Graduate School of Agriculture, Meiji University, Kawasaki, Kanagawa, Japan.
FEMS Microbiol Lett. 2014 Mar;352(2):221-9. doi: 10.1111/1574-6968.12396. Epub 2014 Feb 26.
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.
为了评估 DNA 双链断裂（DSB）对稻瘟病菌体细胞同源重组（HR）的贡献，我们建立了一种新的 DSB 介导的异位 HR 的检测/选择系统。该系统由供体和受体非功能黄色荧光蛋白（YFP）/博来霉素抗性基因（BSD）融合基因和酵母内切酶 I-SceI 基因组成，作为受体特异性 DSB 诱导剂。该系统通过 YFP 荧光和博来霉素抗性的恢复来检测和选择异位 HR 事件。供体和受体转化株的内源性异位 HR 频率较低（>2.1%）。与自发 HR 相比，整合 I-SceI 基因后 HR 增加了约 20 倍，HR 的绝对频率高达 40%，表明 I-SceI 介导的 DSB 通过异位 HR 得到有效修复。此外，为了验证 DSB 对靶向基因替换（TGR）的影响，将携带受体基因的转化株与 DSB 诱导剂一起转染外源供体质粒。没有 DSB 诱导剂时没有观察到 TGR 事件，而有 DSB 诱导剂时则获得了数百个 TGR 事件的菌落。这些结果清楚地表明，引入特异性 DSB 可促进稻瘟病菌的异位 HR 修复。
