OCP3 co-localizes with pTAC2.

<p>(<b>A</b>) Chloroplast localization pattern of PGL34-YFP, PEND-RFP, WHIRLY-GFP, pTAC2-YFP and OCP3-mCHERRY in protoplasts from <i>N. benthamiana</i> evaluated by confocal microscopy. (<b>B</b>) Co-localization patterns of OCP3 with each of the proteins shown in (A). YFP- and GFP-specific fluorescence is shown in green, RFP- and mCHERRY-specific fluorescence is shown in red and chlorophyll-derived fluorescence is shown in blue.</p

Functional OCP3 resides in the chloroplast.

<p>(<b>A</b>) OCP3 amino acid sequence. The 60 amino acid residues conforming the homeodomain are indicated in red letters. The N-terminal signal peptide sequence to chloroplast targeting, as predicted by TargetP, is indicated in green letters. Two canonical bipartite nuclear localization signals (RK-(X)10-KKNKKK and KK-(X)10-RRSKR) are underlined in blue. (<b>B</b>) Fluorescent confocal microscopy evaluation of protein localization in transgenic Arabidopsis plants transformed with a <i>35S::YFP-OCP3</i> construct (upper panel) and a <i>35S::OCP3-YPF</i> constructs (lower panel). YFP-specific fluorescence is shown in green and chlorophyll-derived fluorescence is shown in red. (<b>C</b>) Western blot analysis using anti-GFP antibodies of crude protein extracts derived from Arabidopsis plants transformed with <i>35S::YFP</i>, <i>35S::YFP-OCP3</i> and <i>35S::OCP3-YPF</i> genes constructs, respectively. Molecular mass markers are shown on the left. (<b>D</b>) Characteristic GUS expression pattern, as driven by the <i>Ep5C</i> gene promoter, in leaves of the <i>ocp3</i> mutant. Complementation of this molecular phenotype in <i>ocp3</i> plants upon transformation with <i>35S::OCP3-YFP</i> but not upon transformation with <i>35S::YFP-OCP3</i>. (<b>E</b>) Fluorescent confocal microscopy evaluation of protein localization in transgenic <i>ocp3</i> plants transformed with a <i>35S::OCP3-YPF</i> construct.</p

Comparative immune responses of plastid PPR-related mutants to inoculation with <i>P. cucumerina</i>.

<p>(<b>A</b>) <i>crr2</i>, <i>crr21</i>, <i>ppra</i>, and <i>pprb</i> disease resistance responses to <i>P. cucumerina</i> compared with <i>ocp3</i> and wild-type (Col-0) plants. Lesion diameter of 20 plants per genotype and four leaves per plant were determined 12 d following inoculation with <i>P. cucumerina</i>. Values are means and ± SE (n = 80). ANOVA detected significant differences at the P<0.05 level. Experiments were repeated three times with similar results. (<b>B</b>) Representative leaves from each genotype at 12 days following inoculation with <i>P. cucumerina</i>. Bar represents 5 mm. (<b>C</b>) Aniline blue staining and epifluorescence microscopy was applied to visualize callose accumulation. Micrographs indicate <i>P. cucumerina</i> inoculation and infection site in the different <i>Arabidopsis</i> genotypes at 0 h.p.i (right panel) and at 48 h.p.i. (left panel). Bar represents 500 µm. (<b>D</b>) The number of yellows pixels (corresponding to pathogen-induced callose) per million on digital photographs of infected leaves were used as a means to express arbitrary units (i.e. to quantify the image) at 48 h.p.i. Data are visible microscopy averages from Col-0 and mutant plants (±SE). Different letters above bars indicate statistically significant differences between genotypes, according to one-way ANOVA (P<0.05, n = 15).</p

Monitoring of NDH activity by using chlorophyll fluorescence analysis.

<p>(<b>A</b>) Analysis of the transient increase in chlorophyll fluorescence (apparent Fo) after termination of actinic light (AL) illumination. The bottom curve indicates a typical trace of chlorophyll fluorescence in the wild type (WT). Leaves were exposed to AL (50 µmol photons m⁻² s⁻¹) for 5 min. AL was turned off and the subsequent transient rise in fluorescence caused by plastoquinone reduction based on NDH activity monitored. Insets are magnified traces from the boxed area. The fluorescence levels were normalized by the maximum fluorescence at close PSII centers in the dark (<i>Fm</i>) levels; ML, measuring light; SF, a saturating flash of white light. The fluorescence was monitored using a pulse-amplitude-modulation chlrophyll fluorometer. <i>ocp3-1</i>+<i>OCP3</i>, <i>ocp3-1</i> allele transformed with a wild type <i>OCP3</i> cDNA. (<b>B</b>) Immunoblot analysis of NDH NdhI and NdhJ subunits, the subunit IV of cytochrome <i>b6f</i> (Pet-D) and ascorbate peroxidase (APX). Proteins were extracted from chloroplast preparations from each genotype and lanes loaded with 20 µg protein.</p

OCP3 is co-regulated with a subset of nuclear-encoded chloroplast PPR proteins.

<p>(<b>A</b>) Co-expression gene vicinity network around the <i>OCP3</i> node. This gene cluster was partitioned from the complex network illustrated in Supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003713#ppat.1003713.s003" target="_blank">Figure S3</a> and shows co-expressed genes that are only two steps away from <i>OCP3</i>. Nine out of the 31 genes of the cluster (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003713#ppat.1003713.s010" target="_blank">Table S2</a>) encode PPR proteins and are indicated by circles, while <i>OCP3</i> is indicated by a square. In addition to <i>CRR21</i> (at5g55740), the other 8 <i>PPRs</i> surrounding <i>OCP3</i> node have been tentatively name as <i>PPRa</i> (at4g21190), <i>PPRb</i> (at4g30825), <i>PPRc</i> (at3g29230), <i>PPRd</i> (at3g46610), <i>PPRe</i> (at5g14350), <i>PPRf</i> (at1g15510), <i>PPRg</i> (at3g14330) and <i>PPRh</i> (at3g49140). Nodes indicate individual genes, and edges indicate whether two genes are co-expressed above a certain mutual rank. Color codes for nodes and edges as in Supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003713#ppat.1003713.s003" target="_blank">Figure S3</a>. Green, orange and red edges indicate a mutual rank relationship 10 (green), 10 but 20 (orange) and 20 but 30 (red), respectively, for each connected gene pair. GO characterization of the 31 genes is shown in Table 2. The network was generated, and modified from AraGenNet (<a href="http://aranet.mpimp-golm.mpg.de/aranet" target="_blank">http://aranet.mpimp-golm.mpg.de/aranet</a>). (<b>B</b>) Chloroplast localization pattern of OCP3-CHERRY, PPRa (at4g21190)-YFP, CRR21-GFP, and At1g63680-YFP in protoplasts from <i>N. benthamiana</i> transfected with each respective construct as evaluated by confocal microscopy. (<b>C</b>) Chloroplast co-localization patterns of OCP3-CHERRY with OCP3-YFP and of OCP3-CHERRY with each of the other proteins shown in (B) in protoplast co-transfected with each of the indicated construct pairs. YFP-specific fluorescence is shown in green, CHERRY-specific fluorescence is shown in red and chlorophyll-derived fluorescence is shown in blue.</p

Editing defects in <i>ocp3</i> plants and <i>in vivo</i> association of OCP3 with <i>ndhB</i> RNA.

<p>(<b>A</b>) Nucleotide sequences surrounding the RNA editing sites of ndhB-6 (95644), ndhB-5 (95650), ndhB-4 (96419), ndhB-3 (96579), ndhB-2 (96698) and ndhD-2 (116494) are shown as sequence chromatograms. The editing sites are specified relative to the nucleotide sequence of the complete Arabidopsis chloroplast genome (Genebank accession number AP000423). Editing sites are indicated by a red C residue in the genomic (gDNA) sequence and its conversion or not to a U(T) residue in Col-0 and <i>ocp3</i> derived RNA samples. Editing defects in <i>ocp3</i> are indicated by a blue mark above the corresponding peaks. (<b>B–E</b>) Poisoned primer extension (PPE) assays were conducted on the editing sites ndhB-6 (B), ndhB-5 (C), ndhB-4 (D) and ndhB-3 (E). RT-PCR products were obtained with labeled 6-carboxyfluorescein primers that anneals next to the target editing site (forward PPE primers were used for all sites). Acrylamide gels (below panels) were visualized under UV light, and intensity of bands quantified calculated and plotted. Bars represent mean ± SD, n = 3 independent replicates. Experiments were repeated at least three times with similar results. E, edited; U, unedited, P; primer. (<b>F</b>) Comparative RNA editing efficiency in Col-0 and <i>ocp3</i> plants as quantified from direct DNA sequencing of 100 independent cDNAs per genotype encompassing each of the indicated editing sites. (<b>G</b>) RNA immunoprecipitation (RIP) of anti-HA precipitated protein complexes from leaves derived from Col-0 and a 35S::OCP3:YFP:HA transgenic line. The upper panel shows a Western blot of protein present in crude leaf extracts and proteins immunoprecipitated (IP) with anti-HA antibody. The blot was developed with anti-GFP antibody and shows enrichment of the OCP3:YFP:HA protein in samples derived from the transgenic line. In the lower panel RT-PCR was used to detect association of <i>ndhB</i> transcripts with OCP3-enriched complexes in comparison to the corresponding input sample. Lack of association of <i>ndhD</i> transcripts with OCP3-enriched complexes is shown as a negative control.</p

Pathogen-triggered editing inhibition in plastid <i>ndhB</i> and <i>ndhD</i> transcripts.

<p>(<b>A</b>) Portion of the electrophoretograms from RT-PCR bulk sequencing corresponding to the editable cytosine residue at sites ndhB-7, ndhB-6, ndhB-5, ndhB-4, ndhB-3, ndhB-2, ndhD-1, and ndhD-4 are shown for Col-0 plants at 0,12, 24 and 48 h post-inoculation with <i>P. cucumerina</i>. (<b>B</b>) PPE assays following fungal infection for ndhB-6, ndhB-5 and ndhB-3 sites confirms the reduction of editing extent as detected by bulk sequencing. The PPE products run on acrylamide gels are shown on the right. E, edited; U, unedited; P, primer. (<b>C</b>) Immunoblot analysis of NDH subunit I (NdhI), subunit IV of cytochrome <i>b6f</i> (Pet-D), PSI subunit D-2 (PSAD-2) and ascorbate peroxidase (APX) from Co-0 plants at 0, 6, 12, 24 and 48 h following inoculation with <i>P. cucumerina</i>. Intensity of NdhI immunoreactive bands was quantified and plotted on the right graph. Bars represent mean ± SD, n = 3 independent replicates. The experiment was repeated three times with similar results (<b>D</b>) Extent of <i>P. cucumerina</i> growth on inoculated leaves. At the times indicated DNA was extracted from leaves and the amount of the <i>P. cucumerina β-tubulin</i> gene quantified by qPCR. Data are standardized for the presence of the <i>P. cucumerina β-tubulin</i> gene in Col-0 at time 0. Data represent the mean ± SD; n = 3 biological replicates. (<b>E</b>) Determination of <i>P. cucumerina</i>-induced callose deposition in inoculated leaves of Col-0 plants. (<b>F</b>) <i>P. cucumerina</i>-induced expression of the defense-related <i>PDF1.2</i> gene as determined by RT-qPCR. Data represent the mean ± SD; n = 3 biological replicates. (<b>G</b>) Reduction of NdhI subunit content following inoculation of Col-0 with <i>B. cinerea</i>. NdhI content was quantified as in (C). On the right a Western blot detail revealing early (at 6 h.p.i) fungal-induced down regulation of NdhI subunit accumulation is shown. (<b>H</b>) Early induced down-regulation of NdhI protein accumulation in Arabidopsis seedlings by chitosan. Chitosan was applied for the times indicated to Arabidopsis seedlings and proteins analyzed by Western blot with anti-NhHI, anti-Pet-D and anti-APX antibodies. The experiment was repeated three times with similar results. (<b>I</b>) Chitosan-induced gene activation of <i>MYB51</i> and concomitant gene repression of <i>OCP3</i>, <i>CRR21</i> and <i>PPRa</i> as determined by RT-qPCR. Data represent the mean ± SD; n = 3 biological replicates.</p

Correction: An Extracellular Subtilase Switch for Immune Priming in Arabidopsis

<p>Correction: An Extracellular Subtilase Switch for Immune Priming in Arabidopsis</p

SBT3.3 loss of function increases disease susceptibility to <i>P.</i><i>syringae</i> DC3000 and <i>H. arabidopsidis</i>.

<p>(<b>A</b>) Five-week-old plants were inoculated with <i>Ps</i>DC3000. Zero (white bars), three (grey bars) and five (black bars) days after inoculation, the bacterial growth was measured. Error bars represent standard deviation (n = 12). Asterisks indicate statistical differences to Col-0 (P<0.05) using Student's <i>t</i> test. Below are representatives of inoculated leaves of the indicated genotypes. (<b>B</b>) Quantification of <i>H. arabidopsidis</i> conidia development on Col-0, and <i>sbt3.3-1</i> and <i>sbt3.3-2</i> mutants. Asterisks indicate statistically different distributions of disease severity classes compared with Col-0 plants (χ² test; α = 0.05). (<b>C</b>) Western blots with anti-PR1 antibodies reveals inhibition of PR1 induced accumulation in <i>nrp1</i>, <i>sbt3.3-1</i> and <i>sbt3.3-2</i> mutant plants, compared to Col-0, following inoculation with <i>Ps</i>DC3000. The experiments were repeated three times with similar results. (<b>D–E</b>) Time-course RT-qPCR analysis showing <i>PR-1</i> (D) and <i>SBT3.3</i> (E) gene expression in Col-0, <i>sbt3.3-1</i>, and <i>npr1-1</i> plants after infection with <i>Ps</i>DC3000. Data represent the mean ± SD; n = 3 replicates and gene expression given as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003445#ppat-1003445-g001" target="_blank">Fig. 1</a>.</p

<i>SBT3.3</i> genome organization and induced expression following <i>P.</i>

<p><b>syringae</b><b> DC3000 infection.</b><b>A</b>) Four <i>SBT3.3</i>-like open reading frames sequences (named as <i>SBT3.5, SBT3.4, SBT3.3 and SBT3</i>) are arranged in tandem in chromosome I. The distances are only approximate. Arrowheads indicate direction of transcription. Black arrows above the genes show position of T-DNA insertions rendering the <i>sbt3.4</i> and <i>sbt3.3</i> mutant alleles. (<b>B</b>) Schematic representation of the SBT3.3 preproenzyme structure. Areas marked respectively in black, white and stippled indicate the signal peptide, propeptide, and mature peptide regions. Numbers depict positions of amino acid residues from the N-terminus. The amino acids forming the catalytic triad (D, H, and S) and the conserved N residues are marked. (<b>C</b>) RT-qPCR analyses showing local induction of <i>SBT3.3</i>, <i>PR-1</i>, and <i>PR-2</i> gene expression upon infection with virulent <i>Ps</i>DC3000, and both local and distal induction following infection with the avirulent <i>Ps</i>DC3000 (<i>AvrRpm1</i>) strain. Filled circles represent inoculated plants, and empty circles represent mock-inoculated plants (controls). Data represent mean ± SD, n = 3 replicates. Expression was normalized to the constitutive <i>ACT2</i> gene, then to expression at time 0 in Col-0 plants.</p