Speaker: Dr. Joachim Forner, Max-Planck-Institute for Molecular Plant Physiology, Potsdam

Targeted modification of the plant mitochondrial genome has been a long-standing desired goal of the research community and only in recent years it has become feasible. Key to this are transcription activator-like effectors (TALEs), or more precisely base editors and nucleases (TALENs) that build on these. TALENs are protein-only, freely programmable site-specific DNA endonucleases. TALEN-encoding transgenes can be delivered to the nucleus via classical transformation techniques, while the resulting proteins can be easily targeted to the mitochondria by adding an N-terminal mitochondrial presequence.
Using TALENs, we have managed not only to introduce point mutations, but now also to completely remove the nad9 gene from the mitochondrial genome of Nicotiana tabacum, creating a full knock-out. While the removal of nad9 was coupled to genome rearrangements via recombinations in most of the lines, we also succeeded in isolating a few lines with a clean deletion, without any additional alterations in the mitochondrial genome.
The mutant plants lack the entire complex I in their respiratory chain, but are viable. They display a distinct phenotype, most notably delayed germination and growth as well as altered leaf and flower morphology. They are also male sterile. By allotopic expression of nad9 in the nucleus, we could fully rescue the nad9 knock-out phenotype, including reversion to male fertility. We thus have created an artificial cytoplasmic male sterility (CMS) system.

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