TALEN‐mediated depletion of the mitochondrial gene orf312 proves that it is a Tadukan‐type cytoplasmic male sterility‐causative gene in rice

SUMMARY Cytoplasmic male sterility (CMS) is a trait that causes pollen or anther dysfunctions, resulting in the lack of seed setting. CMS is considered to be caused by the expression of a unique mitochondrial open reading frame referred to as CMS‐associated gene. orf312 has been reported as a CMS‐associated gene of Tadukan‐type CMS (TAA) in rice (Oryza sativa L.), which exhibits impaired anther dehiscence; however, evidence thereof has not yet been reported. Here, we took a loss‐of‐function approach, using a mitochondria‐targeted transcription activator‐like effector nuclease (mitoTALEN) designed to knock out orf312 in TAA, to prove that orf312 indeed is a CMS‐causative gene. Out of 28 transgenic TAA plants harboring the mitoTALEN expression vector, deletion of orf312 was detected in 24 plants by PCR, Southern blot, and sequencing analyses. The 24 plants were grouped into three groups based on the deleted regions. All orf312‐depleted TAA plants exhibited recovery of anther dehiscence and seed setting. The depletion of orf312 and fertility restoration was maintained in the next generation, even in mitoTALEN expression cassette null segregants. In contrast, orf312‐retaining plants were sterile. These results provide robust evidence that orf312 is a Tadukan‐type CMS‐causative gene. Significance Statement Cytoplasmic male sterility (CMS) is caused by the expression of a mitochondrial gene. Here, the mitochondrial CMS‐associated gene orf312 was knocked out using mitochondria‐targeted transcription activator‐like effector nuclease (mitoTALEN), resulting in the recovery of fertility in Tadukan‐type CMS rice, even in mitoTALEN expression cassette null segregants.