves, cell extracts were prepared and incubated for the indicated periods of time. CFP-VFP3 and CFP-VFP5 were detected by anti-CFP antibody, and RuBisCo was detected by Coomassie blue staining. The putative RuBisCo large chain was used as loading control and as reference for normalization of relative protein amounts. Each experiment was performed at least twice with similar results.</p

Specific interaction between VFP3 and VirFdel1 in the yeast two-hybrid system.

<p>(A) Characterization of the VirF-based bait, mutVirFdel1. Yeast cells were grown in the absence of leucine, tryptophan, and histidine. (B) VirFdel1-VFP3 interaction. The indicated dilutions of yeast cells were grown in the absence of histidine, tryptophan, and leucine (top) or in the absence of tryptophan and leucine (bottom). Lane 1, LexA-mutVirFdel1 + Gal4AD-VFP3; lane 2, LexA-mutVirFdel1 + Gal4AD-TMV MP; lane 3, LexA-VirFdel1 + Gal4AD-VFP3; lane 4, LexA-VirFdel1 + Gal4AD-TMV MP; lane 5, LexA-AtCUL1 + Gal4AD-VFP3; Lane 6, LexA-AtCUL1 + Gal4AD-TMV MP. Growth in histidine-deficient medium represents selective conditions for protein-protein interaction.</p