Volume 7, Issue 5, Pages 912-915 (May 2014) A Potential Disease Susceptibility Gene CsLOB of Citrus Is Targeted by a Major Virulence Effector PthA of Xanthomonas citri subsp. citri  Li Zheng , Zou Lifang , Ye Gang , Xiong Li , Ji Zhiyuan , Zakria Muhammad , Hong Ni , Wang Guoping , Chen Gongyou   Molecular Plant  Volume 7, Issue 5, Pages 912-915 (May 2014) DOI: 10.1093/mp/sst176 Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 1 Identification of a Potential C. sinensis Susceptibility Gene Targeted by a Major Virulence Factor PthA of X. citri Subsp. citri. (A) Deletion mutagenesis of four tal genes in Xcc049. Genomic DNAs of the five strains were digested by BamHI and hybridized with a 2.3-kb internal SphI fragment containing the central 102-bp repeat region of pthA. Because the RVDs (repeat-variable diresidues) of TalC and TalD have the same length, Xcc049A (containing talA,B,C,D), Xcc049B(containing talB,C,D), Xcc049C(containing talC,D), and Xcc049D (containing talD) show three, two, one, and one band(s), respectively. Bands below the 2-kb position are the fragments left in the plasmid(s) when the tale genes were gradually deleted. (B) Virulence test of citrus caused by Xcc049A, Xcc049B, Xcc049C, Xcc049D, Xcc049E, and Xcc049E-PthA. Bacterial suspensions (approximately at 1×108 CFU ml–1) were infiltrated into citrus leaves using a needleless syringe, and symptoms were recorded 12 d post inoculation. (C) Bacterial growth in citrus leaves. Numbers represent CFU per square centimeter in leaf tissue investigated at 0, 1, 2, 4, 8, and 12 d after infiltration with strains Xcc049A, Xcc049B, Xcc049C, Xcc049D, Xcc049E, and Xcc049E-PthA. Data represent the mean ± the standard deviation from three replicates and each in three leaves. (D) RVDs of PthA and TalC and their predicted UPT boxes. The optimal UPT boxes are deduced from the known RVD specificities by TAL Binding Codes. RVDs in four different colors (green, blue, red, and dark blue) bind their nucleotides in corresponding colors. The UPT box of CsLOB was screened from the promoter library of C. sinesis genome using the two online programs. Mismatcheed RVD–base pair combinations are shaded in dark gray. (E) Expression profiles of CsLOB in citrus activated by PthA, TalA, TalB, TalC, and TalD. Messenger RNAs of citrus leaves, infiltrated with Xcc049A, Xcc049B, Xcc049C, Xcc049D, Xcc049E, and Xcc049E-PthA strains at 1×108 CFU ml–1, respectively, were collected 24h post inoculation and sequenced by RNA-seq. The upside figure shows the relative expression levels of CsLOB induced by six strains compared to water. The downside displays the log2 values of relative expression of CsLOB induced by six strains compared with each other. (F) CBC Symptoms induced by an artificially designed dTALE that binds a region upstream of the natural UPT box of CsLOB. Xcc049E-PthA, Xcc049E-dTALE, and Xcc049E bacterial suspensions (approximately at 1×108 CFU ml–1) were infiltrated into citrus leaves using a needleless syringe, and CBC symptoms were recorded 12 d post inoculation. Water is used as a negative control. (G) Transcriptional analysis of CsLOB activated by TALEs of X. citri subsp. citri. GUS reporter constructs are codelivered via A. tumefaciens into N. benthamiana with (+) and without (–) constructs TALEs PthA, TalA, TalB, TalC, and TalD, respectively. PthXo1 of X. oryzae pv. oryzae and its target Os8N3 (pOs8N3) were used as a positive control. Promoter of CsSweet1 was used as a control sequence. The GUS activity was determined 2 d post inoculation by stained leaf disks (0.8 cm in diameter) with X-Gluc (5-bromo-4-chloro-3-indolyl-b-D-glucuronide). A blue color indicates positive reaction. Error bars indicate standard deviation (n = 3 samples). 4-MU, 4-methyl-umbelliferone. Molecular Plant 2014 7, 912-915DOI: (10.1093/mp/sst176) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions