Disruption of the mitochondrial gene orf352 partially restores pollen development in cytoplasmic male sterile rice

Abstract

Plant mitochondrial genomes sometimes carry cytoplasmic male sterility (CMS)-associated genes. These genes have been harnessed in agriculture to produce high-yielding F₁ hybrid seeds in various crops. The gene orf352 was reported to be an RT102-type CMS gene in rice (Oryza sativa), although a causal demonstration of its role in CMS is lacking. Here, we employed mitochondrion-targeted transcription activator-like effector nucleases (mitoTALENs), to knock out orf352 from the mitochondrial genome in the cytoplasmic male sterile rice RT102A. We isolated 18 independent transformation events in RT102A that resulted in genome editing of orf352, including its complete removal from the mitochondrial genome in several plants. Sequence analysis around the mitoTALEN target sites revealed the repair of their induced double-strand breaks via homologous recombination. Near the 5ʹ target site, repair involved sequences identical to orf284, while repair of the 3ʹ target site yielded various new sequences that generated new chimeric genes consisting orf352 fragments. Plants with a new mitochondrial gene encoding amino acids 179 to 352 of ORF352 exhibited the same shrunken pollen grain phenotype as RT102A, whereas plants either lacking orf352 or harboring a new gene encoding amino acids 211 to 352 of ORF352 showed partial rescue of pollen viability and germination, although they failed to set seed. These results demonstrated that disruption of orf352 partially restored pollen development, indicating that amino acids 179 to 210 from ORF352 may contribute to the establishment of pollen abortion.