Site-specific DNA double-strand break generated by I-SceI endonuclease enhances ectopic homologous recombination in...

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.