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

Virulence assay in wild-type Dongjin rice and the OsVOZ2 mutant line PFG_3A-07565.

<p><b>A</b>. Schematic representation of the T-DNA insertion in OsVOZ2 T₇ transgenic rice. <i>OsVOZ2</i> consists of four exons (orange boxes) and three introns (line between the orange boxes). The T-DNA was located in the second intron from the translational start site. F and R are the primers used for RT-PCR analysis, which showed the expected size of <i>OsVOZ2</i> in wild-type Dongjin but not in the <i>OsVOZ2</i> mutant rice PFG_3A-07565. Actin1 was used for normalization of the cDNA quantity. <b>B</b>. Virulence assay of the <i>xopN</i>_<i>KXO85</i> mutant in wild-type Dongjin rice and OsVOZ2 mutant rice. 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 21 days after inoculation. <b>C</b>. Measurement of disease severity in flag leaves of wild-type Dongjin rice (□) and OsVOZ2 mutant rice (▨). 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). Lesion lengths were determined 21 days after inoculation. Vertical error bars indicate the standard deviation (SD). The statistical significance was determined using a two-way ANOVA as compared to wild-type Dongjin rice with the post hoc Tukey HSD test (***, P<0.001). <b>D</b>. Growth patterns of the KXO85, <i>xopN</i>_<i>KXO85</i> mutant, and complemented <i>xopN</i>_<i>KXO85</i> mutant in the flag leaves of OsVOZ2 mutant rice (PFG_3A-07565). The data are the average values of 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