Li Chenhao, Ji Chonghui, Huguet-Tapia José C, White Frank F, Dong Hansong, Yang Bing
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.
Mol Plant Pathol. 2019 Oct;20(10):1453-1462. doi: 10.1111/mpp.12820. Epub 2019 Aug 15.
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.
转录激活样效应因子（TALes）是病原菌中最大的III型效应因子家族，在感染过程中起关键作用。水稻白叶枯病菌（Xoo）菌株以及其他一些黄单胞菌病原体的菌株含有大量TALe基因。以往通过传统策略克隆单个或一组TALe基因的技术效率低下且耗时，因为给定基因组中存在多个基因（多达29个拷贝），而且由于单个TALe基因的重复序列（多达33个几乎相同的102核苷酸重复序列），技术上也具有挑战性。因此，只有有限数量的TALe基因被分子克隆和表征，大多数TALe基因的功能仍然未知。在此，我们提出一种简便高效的克隆技术，通过体外同源重组和单链退火选择性克隆TALe基因，并以四种不同的Xoo菌株证明了该方法的可行性。基于吉布森组装策略，设计了两个互补载体，其支架能够优先从基因组片段库中捕获所有TALe基因。与以前使用的方法需要3个月相比，这两种载体系统都能够在大约1个月内从四种Xoo菌株中的每一种克隆出完整的TALe基因，并对水稻中的单个TALes进行功能分析。结果证明了一种推进TALe生物学研究的强大工具，以及该方法在克隆任何感兴趣基因组中多个高度竞争性DNA元件方面广泛应用的潜力。
