Journal
PLANT PHYSIOLOGY
Volume 187, Issue 1, Pages 236-246Publisher
OXFORD UNIV PRESS INC
DOI: 10.1093/plphys/kiab236
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Funding
- Japanese Science and Technology Agency (PRESTO)
- Japan Society for the Promotion of Science [JP16H06182, JP18H02172, JP20H05680, JP21H02169, JP19H02927]
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In this study, orf352 gene was successfully knocked out from the mitochondrial genome of a CMS rice variety using mitoTALENs technology. The results showed that plants with a chimeric mitochondrial gene encoding amino acids 179-352 of ORF352 exhibited the same shrunken pollen grain phenotype, while plants lacking orf352 partially restored pollen development but failed to set seed.
Plant mitochondrial genomes sometimes carry cytoplasmic male sterility (CMS)-associated genes. These genes have been harnessed in various crops to produce high-yielding F-1 hybrid seeds. The gene open reading frame 352 (orf352) was reported to be an RT102-type CMS gene in rice (Oryza sativa), although the mechanism underlying its role in CMS is unknown. Here, we employed mitochondrion-targeted transcription activator-like effector nucleases (mitoTALENs) to knockout orf352 from the mitochondrial genome in the CMS 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 their induced double-strand breaks were repaired via homologous recombination. Near the 5'-target site, repair involved sequences identical to orf284, while repair of the 3'-target site yielded various new sequences that generated chimeric genes consisting of orf352 fragments. Plants with a chimeric mitochondrial gene encoding amino acids 179-352 of ORF352 exhibited the same shrunken pollen grain phenotype as RT102A, whereas plants either lacking orf352 or harboring a chimeric gene encoding amino acids 211-352 of ORF352 exhibited partial rescue of pollen viability and germination, although these plants failed to set seed. These results demonstrated that disruption of orf352 partially restored pollen development, indicating that amino acids 179-210 from ORF352 may contribute to pollen abortion.
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