Targeted knock-out of a conserved plant mitochondrial gene by genome editing

Fusion proteins derived from transcription activator-like effectors (TALEs) haverecently emerged as genome editing tools for mitochondria. TALE nucleases(TALENs) have been applied to delete chimeric reading frames and duplicated(redundant) genes, but produced complex genomic rearrangements, due to theabsence from mitochondria of a pathway for non-homologous end-joining. Herewe report the targeted deletion of a conserved mitochondrial gene, nad9, encodinga subunit of respiratory complex I. By generating a large number of TALENmediated mitochondrial deletion lines, we isolated, in addition to mutants withrearranged genomes, homochondriomic mutants harboring a clean nad9 deletion.Characterization of the nad9 knock-out plants revealed impaired complex Ibiogenesis, male sterility, and defects in leaf and flower development. We showthat these defects can be restored by expressing a functional Nad9 protein from thenuclear genome, thus creating a synthetic cytoplasmic male sterility (CMS) system.Together, our data (i) demonstrate the feasibility of using genome editing to studymitochondrial gene functions by reverse genetics, (ii) highlight the role of complexI in plant development, and (iii) provide proof-of-concept for the construction ofsynthetic CMS systems for hybrid breeding by genome editing.