(A) Deduced amino acid sequence of XB15. Italic bold represents residues that interact with metal cofactors in human PP2Cα [47]. The PP2C catalytic domain is underlined, and the unique insertion sequence in the PP2C catalytic domain is marked with italics. Predicted nuclear targeting signals are double-underlined (http://www.psort.org/).(B) Phylogenetic analysis at POL and PLLs from Arabidopsis and rice.
For determining phylogenetic relationships, protein sequences of the conserved PP2C domains for putative plant proteins related to POL were aligned and then used in ClustalV. Ten thousand bootstrap replicates were performed. Rice PP2Cs with low homology to XB15, Os03g04430, and Os12g39120 were defined as outgroups. Sequences used in this analysis were as follows: POL, PLL1 (AAC3686), PLL2 (NP195860), PLL3 (NP187551), PLL4 (AAL38775), and PLL5 (AAK32783) from Arabidopsis; and XB15, Os03g60650, Os03g25600, Os02g46490, Os05g02110, and Os03g16760 from rice.

(A) Plots of Xoo PR6 populations over 16 d in segregants (F₂) of XA21/XB15 19A-72 and 17A-21, Kitaake, and NTAP lines. For each time point, the bacterial populations were separately determined for three leaves. Capped, vertical bars represent standard deviation of values (cfu/leaf) from three samples. Experiments were repeated over three times with similar results. NTAP-XB15 19A (Myc-Xa21/Ntap-Xb15 [−/+], 19A-72–3, −7, and −12); NTAP-XB15 17A (−/+, 17A-21–4, −5, and −8); XA21/XB15 19A (+/+, 19A-72–5, −6, −8, and −11); XA21/XB15 17A (+/+, 17A-21–2, −3, −7, and 11); Myc-XA21 (+/−, 19A-72–1, −10 and 17A-21–6, −10).
(B) Lesion length development of Xoo PR6 inoculated plants, segregants (F₂) of XA21/XB15 19A-72 and 17A-21, Kitaake, and NTAP lines. Each data point represents the average and standard deviation of at least four samples. NTAP-XB15 19A (Myc-Xa21/Ntap-Xb15 (−/+), 19A-72–12); NTAP-XB15 17A (−/+, 17A-21–8); XA21/XB15 19A (+/+, 19A-72–14); XA21/XB15 17A (+/+, 17A-21–15); Myc-XA21 (+/−, 17A-21–10).

(A) Three-week-old segregants (F₄) of NF9014–1 (Tos17 Xb15) and Myc-XA21 were inoculated with Xoo PR6 and lesion lengths were measured 18 DAI. Each data point represents the average and standard deviation of at least three samples.
(B) Bacterial populations were determined at 0 and 18 DAI. For each time point, the mean of the bacterial cfu/leaf from three plants per genotype is shown. Error bars indicate the standard deviation. The experiment was repeated three times with similar results.

(A) HIS-XB15ΔN was incubated with either glutathione alone (lane 1), glutathione-bound GST-XA21K668 (lane 2), or GST (lane3) in buffer containing 2 mM MgCl₂. After washing three times, proteins were eluted with 2× sample buffer, and analyzed by SDS-PAGE and immunoblotting with anti-GST or anti-HIS antibody. The experiment was repeated three times with similar results.
(B) Immunodetection of Myc-XA21 protein in transgenic plants carrying Myc-Xa21 under control of its native promoter before (Extract) and after immunoprecipitation (IP). Myc-XA21 and Myc-XA21^cp give bands at approximately 140 and 100 kDa, respectively, corresponding to the predicted mass of each protein [42]. A nonspecific band (n.s.) of 95 kDa was detected.
(C) Immunodetection of XB15 in Kitaake (Kit) and transgenic plants overexpressing NTAP-XB15 (17A and 19A). Anti-XB15 antibody detected at band of about 70 kDa of XB15 in Kit and of 95 kDa of in NTAP-XB15 in expressing plants (17A and 19A). Anti-actin antibody was used as a loading control.
(D) Co-immunoprecipitation of XB15 and XA21 after Xoo PR6 inoculation. XA21 was detected after immunoprecipitation with agarose conjugated anti-Myc antibodies. Total protein extracts from 15 g of leaf tissue of Kitaake (Kit), mock-treated Myc-XA21, and Xoo PR6-inoculated Myc-XA21 at the indicated time points were incubated with anti-Myc antibody to immunoprecipitate XA21 complex. The precipitates were used for protein gel blot analysis using anti-Myc antibody (top) or anti-XB15 antibody (bottom). Myc-XA21 and Myc-XA21^cp give bands at about 140 and 100 kDa, respectively, and XB15 is detected as a band of 70 kDa.

The LRR domain of TLR4 recognizes lipopolysaccharide signal (LPS) and transduces the signal across the cell membrane [91]. An adaptor molecule myeloid differentiation factor 88 (MyD88) associated with TLR4 recruits the non-RD serine/threonine kinase, IRAK1, which associates with tumor necrosis factor receptor associated factor 6 (TRAF6) in cytoplasm [102]. TRAF6 forms a complex with TAK1 [93] in which the kinase activity of, TAK1 mediates downstream events, such as inhibitor of κ kinase complex and NF-κB [5,95].
In Arabidopsis, flg22 binds to FLS2, which transduces the pathogen signal through its cytoplasmic non-RD kinase domain, and activates MAPK cascade [15]. Several WRKY transcription factors acting downstream of MAPK cascade to induce defense-related genes including PR genes [97]. KAPP, a PP2C, blocks the activated FLS signaling and attenuates the downstream innate immune response [33]. In rice, AvrXA21 binding to XA21 activates its cytoplasmic non-RD kinase [18,20]. XA21 transphosphorylates proteins such as XB3, which are required for the defense response [42]. In the nucleus, WRKY transcription factors activate defense-related genes such as PR1 and PR10 of XA21 signaling pathway. Recruitment of XB15 to Ser697 in the XA21 JM domain and subsequent dephosphorylation of phosphorylated residue(s) attenuates XA21 signaling.

(A) Schematic diagram of XA21 used as bait and the result of yeast two-hybrid analysis. Domains are as described in previous report [18]. Amino acid residue numbers and the total length of each XA21 constructs are indicated.
(B) Protein gel blot analysis with protein extracted from yeast transformants. The extracts were prepared from the yeast strain pEGY48/p8op-LacZ cotransformed with (1) pAD-GAL4 containing full length of Xb15 (AD-XB15) and pLexA containing XA21K668 (LexA-XA21K668), (2) AD-XB15 and LexA-XA21K(TDG), (3) AD-XB15 and LexA-XA21K668^K736E, (4) AD-XB15 and LexA-XA21JM, (5) AD-XB15 and LexA-XA21K668^S686A, (6) AD-XB15 and LexA-XA21K668^T688A, (7) AD-XB15 and LexA-XA21K668^e, (8) AD-XB15 and LexA-XA21K668^{S686A,T688A,S689A}, (9) AD-XB15 and LexA-XA21K668^S697A, or (10) AD-XB15 and pLexA that serves as a vector control. Using the with anti-LexA antibody, AD-XB15 (unpublished data) and LexA-XA21 mutants give bands corresponding to the expected mass of the fusion proteins. The vector control expresses a 24-kDa band corresponding to the LexA protein.

(A) Immunodetection of Myc-XA21 and NTAP-XB15. Equal amounts (50 μg) of total protein form Kitaake wild type (Kit) and progeny (F₁) from a cross with Myc-XA21 and NTAP-XB15 19A and 17A (19A-72 and 17A-21, respectively) extracted and analyzed by SDS-PAGE, and immunobloted with anti-Myc or PAP antibodies. Equal loading of total proteins was confirmed by Coomassie blue staining of protein (lower panels).
(B) Phenotype/genotype cosegregation analysis of segregants (F₂) of XA21/XB15 19A and 17A. Segregants of 19A-64, 19A-72, 17A-18, and 17A-21, Kitaake wild type (Kit), and transgenic rice carrying Myc-Xa21 (Myc-XA21) were inoculated at 6-wk-old age, and the lesion lengths were measured 12 d after inoculation. Each data point represents the average and standard deviation of at least four samples. Progeny carrying the Xa21 alone or both of Xa21 and Xb15 are labeled with a black bar (+/−) or gray bar (+/+). Progeny missing both genes are labeled with a white bar (−/−). Immunodetection of XB15 in the XA21/XB15 17A and 19A is shown under the graph for each segregant. Total protein was extracted from each plant and protein gel blot analysis was performed with PAP antibody and anti-actin antibody to detect NTAP-XB15 and actin protein, respectively.
(C) Water-soaked disease lesions on two leaves from four genotype of segregants (F₂) (Myc-Xa21/Ntap-Xb15; −/−, +/−, +/+, and −/+) and transgenic plants overexpressing NTAP only (NTAP) 16 d after Xoo PR6 inoculation.

(A) Representation of Xb15 genomic DNA structure and the position of the Tos17 insertion site. Four exons (boxes) are separated by three introns (lines). The protein-coding regions are indicated by shaded boxes with the start codon (ATG) and stop codon (TAA). Gray boxes correspond to untranslated sequences. Arrows indicate locations of primers designed for RT-PCR.
(B) The transcripts levels of Xb15 in Nipponbare wild type (WT), homozygous Tos17 insertion (−/−), heterozygote (−/+), no-insertion plants (+/+). Total RNA was extracted from fully extended 5-wk-old leaves. RT-PCR analysis was performed with Xb15 specific primers indicated in (A). 18S rRNA was used for an internal control.
(C) Morphological phenotype of the Tos17 insertional line (M₅) of Xb15. Photographs were taken 6 wk after germination. Symbols for genotypes for Tos17 in Xb15 are as described above.
(D) PR gene expressions in plants Xb15 Tos17 mutants (M₅). Total RNA was extracted from 5-wk-old wild type and insertion mutant line plants, and RT-PCR was performed with specific primers for PR1a, PR1b, PR10, major birch allergen (Betv1), and PBZ1. Control RT-PCR reactions were carried out with EF1α and 18S rRNA. Symbols for genotypes for Tos17 in Xb15 are same as described above.

(A) XB15 protein is a serine/threonine phosphatase. Five micrograms of GST-XB15FL (full length XB15), GST-XB15ΔN (catalytic domain only), or GST was incubated with: no substrate, 100 μM substrate, 100 μM substrate plus 1 mM sodium vanadate, 100 μM substrate plus 50 mM NaF.
(B) XB15 protein is a PP2C. Five micrograms of GST-XB15FL, GST-XB15ΔN, or GST was incubated with 100 μM substrate in each of the three buffers specific for PP2A, PP2B, and PP2C activity.
(C) The XB15 protein purified from rice plants carries a PP2C activity. NTAP-XB15 and NTAP were purified from transgenic plants and 1 μg of each protein was incubated with 100 μM substrate in PP2C buffer. For the boiled NTAP-XB15, 1 μg of NTAP-XB15 was boiled in water for 20 min. The data are average value of three experiments. Error bars represent standard deviations.

(A) Immobilized GST-XA21K668 was autophosphorylated with [γ-³²P] ATP in kinase buffer. After washing, activated GST-XA21K668 was incubated with HIS-XB15ΔN (1 μg) at 37 °C for the indicated time (0, 1, 5, and 20 min). The samples were subsequently boiled in the presence of 2× sample buffer, separated by SDS-PAGE, stained with Coomassie brilliant blue R250 and destained (Coomassie). For the autoradiography, the dried gel was exposed to an image plate (Autorad).
(B) Activated GST-XA21K668 was incubated with 1 μg (+) or 10 μg (++) of HIS-XB15ΔN at 37 °C for 5 min. The samples were subsequently boiled in the presence of 2× sample buffer, separated by SDS-PAGE, stained with Coomassie brilliant blue R250 and destained (Coomassie). For the autoradiography, the dried gel was exposed to an image plate (Autorad).