Effect of exogenous chemical treatments (SA, MeJA, or COR) on the flg22-triggered oxidative burst.

<p>(A-C) Arabidopsis seedlings were pre-incubated with various concentrations of chemicals for the indicated time periods (0 and 24 h) before the start of ROS measurements. Flg22 (1 µM) was added at zero time. Error bars represent the SD of five independent samples (n = 10) and similar results were obtained in three independent experiments.</p

The Activated SA and JA Signaling Pathways Have an Influence on flg22-Triggered Oxidative Burst and Callose Deposition

<div><p>The first line of defense in plants against pathogens is induced by the recognition of microbe-associated molecular patterns (MAMP). Perception of bacterial flagellin (flg22) by the pattern recognition receptor flagellin-sensing 2 (FLS2) is the best characterized MAMP response, although the underlying molecular mechanisms are not fully understood. Here we studied the relationship between salicylic acid (SA) or jasmonic acid (JA) signaling and FLS2-mediated signaling by monitoring flg22-triggered responses in known SA or JA related mutants of <i>Arabidopsis thaliana</i> (L.) Heynh. The <i>sid2</i> mutant, impaired in SA biosynthesis, had less basal <i>FLS2</i> mRNA accumulation than the wild type, which correlated with suppression of early flg22 responses such as ROS production and induction of marker genes, <i>WRKY29</i> and <i>FRK1</i>. The JA-signaling mutants, <i>jar1</i> and <i>coi1,</i> exhibited an enhanced flg22-triggered oxidative burst and more callose accumulation than the wild type, and pretreatment with SA or coronatine (COR), a structural mimic of JA-isoleucine, altered these flg22-induced responses. Nonexpressor of pathogenesis-related genes 1 (NPR1) acted downstream of SID2 and required SA-dependent priming for the enhanced flg22-triggered oxidative burst and callose deposition. Activation of JA signaling by COR pretreatment suppressed the flg22-triggered oxidative burst and callose accumulation in a coronatine insensitive 1 (COI1) dependent manner. COR had a negative effect on flg22 responses but only the flg22-triggered oxidative burst depended on SA-JA/COR signaling antagonism. Thus the activated SA and JA signaling pathways have an influence on flg22-triggered oxidative burst and callose deposition. These results may explain how SA and JA signaling are cross talked for regulation of flg22-triggered responses.</p></div

SA and JA signaling are required for flg22-triggered oxidative burst.

<p>Flg22-induced ROS generation was monitored in liquid-grown intact seedlings of indicated Arabidopsis genotypes after treatment with 1 µM flg22. Error bars represent the SD of five independent samples (n = 10) and similar results were obtained in multiple independent experiments.</p

Effect of SA or COR pretreatment on flg22-induced <i>MYB51</i> transcript accumulation and callose deposition of Arabidopsis seedlings.

<p>(A) <i>MYB51</i> transcripts were measured in 8-day-old seedlings 1 h after treatment with 1 µM flg22. Data represent SD. All quantitative gene expression measurements were performed using technical triplicates and biological duplicates. (B–C) Eight-day-old seedlings were pre-incubated with SA (100 µM) or COR (0.5 µM) for 24 h, after which the seedlings treated with flg22 for 1 h were stained with aniline blue. Relative callose intensities were quantified as the number of fluorescent callose-corresponding pixels relative to the total number of pixels covering plant material. Values represent SE, n>6. Differential letter types indicated significant differences (α = 0.05) by one-way ANOVA and Tukey HSD test of comparisons between plant genotypes with individual treatment.</p

The effect of SA and COR in the flg22-triggered oxidative burst is dependent on NPR1 and COI1, respectively.

<p>(A–D) Effect of pretreatment with SA (100 µM) or COR (0.5 µM) for 24 h on the flg22-triggered oxidative burst in mutant [<i>pad4</i> (A), <i>npr1</i> (B), <i>jar1</i> (C), <i>coi1</i> (D)] and wild-type Columbia seedlings. Flg22 (1 µM) was added at zero time. Error bars represent the SD of five independent samples (n = 10) and similar results were obtained in three independent experiments.</p

COR is required to overcome the SA effect during the flg22-triggered oxidative burst.

<p>(A) Effect of pretreatment with SA (100 µM) or COR (0.5 µM) for 24 h on the flg22- triggered oxidative burst in <i>cim6</i> and wild-type Columbia seedlings. Flg22 (1 µM) was added at zero time. (B) COR did not suppress flg22-induced ROS generation when applied simultaneously with SA. Eight-day-old seedlings were pre incubated with SA (100 µM), COR (0.5 µM), or SA plus COR for 24 h. Flg22 (1 µM) was added at zero time. Error bars represent the SD of five independent samples (n = 10) and similar results were obtained in at least two independent experiments.</p

Down regulation of the flg22 response genes in <i>sid2</i> plants.

<p>For Quantitative RT-PCR analysis, 8-day-old seedlings were pre-treated with 100 µM of salicylic acid for 24 h and then incubated in 1 µM flg22 solution for 1 h. <i>ACT2 </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088951#pone.0088951-Qiu1" target="_blank">[74]</a> was used as a control. Data represent SD. All quantitative gene expression measurements were performed using technical triplicate and biological duplicates. Differential letter types indicated significant differences (α = 0.05) by one-way ANOVA and Tukey HSD test of comparisons between plant genotypes with individual treatment.</p

Localization of XopN_KXO85, OsVOZ2, and OsXNP in plant cells.

<p>Subcellular localization of the XopN_KXO85-GFP, OsVOZ2-GFP, and OsXNP-GFP fusion proteins in maize mesophyll cells. OsABF1-RFP was used as a nuclear marker. GFP (green) fluorescence was merged with RFP (red) fluorescence. Bars = 10 µm.</p

Pathogenicity test for <i>xop</i> mutants of <i>Xoo</i> KXO85 in rice.

<p><b>A</b>. Disease severity of each <i>xop</i> mutant in 3-month-old rice leaves. W, water; 85, wild-type KXO85; Q, KXO85 <i>xopQ</i>_<i>KXO85</i>::EZ-Tn<i>5</i>; X, KXO85 <i>xopX</i>_<i>KXO85</i>::EZ-Tn<i>5</i>; P1, KXO85 <i>xopP1</i>_<i>KXO85</i>::EZ-Tn<i>5</i>; P2, KXO85 <i>xopP2</i>_<i>KXO85</i>::EZ-Tn<i>5</i>; N, KXO85 <i>xopN</i>_<i>KXO85</i>::EZ-Tn<i>5</i>. <b>B</b>. Disease severity of the <i>xopN</i>_<i>KXO85</i> mutants in the flag leaves of rice grown in a paddy field. W, water; 85, KXO85; N, KXO85 <i>xopN</i>_<i>KXO85</i>::EZ-Tn<i>5</i>; and N^C, KXO85 <i>xopN</i>_<i>KXO85</i>::EZ-Tn<i>5</i> (pML122G2). Photographs were taken and lesion lengths were determined 21 days after inoculation. Vertical error bars indicate the standard deviations (SD). The data are the averages of 12–15 replicates for each treatment. Columns and lines not connected by the same letter are significantly different (P<0.05) as determined by a one-way ANOVA (P<0.001) followed by post hoc Tukey HSD analysis. <b>C</b>. Bacterial growth patterns of the KXO85, <i>xopN</i>_<i>KXO85</i> mutant, and complemented <i>xopN</i>_<i>KXO85</i> mutant strains in flag leaves of wild-type Dongjin. The data are shown as the average values for three replicates; vertical bars indicate the error ranges (±SD). The bacterial populations were assessed every 3 days after inoculation. Different letters at day 21 indicate significant differences (P<0.05) as determined by a one-way ANOVA (P<0.001) followed by post hoc Tukey HSD analysis.</p

Interactions between XopN_KXO85 and OsVOZ2 and OsXNP.

<p><b>A</b>. Screening for interactors of XopN_KXO85 in rice using a yeast two-hybrid system. S (strong: pEXP ^TM32/Krev1 + pEXP ^TM22/RalGDS-wt), W (weak: pEXP ^TM32/Krev1 + pEXP ^TM22/RalGDS-m1), and A (absent: pEXP ^TM32/Krev1 + pEXP ^TM22/RalGDS-m2) indicate the strength of each interaction. Three independent and representative colonies are shown for each bait–prey combination. <b>B</b>. <i>In vivo</i> pull-down analysis of XopN_KXO85 and OsVOZ2 (left panel) and XopN_KXO85 and OsXNP (right panel). Total proteins from <i>N</i><i>. benthamiana</i> leaves co-expressing XopN_KXO85-6× His and Flag-OsVOZ2 or XopN_KXO85-6× His and OsXNP-Flag protein were purified by Ni⁺ affinity chromatography followed by Western blotting using anti-His and anti-Flag antibodies. The expected molecular weights were as follows: XopN_KXO85-6× His = 78.7 kDa; Flag-OsVOZ2 = 74.6 kDa; OsXNP-Flag = 40.1 kDa; +, protein expressed; and -, vector control. <b>C</b>. BiFC analysis of XopN_KXO85 -OsVOZ2, XopN_KXO85 -OsXNP, and XopN_KXO85 -OsVOZ1 interactions in <i>N</i><i>. benthamiana</i> leaves. Negative, pDEST-SCYNE(R)^GW + pDEST-SCYCE(R)^GW; positive, pEXP-SCYNE(R)-Cnx7 + pEXP-SCYCE(R)-Cnx6. Bars = 50 µm.</p