RADA-dependent branch migration has a predominant role in plant mitochondria and its defect leads to mtDNA instability and cell cycle arrest

Abstract

Mitochondria of flowering plants have large genomes whose structure and segregation are modulated by recombination activities. The post-synaptic late steps of mitochondrial DNA (mtDNA) recombination are still poorly characterized. Here we show that RADA, a plant ortholog of bacterial RadA/Sms, is an organellar protein that drives the major branch-migration pathway of plant mitochondria. While RadA/Sms is dispensable in bacteria, RADA-deficient Arabidopsis plants are severely impacted in their development and fertility, correlating with increased mtDNA recombination across intermediate-size repeats and accumulation of recombination-generated mitochondrial subgenomes. The radA mutation is epistatic to recG1 that affects the additional branch migration activity. In contrast, the double mutation radA recA3 is lethal, underlining the importance of an alternative RECA3-dependent pathway. The physical interaction of RADA with RECA2 but not with RECA3 further indicated that RADA is required for the processing of recombination intermediates in the RECA2-depedent recombination pathway of plant mitochondria. Although RADA is dually targeted to mitochondria and chloroplasts we found little to no effects of the radA mutation on the stability of the plastidial genome. Finally, we found that the deficient maintenance of the mtDNA in radA apparently triggers a retrograde signal that activates nuclear genes repressing cell cycle progression.

Disruption of recombination machinery alters the mutational landscape in plant organellar genomes

“…The repeat mediated mtDNA recombination activity we documented in the msh1, radA and recA3 mutants is consistent with the previously documented recombination increases of these mutant backgrounds (Shedge et al 2007;Arrieta-Montiel et al 2009;Rowan et al 2010;Davila et al 2011;Miller-Messmer et al 2012;Zampini et al 2015;Wu et al 2020;Chevigny et al 2022;Zou et al 2022). The absence of an effect in the why2 mutants is interesting given that why2 is the most abundant protein in mitochondrial nucleoids (Fuchs et al 2020) and plants lacking why2 display aberrant mitochondrial morphology (Golin et al 2020;Negroni et al 2024).…”

“…Interestingly, RECA2 is thought to initiate recombination in both pathways and is essential in plants (Miller-Messmer et al 2012;Chevigny et al 2022). The larger SNV and indel increases in the radA mutants than in the recA3 mutants may reflect the relative utilization (and importance) of these two partially redundant HR pathways (Chevigny et al 2022).…”

“…WHY2 is a mitochondrially targeted whirly protein that binds single-stranded DNA to inhibit recombination between small repeated sequences via micro-homology mediated end joining (MMEJ) ( Cappadocia et al 2010 ) and is also the most abundant protein in mitochondrial nucleoids (as measured in A. thaliana cell culture; Fuchs et al 2020 ). RADA is a dual-targeted DNA helicase, which has been shown to accelerate the processing of recombination intermediates and promote mtDNA stability in A. thaliana ( Chevigny et al 2022 ). RECA1 is a plastid-targeted protein that has been proposed to act synergistically with plastid whirly proteins to promote plastid genome integrity either by facilitating polymerase lesion bypass or by reversing stalled replication forks ( Rowan et al 2010 ; Zampini et al 2015 ).…”

Long‐read sequencing characterizes mitochondrial and plastid genome variants in Arabidopsis msh1 mutants

“…The mt genomes of Col‐0 and An‐1 differ by five rearrangements mediated by repeat groups B, I, H‐1/2, L, and H‐1/3, while that of L er has an additional rearrangement mediated by repeat group LL (Figure 5a). The small circles generated by closely located direct repeats (H‐1/2, H‐1/3, and LL) were lost during evolution, but in certain cases they can behave like autonomously replicating episomes (Chevigny et al., 2022; Wallet et al., 2015). Despite this apparent reproducibility, there were also examples of accession‐specific large indels, including a 1105‐bp sequence absent in Col‐0, a 1790‐bp sequence absent in C24, a 981‐bp sequence absent in Kyoto, and a 1407‐bp sequence uniquely present in Sha.…”

Long-read sequencing characterizes mitochondrial and plastid genome variants in Arabidopsismsh1mutants

“…The mt genomes of Col-0 and An-1 differ by five rearrangements mediated by repeat group B, I, H-1/2, L and H-1/3, while that of L er has an additional rearrangement mediated by repeat group LL (Figure 5a). The small circles generated by closely located direct repeats (H-1/2, H-1/3 and LL) were lost during evolution, but in certain cases they can behave like autonomously replicating episomes (Wallet et al 2015; Chevigny et al, 2022). Despite this apparent reproducibility, there were also examples of accession-specific large indels, including a 1105-bp sequence absent Col-0, a 1790-bp sequence absent in C24, a 981-bp sequence absent in Kyoto, and a 1407-bp sequence uniquely present in Sha.…”