Analysis of marker gene transfer from chloroplasts to mitochondria in heat-shocked and selection-pressured tobacco

Angiosperm mitochondrial genomes have highly complex and diverse structures that are partly due to frequent insertions of nuclear and chloroplast DNA (cpDNA) into mitochondrial DNA (mtDNA). This suggests the existence of mechanisms for gene transfer from chloroplasts to mitochondria, but these have yet to be discovered. In this study, we aimed to capture chloroplast-to-mitochondrion gene transfer by analyzing the translocation of a marker gene, sul, encoding a bacterial dihydropteroate synthase that confers sulfonamide resistance in tobacco (Nicotiana tabacum), to mtDNA. First, we created tobacco chloroplast transformants in which sul, surrounded on both sides by ∼1 kb of mitochondrial homologous sequences that enable targeted integration into mtDNA, was introduced into the chloroplast genome. Heat shock enhanced sul expression in the transformants, suggesting that chloroplast degradation can stimulate gene transfer from chloroplasts to mitochondria. Shoot regeneration using the heat-shocked chloroplast transformants under sulfadiazine selection resulted in several transformants with moderate resistance to sulfadiazine. Deep sequencing analysis of the target mitochondrial locus detected sul in the sulfadiazine-resistant (SR) plants, but an integration efficiency was 0.0011–0.0051 %. We validated the results by ruling out sul integration into nuclear mitochondrial DNA (NuMT). From these results, we propose the established system is capable of capturing gene transfer from chloroplasts to mitochondria in tobacco, but the transfer efficiency is substantially lower than those from organelles to nucleus.