Site-specific DNA double-strand break generated by I-SceI endonuclease enhances ectopic homologous recombination in Pyricularia oryzae
Site-specific DNA double-strand break generated by I-SceI endonuclease enhances ectopic homologous recombination in Pyricularia oryzae
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DOI：
10.1111/1574-6968.12396
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发表时间：
2014-03-01
影响因子：
2.1
通讯作者：
Kuwata, Shigeru
中科院分区：
文献类型：
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作者：
Arazoe, Takayuki;Younomaru, Tetsuya;Kuwata, Shigeru
为了评价DNA双链断裂(DSBs)在稻瘟病菌体细胞同源重组(HR)中的作用，我们建立了DSBs介导的异位同源重组检测/选择系统。该系统由供体和受体的无功能黄色荧光蛋白/杀菌素S脱氨酶融合基因和酵母内切酶I-SCEI基因作为受体特异的DSB诱导子组成。该系统通过恢复YFP荧光和杀菌素S抗性来检测和选择异位HR事件。供受体转化系的内源异位频率较低(>；2.1%)。与自发性HR相比，整合I-SCEI基因后HR增加20倍，HR绝对频率高达40%，表明I-SCEI介导的DSB可通过异位HR有效修复。此外，为了验证DSB对靶向基因替换(TGR)的影响，将外源供体质粒与DSB诱导剂一起导入含有受体基因的转化系。在没有DSB诱导剂的情况下没有观察到TGR事件，而使用DSB诱导剂获得了数百个由TGR事件产生的集落。这些结果清楚地表明，位点特异性DSB的引入促进了米曲霉的异位HR修复。
To evaluate the contribution of DNA double-strand breaks (DSBs) to somatic homologous recombination (HR) in Pyricularia oryzae, we established a novel detection/selection system of DSBs-mediated ectopic HR. This system consists of donor and recipient nonfunctional yellow fluorescent protein (YFP)/blasticidin S deaminase (BSD) fusion genes and the yeast endonuclease I-SceI gene as a recipient-specific DSB inducer. The system enables to detect and select ectopic HR events by the restoration of YFP fluorescence and blasticidin S resistance. The transformed lines with donor and recipient showed low frequencies of endogenous ectopic HR (>2.1%). Compared with spontaneous HR, c. 20-fold increases in HR and absolute frequency of HR as high as 40% were obtained by integration of I-SceI gene, indicating that I-SceI-mediated DSB was efficiently repaired via ectopic HR. Furthermore, to validate the impact of DSB on targeted gene replacement (TGR), the transformed lines with a recipient gene were transfected with an exogenous donor plasmid in combination with the DSB inducer. TGR events were not observed without the DSB inducer, whereas hundreds of colonies resulting from TGR events were obtained with the DSB inducer. These results clearly demonstrated that the introduction of site-specific DSB promotes ectopic HR repair in P.oryzae.