mRNA granules localize to P-bodies (related to Fig 9).

<p>Asterisks represent statistically significant changes in comparison to nonstress conditions (Wilcoxon test; <i>p</i> < 0.05).</p><p>mRNA accumulations overlap with P-bodies under nonstress (-NaCl) and salt stress (+NaCl) conditions. Data denote the average (in %) of 65 cells (RD29B) or 120 cells (TZF3).</p

The SPI protein interacts with DCP1.

<p>(A) Schematic presentation of the domain organization of the SPI protein: the ARMADILLO repeats (ARM), the Concanavalin A-like lectin domain (ConA), and the C-terminal PBW module (SPI-PBW). (B) Yeast two-hybrid interactions. Upper part: double transformed yeast cells on selective dropout medium lacking leucine (-L) and tryptophan (-W). Bottom part: interaction between SPI-PBW, N-terminally fused to the GAL4 Binding Domain (BD), and DCP1 and other P-body core components fused to the GAL4 Activation Domain (AD), on selective dropout medium lacking leucine (-L), tryptophan (-W), and histidine (-H), supplemented with 5 mM 3-Aminotrizole (3AT). The Green Fluorescent Protein (GFP), N-terminally fused to the GAL4-AD, has been included as negative control. (C) Coprecipitation of SPI-PBW-His₆ with DCP1-MBP. Throughputs (TP), wash fractions (WF), and resin-bound MBP fusions (B) were detected by α-MBP (upper part) and α-His₆ (lower part) antibody staining. GST-SPI-PBW-His₆ (~110 kDa, arrowhead) coprecipitated with MBP-DCP1 (~ 83 kDa), but not with MBP (~42 kDa). Samples detected on different blots are separated by lines. (D) FRET_E (in %) was measured in whole leaf epidermis cells (whole cells) and stationary P-bodies (PBs). YFP was bleached in whole cells (for details see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002188#sec016" target="_blank">Materials and Methods</a>). Mean FRET_E’s for 35S_pro:YFP-gSPI and 35S_pro:DCP1-CFP (n = 11 cells) or 35S_pro:YFP and 35S_pro:DCP1-CFP (n = 10 cells) are shown. Error bars represent standard deviations for whole cells, and the standard deviation of the mean for PBs (<i>n</i> = 31 stationary PBs derived from whole cell samples). Two-tailed student’s <i>t</i> test was performed to compare FRET_E between 35S_pro:YFP-gSPI/35S_pro:DCP1-CFP and 35S_pro:YFP/35S_pro:DCP1-CFP for each group (*** <i>p</i> < 0.001). (E) Representative images of 35S_pro:DCP1-CFP in a transiently transfected leaf epidermis cell prior to (left) and after (middle) Acceptor-photobleaching (AP). For a better visualization, the increase of fluorescence intensity of DCP1-CFP after AP is presented in pseudocolors (right), see color scale for comparison. A group of stationary PBs is highlighted by the boxed area and magnified (lower row). Yellow arrowheads in magnifications mark stationary PBs used for FRET quantifications. Scale bars: 30 μm.</p

mRNAs of RD29B and TZF3 form cytoplasmic granules.

<p>16BoxB-gRD29B (A) and 16BoxB-gTZF3 (B) are indirectly visualized by the LambdaN22-VENUS reporter in transiently transfected leaf epidermis cells (related to Tables <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002188#pbio.1002188.t003" target="_blank">3</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002188#pbio.1002188.t004" target="_blank">4</a>). Column I presents the LambdaN22-mVENUS reporter, column II DCP1-mCHERRY, column III the overlay of column I (green) and II (magenta). Rectangular image magnifications present P-bodies nonoverlapping (white arrowheads) and P-bodies overlapping (yellow arrowheads) RNA accumulations. Scale bar: 25 μm.</p

Blocking translation leads to a reduction of P-body number.

<p>Changes of P-body number were analyzed in whole leaf areas of transgenic plants expressing DCP1-YFP. (A) Whole leaf areas are presented before (0 min) and after continuous CHX treatments (45 min and 90 min). Scale bar: 50 μm. (B) Reduction of P-body number (in %) relative to untreated samples (time point 0) determined 45 min and 90 min after CHX treatment. Data denote the average from seven biological replicates. SD values represent standard deviations. No statistical differences were found between wild-type and <i>spi</i> mutants (Two-tailed student’s <i>t</i> test).</p

SPI and DCP1 interact at P-bodies in BiFC assays.

<p>Fusion proteins are expressed under the control of the 35S promoter. (A) Interaction of YFP_N-SPI and YFP_C-DCP1 (left) in transiently transformed <i>N</i>. <i>benthamiana</i> leaves 72 h post-transfection at DCP2-mCHERRY labeled P-bodies (middle). Right pictures show the overlay of the left (green) and middle (magenta) pictures. (B) Interaction of YFP_N-SPI and YFP_C-DCP1 in cytoplasmic dots (left) in <i>N</i>. <i>benthamiana</i> leaves coexpressing free RFP as transformation control (middle). Right pictures show the corresponding transmission picture. (C–F) Representative images of BiFC negative controls. No YFP signal in leaf epidermis cells coexpressing YFP_N-SPI-PBW and YFP_C-AtMYC1 in combination with DCP2-mCHERRY (C) and free RFP (D). No YFP signal in leaf epidermis cells coexpressing YFP_C-DCP1 and VPS25-YFP_N in combination with DCP2-mCHERRY (E) and free RFP (F). Scale bar: 50 μm.</p

Interspecific interactions between SPI-PBW and DCP1 homologs from mammals and yeast.

<p>(A) Yeast two-hybrid interactions. Top part: double transformed yeast cells on selective dropout medium lacking leucine (-L) and tryptophan (-W). Bottom part: interactions between the SPI-PBW N-terminally fused to the GAL4 Binding Domain (BD) and the human DCP1 isoforms (DCP1a and b) and yeast DCP1p, N-terminal fused to the GAL4 Activation Domain (AD) on selective dropout medium lacking leucine (-L), tryptophan (-W) and histidine (-H), supplemented with 3 mM 3-Aminotrizole (3AT). GFP N-terminal fused to the GAL4-AD has been included as negative control. (B) Coprecipitations of bacterially expressed proteins. GST-SPI-PBW-His₆ (arrowhead, ~110 kDa) coprecipitated with MBP-DCP1a (~90 kDa), MBP-DCP1b (~92kDa) and MBP-DCP1p (~60 kDa), but not with MBP (~42 kDa) as negative control. Throughputs (TP), last wash fractions (WF), and resin bound fractions (B) are visualized by α-MBP (upper row) and α-His₆ antibody staining (lower row). Samples detected on different blots are separated by lines.</p

P-body number in leaf epidermis cells transiently expressing DCP1-mCHERRY.

<p>* = <i>p</i> < 0.05</p><p>Average numbers of P-bodies are provided for Col-0, <i>spi-2</i> and <i>spi-4</i> under nonstress (½MS) and salt stress (½MS supplemented with 140mM NaCl for 10 h) conditions. Each biological replicate (n) comprises at least 30 cells. SD values represent standard deviations. Two-tailed student’s <i>t</i> tests were performed to compare stress and nonstress conditions.</p

Transcriptome-wide analysis.

<p>(A) Mapman visualization of log2-fold changes in Col-0 upon salt treatment and (B) the corresponding 25 most strongly enriched GO terms. Darker colors in GO term categories represent higher q-values (BH-corrected). (C) Mapman visualization of log2-fold changes in <i>spi</i> upon salt stress induction and (D) the corresponding 25 most strongly enriched GO terms in <i>spi</i>. For color codes see 6C. (E) Venn diagram comparing the salt stress-dependent up-regulation of transcripts in Col-0 (wt) and <i>spi</i> (see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002188#pbio.1002188.s023" target="_blank">S8 Table</a>).</p

Significantly changed gene expression between Col-0 and <i>spi</i>.

<p>Data present only those with a q-value < 0.01, Benjamini Hochberg (related to <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002188#pbio.1002188.g006" target="_blank">Fig 6</a>).</p

Numbers of mRNA granules per cell (related to Fig 9).

<p>Asterisk represents statistically significant changes in comparison to non stress conditions (Wilcoxon test; p < 0.05).</p><p>Average number of RD29B (<i>n</i> = 65 cells) and TZF3 (<i>n</i> = 120 cells) mRNA granules under non stress (-NaCl) and salt stress (+NaCl) conditions. SD values represent standard deviations.</p