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An efficient method to clone TAL effector genes from Xanthomonas oryzae using Gibson assembly.
Mol Plant Pathol. 2019 10; 20(10):1453-1462.MP

Abstract

Transcription Activator-Like effectors (TALes) represent the largest family of type III effectors among pathogenic bacteria and play a critical role in the process of infection. Strains of Xanthomonas oryzae pv. oryzae (Xoo) and some strains of other Xanthomonas pathogens contain large numbers of TALe genes. Previous techniques to clone individual or a complement of TALe genes through conventional strategies are inefficient and time-consuming due to multiple genes (up to 29 copies) in a given genome, and technically challenging due to the repetitive sequences (up to 33 nearly identical 102-nucleotide repeats) of individual TALe genes. Thus, only a limited number of TALe genes have been molecularly cloned and characterized, and the functions of most TALe genes remain unknown. Here, we present an easy and efficient cloning technique to clone TALe genes selectively through in vitro homologous recombination and single-strand annealing, and demonstrate the feasibility of this approach with four different Xoo strains. Based on the Gibson assembly strategy, two complementary vectors with scaffolds that can preferentially capture all TALe genes from a pool of genomic fragments were designed. Both vector systems enabled cloning of a full complement of TALe genes from each of four Xoo strains and functional analysis of individual TALes in rice in approximately 1 month compared to 3 months by previously used methods. The results demonstrate a robust tool to advance TALe biology and a potential for broad usage of this approach to clone multiple copies of highly competitive DNA elements in any genome of interest.

Authors+Show Affiliations

Department of Plant Pathology, Nanjing Agricultural University, Nanjing, 210095, Jiangsu Providence, P.R. China. Division of Plant Sciences, University of Missouri, Columbia, MO, 65211, USA.Division of Plant Sciences, University of Missouri, Columbia, MO, 65211, USA.Department of Plant Pathology, University of Florida, Gainesville, FL, 32611, USA.Department of Plant Pathology, University of Florida, Gainesville, FL, 32611, USA.Department of Plant Pathology, Nanjing Agricultural University, Nanjing, 210095, Jiangsu Providence, P.R. China.Division of Plant Sciences, University of Missouri, Columbia, MO, 65211, USA. Donald Danforth Plant Science Center, St. Louis, MO, 63132, USA.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

31414714

Citation

Li, Chenhao, et al. "An Efficient Method to Clone TAL Effector Genes From Xanthomonas Oryzae Using Gibson Assembly." Molecular Plant Pathology, vol. 20, no. 10, 2019, pp. 1453-1462.
Li C, Ji C, Huguet-Tapia JC, et al. An efficient method to clone TAL effector genes from Xanthomonas oryzae using Gibson assembly. Mol Plant Pathol. 2019;20(10):1453-1462.
Li, C., Ji, C., Huguet-Tapia, J. C., White, F. F., Dong, H., & Yang, B. (2019). An efficient method to clone TAL effector genes from Xanthomonas oryzae using Gibson assembly. Molecular Plant Pathology, 20(10), 1453-1462. https://doi.org/10.1111/mpp.12820
Li C, et al. An Efficient Method to Clone TAL Effector Genes From Xanthomonas Oryzae Using Gibson Assembly. Mol Plant Pathol. 2019;20(10):1453-1462. PubMed PMID: 31414714.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - An efficient method to clone TAL effector genes from Xanthomonas oryzae using Gibson assembly. AU - Li,Chenhao, AU - Ji,Chonghui, AU - Huguet-Tapia,José C, AU - White,Frank F, AU - Dong,Hansong, AU - Yang,Bing, Y1 - 2019/08/15/ PY - 2019/8/16/pubmed PY - 2020/6/23/medline PY - 2019/8/16/entrez KW - Xanthomonas KW - Gibson assembly KW - TAL effectors KW - rice SP - 1453 EP - 1462 JF - Molecular plant pathology JO - Mol Plant Pathol VL - 20 IS - 10 N2 - Transcription Activator-Like effectors (TALes) represent the largest family of type III effectors among pathogenic bacteria and play a critical role in the process of infection. Strains of Xanthomonas oryzae pv. oryzae (Xoo) and some strains of other Xanthomonas pathogens contain large numbers of TALe genes. Previous techniques to clone individual or a complement of TALe genes through conventional strategies are inefficient and time-consuming due to multiple genes (up to 29 copies) in a given genome, and technically challenging due to the repetitive sequences (up to 33 nearly identical 102-nucleotide repeats) of individual TALe genes. Thus, only a limited number of TALe genes have been molecularly cloned and characterized, and the functions of most TALe genes remain unknown. Here, we present an easy and efficient cloning technique to clone TALe genes selectively through in vitro homologous recombination and single-strand annealing, and demonstrate the feasibility of this approach with four different Xoo strains. Based on the Gibson assembly strategy, two complementary vectors with scaffolds that can preferentially capture all TALe genes from a pool of genomic fragments were designed. Both vector systems enabled cloning of a full complement of TALe genes from each of four Xoo strains and functional analysis of individual TALes in rice in approximately 1 month compared to 3 months by previously used methods. The results demonstrate a robust tool to advance TALe biology and a potential for broad usage of this approach to clone multiple copies of highly competitive DNA elements in any genome of interest. SN - 1364-3703 UR - https://www.unboundmedicine.com/medline/citation/31414714/An_efficient_method_to_clone_TAL_effector_genes_from_Xanthomonas_oryzae_using_Gibson_assembly_ DB - PRIME DP - Unbound Medicine ER -