Interventions used in this study to manipulate the jasmonate pathway.

<p>Shown in red are: 1. loss-of-function mutation of the JA synthesis gene <i>allene oxide synthase</i> (<i>aos</i>); 2. treatment with exogenous JA; 3. loss-of-function mutations in the negative regulator <i>NINJA</i>; 4. gain-of-function mutation of MYC transcription factors. The dashed box surrounding MYC indicates that it is conceptually possible to use an overactive MYC to drive JA responses in the absence of JA synthesis (step 1) and of negative regulators like NINJA (steps 1 and 3 combined). This was achieved using a novel <i>myc2</i> mutant that amplifies JA responses.</p

MYC2 ^E165K confers extreme hypersensitivity to exogenous JA.

<p>(A) Representative 7-do seedlings of WT and <i>myc2-322B</i> (<i>m2-322B</i>) mutants grown in control conditions (ctrl) or on media supplemented with 25 μM MeJA. Scale bar = 0.5 cm (B) Confocal microscopy images of propidium iodide stained primary root meristems of WT and <i>myc2-322B</i> 5-do seedlings grown in the absence (ctrl) or presence of 25 μM MeJA. Scale bar = 50 μm. Vertical white bars represent the root division zone and the horizontal yellow dashed line marks the root—hypocotyl boundary of <i>myc2-322B</i> grown in the presence of MeJA. (C) Root length of 7-do seedlings of the indicated genotype grown in the absence (control) or presence of 25 μM MeJA. <i>n-1</i> refers to <i>ninja-1</i>. Data shown are means (± SD) from 20–49 plants. Letters above bars indicate statistically significant differences between samples as determined by Tukey’s HSD test (P < 0.01).</p

Root growth reduction after repeated shoot wounding is JA dependent.

<p>(A-D) Root measurements of 5-day-old (do) WT and <i>aos</i> seedlings grown in control conditions or subjected to repetitive cotyledon wounding: (A) representative control (ctrl) and wounded (w) seedlings; (B) primary root length; (C) cortex cell number in the primary root meristem; (D) box plot of cortex cell length in the differentiation zone of the primary root in control and wounded (w) samples of the indicated genotype. Data shown are means (± SD) from 27–55 (B) or 10 plants (C-D). Asterisks: Student’s t test significance compared with untreated controls (*P < 0.001) and letters in (D) indicate statistically significant differences between pairs as determined by Tukey’s HSD test (P < 0.001). (E-F) qRT-PCR of cell cycle marker genes (E) <i>CYCB1;1</i> and (F) <i>PCNA1</i> in 5-do roots of WT and <i>aos</i> seedlings grown in control conditions or subjected to repetitive cotyledon wounding. Root samples were collected 1 h after the 5^th cotyledon wound. Transcript levels were normalized to <i>UBC21</i> and displayed relative to the expression of the WT control. Bars represent means of three biological replicates (±SD), each containing a pool of roots from ~60 individuals. Asterisks: Student’s t test significance compared with untreated controls (*P < 0.01).</p

<i>myc2-322B</i> exhibits enhanced root JA responses in a NINJA-dependent manner.

<p><i>JGP</i> expression in 5-do seedlings of WT (A), <i>myc2-322B</i> (B, <i>m2-322B</i>) and <i>ninja-1 myc2-322B</i> (C, <i>n-1 m2-322B</i>). Note the constitutive reporter activity stained in blue (Scale bars = 0.5 mm). (D) qRT-PCR of basal <i>JAZ10</i> expression in 5-do roots of <i>myc2-322B</i>, <i>ninja-1</i> (<i>n-1</i>), <i>ninja-1 myc2-322B</i> (<i>n-1 m2-322B</i>), <i>ninja-2 myc2-322B</i> (<i>n-2 m2-322B</i>), <i>ninja-1 myc2-322B/+</i> (<i>n-1 m2-322B/+</i>) and <i>ninja-2 myc2-322B/+</i> (<i>n-2 m2-322B/+</i>). <i>JAZ10</i> transcript levels were normalized to those of <i>UBC21</i> and displayed relative to the expression of unwounded WT samples, which are set to 1. Bars represent the means of three biological replicates (±SD), each containing a pool of ~60 roots. Complete qRT-PCR data are in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005300#pgen.1005300.s027" target="_blank">S1 File</a>. (E) Root length quantification of WT and 7-do mutant lines. Data are the means (±SD) from 20–48 plants. Primary root meristem cell number (F) and box plot summary of cortex-cell length (G) in 5-do seedlings of WT and mutant lines grown in control conditions, and of WT grown in presence of 25 μM MeJA (n = 10). Letters above bars and box plots (E-G) indicate statistically significant differences between samples as determined by Tukey’s HSD tests (P < 0.01).</p

Spatial localization of NINJA, MYC2, MYC3 and MYC4 in the primary root meristem and contribution of the three TFs to the <i>ninja</i> mutant phenotype.

<p>Expression pattern overviews of (A) <i>NINJA</i>_<i>pro</i><i>-NLS3xVENUS</i>, (B) <i>MYC2</i>_<i>pro</i><i>-NLS3xVENUS</i>, (C) <i>MYC3</i>_<i>pro</i><i>-NLS3xVENUS</i> and (D) <i>MYC4</i>_<i>pro</i><i>-NLS3xVENUS</i> fluorescent reporters in 5-do WT primary roots. Close-ups of <i>MYC2</i>_<i>pro</i><i>-NLS3xVENUS</i> (E and F), <i>MYC3</i>_<i>pro</i><i>-NLS3xVENUS</i> (G and H) and <i>MYC4</i>_<i>pro</i><i>-NLS3xVENUS</i> (I and J) expression in, respectively, the elongation and division zones of the primary root. Confocal microscopy images (A-J) represent merged overlays of the fluorescent (yellow) and propidium iodide (red) stained roots. Scale bars = 50 μm. (K-N) Distribution maps of NINJA, MYC2, MYC3 and MYC4 expression patterns (green) in the primary root meristem based on promoter and protein fusion reporters (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005300#pgen.1005300.s004" target="_blank">S4 Fig</a>). (O) qRT-PCR of basal <i>JAZ10</i> expression in 5-do roots of <i>ninja-1</i> (<i>n-1</i>), <i>ninja-2</i> (<i>n-2</i>), <i>myc2</i> (<i>m2</i>), <i>myc3</i> (<i>m3</i>), <i>myc4</i> (<i>m4</i>), <i>ninja-1 myc2</i> (<i>n-1 m2</i>), <i>ninja-1 myc3</i> (<i>n-1 m3</i>), <i>ninja-1 myc4</i> (<i>n-1 m4</i>), <i>myc2 myc3</i> (<i>m23</i>), <i>myc2 myc4</i> (<i>m24</i>), <i>myc3 myc4</i> (<i>m34</i>), <i>ninja-1 myc2 myc3</i> (<i>n-1 m23</i>), <i>ninja-1 myc2 myc4</i> (<i>n-1 m24</i>), <i>ninja-1 myc3 myc4</i> (<i>n-1 m34</i>), <i>myc2 myc3 myc4</i> (<i>m234</i>), <i>ninja-1 myc2 myc3 myc4</i> (<i>n-1 m234</i>), and <i>ninja-2 myc2 myc3 myc4</i> (<i>n-2 m234</i>). <i>JAZ10</i> transcript levels were normalized to those of <i>UBC21</i> and displayed relative to the expression of WT controls that are set to 1 and indicated with a dashed line. Bars represent the means of three biological replicates (±SD), each containing a pool of ~60 roots. Complete qRT-PCR data are in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005300#pgen.1005300.s027" target="_blank">S1 File</a>. (P) Root length of 7-do seedlings of the same genotype as indicated in (O). Data shown are means (± SD) from 22–48 plants; letters above bars indicate statistically significant differences between samples as determined by Tukey’s HSD test (P < 0.01).</p

Overview of denitrification capacity of <i>P</i>. <i>veronii</i> 1YdBTEX2.

<p>(A) Overnight growth of <i>P</i>. <i>veronii</i> 1YdBTEX2 wild type (WT) and the Δ<i>nar</i> mutant in presence (+O₂, left) or absence of air but with 15 mM nitrate supplemented medium (+NO₃,–O₂, right panel) conditions. Note the gas formation in the right panel of the WT incubation. (B) Gene regions predicted for denitrification in the <i>P</i>. <i>veronii</i> 1YdBTEX2 chromosome 1 with trivial gene names indicated. Black bar represents the deleted region in <i>P</i>. <i>veronii</i> Δ<i>nar</i>.</p

Circular maps of the replicons encompassing the <i>P</i>. <i>veronii</i> 1YdBTEX2 genome.

<p>(A) Chromosome 1 (chr1) with indication of possible genomic islands (GEI) and prophages (pf). The outermost circles show the location and orientation of predicted coding regions (blue and cyan), followed by tRNA (olive green) and rRNA genes (black), predicted regions of genome plasticity (blue-green-brown) islands and prophages (grey). The inner circles represent BLASTN comparisons with the close relatives <i>P</i>. <i>fluorescens</i> SBW25 (red, Acc. No. AM181176.4), <i>P</i>. <i>trivialis</i> strain IHBB745 (deep pink, CP011507.1), <i>P</i>. <i>syringae</i> pv. syringae B728a (dark purple, CP000075.1), <i>P</i>. <i>putida</i> KT2440 (light purple, AE015451.1) and <i>P</i>. <i>knackmussii</i> B13 (persian green, HG322950). GC skew (dark magenta and yellow green) is shown in the most central circle. (B) As A, but for the chromosome 2 replicon (chr2). Inner circles, from outwards to inwards, predicted transposons (dark purple) and <i>tra</i> genes (green), regions of genome plasticity (blue-green-brown) and prophages (grey), followed by BLASTN comparisons to <i>P</i>. <i>fluorescens</i> SBW25 plasmid pQB103 (red, AM235768.1, NC_009444.1), <i>Pseudomonas stutzeri</i> strain 19SMN4 plasmid pLIB119 (deep pink, CP007510.1), <i>Pseudomonas mandelii</i> JR-1 plasmid (dark purple, CP005961.1) and <i>Pseudomonas resinovorans</i> NBRC 106553 plasmid pCAR1.3 (Persian green, AP013069.1). (C) As B, but for the plasmid replicon. The inner circles represent the BLASTN comparisons with <i>P</i>. <i>putida</i> S12 plasmid pTTS12 (red, CP009975.1), and <i>Pseudomonas</i> sp. VLB120 plasmid pSTY (purple, CP003962.1). Plots generated with DNAPlotter [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165850#pone.0165850.ref046" target="_blank">46</a>].</p

Comparison of catabolic gene transcription involved in toluene or <i>meta-</i>cleavage metabolism by <i>P</i>. <i>veronii</i> 1YdBTEX2 in liquid culture with succinate (Li-Su) or toluene (Li-To).

<p>(A) Normalized read counts across the <i>ipb</i> gene cluster (PVE_r2g0739-0753). Note the decrease as a result of the transposon insertion (white arrow). (B) Expression level (reads per kilobase per million, RPKM, ¹⁰log scale) of the <i>ipb</i> cluster genes (numbers refer to PVE_r2g loci). (C) as B, for the <i>dmp</i> cluster genes (PVE_r2g0708-0719), and the proposed gene encoding for the dihydrodiol dehydrogenase (PVE_r2g0805). (D) as B, for the <i>nah</i> cluster genes (PVE_r2g0834-0847).</p

Genome-wide gene expression differences in <i>P</i>. <i>veronii</i> 1YdBTEX2 after 1 h exposure to different carbon sources or growth environment.

<p>(A) Two-dimensional Principal Component Analysis of quadruplate global RNA-sequencing data sets of <i>P</i>. <i>veronii</i> 1YdBTEX2 incubated in liquid medium with succinate (Li-Su), or toluene (Li-To), or in sand with succinate (Sa-Su) or with toluene (Sa-To). (B) Venn diagram with the number of unique and common genes significantly differentially expressed (2-way ANOVA, ²log-fold-change [logFC] >1, false-discovery rate [FDR] <0.05, <i>P</i> <0.01) as result of change of carbon source (succinate to toluene) or environment (liquid to sand). (C) Smear-plot of global gene expression intensity (²log CPKM) versus expression changes (²log fold change) compared between cells incubated with toluene (Li-To) versus succinate (Li-Su); in grey, genes not statistically differentially expressed (logFC<1, FDR>0.05, <i>P</i> >0.01); magenta, genes with lower, and dark purple, genes with higher expression in presence of toluene (+). Blue, <i>ipb</i> genes; yellow, <i>dmp</i> genes; green, <i>ttg</i> genes (toluene efflux pump). (D) Gene expression changes as an effect of carbon source (succinate versus toluene, left) or of environment (liquid versus sand, right), and plotted as function of genomic location (chromosome 1, chr1; chromosome 2, chr2 and plasmid, plm; organized according to locus_tag number). Bars indicate ²log-fold change. Dark purple, statistically significantly higher expressed genes in presence of toluene (+, left) or sand (+, right); cyan, lower expressed genes in pink. Positions of the <i>ipb</i>, <i>dmp</i> and <i>ttg</i> genes are highlighted.</p