Characterization and of Ralstonia TAL-Like DNA-Binding Specificities Effectors  

作　　者：Lixin Li Ahmed Atef Agnieszka Piatek Zahir Ali Marek Piatek Mustapha Aouida Altanbadralt Sharakuu Ali Mahjoub Guangchao Wang Suhail Khan Nina V. Fedoroff Jian-Kang Zhu Magdy M. Mahfouz 

机构地区：[1]Center for Desert Agriculture, King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia [2]Department of Biological Sciences, Faculty of Sciences, King Abdulaziz University, PO Box 80203, Jeddah 21589, Kingdom of Saudi Arabia [3]Imaging Core Laboratory, King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia [4]Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907, USA

出　　处：《Molecular Plant》2013年第4期1318-1330,共13页分子植物（英文版）

摘　　要：Transcription activator-like effectors （TALEs） from Xanthomonas sp. have been used as customizable DNA- binding modules for genome-engineering applications, Ralstonia solanacearum TALE-like proteins （RTLs） exhibit similar structural features to TALEs, including a central DNA-binding domain composed of 35 amino acid-long repeats. Here, we characterize the RTLs and show that they localize in the plant cell nucleus, mediate DNA binding, and might function as transcriptional activators. RTLs have a unique DNA-binding architecture and are enriched in repeat variable di-residues （RVDs）, which determine repeat DNA-binding specificities. We determined the DNA-binding specificities for the RVD sequences ND, HN, NP, and NT. The RVD ND mediates highly specific interactions with C nucleotide, HN interacts spe- cifically with A and G nucleotides, and NP binds to C, A, and G nucleotides. Moreover, we developed a highly efficient repeat assembly approach for engineering RTL effectors. Taken together, our data demonstrate that RTLs are unique DNA-targeting modules that are excellent alternatives to be tailored to bind to user-selected DNA sequences for targeted genomic and epigenomic modifications. These findings will facilitate research concerning RTL molecular biology and RTL roles in the pathogenicity of Ralstonia spp.Transcription activator-like effectors （TALEs） from Xanthomonas sp. have been used as customizable DNA- binding modules for genome-engineering applications, Ralstonia solanacearum TALE-like proteins （RTLs） exhibit similar structural features to TALEs, including a central DNA-binding domain composed of 35 amino acid-long repeats. Here, we characterize the RTLs and show that they localize in the plant cell nucleus, mediate DNA binding, and might function as transcriptional activators. RTLs have a unique DNA-binding architecture and are enriched in repeat variable di-residues （RVDs）, which determine repeat DNA-binding specificities. We determined the DNA-binding specificities for the RVD sequences ND, HN, NP, and NT. The RVD ND mediates highly specific interactions with C nucleotide, HN interacts spe- cifically with A and G nucleotides, and NP binds to C, A, and G nucleotides. Moreover, we developed a highly efficient repeat assembly approach for engineering RTL effectors. Taken together, our data demonstrate that RTLs are unique DNA-targeting modules that are excellent alternatives to be tailored to bind to user-selected DNA sequences for targeted genomic and epigenomic modifications. These findings will facilitate research concerning RTL molecular biology and RTL roles in the pathogenicity of Ralstonia spp.

关 键 词：Ralstonia solanacearum genome engineering TAL effectors TALE activators and repressors TALE nucleases （TALENs） targeted genome modifications. 

分 类 号：Q78[生物学—分子生物学] S513[农业科学—作物学]