Recombination establishes the chiasmata that physically link pairs of homologous chromosomes in meiosis, ensuring their balanced segregation at the first meiotic division and generating genetic variation. The visible manifestation of genetic crossing-overs, chiasmata are the result of an intricate and tightly regulated process involving induction of DNA double-strand breaks and their repair through invasion of a homologous template DNA duplex, catalysed by RAD51 and DMC1 in most eukaryotes. We describe here a RAD51-GFP fusion protein that retains the ability to assemble at DNA breaks but has lost its DNA break repair capacity. This protein fully complements the meiotic chromosomal fragmentation and sterility of Arabidopsis rad51, but not rad51 dmc1 mutants. Even though DMC1 is the only active meiotic strand transfer protein in the absence of RAD51 catalytic activity, no effect on genetic map distance was observed in complemented rad51 plants. The presence of inactive RAD51 nucleofilaments is thus able to fully support meiotic DSB repair and normal levels of crossing-over by DMC1. Our data demonstrate that RAD51 plays a supporting role for DMC1 in meiotic recombination in the flowering plant, Arabidopsis. Recombination ensures coordinated disjunction of pairs of homologous chromosomes and generates genetic exchanges in meiosis and, with some exceptions, involves the co-operation of the RAD51 and DMC1 strand-exchange proteins. We describe here a RAD51-GFP fusion protein that has lost its DNA break repair capacity but retains the ability to assemble at DNA breaks in the plant, Arabidopsis - fully complementing the meiotic chromosomal fragmentation and sterility of rad51 mutants, and this depends upon DMC1. No effect on genetic map distance was observed in complemented rad51 plants even though DMC1 is the only active strand transfer protein. The inactive RAD51 nucleofilaments are thus able to fully support meiotic DSB repair and normal levels of crossing-over by DMC1 in Arabidopsis. The RAD51-GFP protein confers a dominant-negative inhibition of RAD51-dependent mitotic recombination, while remaining fully fertile - a novel and valuable tool for research in this domain. These phenotypes are equivalent to those of the recently reported yeast rad51-II3A mutant, (Cloud et al. 2012), carrying the implication of their probable generality in other eukaryotes and extending them to a species with a very different relation between numbers of meiotic DNA double-strand breaks and crossing-overs (∼2 DSB/CO in yeast; ∼25–30 DSB/CO in Arabidopsis; ∼15 DSB/CO in mice).

Summary: In addition to the recombinase Rad51, vertebrates have five paralogs of Rad51, all members of the Rad51‐dependent recombination pathway. These paralogs form two complexes (Rad51C/Xrcc3 and Rad51B/C/D/Xrcc2), which play roles in somatic recombination, DNA repair and chromosome stability. However, little is known of their possible involvement in meiosis, due to the inviability of the corresponding knockout mice. We have recently reported that the Arabidopsis homolog of one of these Rad51 paralogs (AtXrcc3) is involved in DNA repair and meiotic recombination and present here Arabidopsis lines carrying mutations in three other Rad51 paralogs (AtRad51B, AtRad51C and AtXrcc2). Disruption of any one of these paralogs confers hypersensitivity to the DNA cross‐linking agent Mitomycin C, but not to γ‐irradiation. Moreover, the atrad51c‐1 mutant is the only one of these to show meiotic defects similar to those of the atxrcc3 mutant, and thus only the Rad51C/Xrcc3 complex is required to achieve meiosis. These results support conservation of functions of the Rad51 paralogs between vertebrates and plants and differing requirements for the Rad51 paralogs in meiosis and DNA repair.

The eukaryotic RecA homologue Rad51 is a key factor in homologous recombination and recombinational repair. Rad51‐like proteins have been identified from yeast (Rad55, Rad57 and Dmc1) to vertebrates (Rad51B, Rad51C, Rad51D, Xrcc2, Xrcc3 and Dmc1). These Rad51‐like proteins are all members of the genetic recombination and DNA damage repair pathways. The sequenced genome of Arabidopsis thaliana encodes putative homologues of all six vertebrate Rad51‐like proteins. We have identified and characterized an Arabidopsis mutant defective for one of these, AtXRCC3 , the homologue of XRCC3 . atxrcc3 plants are sterile, while they have normal vegetative development. Cytological observation shows that the atxrcc3 mutation does not affect homologous chromosome synapsis, but leads to chromosome fragmentation after pachytene, thus disrupting both male and female gametogenesis. This study shows an essential role for AtXrcc3 in meiosis in plants and possibly in other higher eukaryotes. Furthermore, atxrcc3 cells and plants are hypersensitive to DNA‐damaging treatments, supporting the involvement of this Arabidopsis Rad51‐like protein in recombinational repair.

Summary: The ability to address the CRISPR‐Cas9 nuclease complex to any target DNA using customizable single‐guide RNAs has now permitted genome engineering in many species. Here, we report its first successful use in a nonvascular plant, the moss Physcomitrella patens. Single‐guide RNAs (sgRNAs) were designed to target an endogenous reporter gene, PpAPT, whose inactivation confers resistance to 2‐fluoroadenine. Transformation of moss protoplasts with these sgRNAs and the Cas9 coding sequence from Streptococcus pyogenes triggered mutagenesis at the PpAPT target in about 2% of the regenerated plants. Mainly, deletions were observed, most of them resulting from alternative end‐joining (alt‐EJ)‐driven repair. We further demonstrate that, in the presence of a donor DNA sharing sequence homology with the PpAPT gene, most transgene integration events occur by homology‐driven repair (HDR) at the target locus but also that Cas9‐induced double‐strand breaks are repaired with almost equal frequencies by mutagenic illegitimate recombination. Finally, we establish that a significant fraction of HDR‐mediated gene targeting events (30%) is still possible in the absence of PpRAD51 protein, indicating that CRISPR‐induced HDR is only partially mediated by the classical homologous recombination pathway.