Experimental design.

<p><b>A.</b> Three biological replicate pools of 48 ten day-old wild-type (Col-0) seedlings were pre-treated for two hours with either 3AB, 3-MB, or vehicle (DMSO), and then treated for one hour with either flg22 flagellin peptide or sterile H₂O. <b>B.</b> Three biological replicate pools of 48 ten day-old wild-type (Col-0) or <i>parg1-2</i> knockout seedlings were treated for one hour with either elf18 EF-TU peptide or sterile H₂O.</p

Gene ontology over-representation in genes differentially regulated by either flg22 or elf18.

<p>Gene ontology over-representation in genes differentially regulated by either flg22 or elf18.</p

Hierarchical clustering of expression patterns for all 30,387 genes present on 1-plex Nimblegen <i>Arabidopsis thaliana</i> array.

<p>Standardized transcript abundances (mean = 0, standard deviation = 1) for three biological replicates of nine treatments (Col-0 untreated, Col-0 + flg22, Col-0 + 3AB, Col-0 + 3MB, Col-0 + flg22 + 3AB, Col-0 + flg22 + 3MB, Col-0 + elf18, <i>parg1-2</i> untreated, and <i>parg1-2</i> + elf18) were used to determine Euclidean distances between treatments and genes, represented by the left and top dendrograms, respectively. f = 1μM flg22, e = 1μM elf18, A = 2.5mM 3-aminobenzmide (3AB), M = 2.5mM 3-methoxybenzamide (3MB), C = Col-0 (wild-type), p = <i>parg1-2</i>.</p

Determination of differentially regulated genes.

<p>After initial analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190268#sec002" target="_blank">methods</a>), lists of genes differentially regulated between flg22 and flg22 + 3AB (<b>A-D</b>) or between Col-0 elf18 and <i>parg1-2</i> elf18 (<b>E-H</b>) were assembled. Upregulated (red) (<b>A, C, E, F</b>) and downregulated (blue) (<b>B, D, G, H</b>) genes were determined separately. “Broken” genes were defined for this study as those genes that displayed statistically significant differences in mRNA abundance after treatment with MAMP (flg22 or elf18) (stringent cutoff = FDR <0.05, fold-change>1.3 or <-1.3) versus untreated, but for which MAMP treatment in the presence of 3AB or <i>parg1-2</i> did not cause a statistically significant difference (even at the relatively permissive cutoff of p<0.05, fold-change>1.0 or <-1.0). “Misregulated” genes were defined as those genes upon which MAMPs had no statistically significant impact on mRNA abundance in wild-type plants, even using non-stringent cutoff values (p<0.05 and fold-change>1.0 or <-1.0), but for which MAMP treatment did cause significant differences (at stringent cutoff values) in the presence of either 3AB or <i>parg1-2</i>. To further reduce the occurrence of Type I false positives, the highlighted gene lists were then filtered once more for flg22 v flg22 + 3AB (p<0.05, fold-change>1.3 or <-1.3) or Col-0 elf18 v <i>parg1-2</i> elf18 (p<0.05, fold-change>1.3 or <-1.3).</p

Seedling growth inhibition assay.

<p>Five-day-old seedlings of the indicated genotypes were treated with 0.05uM (low) or 1.0uM (high) flg22 peptide and grown for an additional 14 d. Fresh seedlings weights were then recorded and normalized to the average untreated weight within each genotype. A. Pectin methylesterase inhibitor (<i>PMEI</i>) (At5g64640) knockouts versus untreated, three (<i>pmei-1</i>) and four (<i>pmei-2</i>) biological replicates of 12 seedlings per treatment. B. F-box domain-containing gene (At5g15660) knockouts versus untreated, three (<i>f-box-1</i>) and two (<i>f-box-2</i>) biological replicates of 12 seedlings each. C. <i>Folylpolygutamate synthetase 3 (FPGS3)</i> (At3g55630) knockout versus untreated, three biological replicates. D. TIR-X domain-containing gene (At1g47370), three biological replicates. Asterisks summarize ANOVA results across all experiments for tests of similarity of means between the mutant genotype and wild-type plants treated with the same concentration of flg22. E. <i>FPGS1</i> (At5g05980), <i>FPGS2</i> (At3g10160), and <i>FPGS3</i> (At3g55630) versus untreated, three biological replicates. (Tukey's simultaneous test: * <i>P</i> < 0.05; **<i>P</i> < 0.005; no asterisk, <i>P</i> > 0.05).</p

Identification of genes differentially regulated between flg22 and elf18 in wild-type plants.

<p><b>A</b>, After initial analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190268#sec002" target="_blank">methods</a>), lists of genes differentially regulated between flg22 and elf18 were assembled. Upregulated (red) and downregulated (blue) genes were determined separately. Flg22-regulated genes not differentially regulated by elf18 in these experiments were defined as those that displayed statistically significant differences in mRNA abundance after treatment with flg22 versus untreated (FDR < 0.05, fold-change > 1.3 or < -1.3), which did not display statistically significant difference in mRNA abundance after treatment with elf18 versus untreated even using more permissive cutoff values (p < 0.05, fold-change > 1.0 or <-1.0). The reciprocal calculations were used to determine elf18-regulated genes not differentially regulated by flg22 in these experiments. <b>B</b>, Hierarchical clustering of transcript abundance for MAMP-regulated genes identified in A as exhibiting differential regulation by only one of the two studied MAMPs (A, n = 1897). Each column represents a single gene and each row a single treatment (all treatments replicated three times); gene abundances standardized to mean = 0, standard deviation = 1.0; red = more abundant, blue = less abundant. Clustering was performed by calculating Euclidean distances on columns, using average linkage scores. <b>C</b>, Average transcript abundance (y-axis) for each treatment (x-axis), for each gene within the designated color-coded clusters shown at the top of A. Red lines denote overall mean mRNA abundance for all genes within the cluster, grayscale lines represent each individual gene within the cluster.</p

Callose deposition assay.

<p>A. 10-day-old Arabidopsis seedlings were treated with distilled, deionized water (H₂O) or 1 μm flg22, fixed 24 h after flg22 elicitation, and visualized for callose deposition by aniline blue staining and epifluorescence microscopy. Degree of callose deposition was categorized using a scale of 0 to 5, 0 = no callose deposits, 5 = dense callose deposits over entire field of view. Twelve cotyledons per genotype were examined and compared to wild-type (Col-0) responses per biological replicate (n). <i>pmei-2</i> (At5g64640) n = 1; <i>rba-2</i> (At1g47370) n = 5; <i>f-box-1</i> (At5g15660) n = 4; <i>fpgs3-1</i> and <i>fpgs3-2</i> (At3g55630) n = 4, n = 1, respectively. Asterisks summarize ANOVA results across all experiments for tests of similarity of means between the mutant genotype and wild-type plants treated with flg22 (Tukey's simultaneous test: †P<0.15; ††P<0.1; *P<0.05; **P<0.005; no asterisk, P > 0.05). B. For selected lines, callose deposits were quantified as average percent area covered by white pixels within the viewfield, corresponding to flg22-induced callose, +/- standard error. Ten cotyledons per genotype were examined for each of four biological replicates. Asterisks summarize ANOVA results across all experiments for tests of similarity of means between the mutant genotype and wild-type plants treated with flg22 (Tukey's simultaneous test: *P < 0.055; **P < 0.005; no asterisk, P > 0.05). Representative flg22-treated cotyledons for each genotype in B are shown in C.</p

Elf18-regulated genes broken by <i>parg1-2</i> knockout that are involved in innate immunity.

<p>Elf18-regulated genes broken by <i>parg1-2</i> knockout that are involved in innate immunity.</p

Hierarchical clustering of gene products whose regulation by MAMPs are broken or misregulated by PARP inhibitor or <i>parg1-2</i> knockout.

<p><b>A, C</b>, Hierarchical clustering of transcript abundance for MAMP-regulated genes identified (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190268#pone.0190268.g003" target="_blank">Fig 3</a>) as exhibiting broken or misregulated expression due to 3AB treatment (A, n = 102) or due to <i>parg1-2</i> mutation (C, n = 78). Each column represents a single gene and each row a single treatment (all treatments replicated three times); gene abundances standardized to mean = 0, standard deviation = 1.0; red = more abundant, blue = less abundant. Clustering was performed by calculating Euclidean distances on columns, using average linkage scores. <b>B, D</b>, Average transcript abundance (y-axis) for each treatment (x-axis), for each gene within the designated color-coded clusters shown at the top of A and C. Red lines denote overall mean mRNA abundance for all genes within the cluster, and grayscale lines represent each individual gene within the cluster.</p