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

Table_2_Lack of a Cytoplasmic RLK, Required for ROS Homeostasis, Induces Strong Resistance to Bacterial Leaf Blight in Rice.DOCX

<p>Many scientific findings have been reported on the beneficial function of reactive oxygen species (ROS) in various cellular processes, showing that they are not just toxic byproducts. The double-edged role of ROS shows the importance of the regulation of ROS level. We report a gene, rrsRLK (required for ROS-scavenging receptor-like kinase), that encodes a cytoplasmic RLK belonging to the non-RD kinase family. The gene was identified by screening rice RLK mutant lines infected with Xanthomonas oryzae pv. oryzae (Xoo), an agent of bacterial leaf blight of rice. The mutant (ΔrrsRLK) lacking the Os01g02290 gene was strongly resistant to many Xoo strains, but not to the fungal pathogen Magnaporthe grisea. ΔrrsRLK showed significantly higher expression of OsPR1a, OsPR1b, OsLOX, RBBTI4, and jasmonic acid-related genes than wild type. We showed that rrsRLK protein interacts with OsVOZ1 (vascular one zinc-finger 1) and OsPEX11 (peroxisomal biogenesis factor 11). In the further experiments, abnormal biogenesis of peroxisomes, hydrogen peroxide (H₂O₂) accumulation, and reduction of activity of ROS-scavenging enzymes were investigated in ΔrrsRLK. These results suggest that the enhanced resistance in ΔrrsRLK is due to H₂O₂ accumulation caused by irregular ROS-scavenging mechanism, and rrsRLK is most likely a key regulator required for ROS homeostasis in rice.</p

Table_4_Lack of a Cytoplasmic RLK, Required for ROS Homeostasis, Induces Strong Resistance to Bacterial Leaf Blight in Rice.DOCX

<p>Many scientific findings have been reported on the beneficial function of reactive oxygen species (ROS) in various cellular processes, showing that they are not just toxic byproducts. The double-edged role of ROS shows the importance of the regulation of ROS level. We report a gene, rrsRLK (required for ROS-scavenging receptor-like kinase), that encodes a cytoplasmic RLK belonging to the non-RD kinase family. The gene was identified by screening rice RLK mutant lines infected with Xanthomonas oryzae pv. oryzae (Xoo), an agent of bacterial leaf blight of rice. The mutant (ΔrrsRLK) lacking the Os01g02290 gene was strongly resistant to many Xoo strains, but not to the fungal pathogen Magnaporthe grisea. ΔrrsRLK showed significantly higher expression of OsPR1a, OsPR1b, OsLOX, RBBTI4, and jasmonic acid-related genes than wild type. We showed that rrsRLK protein interacts with OsVOZ1 (vascular one zinc-finger 1) and OsPEX11 (peroxisomal biogenesis factor 11). In the further experiments, abnormal biogenesis of peroxisomes, hydrogen peroxide (H₂O₂) accumulation, and reduction of activity of ROS-scavenging enzymes were investigated in ΔrrsRLK. These results suggest that the enhanced resistance in ΔrrsRLK is due to H₂O₂ accumulation caused by irregular ROS-scavenging mechanism, and rrsRLK is most likely a key regulator required for ROS homeostasis in rice.</p

Table_3_Lack of a Cytoplasmic RLK, Required for ROS Homeostasis, Induces Strong Resistance to Bacterial Leaf Blight in Rice.DOCX

<p>Many scientific findings have been reported on the beneficial function of reactive oxygen species (ROS) in various cellular processes, showing that they are not just toxic byproducts. The double-edged role of ROS shows the importance of the regulation of ROS level. We report a gene, rrsRLK (required for ROS-scavenging receptor-like kinase), that encodes a cytoplasmic RLK belonging to the non-RD kinase family. The gene was identified by screening rice RLK mutant lines infected with Xanthomonas oryzae pv. oryzae (Xoo), an agent of bacterial leaf blight of rice. The mutant (ΔrrsRLK) lacking the Os01g02290 gene was strongly resistant to many Xoo strains, but not to the fungal pathogen Magnaporthe grisea. ΔrrsRLK showed significantly higher expression of OsPR1a, OsPR1b, OsLOX, RBBTI4, and jasmonic acid-related genes than wild type. We showed that rrsRLK protein interacts with OsVOZ1 (vascular one zinc-finger 1) and OsPEX11 (peroxisomal biogenesis factor 11). In the further experiments, abnormal biogenesis of peroxisomes, hydrogen peroxide (H₂O₂) accumulation, and reduction of activity of ROS-scavenging enzymes were investigated in ΔrrsRLK. These results suggest that the enhanced resistance in ΔrrsRLK is due to H₂O₂ accumulation caused by irregular ROS-scavenging mechanism, and rrsRLK is most likely a key regulator required for ROS homeostasis in rice.</p