Markerless gene deletion in Ralstonia solanacearum based on its natural transformation competence.
Markerless gene deletion in Ralstonia solanacearum based on its natural transformation competence.
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基于自然转化能力的青枯菌无标记基因缺失
DOI：
10.3389/fmicb.2022.977580
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发表时间：
2022
影响因子：
5.2
通讯作者：
中科院分区：
文献类型：
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青枯雷尔斯顿菌复合体（RSSC）是一组革兰氏阴性细菌病原体，能够感染多种植物和农作物，引起严重的维管束枯萎病。与细菌毒力相关的基因的功能分析对于阐明控制细菌致病性的分子机制至关重要。为此，一种有效的基因删除方法将有很大帮助。在本研究中，我们通过利用其自然转化能力和 FLP/FRT 重组系统，开发一种高效且简单的无标记基因删除方法。我们发现使用 PCR 产物的自然转化比基于质粒的三亲交配和电穿孔提供更高的转化频率。因此，我们通过整合目标基因的上游和下游DNA片段以及侧翼为FRT位点的抗生素抗性基因来生成基因缺失融合PCR片段，并通过自然转化将PCR产物递送至青枯菌细胞中。使用这种方法，我们敲除了从不同寄主植物分离的几个青枯菌菌株中分别与胞外多糖（EPS）生物合成和调控相关的epsB和phcA基因，频率从5（1E-08）到45（1E-08）。为了去除抗生素标记基因，将表达FLP酶的质粒引入上述敲除突变体中，从而能够去除标记基因。因此，自然转化和FLP/FRT重组系统的有效结合为假定毒力基因的功能研究和阐明青枯菌致病机制提供了一种简单有效的方法。
Ralstonia solanacearum species complex (RSSC) is a group of Gram-negative bacterial pathogen capable of infecting numerous plants and crops, causing severe vascular wilt diseases. Functional analysis of the genes associated with bacterial virulence is critical for elucidating the molecular mechanisms that govern the bacterial pathogenicity. To this end, an efficient gene deletion method would be of great help. In this study, we set to develop an efficient and simple markerless gene deletion method by exploiting its natural transformation competence and the FLP/FRT recombination system. We found that natural transformation using PCR products provided much higher transformation frequency than the plasmid-based triparental mating and electroporation. We thus generated the gene deletion fusion PCR fragments by incorporating the upstream and downstream DNA fragments of the target gene and an antibiotic resistance gene flanked by FRT sites, and delivered the PCR products into R. solanacearum cells through natural transformation. Using this method, we knocked out the epsB and phcA genes, which are associated with exopolysaccharide (EPS) biosynthesis and regulation, respectively, in several R. solanacearum strains isolated from different host plants at a frequency from 5 (1E-08) to 45 (1E-08). To remove the antibiotic marker gene, the plasmid expressing the FLP enzyme was introduced into the above knockout mutants, which enabled removal of the marker gene. The effective combination of natural transformation and the FLP/FRT recombination system thus offers a simple and efficient method for functional study of putative virulence genes and for elucidation of R. solanacearum pathogenic mechanisms.