Abstract
SummaryTranscription 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.
Highlights
Phytopathogenic bacteria of the genus Xanthomonas cause a variety of plant diseases including those inflicting severe losses on the economically important crop plants worldwide (Leyns et al, 1984)
The Transcription Activator-Like effector (TALe) gene sequences were retrieved from genome data of thirty-three Xanthomonas oryzae pv. oryzae (Xoo), ten X. oryzae pv. oryzicola (Xoc), two X. citri pv. vignicola and one X. citri pv. malvacearum strains in the NCBI (National Center for Biotechnology Information) database (Supplementary Table S1)
We present two complementary vector systems for cloning TALe gene fragments selectively from a genomic pool of fragments through in vitro homologous recombination and single strand annealing, a protocol known as the Gibson assembly method
Summary
Phytopathogenic bacteria of the genus Xanthomonas cause a variety of plant diseases including those inflicting severe losses on the economically important crop plants worldwide (Leyns et al, 1984). To make a cloning vector for selectively cloning of the SphI fragments of TALe genes, the previously cloned TALe gene pthXo1, which encodes the major virulence effector for the Asian Xoo strain PXO99A (Yang and White, 2004), was used as the backbone of TALe gene vector without the central SphI repetitive region.
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