MitoTALEN-mediated mitochondrial gene-knockout revealed a cytoplasmic male sterility-causative gene in Oryza sativa cv. Tadukan. Ayumu Takatsuka 1, Tomohiko Kazama 2, Shinichi Arimura 3, Kinya Toriyama 1 (1 Grad. Sch. Agr. Sci.,Tohoku Univ., 2 Fac. Agr., Kyushu Univ., 3 Grad. Sch. Life Sci., Univ. Tokyo) Cytoplasmic male sterility (CMS) is a powerful tool to produce F1 hybrid seeds preventing self-pollination. CMS plants produce unfunctional anthers or pollen caused by the interaction between mitochondrial and nuclear genes. A CMS line, TAA was obtained by successive backcrossing between Oryza sativa cv. Tadukan (a cytoplasm donor) and cv. Taichung 65 (T65, a recurrent pollen parent). In TAA, mature pollen looked normal, but anthers did not dehisce, resulting in no seed set or extremely low rates of seed set at a few panicles. Up to now, we have identified a TAA-specific mitochondrial gene, named orf312, as a strong candidate gene causing Tadukan-type CMS. In this study, we knocked out orf312 using 'mitoTALEN', transcription activator-like effector nuclease (TALEN) with mitochondrial localization signals. The mitoTALEN is expected to cause double strand breaks in the orf312 coding sequence, leading to the deletion of orf312 by homologous recombination in the mitochondrial genome. Among 24 regenerated T0 plants, investigation of orf312 by PCR and Southern blot analysis demonstrated that 19 plants lost orf312. All these plants exhibited anther dehiscence, resulting in restoration of seed set. Other plants, in which the orf312 coding sequence was detected, anther did not dehisce and remained sterile. The adjacent fragments connected to the downstream of orf312 were amplified by fusion primer and nested integrated PCR. Sequencing analysis revealed that fertility-restored plants were classified into three kinds of recombination events via different homologous sequences. Through loss-of-function approach, we concluded that orf312 was a CMS-causative gene in Tadukan-type CMS.