Supporting InformationChen et al. 10.1073/pnas.0912311107SI Materials and MethodsPlasmid Construction for Yeast Two-Hybrid Analyses. XA21K668, atruncated XA21 containing its entire juxtamembrane (JM) andentirekinasedomains,wasPCRamplifiedandclonedintopENDR/D-TOPO (Invitrogen). The specific primer pair used is 5′-CAC-CATGTCATCACTCTACTTGCTTATA-3′/5′-TCAGAATTC-AAGGCTCCCACCTTC-3′. After the sequence was verified, it wassubcloned into pNLexAgtw [a gateway compatible vector modifiedfrompNLexA(Clontech)]byusingGatewayLRClonaseEnzymeMix(Invitrogen). The resulting plasmid was designated as a BD vector,pNlexAXA21K668. The single amino acid mutant pNlexA-XA21K668K736E in pNlexA-XA21K668 was introduced by muta-genesis using the QuikChange site-directed mutagenesis kit (Stra-tagene) following the manufacture’s protocol. The primer pair usedfor the site-directed mutagenesis is 5′-GTTGCAGTGGAAGTAC-TAAAGCTTGAAAATCC-3′/5′-GGATTTTCAAGCTTTAGTA-CTTCCACTGCAAC-3′. XB24, XB24(1-146), and XB24(146-198)were PCR amplified using primer pairs, 5′-CACCATGGGTTGG-CGTTGGCACGAC-3′/5′-TTACACATCTGTAATCTTGCTGC-3′, 5′-CACCATGGGTTGGCGTTGGCACGAC-3′/5′-TTAGA-CACCAAAAGACTTCAAGAAATC-3′, and 5′-CACCGTCCC-TTCCATCAATGAAAGG-3′/5′-TTAGACACCAAAAGACTT-CAAGAAATC-3′, respectively, and cloned into pENDR,generating pENTR-XB24, pENTR-XB24(1-146), and pENTR-XB24(146-198). They were recombined with pB42ADgc [a gatewaycompatible vector modified from pB42AD (Clontech)] to yield theADvectors, pB42AD-XB24, pB42AD-XB24(1-146), andpB42AD-XB24(146-198).

Plasmid Construction for Producing GST- or His-Fusion Protein.XA21K668, was amplified by the primer pair 5′-TCTAGAATG-TCATCACTCTACTTGCTTATA-3′/5′- GTCGACTCAGAATT-CAAGGCTCCCACC-3′ (XbaI and SalI recognition sites were un-derlined) and cloned into pGEM-Teasy vector (Promega) to obtainpGEM-T-XA21K668. pGEM-T-XA21K668 was cut by XbaI/SalIand cloned into pGEX-KG-1 vector precut by XbaI/SalI, givingpEGX-KG-1-XA21K668 (for GST-XA21K668). The pGEX-KG-1wasmodified from thepGEX-KG.ForHis fusion vectors, pGEM-T-XA21K668 K736E (for GST-XA21K668K736E) was created by site-directed mutagenesis using pGEM-T-XA21K668K736E as DNAtemplate and primer pair 5′-GTTGCAGTGGAAGTACTAAAGC-TTGAAAATCC-3′/5′-GGATTTTCAAGCTTTAGTACTTCCA-CTGCAAC-3′.XB24andGFPwerePCRamplifiedbyprimerpairs,5′-TCTAGAATGGGTTGGCGTTGGCACGAC-3′/5′-GTCGAC-TTACACATCTGTAATCTTGCTGC-3′and5′-TCTAGAATGG-CGAGTAAAGGAGAAGAAC-3′/5′-GTCGACTTATTTGTA-TAGTTCATCCATGC-3′, respectively, and cloned into pGEM-Teasy vector to generate pGEM-T-XB24 and pGEM-T-GFP. Thenthey were cloned into pHis vector precut by XbaI/SalI to yield pHis-XB24andpHis-GFP.The singleaminoacidmutantpHis-XB24S154A

was created by site-directed mutagenesis from pHis-XB24 usingprimer pair 5′-GAAAGGGAGGCGAGCTCATTTG-3′/5′-CAAA-TGAGCTCGCCTCCCTTTC-3′.

Plasmid Construction for RNA Interference (RNAi) of Xb24. ForRNAiconstruction, a 397-bp fragment was amplified from Xb24 withprimer pair 5′-CACCCTGCGTCGGCAGGCCCTCTG-3′/5′-TT-ACACATCTGTAATCTTGCTGC-3′ and cloned into pENDR tomake pENTR-Xb24-397bp. pENTR-Xb24-397bp was recombinedwith a binary vector pANDA (kindly provided by Ko Shimamoto,Nara Institute of Science and Technology, Japan) to generatepANDA-Xb24RNAi (Xb24Ri).

Plasmid Construction for Overexpression of XB24 and XB24S154A inRice. pENTR-Xb24 was recombined with Ubi-C4300 gateway vec-tor, generating Ubi-C4300-XB24ox. pENTR-Xb24 and pENTR-Xb24S154Awere recombined withUbi-C1300Ntap-new intron vector(1) to make pC1300-Ntap-Xb24 (Ubi-Ntap-Xb24) and pC1300-Ntap-Xb24S154A (Ubi-Xb24S154A). pENTR-Xb24S154Awas created bysite-directed mutagenesis from pENTRXb24 using the primer pairas described above. pC1300-Ntap (Ubi-Ntap) was obtained throughsubcloning the Ntap fragment amplified into Ubi-pC1300 directly.

Plasmid Construction for ProA-Tagged Xa21 Transgenic Plants. Theconstruct pCAMBIA4300-ProA-Xa21 (ProA-Xa21), which isunder the native promoter of Xa21, was created according to thestrategy described in ref. 2. ProA-Xa21 construction was se-quence verified before use.

Yeast Two-Hybrid Assays. The Matchmaker LexA two-hybrid sys-tem (Clontech) was used for yeast two-hybrid assays. YeastpEGY48/p8op-lacZ (Clontech) was cotransformed with the BDand AD vectors by using the Frozen-EZ yeast transformation IIkit (Zymo Research) and spread on an appropriate medium.

Generation of Rice Transgenic and F1 Plants.Rice transformationwasperformed as described in ref. 3. pANDA-Xb24RNAi, ProA-Xa21,Ubi-Ntap-Xb24, Ubi-Ntap-Xb24S154A, and Ubi-Ntap were trans-formed into rice Kitaake. Ubi-Xb24/C4300 was transformed intoXA21 (TP309 background). All crosses were performed using theXa21 as pollen donor unless otherwise indicated. TransformantsofUbi-Xb24/C4300 inProA-Xa21were selectedwithmannose, andtransformants of all other genes were selected with hygromycin.

RNA Extraction, RT-PCR, and Real-Time RT-PCR Analyses. Total RNAwasextracted from leaf tissueusingTRIzol reagent (Invitrogen) andwas reverse-transcribed to cDNA using M-MLV reverse tran-scriptase (Invitrogen). RT-PCR used primer pairs, 5′-TTGTAC-ACTGTGATATTAAATC-3′/5′-TCAGAATTCAAGGCTCCCA-CCTTC-3′ (forXa21), and 5′-CACCCTGCGTCGGCAGGCCC-TCTG-3′/5′-TTACACATCTGTAATCTTGCTGC-3′, (forXb24-RNAi, Xb24ox, Ntap-Xb24, or Ntap-Xb24S154A), 5′-ATGGTGG-TCGACAACAAGTTC-3′/5′-CCCTCCACTAGACAGTGCGC-CG-3′ (forNtap) and 5′-ACGGCGATAACAGCTCCTCTT-3′/5′-CCTCTTCCAGCCTTCCTTCAT-3′ (for actin gene). Real-timeRT-PCRwas performed using the SYBRGREENPCRMaterMixKit (AppliedBiosystems).Theprimer pair 5′-GTGTCCCTTCCAT-CAATGAAAG-3′/5′-TGCTGCTGAATTCTGCGTAGTC-3′ wasused for analysis on the transcripts accumulation of Xb24. The finalresults obtained for the different samples analyzed were stand-ardized to the Ubiquitin 5 RT-PCR product level using the primerpair5′-ATGGCCAACCACTTCGACCG-3′/5′-TAAGCCTGCTG-GTTGTAGACGT-3′.

Plant Growth, Xoo Inoculation, and Disease Resistance Determination.Rice plants were grown in the greenhouse until 3 or 6 weeks of ageand transferred to the growth chamber before Xoo inoculation.Bacterial suspensions (OD600 of 0.5) of Xoo strains PXO99(PXO99AZ, expressing Ax21 activity) and PXO99ΔRaxST(lacking Ax21 activity) were used to inoculate rice by the scis-sors-dip method (4). The disease lesion length and bacterialpopulation accumulated in rice leaf were evaluated as reportedbefore (3). The t test was performed to assess significance.

Expression and Purification of Recombinant Proteins. BL21 codonplus E. coli cells (Invitrogen) were transformed with the recombi-

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nantGST orHis plasmids, and protein expression was induced with120 μM isopropyl β-D-thiogalactopyranoside (OD600 = 0.6) for 7 h(for GST-fusion protein) or 4 h (for His-fusion protein) at 30 °C.GST-fusion proteins were purified using glutathione Sepharose 4B(Amersham) according to the manufacturer’s instructions. His-fusion proteins were purified using Ni-NTA beads (Qiagen) ac-cording to the manufacturer’s instructions with some modificationsto remove the possible ATPases from E. coli extracts. The mod-ifications mainly include the following: (i) we reduced sonicationtime (10 sec, three times) on the resuspended cells; (ii) beforeprotein elution, we performed two washes using 10 mL of 100 mMimidazole with 50 mM NaH2PO4, and 300 mM NaCl after theregular wash; and (iii) we dialyzed the eluted proteins in 5 mMTris·HCl (pH 7.6) containing 0.5 mM phenylmethanesulfonyl flu-oride (PMSF) at 4 °C overnight using dialysis cassettes (ThermoScientific). The proteins were then used for analyses after the pu-rification was confirmed by using SDS/PAGE separating and Coo-massie brilliant blue staining. For ProA-XA21 or Ntap-fusionprotein purification from rice plants, protein extracts were preparedfrom 5 g of leaf tissue in 25mL of ice-cold extraction buffer [20mMTris·HCl (pH 8.0), 150 mM NaCl, 0.1% Triton X-100, 2.5 mMEDTA, 2 mM benzamidine (Sigma-Aldrich), 10 mM β-mercap-toethanol, 20mMNaF, 1mMPMSF, 1%ProteaseMixture (Sigma-Aldrich), 10 μM leupeptin, 5% glycerol]. After filtering throughdouble layers ofMiracloth (Calbiochem) followedby centrifugationtwice at 13,000× g for 10minat 4 °C, the supernatantwasmixedwith400 μL of IgG Sepharose beads (Amersham Biosciences) and in-cubated at 4 °C for 1 h. The beads bond ProA-XA21 protein werethen washed four times in 1 mL of protein extraction buffer lackingprotease inhibitors before use. The beads bondNtap-fusion protein

were washed four times in 1 mL of protein extraction buffer lackingprotease inhibitors and twice in 0.4mLof 5mMammoniumacetate(pH 5.0). The protein was eluted with 2 mL of 0.5 M HOAC (pH3.4), neutralized with one-tenth volume of 1 M Tris·HCl (pH 8.0),and dialyzed overnight in 5 mM Tris·HCl (pH 7.6) containing 0.5mMPMSF. After dialysis, the protein was concentrated by acetoneprecipitation and then resuspended in 5 mM Tris·HCl (pH 7.6) foranalyses.

Coimmunoprecipitation of Rice Proteins. To coimmunoprecipitateProA-XA21 and XB24, the supernatant from protein extracts wasmixed with 400 μL of IgG Sepharose beads (Amersham Bio-sciences) according the method described in ref. 2.

Phylogenetic and Molecular Evolutionary Analyses. Proteinmotif waspredicted by ScanPrositeResultsViewer (www.expasy.ch/prosite).Phylogenetic andmolecular evolutionary analyses were conductedusing MEGA version 4 (5). Briefly, amino acid sequences fromentire proteins were aligned by ClustalWusing default parameterswithout manual editing, and the maximum parsimony phyloge-netic tree was constructed using default parameters with the ex-ception that “use all sites” was chosen for the parameter of gaps/missing data.

Antibodies. Anti-LexA (Clontech), anti-HA (Covance), anti-Myc(SantaCruzBiotechnology), andprobePeroxidaseAnti-Peroxidase(PAP)(Sigma)werepurchasedasindicated.Theanti-XB24antibodywas generated by Pacific Immunology (www.pacificimmunology.com) using the synthetic peptide SELVESKTENTEEDVand theirestablished procedures.

1. Rohila JS, et al. (2006) Protein-protein interactions of tandem affinity purification-tagged protein kinases in rice. Plant J 46:1–13.

2. Wang YS, et al. (2006) Rice XA21 binding protein 3 is a ubiquitin ligase required for fullXa21-mediated disease resistance. Plant Cell 18:3635–3646.

3. Chern M, Canlas PE, Fitzgerald HA, Ronald PC (2005) Rice NRR, a negative regulator ofdisease resistance, interacts with Arabidopsis NPR1 and rice NH1. Plant J 43:623–635.

4. Song WY, et al. (1995) A receptor kinase-like protein encoded by the rice diseaseresistance gene, Xa21. Science 270:1804–1806.

5. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary GeneticsAnalysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599.

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MGWRWHDDGDDGGRGLGDIPDLAGGGGGGDGERCATRR

VVQSRCHTEEVEPGRFVRKCEKTEQLLRDCVGRPSELVESK

TENTEEDVTDEMKSGSLSLGFPTNEPFAFPGLRSDIEALEKG

LFGSIGSFLDDAERMTNDFLKSFGVPSINERESSSFDGQPTG

R H I G G Q P A G R H I E E G T A K D T K Q N D Y A E F S S K I T D V

B

Genomic sequence1 1888 bp

Untranslated regionCoding region Intron

5’ 3’

A

P-[SAP]-[LIV]-[DNH]-{LKGN}-{F}-{S}-S-{DCPH}-S

C

Fig. S1. Coding region of XB24 and its amino acid sequence. (A) Transcriptional and translational pattern of the Xb24 gene. (B) Deduced XB24 amino acidssequence. The ATP synthase α and β subunits signature motif is underlined, and the predicted catalytically active residue S154 is in boldface. (C) ATPaseconsensus pattern. The active residue is in boldface. The amino acids in square brackets are conserved and replaceable by one another. The amino acids in curlybraces are not conserved but preferred. The others are conserved.

Fig. S2. Phylogenetic relationships among XB24 and other proteins containing the ATPase motif from rice, Arabidopsis, and human. Os, At, and Hs denoterice, Arabidopsis, and human, respectively.

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C B ) m

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K

Fig. S3. Development of homozygous line A114 from the transgenic ProA-Xa21 plant 13. (A) Lesion lengths of T1 plants were measured after inoculation withthe Xoo strain PXO99. T1 plants were generated from the T0 transgenic ProA-Xa21 plant 13 that carried a single locus insertion. Xa21 and Kitaake rice wereused as controls. The lesion length difference between the rice plants carrying the transgene ProA-Xa21 and the plants lacking the transgene is significant (P =2.96 × 10−7). (B) Photograph of rice leaves from Kitaake, a representative resistant plant (labeled “R”) and a representative susceptible plants (labeled “S”),respectively, at 14 days after PXO99 inoculation. (C) Lesion length measurements of A114 at 14 days after PXO99 inoculation. A114-1 and A114-2 represent twoindividuals from the line A114. The lesion length difference between A114-1 and Kitaake plants is significant (P = 8.35 × 10−6) as well as the difference betweenA114-2 and Kitaake (P = 2.36 × 10−5). (D) ProA-XA21 protein expression was determined on plants A114-1 and A114-2. (Upper) ProA-XA21 as detected by theprobe Peroxidase Anti-Peroxidase (PAP). (Lower) SDS/PAGE gel after staining with Coomassie brilliant blue (CBB) as a loading control. In both (A) and (B), eachdata point represents the average and standard deviation of three leaves from each inoculated individual.

Pi

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Fig. S4. ATPase activity assays on E. coli-produced proteins His-XB24 and His-XB24S154A. His-GFP protein purified from E. coli and commercial ATPase fromporcine cerebral cortex (Sigma) are included as negative and positive controls, respectively. The different dosages (0.6, 1.8, 5.5, and 16.5 μg) of His-XB24 proteinwith same incubation time or the same dosage of His-XB24 protein with different incubation time (5, 15, 30, 60, and 120 min) were analyzed respectively. Pi,free phosphate group, a product of the ATP hydrolysis reaction.

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ProA-Xa21

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Light IgG chain

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e k a a t i K

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Fig. S5. In vitro kinase autophosphorylation analysis on ProA-XA21 in the presence or absence of His-XB24. The ProA-XA21 proteins immunoprecipitated fromrice leaf tissue 0 days, 1 day (1d), and 2 days (2d) after inoculation with PXO99 were used for kinase autophosphorylation analyses in the presence or absence ofthe purified His-XB24 protein. Kinase autophosphorylation was carried out as described [Liu GZ, et al. (2002) J Biol Chem 277:20264–20269], with the exceptionthat the exposure time is 3 days. (Upper) autoradiogram from the XA21 autophosphorylation experiment. (Lower) CBB-stained gel containing im-munoprecipitated ProA-XA21 and His-XB24 proteins. His-XB24 and the heavy and light chains of IgG (from IgG Sepharose 6 Fast Flow that was used forpurification of ProA-XA21) are indicated. The ProA-XA21 bands are not visible because of the low quantity obtained from the purification.

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Fig. S6. Effects of XB24 ATPase on XA21 autophosphorylation. (A) In vitro autophosphorylation assay on GST-XA21K668 (labeled GST-K668) in the presence ofE. coli-expressed His-XB24 (wild type) or His-GFP. (B) In vitro autophosphorylation assay on GST-XA21K668 in the presence of the E. coli-expressed, ATPasecatalytically inactive mutant His-XB24S154A. The same amount of GST-XA21K668 and the same amount of His-XB24, His-GFP, and His-XB24S154A were usedfor these experiments. (A Upper and B Upper) Representative autoradiograms. (A Lower and B Lower) Quantitative results from three independent experi-ments. The autophosphorylation assays on GST-XA21K668 in the absence of XB24, XB24S154A, or GFP proteins are the control experiments (labeled with CK)and their autophosphorylation levels were arbitrarily considered as “1.”

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Fig. S7. Correlation of reduced expressions of Xb24 with enhanced XA21-mediated resistance. (A) Identification of two independent Xb24RNAi plants thathave reduced expression of Xb24. (Upper) Results of RT-PCR using the primer pair specific for Xb24. (Lower) Results of RT-PCR using a primer pair specific forthe actin gene. Ri-3 and Ri-9 represent Xb24RNAi-3 and Xb24RNAi-9, respectively. (B) Correlation of lesion length with levels of Xb24 expression. Lesion lengthswere measured on 3-week-old progeny from the crosses of Xb24RNAi-3/Xa21 and Xb24RNAi-9/Xa21, respectively, at 14 days after PXO99 inoculation. Threeleaves of each plant were inoculated and measured (Top). The lesion length difference between Xa21 and Xa21 in the presence of Xb24RNAi (containing bothXa21 gene and the Xb24RNAi) plants is significant (P = 3.66 × 10−7). The relative level of transcription of Xb24 in the plants as indicated was derived from themeans of real-time RT-PCR experiments with duplicate independent samples. The results obtained for the different plants were normalized to ubiquitin 5expression and the Kitaake plant (Middle). (Bottom) PCR-based genotyping results of the Xa21 gene (indicated by Xa21-specific amplification) and theXb24RNAi construct (indicated by the Gus linker amplification). Error bars indicate SDs.

Xa21 TP309

Xa21/Xb24ox-1, -2, -3, -4, -5

TP309/Xb24ox-1, -2

)M

c(

ht

gn

el

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oi

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Fig. S8. Overexpression of Xb24 compromises XA21-mediated resistance. Lesion length measurements of rice leaves 14 days after Xoo inoculation. Xa21/Xb24ox-1, -2, -3, -4, and -5 represent five independent T0 transgenic plants of Xb24 overexpressed in the in Xa21 TP309 genetic background. TP309/Xb24ox-1and -2 represent two independent T0 transgenic plants of Xb24 overexpressed in TP309. Each bar represents the average and SD of three leaves.

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) m

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Fig. S9. Requirement for the XB24 ATPase in regulation of XA21 biological function. Lesion lengths were measured for Xa21, Kitaake, Xa21/Xb24, and Xa21/Xb24S154A plants at 14 days after Xoo strain PXO99 inoculation. Xa21/Xb24 and Xa21/Xb24S154A plants were generated using NtapXb24ox and Ntap-Xb24S154Aox plants, as parents to cross with ProA-Xa21 plants. Protein levels of ProA-XA21, Ntap-XB24, and Ntap-XB24S154A were separately determined byWestern blotting using probe Peroxidase Anti-Peroxidase (PAP). Error bars indicate SDs.

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