Auxin promotes hypocotyl elongation in light-grown seedlings.

<p>Auxin promotes hypocotyl elongation in a range of day-length conditions. Average hypocotyl length of wild-type seedlings grown in short days (SD; 8/16), long days (LD; 16/8) or constant light (LL) and treated with 5 µM picloram was determined following 24, 48, or 72 hours of auxin treatment. Hypocotyl length on auxin is shown as a percentage of length on control medium. Error bars indicate standard error. (B) Auxin response in seedlings increases with auxin concentration. Images of aerial portions of individual 7 day-old seedlings were captured following 48 hours of IAA treatment at the indicated concentrations. (C) Hypocotyl auxin response requires auxin signaling. Average hypocotyl length of wild-type or <i>aux/iaa</i> mutant seedlings treated with 5 µM picloram (red bars) or IAA (blue bars) was measured following 48 hours of auxin treatment. Hypocotyl length on auxin relative to the untreated control is shown as in (a). <i>Error</i> bars indicate standard error. Statistical significance was determined using a Tukey HSD post hoc comparison among the means on the analysis of variance using type III sums of squares (p<0.05).</p

The <i>afb5</i> mutant fails to respond to picloram.

<p>The <i>afb5-5</i> mutant fails to regulate transcription in response to picloram. Differential gene expression between hypocotyl samples of solvent-treated wild-type (Col-0) or <i>afb5-5</i> seedlings (<i>afb5</i>) or seedlings treated with picloram (+ pic) for 30 minutes (30) or 120 minutes (120), as determined by analysis of microarray data. The number of genes differentially expressed between samples is shown in lines connecting each sample pair. Data from microarray experiments (a) and (b) were combined for identification of 1193 picloram-responsive genes. (B) Aver<i>age e</i>xpression values of 740 auxin-induced (upper panel) or 453 auxin-repressed (lower panel) genes are not different in hypocotyls from control seedlings (Col-0), co<i>ntrol-t</i>reated <i>afb5-5</i> mutant seedlings (control <i>afb5</i>) or picloram-treated <i>afb5-5</i> mutant seedlings (pic <i>afb5</i>). Differentially expressed genes identified using the RankProd package were selected and average expression values for microarray ‘b’ are shown.</p

Model for the transcriptional auxin response preceding hypocotyl elongation.

<p>Auxin levels in hypocotyl tissue elevate in response to growth-promoting conditions or exogenous auxin, which activates transcriptional auxin signaling. Early auxin-responsive genes include those encoding GA oxidases, cell wall modifying enzymes, and other factors that may contribute directly to cell elongation or regulate expression of additional growth-promoting genes. These pathways may <i>be reinf</i>orced by activity of PIF4 and PIF5, which are liberated from DELLA repression due to auxin-mediated modulation of GA levels. In growth-promoting conditions, auxin-responsive genes may be PIF-dependent due to PIF regulation of auxin biosynthesis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036210#pone.0036210-Nozue1" target="_blank">[39]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036210#pone.0036210-Niwa1" target="_blank">[42]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036210#pone.0036210-Nozue2" target="_blank">[77]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036210#pone.0036210-Franklin1" target="_blank">[87]</a>.</p

Hypocotyl auxin response requires TIR1/AFB auxin receptors.

<p>(A–C) Hypocotyl length of wild-type or <i>tir1/afb</i> single or multiple mutant seedlings grown in short days and treated with IAA at the indicated concentrations was measured following 48 hours of auxin treatment. Asterisks represent mutants showing a significantly different response to hormone treatment compared to wildtype. A general linear model (glm performed in R using the car package <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036210#pone.0036210-Fox1" target="_blank">[104]</a>) was used to determine significance and main effects for genotype were confirmed using ANOVA type III sums of squares. All assumptions for GLM were fulfilled. Error bars indicate standard error.</p

Auxin promotes hypocotyl elongation through PIF-dependent and –independent pathways.

<p>PIF4 and PIF5 are not required for transient auxin response. Average hypocotyl length of wild-type (Col-0) or <i>pif4pif5</i> mutant seedlings treated with 5 µM IAA for two hours was measured each hour for 7 hours. Error bars indicate standard error. Hypocotyl length at each time point is shown as a percentage of length at time 0. (B) Auxin-responsive genes are associated with growth conditions. Picloram-induced genes are also induced in the dark (column 1, up in WTD) and during growth (2, upG; 6, upG PIF4/5), and repressed by light (3, down in WTRc; 7, DL) and in the pifq mutant (4, down in pifqR1; 5, down in pifqD). Picloram-repressed genes are also repressed in the d<i>ark (colu</i>mn 9, down in WTD) and upregulated by light (10, upL; 15 up in WTRc), during stationary phase (11, upS; 13 Nozue upS PIF4/5), and in the pifq mutant (12, up in pifqR1; 14, up in pifqD) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036210#s3" target="_blank">Methods</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036210#pone-0036210-t001" target="_blank">Table 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036210#pone.0036210.s011" target="_blank">Table S6</a> for complete description of array conditions shown). CW indicates genes associated with cell wall metabolism (column 8). (C) PIF4/5-dependent genes are regulated by auxin in seedlings. Wild-type (Col-0) or <i>pif4pif5</i> mutant 5-day-old seedlin<i>gs were t</i>reated with 5 µM IAA or a solvent control for 2 hours and used for RNA isolation. Expression value of each gene shown, relative to a control gene, was determined by qRT-PCR.</p

Microarray data selected for comparison to auxin-regulated gene sets.

<p>Microarray data selected for comparison to auxin-regulated gene sets.</p

Gibberellin biosynthesis is required for hypocotyl auxin response.

<p>(A,B,C) Asterisk represents mutants showing a significantly different response to hormone treatment compared to wildtype or control treatment. A general linear model (glm performed in R using the car package <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036210#pone.0036210-Czechowski1" target="_blank">[101]</a>) was used to determine significance and main effects for genotype were confirmed using ANOVA type III sums of squares. All assumptions for GLM were fulfilled. (A)_Paclobutrazol inhibits hypocotyl auxin response. Hypocotyl length of wild-type seedlings grown in short-day conditions and treated with paclobutrazol at the indicated concentrations (black line) or paclobutrazol plus 5 µM picloram (red line) was measured following 48 hours of treatment. Error bars indicate standard error. (B) Paclobutrazol-mediated inhibition of hypocotyl elongation is not overcome by higher auxin concentration. Hypocotyl length of wild-type seedlings treated with IAA (blue line) or picloram (red line) at the indicated concentrations or IAA and 2.5 µM paclobutrazol (PAC; green line) was measured following 48 hours of treatment. Error bars indicate standard error. (C) RGA protein degrades in response to auxin treatment in hypocotyls of auxin-treated seedlings. Abundance of RGA-GFP protein in hypocotyl tissues was analyzed by epifluorescence microscopy over a 0–24 hour time course. Three day-old seedlings were treated for 2, 4, or 24 hours with 50 µM GA₃, 5 µM IAA, 5 µM IAA +2.5 µM paclobutrazol, or a solvent control, prior to imaging. (D) A GA biosynthesis mutant is partially auxin-resistant. Hypocotyl length of wild-type seedlings (Col-0) or the <i>ga20ox1/ox2</i> mutant <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036210#pone.0036210-Rieu1" target="_blank">[98]</a> treated with IAA at the indicated concentrations was measured following 48 hours of auxin treatment. Hypocotyl length on auxin is shown as a percentage of length on control medium. Error bars indicate standard error.</p

Small RNA from protein-coding gene and pseudogene loci in <i>P. infestans</i>.

<p>(A) Heatmap-based size profile of 18-30-nucleotide small RNA reads mapping to 1,166 <i>P. infestans</i> genes that overlapped at least 100 total small RNA reads. Gene annotation groups with at least four genes are labeled with alternating black and gray boxes. (B and C) Regional metaplots with average 18-27-nucleotide small RNA reads per position. X-axis positions are relative scale (0–100%) for each region. Genes from (A) with more 24-30- than 18-23-nucleotide small RNA reads, LTR retrotransposons and transposons are shown (B). Genes from (A) with more 18-23- than 24-30-nucleotide small RNA reads, crinklers and type III fibronectins are shown (C).</p

Distribution of small RNA in the <i>P. infestans</i> genome.

<p>(A–D) Two-dimensional binning of <i>P. infestans</i> genes based on the length of 5′ and 3′ flanking intergenic regions. Plots are divided into quadrants with dashed lines: gene-dense regions (GDRs), gene-sparse regions (GSRs), and border regions (BRs). (A) Heatmap color scale represents the number of genes per bin for all genes (left) and genes without transposable elements in flanking intergenic regions (Data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077181#pone.0077181-Haas1" target="_blank">[9]</a>). (B) Genes were binned as in (A), but the heatmap color scale represents the percentage of base pairs in both the 5′ and 3′ flanking intergenic regions that were occupied by transposable elements. (C and D) Genes were binned as in (A), but the heatmap color scale represents the repeat-normalized small RNA reads per base pair in either both the 5′ and 3′ flanking intergenic regions (C) or the gene body (D).</p

RNA silencing machinery in <i>Phytophthora</i>.

<p>(A–C) Phylogenetic analysis of Rdr, Dcl and Ago proteins, respectively. Protein domains used for analysis were the Rdr RdRP domain, the Dcl RNaseIII A and B domains and the Ago DUF1785-PAZ-PIWI region. Maximum Likelihood bootstrap support values are shown. In (A) and (B), taxonomic groups are color-coded: viridiplantae (green), alveolates (blue) and oomycetes (orange). (D) Synteny maps of regions containing Ago3–5 (top) and Ago1/2 (bottom) in <i>P. ramorum</i> (<i>Pr</i>), <i>P. sojae</i> (<i>Ps</i>), and <i>P. infestans</i> (<i>Pi</i>). Regions are labeled with scaffold numbers, alignment orientation (+/−) and coordinate ranges. Genes (dark arrows), Ago genes (orange arrows), and transposable elements (green arrows) are depicted. Conserved intervals are connected by gray (direct) or blue (inverse) boxes (E) MAFFT alignment with ClustalX coloring of amino acid residues from the Ago MID (5′ phosphate binding) and PIWI (catalytic) domains. Positions shown to interact with the guide-strand 5′ monophosphate (MID), and the Mg²⁺-coordinating catalytic residues (PIWI) are marked with an asterisk <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077181#pone.0077181-Elkayam1" target="_blank">[40]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077181#pone.0077181-Schirle1" target="_blank">[41]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077181#pone.0077181-Ma1" target="_blank">[42]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077181#pone.0077181-Parker1" target="_blank">[43]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077181#pone.0077181-Parker2" target="_blank">[44]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077181#pone.0077181-Wang1" target="_blank">[45]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077181#pone.0077181-Jinek1" target="_blank">[46]</a>.</p