mitoTALENs to explore mitochondrial DNA segregation and repair

Abstract

Plant mitochondrial genome (mtDNA) is far more complex and larger than its animal or fungi counterparts. The complexity of the plant mtDNA originates from very active homologous recombination (HR) that generates alternative mtDNA configurations and promotes rapid genome evolution. Several key factors of mitochondrial HR have been identified, yet many have multiple paralogs, or have not been identified yet.
We use the genome editing tool TALENs (Transcription Activator Like Effector Nuclease) to further explore plant mtDNA replication and repair. TALENs are designed to have a Fok1 endonuclease domain to make targeted double strand breaks. DNA damage leads to the loss of the target mtDNA sequence and an increase in HR. Targeting one of the two atp6 gene copies, which encodes ATP synthase subunit 6, caused a phenotype with developmental difficulties similar to the one observed in mutants of the HR repair system. Complete NGS sequencing of the mtDNA following TALEN activity suggests ambiguity in the choice of the HR site to repair DSB, creating heteroplasmy of different repair variants.
To better understand the roles of known HR key factors in repair, their Arabidopsis mutants were transformed with the TALEN constructs. NGS analyses of the mutants’ mtDNA revealed that different major mitotypes are created due to the discrete preference of repeats mobilized for homology-based repair: homology recombination, single-strand annealing, and microhomology-mediated repair. Furthermore, some mutants did not show any trace of repair, suggesting plants can reconstitute the viable mitochondrial genome from pre-existing mitotypes via autonomous replication of sub-genomes. Finally, NGS give clues that there is a mechanism of surveillance over homology recombination activity and genomic equilibrium.

Rokas Kubilinskas1, Elsa Lavigne1, Shin-ichi Arimura2, Jose M. Gualberto1
1Institute of Molecular Biology of Plants-CNRS, University of Strasbourg, France
2Laboratory of Plant Molecular Genetics, The University of Tokyo, Japan