Volume 8, Issue 2, Pages 290-302 (February 2015) XA23 Is an Executor R Protein and Confers Broad-Spectrum Disease Resistance in Rice  Chunlian Wang, Xiaoping Zhang, Yinglun Fan, Ying Gao, Qinlong Zhu, Chongke Zheng, Tengfei Qin, Yanqiang Li, Jinying Che, Mingwei Zhang, Bing Yang, Yaoguang Liu, Kaijun Zhao  Molecular Plant  Volume 8, Issue 2, Pages 290-302 (February 2015) DOI: 10.1016/j.molp.2014.10.010 Copyright © 2015 The Author Terms and Conditions

Figure 1 Map-Based Cloning of Xa23. (A) Genetic map of the Xa23 locus. (B) Physical map of the Xa23 locus in BAC clone B608 of CBB23 (72114-bp insert) relative to the corresponding chromosomal region of Nipponbare (Nip). Green bars represent two transposon insertions in B608. Numbers above and below the bars indicate the nucleotide positions of Nip chromosome 11 (GenBank accession: NC_008404.2) and B608, respectively. Relative positions of the transformation-competent artificial chromosome (TAC) clones derived from B608 are indicated by vertical dotted lines. (C) Genomic organization of the Xa23 locus. The colinear regions of B608 (CBB23) and Nip were aligned and regions with 98%–100% nucleotide sequence homology are indicated by the light blue background. Numbers above or below the bars indicate the nucleotide positions. Green boxes represent predicted exons of the inserted 7.6-kb transposon flanked by the 245-bp terminal inverted repeats TIR-L and TIR-R (yellow arrows) in B608. The predicted 342-bp ORF (ORF113) is depicted as a red box. (D) Leaves of transgenic rice lines T189-11, W17-17, and the wild-type MDJ8 show the lesions caused by PXO99A. Statistical analysis on the lesion lengths is shown in Supplemental Figure 2. Molecular Plant 2015 8, 290-302DOI: (10.1016/j.molp.2014.10.010) Copyright © 2015 The Author Terms and Conditions

Figure 2 Deduced Amino Acid Sequence of XA23 Aligned with XA10 by the Program Clustal X. The output of alignment was shaded by GENEDOC 2.1 according to the following amino acid property similarity groups: 1 = D/N, 2 = E/Q, 3 = S/T, 4 = K/R, 5 = F/Y/W, and 6 = L/I/V/M. Within the aligned region, the identity and similarity ratios are 49.6% and 63.7%, respectively. Dashes indicate the missing sequence or gaps generated by the alignment. The three transmembrane helices (underlined with red lines) of XA23 were predicted by the SOSUI program (http://bp.nuap.nagoya-u.ac.jp/sosui/sosui_submit.html). Four transmembrane helices (M1–M4) of XA10 are underlined in green. Asterisks indicate the amino acid positions 10, 30, 50, 70, 90, 110 and 130. Molecular Plant 2015 8, 290-302DOI: (10.1016/j.molp.2014.10.010) Copyright © 2015 The Author Terms and Conditions

Figure 3 Comparison of Nucleotide Sequences between the Resistant Xa23 Allele and Susceptible xa23 Alleles. The nucleotide sequence of CBB23 was used as the reference sequence. The 26 bp (partially) of the TIR-L (totally 245 bp) is highlighted in yellow. The 28-bp EBEAvrXa23 is highlighted in green. The 7-bp polymorphism in JG30 is highlighted in purple. The 21-bp EBEJG30 is boxed. Three tandem repeats in the Xa23 promoter are indicated by arrows. The cDNA of Xa23 (partially) is shown and underlined, including the 56-bp 5′-untranslated region (UTR), and the first 42 bp of ORF113 highlighted in blue. Dashed lines indicate regions of nucleotide deletions. Asterisks indicate nucleotides identical to those of CBB23. Molecular Plant 2015 8, 290-302DOI: (10.1016/j.molp.2014.10.010) Copyright © 2015 The Author Terms and Conditions

Figure 4 Association of Xa23 Transcription and Bacterial Blight Resistance in Rice. (A) Transcription assays of Xa23 (ORF113) in rice lines JG30, CBB23, and Nipponbare (Nip). RNAs from leaves inoculated with PXO99A were used for quantitative (q) RT-PCR with gene-specific primers. (B) Transcription assays of Xa23 in transgenic rice lines T189-11, W17-17, and the wild-type MDJ8. RNAs from leaves 3 days post inoculation with water (Mock), PXO99A, PXO99A-avrXa23-knockout mutant P99M5, and its avrXa23-complementary strain P99M5-L were used for qRT-PCR with gene-specific primers. (C) Disease reactions of RNAi rice R350 and the wild-type ZW1 to PXO99A using the leaf-clipping method. Leaf lesions 14 days post inoculation are indicated by arrows and shown by representative leaves. Statistical analysis of the lesion length is shown in Supplemental Figure 2. (D) Transcription assays of Xa23 in RNAi rice R350 and wild-type ZW1. RNAs from leaves 3 days post inoculation with water (Mock), PXO99A, avrXa23-knockout mutant P99M5 and avrXa23-complementary strain P99M5-L of Xoo were used for qRT-PCR with gene-specific primers. (E) Design and architecture of the designed TALE dTE-JG30. Twenty-one nucleotides (arrowed) including the 7-bp polymorphism (red letters) in the promoter of xa23 allele in JG30 were chosen as the TALE binding element (EBEJG30) of dTE-JG30. Numbers above the sequence indicate the nucleotide positions relative to the translation start codon of ORF113 (blue letters) in CBB23. The transposon terminal inverted repeat TIR-L is highlighted in yellow. Grey boxes represent the N- and C-terminal regions of dTE-JG30. Green, red, yellow, and blue ovals represent the 34-aa repeats with RVDs NI, HD, NG and NN, respectively. Blue bars and the red triangle represent the nuclear localization motifs (NLS) and acidic activator domain (AD), respectively. (F) Disease reactions of JG30 (J), CBB23(C), and MDJ8 (M) to dTE-JG30-containing Xoo strain P99_dTE-JG30 and the wild-type PXO99A inoculated using the leaf-clipping method. Resistant and susceptible lesions 14 days post inoculation are shown by representative leaves. Statistical analysis of the lesion length is shown in Supplemental Figure 2. (G) dTE-JG30 specifically triggers HR (brown areas) in JG30. Bacterial suspensions of P99_dTE-JG30 and the wild-type PXO99A were infiltrated into rice leaves using a needleless syringe. MDJ8 was used as a negative control. Photographs were taken 4 days post infiltration. (H) Transcription assays of xa23 in JG30 and MDJ8. RNAs from leaves infiltrated with P99_dTE-JG30 were used for qRT-PCR with gene-specific primers. The rice ubiquitin gene was used as an internal control for qRT-PCR. Each value in (A), (B), (D), and (H) represents the mean ± SD (n = 3 replicates). Student’s t-test analysis indicates a significant difference (compared with control, ** P < 0.01). Molecular Plant 2015 8, 290-302DOI: (10.1016/j.molp.2014.10.010) Copyright © 2015 The Author Terms and Conditions

Figure 5 XA23 Triggers Strong HR in Dicot Plants. (A) Schematic representations of pCAMBIA1305-based constructs harboring different lengths of B608-derived DNA fragments containing ORF113 (red arrow) with indications of the start and stop codons. The grey boxes indicate the 5′ and 3′ UTRs. The region of EBEAvrXA23 was depicted as a small yellow box. Numbers above the bar indicate nucleotide positions of B608. Orange, green, and black bars represent ORF113-containing fragments triggering strong, weak, and no HRs, respectively. Blue arrows represent the CaMV35S promoter (35S). The translational start codon ATG of ORF113 was mutated to ACG in construct HR25 and AGG in HR27. Constructs HP26, HP29, HP38, and HP40 were modified from P26, P29, P38, and P40, respectively, by deleting the enhancer of the 35S promoter upstream of the HYG (R) gene in pCAMBIA1305. (B) HR assays by transient transformation (Agroinfiltration) of the indicated constructs in N. benthamiana. (C) HR assays of constructs with the 35S enhancer removed. Cell suspensions of A. tumefaciens strains harboring the constructs were individually infiltrated (AvrXa23-) or co-infiltrated (AvrXa23+) with avrXa23-expressing A. tumefaciens strain into N. benthamiana leaves. For all the HR analysis, dashed cycles mark the inoculated areas. Leaves were sampled 4 days post infiltration and cleared in ethanol to visualize the HR (brown areas). Molecular Plant 2015 8, 290-302DOI: (10.1016/j.molp.2014.10.010) Copyright © 2015 The Author Terms and Conditions

Figure 6 Interactions of the TALE AvrXa23 and the Xa23 and xa23 Promoter Fragments. (A) Alignment of predicted EBE in Xa23 and the RVDs of AvrXa23. The central repeats of AvrXa23 are represented by the RVDs. An asterisk (*) represents the missing 13th amino acid. The vertical lines indicate the matching between the RVDs and the nucleotides of target DNA in the promoter of Xa23, or lack thereof, for mismatches. (B) Nucleotide sequences of Xa23 promoter fragments B28, B43, and B43m (mutant of B43, the mutated nucleotides are indicated by italic letters) from the resistant rice CBB23, and the xa23 promoter fragments J34 and J43 from the susceptible rice JG30. The 7-bp indel (insertion/deletion) is indicated by red letters. (C) Electrophoretic mobility shift assay with the His-AvrXa23 fusion protein and biotin-labeled Xa23 and xa23 promoter fragments (probes) in a 4% nondenaturing polyacrylamide gel. Protein amounts are in femtomoles. Positions of the bound and free probe are indicated at the left. (D) Electrophoretic mobility shift assay competition experiment between AvrXa23 and different amounts (in femtomoles) of the unlabeled competing DNA (B43, cold probe). Molecular Plant 2015 8, 290-302DOI: (10.1016/j.molp.2014.10.010) Copyright © 2015 The Author Terms and Conditions