Targeted gene disruption of ATP synthases 6‐1 and 6‐2 in the mitochondrial genome of Arabidopsis thaliana by mitoTALENs

Abstract

We recently achieved targeted disruptions of cytoplasmic male sterility (CMS)-associated genes in the mitochondrial genomes of rice and rapeseed by using mitochondria-targeted transcription activator-like effector nucleases (mitoTALENs). It was the first report of stable and heritable targeted gene modification of plant mitochondrial genomes. Here, we attempted to use mitoTALENs to disrupt two mitochondrial genes in the model plant Arabidopsis thaliana(Arabidopsis) using three different promoters and two types of TALENs. The targets were the two isoforms of the ATP synthase subunit 6 gene, atp6-1 and atp6-2. Each of these genes was successfully deleted and the mitochondrial genomes were recovered in a homoplasmic state. The nuclear genome also has a copy of atp6-1, and we were able to confirm that it was the mitochondrial gene and not the nuclear pseudogene that was knocked out. Among the three mitoTALEN promoters tried, the RPS5A promoter was the most effective. Conventional mitoTALENs were more effective than singlemolecule mito-compactTALENs. Targeted mitochondrial gene deletion was achieved by crossing as well as by floral-dip transformation to introduce the mitoTALEN constructs into the nucleus. The gene disruptions were caused by large (kb-size) deletions. The ends of the remaining sequences were connected to distant loci, mostly by illegitimate homologous recombinations between repeats.

Targeted editing of the Arabidopsis mitochondrial genome

“…And despite the popularity of CRISPR, these older techniques are still being used. In this issue, Shin‐Ichi Arimura and his colleagues report the first stable transformation of the Arabidopsis mitochondrial genome using TALENs (Arimura et al , 2020).…”

Organelle genome assembly uncovers the dynamic genome reorganization and cytoplasmic male sterility associated genes in tomato

“…In many cases, fusion genes have been reported to be CMS-associated genes, e.g., orf307 in Oryza sativa 21 and orf72 in Brassica oleracea 22 , and to produce cytotoxic proteins, which lead to male sterility. Knockout mutagenesis with mitoTALENs 14,15 targeting the candidate genes would be useful to identify CMS-associated genes and to generate CMS lines from normal tomato cultivars.…”

“…More recently, it has become possible to alter or edit gene sequences of mitochondrial genomes with TALEN technology 14 . This technology has been used to disrupt CMS-associated genes in mitochondrial genomes and thereby generate Arabidopsis thaliana , Oryza sativa , and Brassica napus with CMS 14,15 .…”

Effects of mitoTALENs-Directed Double-Strand Breaks on Plant Mitochondrial Genomes

“…The recombinations altered the configuration of the genome by introducing or deleting repeats and changing the gene order. We also used mitoTALENs to knock out two redundant mitochondrial genes (atp6-1 and atp6-2) in Arabidopsis, which resulted in similar alterations of the genome [39]. Homologous recombinations usually faithfully repair DSBs by using templates.…”

“…genes (atp6-1 and atp6-2) in Arabidopsis, which resulted in similar alterations of the genome [39]. Homologous recombinations usually faithfully repair DSBs by using templates.…”