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Open Access

Peer-reviewed

Research Article

Arabidopsis RAD51, RAD51C and XRCC3 proteins form a complex and facilitate RAD51 localization on chromosomes for meiotic recombination

  • Hang Su ,

    Contributed equally to this work with: Hang Su, Zhihao Cheng

    Affiliation State Key Laboratory of Genetic Engineering and Collaborative Innovation Center of Genetics and Development, Ministry of Education Key Laboratory of Biodiversity Sciences and Ecological Engineering, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai, China

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  • Zhihao Cheng ,

    Contributed equally to this work with: Hang Su, Zhihao Cheng

    Affiliations State Key Laboratory of Genetic Engineering and Collaborative Innovation Center of Genetics and Development, Ministry of Education Key Laboratory of Biodiversity Sciences and Ecological Engineering, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai, China, Institute of Banana and Plantain, Chinese Academy of Tropic Agriculture Science, Haikou, Hainan province, China

    ⨯
  • Jiyue Huang,

    Affiliation State Key Laboratory of Genetic Engineering and Collaborative Innovation Center of Genetics and Development, Ministry of Education Key Laboratory of Biodiversity Sciences and Ecological Engineering, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai, China

    ⨯
  • Juan Lin,

    Affiliation State Key Laboratory of Genetic Engineering and Collaborative Innovation Center of Genetics and Development, Ministry of Education Key Laboratory of Biodiversity Sciences and Ecological Engineering, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai, China

    ⨯
  • Gregory P. Copenhaver,

    Affiliations Department of Biology and the Integrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America, Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina, United States of America

    ⨯
  • Hong Ma,

    Affiliations State Key Laboratory of Genetic Engineering and Collaborative Innovation Center of Genetics and Development, Ministry of Education Key Laboratory of Biodiversity Sciences and Ecological Engineering, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai, China, Center for Evolutionary Biology, Institutes of Biomedical Sciences, School of Life Sciences, Fudan University, Shanghai, China

    ⨯
  • Yingxiang Wang

    yx_wang@fudan.edu.cn

    Affiliation State Key Laboratory of Genetic Engineering and Collaborative Innovation Center of Genetics and Development, Ministry of Education Key Laboratory of Biodiversity Sciences and Ecological Engineering, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai, China

    ⨯

Abstract

Meiotic recombination is required for proper homologous chromosome segregation in plants and other eukaryotes. The eukaryotic RAD51 gene family has seven ancient paralogs with important roles in mitotic and meiotic recombination. Mutations in mammalian RAD51 homologs RAD51C and XRCC3 lead to embryonic lethality. In the model plant Arabidopsis thaliana, RAD51C and XRCC3 homologs are not essential for vegetative development but are each required for somatic and meiotic recombination, but the mechanism of RAD51C and XRCC3 in meiotic recombination is unclear. The non-lethal Arabidopsis rad51c and xrcc3 null mutants provide an opportunity to study their meiotic functions. Here, we show that AtRAD51C and AtXRCC3 are components of the RAD51-dependent meiotic recombination pathway and required for normal AtRAD51 localization on meiotic chromosomes. In addition, AtRAD51C interacts with both AtRAD51 and AtXRCC3 in vitro and in vivo, suggesting that these proteins form a complex (es). Comparison of AtRAD51 foci in meiocytes from atrad51, atrad51c, and atxrcc3 single, double and triple heterozygous mutants further supports an interaction between AtRAD51C and AtXRCC3 that enhances AtRAD51 localization. Moreover, atrad51c-/+ atxrcc3-/+ double and atrad51-/+ atrad51c-/+ atxrcc3-/+ triple heterozygous mutants have defects in meiotic recombination, suggesting the role of the AtRAD51C-AtXRCC3 complex in meiotic recombination is in part AtRAD51-dependent. Together, our results support a model in which direct interactions between the RAD51C-XRCC3 complex and RAD51 facilitate RAD51 localization on meiotic chromosomes and RAD51-dependent meiotic recombination. Finally, we hypothesize that maintenance of RAD51 function facilitated by the RAD51C-XRCC3 complex could be highly conserved in eukaryotes.

Author summary

Meiotic recombination and sister chromatid cohesion are important for maintaining the association between homologous chromosomes and ensuring their accurate segregation. Meiotic recombination starts with a set of programmed DNA double-strand breaks (DSBs), catalyzed by the SPO11 endonuclease. Processing of DSB ends produces 3′ single-stranded DNA tails, which form nucleoprotein filaments with RAD51 and DMC1, homologs of the prokaryotic RecA protein. The eukaryotic RAD51 gene family has seven ancient paralogs, in addition to RAD51 and DMC1, the other five members in mammals form two complexes: RAD51B-RAD51C-RAD51D- XRCC2 (BCDX2) and RAD51C-XRCC3 (CX3). To date, the molecular mechanism of CX3 in animal meiosis remains largely unknown due to the essential roles of these two proteins in embryo development. In Arabidopsis, RAD51C and XRCC3 are required for meiosis and fertility, but their specific mechanisms are unclear. Here we present strong evidence that Arabidopsis RAD51 forms a protein complex with AtRAD51C-AtXRCC3 in vivo. Our data also support the previous hypothesis that CX3 promotes RAD51-denpendet meiotic recombination by affecting its localization on chromosomes. Given that the RAD51, RAD51C and XRCC3 proteins are highly conserved in plants and vertebrates, the mechanism we present here could be important for the regulation of meiotic recombination in both plants and vertebrate animals.

Citation: Su H, Cheng Z, Huang J, Lin J, Copenhaver GP, Ma H, et al. (2017) Arabidopsis RAD51, RAD51C and XRCC3 proteins form a complex and facilitate RAD51 localization on chromosomes for meiotic recombination. PLoS Genet 13(5): e1006827. https://doi.org/10.1371/journal.pgen.1006827

Editor: Wojciech P. Pawlowski, Cornell University, UNITED STATES

Received: January 17, 2017; Accepted: May 17, 2017; Published: May 31, 2017

Copyright: © 2017 Su et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This work was supported by grants from the National Natural Science Foundation of China (31370347 and 31570314), and by funds from the State Key Laboratory of Genetic Engineering, Fudan University and Rijk Zwaan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Homologous recombination (HR) is important for repairing DNA damage and maintaining genomic stability. Meiotic HR and sister chromatid cohesion are required for maintaining physical associations between homologous chromosomes (homologs) and ensuring their accurate segregation. Meiotic HR is initiated by programmed DNA double-strand breaks (DSBs) that are catalyzed by SPO11, a topoisomerase-like protein [1]. The resulting DSB ends are processed by the MRE11- RAD50-NBS1 (MRN) protein complexes to generate 3′ single-stranded DNA (ssDNA) tails [2,3], which are subsequently protected by replication protein A (RPA) [4]. Functional homologs of the E. coli RecA protein, RAD51 and DMC1 [5,6] bind to the 3ʹ tails to form nucleoprotein filaments with the help of several proteins identified in multiple species, including Saccharomyces cerevisiae (Rad52 [7], Rad54 [8], Tid1/Rhd54 [9], Rad55-Rad57 [10], Swi5-Sfr1 [11] and PCSS complex [12]), Arabidopsis thaliana (RAD51C [13], XRCC3 [14], MND1-HOP2[15] and ATR/ATRIP [16]), and mammals (Mnd1-Hop2 [17] and Brca2-Dss1 [18]). The nucleoprotein filaments facilitate single-end invasion of a non-sister chromatid, resulting in the formation of a recombination intermediate called a D-loop, which is then processed to ultimately produce either crossovers (COs) or non-crossovers (NCOs) [19].

In vertebrate animals and plants, the RAD51 gene family is highly conserved with seven members: DMC1, RAD51, RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3 [20–23], which share Walker A and Walker B motifs with over 37.5% similarity [24]. In mice, mutations in any of the paralogs, except DMC1, lead to embryonic lethality following spontaneous DNA damage or errors [25–29]. In the model plant Arabidopsis thaliana, all seven genes are dispensable for vegetative growth [13,14,24,30–33]. However, AtRAD51, AtRAD51C and AtXRCC3 are required for somatic and meiotic recombination, as well as plant fertility. Mutations in any of these three genes result in a meiotic chromosome fragmentation phenotype [13,14,24,30–32]. Moreover, AtDMC1 is specifically required for meiotic homolog pairing and recombination [34,35]. In contrast to atrad51, atrad51c and atxrcc3 mutants, atdmc1 mutants do not suffer meiotic chromosome fragmentation; instead their DSBs are thought to be repaired using sister chromatids as templates [34,35]. The three other paralogs, AtRAD51B, AtRAD51D and AtXRCC2, seem to be unnecessary for meiotic DSB repair, because the triple mutant has normal chromosome morphology and fertility [33]. Except for slight differences in synapsis, the chromosome morphology using light microscopy for DAPI-stained chromosomes and fertility phenotypes of atrad51c and atxrcc3 mutants are similar to those of atrad51, suggesting that their functions are related, but further analyses are needed to understand their mechanistic roles in meiotic DSB repair.

Biochemical studies in human cells demonstrate that RAD51 paralogs associate with one another in two distinct complexes: RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2) and RAD51C-XRCC3 (CX3) [36,37]. The CX3 complex stabilizes RAD51 binding to ssDNA [36–39] in vitro, thus promoting single-end invasion. Moreover, RAD51C and XRCC3 also help mediate Holliday junction (HJ) resolution in vitro [40], suggesting a later role in meiotic recombination. A yeast two-hybrid assay demonstrated that the Arabidopsis RAD51 paralogs also interact with each other [41], supporting the idea that RAD51 paralogs function by formation of distinct protein complexes in both animals and plants. However, whether the RAD51 paralogs associate with each other in planta has not been tested.

In this study, we report that Arabidopsis homologs of RAD51, RAD51C and XRCC3 show highly similar meiotic chromosome morphological defects using immune-localization for key markers. We also provide evidence that AtRAD51C and AtXRCC3 are required for AtRAD51 localization on chromosomes. Both in vitro and in vivo data demonstrate that AtRAD51C interacts with AtRAD51 and AtXRCC3. Furthermore, observation of AtRAD51 foci in atrad51, atrad51c and atxrcc3 single, double and triple heterozygotes reveals that AtRAD51C and AtXRCC3 both are involved in AtRAD51 loading. Triple heterozygotes also experience non-homolog chromosome associations and have reduced CO frequencies. Together, these results demonstrate that AtRAD51C, AtXRCC3 and AtRAD51 form a complex in planta and are required for AtRAD51 loading on chromosomes.

Results

AtRAD51, AtRAD51C and AtXRCC3 have non-redundant roles in meiotic recombination

Previous studies have found that AtRAD51, AtRAD51C and AtXRCC3 are required for meiotic DSB repair and plant fertility and mutation of individual genes cause indistinguishable chromosome entanglement and fragmentation phenotypes [13,14,31,32]. The similarity of the phenotypes suggests that these genes might function in the same genetic pathway or process. To test this hypothesis, we generated double mutants between atrad51-3 (SAIL_873_C08) [42], atrad51c (SALK_021960) [13], and atxrcc3 (SALK_045564) [14] and found that the chromosome morphologies of atrad51 atrad51c (48 cells), atrad51 atxrcc3 (65 cells), and atrad51c atxrcc3 (54 cells) double mutants showed no obvious differences compared with each of the single mutants (S1 Fig). The lack of an additive phenotype in the double mutants further supports the hypothesis that they act together in the same biological process.

To search for subtle chromosomal phenotypes that could discriminate between the three mutants, we used FISH with a centromere probe for atrad51 (82 cells); atrad51c (96 cells) and atxrcc3 (81cells) and a bacterial artificial chromosome (BAC-F19K16) probe that targets a telomere proximal region on chromosome 1 for atrad51 (31 cells); atrad51c (45 cells) and atxrcc3 (22 cells) [43]. Wild-type (WT) meiocytes had three to five centromere signals at pachytene, indicative of paired homologous centromeres in a cluster (Fig 1A). Although the three mutants had no typical pachytene chromosomes, they all displayed similar centromere clusters or numbers of signals at a stage similar to that of WT, suggesting that AtRAD51, AtRAD51C and AtXRCC3 are not required for early centromere pairing or clustering (Fig 1D, 1G and 1J). At diakinesis and metaphase I, WT meiocytes had five bivalents, each with two paired centromere signals corresponding to the associated homologs (Fig 1B). In contrast, the three mutants each had 10 centromere signals located on abnormally associated chromosomes (multivalents-with more than two chromosomes) (Fig 1E, 1H and 1K), indicating a failure to maintain homolog association, at least at the centromere regions. We next examined homolog pairing on the chromosome arms using the telomere-proximal BAC probe. Unlike the single focus observed on WT pachytene chromosomes, indicative of fully synapsed homologs, meiocytes from each of the three mutants showed two separate signals, indicating a failure to pair properly (Fig 1M–1P). We also performed ASY1 and ZYP1 immuno-localization in WT and mutants. No obvious difference of ASY1 signals at zygotene was found between WT and mutants (S2 Fig). However, unlike WT with linear ZYP1 distribution on pachytene chromosomes, ZYP1 was completely disappeared in rad51, while some punctate or discontinuous ZYP1 signals were observed in xrcc3 and rad51c (S2 Fig). Together, these results demonstrate that AtRAD51, AtRAD51C and AtXRCC3 are not required for recombination-independent centromere clustering, but are necessary for homolog pairing, consistent with previous findings obtained using FISH experiment [44]. The similarities of the mutant phenotypes further support the idea that they act in the same process.

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Fig 1. Fluorescence in situ hybridization (FISH) analysis of chromosome interactions in atrad51, atrad51c and atxrcc3 mutants.

(A-L) Chromosome morphologies of wild type (WT), atrad51, atrad51c and atxrcc3 mutants at pachytene, diakinesis and anaphase I. Centromeres appear as red dots. (M-P) WT pachytene chromosomes showing a single signal from the BAC-F19K16 probe and atrad51, atrad51c, and atxrcc3 chromosomes showing two signals from the same probe. (Q-S) Equal segregation of BAC-F19K16 signals at metaphase I in WT and unequal segregation in atrad51 and atrad51c mutants. (T) BAC-F19K16 signals located on atxrcc3 chromosome fragments at metaphase I. For FISH analysis of chromosomes using the BAC-F19K16 probe, the left panels show the chromosome morphology following staining with 6-diamidino-2-phenylindole (DAPI), the middle panels show the BAC-F19K16 signals (red dots) and the right panels merge the DAPI staining and BAC-F19K16 signals. Scale bar: 5 μm.

https://doi.org/10.1371/journal.pgen.1006827.g001

Arabidopsis RAD51C and XRCC3 are required for normal localization of RAD51 on chromosomes

Loading of RAD51 on ssDNA is aided by several proteins, including Rad52 [45], Rad55-57 (Rad51 paralogs) [46] and Sfr1-Swi5 [11] in yeast, the Brca2-Dss1 complex in mammalian cells [47], and also by AtBRCA2 in Arabidopsis [48]. The similarity of meiotic defects in Arabidopsis rad51, rad51c and xrcc3 mutants suggests the RAD51 paralogs RAD51C and XRCC3 may function in meiotic recombination by affecting RAD51 function. To test this hypothesis, we performed an immunofluorescence assay using an AtRAD51 antibody [49]. In Arabidopsis, formation of DSBs is thought to occur at leptotene [50]. At a similar stage, we found that WT plants had 187.7±24.5 AtRAD51 foci per meiocyte (n = 14), but the number of foci was greatly reduced in atrad51c (36.1±9.7, n = 17; P = 1.5E-13) and atxrcc3 (33.7±10.3, n = 34; P = 5.7E-13) mutant meiocytes (Fig 2A, 2C, 2D and 2Q). In contrast, a parallel experiment did not detect any AtRAD51 foci in atrad51 mutant meiocytes at zygotene (Fig 2B). A similar pattern was also observed using pachytene meiocytes (Fig 2E–2H). These results provide evidence that Arabidopsis RAD51C and XRCC3 are required for formation of wild type level of RAD51 foci on meiotic chromosomes. This is consistent with the previous findings for Rad51 paralogs in yeast [46]. Nevertheless, the reduction of AtRAD51 foci in atrad51c and atxrcc3 homozygous mutants does not preclude the possibility that normal level DSBs are formed in these mutants. To test whether DSB frequency is altered in atrad51c and atxrcc3 mutants, we examined the distribution of a DSB marker, phosphorylated histone H2AX (γ-H2AX) [51]. At zygotene, after DSBs have been formed, no significant differences in the number of γ-H2AX foci were detected between WT (189.3±26.5, n = 39), atrad51 (176.7±15.5, n = 19; P = 0.062), atrad51c (183.6±18.0, n = 18; P = 0.41) and atxrcc3 (178.3±13.5, n = 19; P = 0.097) mutants (Fig 2I–2L and 2R). In Arabidopsis, most meiotic DSBs are thought to be repaired during zygotene-pachytene. We found that γ-H2AX foci were obviously reduced in WT (56.9±15.2, n = 55) pachytene meiocytes compared those of atrad51 (132.1±15.4, n = 13; P = 1.5E-11), atrad51c (120.6±16.6, n = 14; P = 7.2E-11) and atxrcc3 (122.2±18.8, n = 18; P = 1.7E-11) mutants (Fig 2M–2P and 2S). The presence of normal numbers of zygotene γ-H2AX foci and reduced AtRAD51 foci suggests that AtRAD51C and AtXRCC3 are not required for meiotic DSB formation, but are necessary for AtRAD51 loading.

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Fig 2. Immunofluorescence of AtRAD51 and γ-H2AX in wild type, atrad51, atrad51c and atxrcc3 mutants.

(A, E) Localization of RAD51 on wild-type (WT) chromosomes at zygotene and pachytene stages, respectively. (B, F) No signal was detected in the atrad51 mutant at similar stages. (C, D, G, H) The numbers of AtRAD51 foci were significantly reduced in the atrad51c and atxrcc3 mutants compared with WT at zygotene and pachytene, respectively. (I, M) Localization of γ-H2AX on wild-type (WT) chromosomes at zygotene and pachytene stages, respectively. (J-L) No significant differences were detected between atrad51, atrad51c and atxrcc3 mutants at zygotene. (N-P) More γ-H2AX foci were detected at pachytene in atrad51, atrad51c and atxrcc3 mutants compared to WT. (Q) The number of RAD51 foci on chromosomes from wild type and mutants at zygotene. (R-S) The number of γ-H2AX foci on chromosomes from wild type and mutants at zygotene and pachytene. Left panels show the chromosome morphology following staining with 6-diamidino-2-phenylindole (DAPI), middle panels show AtRAD51 foci (red dots), and right panels merge the DAPI-stained images with the AtRAD51 foci images. Scale bar: 5 μm. ** p<0.01 (two-tailed Student’s t-test).

https://doi.org/10.1371/journal.pgen.1006827.g002

Arabidopsis RAD51C interacts with RAD51 and XRCC3 in vitro and in vivo

In yeast, the Rad51 paralogs Rad55 and Rad57 form a heterodimeric complex to stimulate RAD51 activity [10]. Vertebrate Rad51 paralogs interact with one another to form two distinct complexes: BCDX2 and CX3 [52]. Like vertebrates, Arabidopsis has seven RAD51 paralogs, and previous yeast two-hybrid assays have shown that XRCC3 interacts with both RAD51C and RAD51 [41]. However, whether these proteins interact in planta has not been investigated.

As an initial test for potential interactions we used a yeast two-hybrid assay (Y2H) and found that AtXRCC3 interacts with both AtRAD51 and AtRAD51C (Fig 3A), consistent with the previously identified interactions in Y2H system [41]. The interaction between AtRAD51C and AtXRCC3 was further supported by a pull-down assay using recombinant fusion protein of glutathione S-transferase (GST) with AtRAD51C and an AtXRCC3-His tag fusion protein (Fig 3B). In addition to the previously identified interactions, we also found that GST-AtRAD51 interacts with AtRAD51C-His (Fig 3B). To explore whether these associations also occurred in planta, we examined the interactions using bimolecular fluorescence complementation (BiFC) in tobacco (Nicotiana benthamiana) cells. Strong nuclear signals, indicating interaction, were observed for AtRAD51C with either AtRAD51 or AtXRCC3 (Fig 3C). These results provide the first direct evidence that plant RAD51 paralogs RAD51C and XRCC3 interact directly with RAD51 in vitro and in planta.

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Fig 3. AtRAD51C interacts with AtRAD51 and AtXRCC3 in vitro and in vivo.

(A) Yeast two-hybrid assay showing AtXRCC3 interaction with AtRAD51 and AtRAD51C. The known DYT1-DYT1 interaction is used as a positive control [82]. AD refers to the activation domain, BD refers to the DNA-binding domain, SD-2 refers to SD-Leu-Trp and SD-4 refers to SD-Leu-Trp-His-Ade+X-gal. Blue indicates a positive interaction. (B) Pull-down assay showing that AtRAD51C interacts with AtRAD51 and AtXRCC3. (C) BiFC assay in tobacco cells showing strong nuclear signals for AtRAD51-AtRAD51C and AtRAD51C-AtXRCC3 interactions. Left panels are yellow fluorescent protein (YFP) signals, middle panels are nuclei stained with 6-diamidino-2-phenylindole (DAPI), and right panels merge the DAPI-stained images with the YFP signals and bright field images.

https://doi.org/10.1371/journal.pgen.1006827.g003

Male meiotic defects are observed in atrad51c-/+ atxrcc3-/+ double and atrad51-/+ atrad51c-/+ atxrcc3-/+ triple heterozygous mutants

A recent study identified a weak atrad51 allele, atrad51-2 [42], with a T-DNA insertion in the 3′-untranslated region (UTR) that results in reduced AtRAD51 protein levels. This mutant had mild chromosome fragmentation and partial synapsis, as well as some bivalent formation with homologs and non-homologs [42]. In contrast, the atrad51-1 null mutant had severe chromosome fragmentation and formed multivalents during meiotic prophase I [31]. These findings suggest that reducing AtRAD51 level might be a strategy for investigating its meiotic function. Alternatively, analysis of double heterozygous mutants in genes encoding components of a complex can reveal phenotypic defects, even though the corresponding single heterozygotes are phenotypically normal [53,54]. We hypothesized that double/triple heterozygotes of atrad51, atrad51c and atxrcc3 might reduce, but not abolish, their interactions in a complex and reveal informative meiotic phenotypes

To test this hypothesis, we generated atrad51-/+, atrad51c-/+ and atxrcc3-/+ double and triple heterozygous mutants and compared their meiotic phenotypes with WT. Analysis of meiotic chromosome morphology after DAPI staining showed that atrad51-/+, atrad51c-/+ and atxrcc3-/+ single heterozygote meiocytes and atrad51-/+ atrad51c-/+ and atrad51-/+ atxrcc3-/+ double heterozygotes had similar phenotypes compared to WT (Fig 4A–4L; S3 Fig). In addition, meiocytes from atrad51c-/+ atxrcc3-/+ double heterozygotes had chromosome morphology similar to WT at pachytene (Fig 4M), but at diakinesis, WT formed five bivalents, whereas 32.8% (20 of 61, n = 61) of the atrad51-/+ atxrcc3-/+ double heterozygote meiocytes had non-homologous chromosome associations (Fig 4N). The cell appears to be able to resolve these associations since equal division of chromosomes was observed at anaphase I and II (Fig 4O and 4P). Meiocytes from atrad51-/+ atrad51c-/+ atxrcc3-/+ triple heterozygotes had a more severe non-homolog association phenotype (47.8% at diakinesis, 22 of 46, n = 46, Fig 4R) and had unequal chromosome segregation at metaphase II (16.7%, 2 of 12, n = 12, Fig 4T). No chromosome fragments were observed in the triple heterozygote, suggesting it is still capable of DSB repair. The results also suggest that RAD51C and XRCC3 are functionally more related to each other than either is to RAD51. Previous studies showed that T-DNA translocation can cause a similar pattern of chromosome association using light microscopy because the translocated chromosome can associated with two normal chromosomes [55,56]. We have verified the T-DNA insertion site by sequencing the junction with flanking genomic DNAs and the results indicated that these mutations are not associated with translocations.

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Fig 4. Centromere-fluorescence in situ hybridization analysis of chromosome morphology in double and triple heterozygous mutants.

(A-L) Wild type (WT), atrad51-/+ atrad51c-/+ and atrad51-/+ atxrcc3-/+ mutant chromosome morphologies at pachytene, diakinesis, metaphase I and metaphase II. Compared with WT, atrad51-/+ atrad51c-/+ and atrad51-/+ atxrcc3-/+ mutants had similar chromosome morphologies. (M) atrad51c-/+ atxrcc3-/+ chromosome morphology at pachytene was similar to WT, but showed 32.8% (n = 61) non-homolog association at diakinesis (N). (O, P) atrad51c-/+ atxrcc3-/+ chromosome morphology at metaphase I and II, respectively. Chromosome morphology of atrad51-/+ atrad51c-/+ atxrcc3-/+ at pachytene (Q) and diakinesis (R), metaphase I (S) and metaphase II (T). The atrad51-/+ atrad51c-/+ atxrcc3-/+ triple chromosome morphology showed more severe defects than atrad51c-/+ atxrcc3-/+. Red dots indicate centromere signals. Yellow arrows show non-homologous chromosome associations (N, R, S). The red numbers in N, R, S refer to the number of abnormal cells out of all cells observed. Scale bar: 5 μm.

https://doi.org/10.1371/journal.pgen.1006827.g004

Repair of meiotic DSBs is delayed in atrad51-/+, atrad51c-/+ and atxrcc3-/+ single, double and triple heterozygous mutants

To test whether meiotic DSB repair is delayed in the heterozygotes, we performed immunostaining experiments using a γH2AX antibody. As mentioned above, WT meiocytes had 189.3±26.5 (n = 39) and 56.9±15.2 (n = 55) γH2AX foci at zygotene and pachytene, respectively (Fig 5A, 5I, 5Q and 5R and Table 1). All single, double and triple heterozygotes had no obvious differences in the number of γH2AX foci at zygotene, but had significantly more foci at pachytene (Fig 5A–5R, S1 Table). Moreover, the double and triple heterozygotes had more foci at pachytene than the single heterozygotes. There are significantly fewer foci in atrad51-/+ (78.1±19.4, n = 17), atrad51c-/+ (83.3±10.8, n = 12) and atxrcc3-/+ (82.0±25.9, n = 24) (S1 Table) compared to the double mutants atrad51-/+ atrad51c-/+ (96.3±15.4, n = 30), atrad51-/+atxrcc3-/+ (100.4±14.8, n = 21) and atrad51c-/+atxrcc3-/+ (105.2±24.1, n = 15), which in turn have significantly fewer foci (S1 Table) than the triple atrad51-/+ atrad51c-/+ atxrcc3-/+ (113.3±14.8, n = 46) (Fig 5J–5P and 5R and Table 1). These data suggest that DSB formation is normal in the heterozygotes, but there is a defect in the progression of DSB repair, and that AtRAD51, AtRAD51C and AtXRCC3 function in this process.

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Fig 5. Immunostaining of γ-H2AX signals in single, double and triple heterozygous mutant chromosomes at zygotene and pachytene stages.

(A-H) The distribution of γ-H2AX on zygotene chromosomes showed no obvious differences among the eight genotypes examined. (I-P) The γ-H2AX distribution in pachytene chromosomes from single, double and triple heterozygous mutants. Left panels show the chromosome morphology following staining with 6-diamidino-2-phenylindole (DAPI), middle panels show γ-H2AX foci (red dots), and right panels merge the DAPI-stained images with the γ-H2AX foci images. (Q-R) The number of γ-H2AX foci in chromosomes from the eight genotypes at zygotene and pachytene in A-P. Scale bar: 5 μm. * p<0.05, ** p<0.01 (two-tailed Student’s t-test).

https://doi.org/10.1371/journal.pgen.1006827.g005

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Table 1. Numbers of γ-H2AX and AtRAD51 foci in single, double and triple heterozygous mutants at zygotene and pachytene.

https://doi.org/10.1371/journal.pgen.1006827.t001

Because AtRAD51C and AtXRCC3 are required for normal AtRAD51 localization, we next examined AtRAD51 localization in heterozygous mutant meiocytes. As described above, WT meiocytes have 187.7±24.5 (n = 14) AtRAD51 foci at zygotene and 51.2±14.0 (n = 65) foci at pachytene (see Fig 6A and 6I for examples and Table 1). In contrast, single, double and triple heterozygous mutant meiocytes have significantly fewer AtRAD51 foci at zygotene (p<0.05; Fig 6A–6H and 6Q). At pachytene, the three single mutant heterozygotes show no obvious differences in the number of AtRAD51 foci compared with WT (Fig 6I–6L and 6R), but the double and triple heterozygotes exhibited reduced AtRAD51 foci (p<0.05; Fig 6M–6P and 6R). These findings are consistent with the earlier results, suggesting that AtRAD51C and AtXRCC3 play related roles in AtRAD51 loading on chromosomes, likely in a protein complex.

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Fig 6. Immunostaining of AtRAD51 signals in single, double and triple heterozygous mutant chromosomes at zygotene and pachytene.

(A-H) The locations and (Q) numbers of AtRAD51 foci on zygotene chromosomes from atrad51-/+, atrad51c-/+, atxrcc3-/+, atrad51-/+ atrad51c-/+, atrad51-/+ atxrcc3-/+, atrad51c-/+ atxrcc3-/+ and atrad51-/+ atrad51c-/+ atxrcc3-/+ heterozygous mutant meiocytes showed reductions compared to wild type (WT). (I-L) The location and (R) number of AtRAD51 foci on pachytene chromosomes in the three single heterozygotes showed no obvious differences compared with WT. (M-P) The location and (R) number of AtRAD51 foci on pachytene chromosomes in the double and triple heterozygous mutants were significantly reduced relative to WT. Left panels show the chromosome morphology following staining with 6-diamidino-2-phenylindole (DAPI), middle panels show AtRAD51 foci (red dots), and right panels merge the DAPI-stained images with the AtRAD51 foci images. Scale bar: 5 μm. ** p<0.01 (two-tailed Student’s t-test).

https://doi.org/10.1371/journal.pgen.1006827.g006

CO number is reduced in atrad51-/+ atrad51c-/+ atxrcc3-/+ triple heterozygotes

As described earlier, the weak atrad51-2 allele is capable of forming bivalents and executing recombination [42]. Similarly, the heterozygous plants analyzed here also completed meiotic recombination to some extent and had partial fertility. To examine CO frequencies in comparison between the various genotypes, we counted the number of chiasmata, the physical manifestation of crossing-over, in WT and mutant meiocytes at both diplotene and metaphase I. On average, WT had 10.1±1.1 (n = 52) chiasmata per meiocyte and no obvious significant differences were observed in the single heterozygotes: atrad51-/+ with 9.6±0.7 (n = 20; P = 0.072) per meiocyte, atrad51c-/+ with 9.6±0.7 (n = 21; P = 0.052) per meiocyte and atxrcc3-/+ with 9.6±0.8 (n = 24; P = 0.066) per meiocyte. The atrad51-/+ atrad51c-/+, atrad51-/+ atxrcc3-/+ and atrad51c-/+ atxrcc3-/+ double heterozygotes showed a slight, but statistically significant, reduction of chiasmata with 8.4±1.2 (n = 14; P = 6.0E-05), 8.0±0.8 (n = 10; P = 2.9E-06) and 7.1±1.0 (n = 34; P = 1.2E-20) per meiocyte, respectively (Fig 7A–7D and 7K). The atrad51-/+ atrad51c-/+ atxrcc3-/+ triple heterozygous mutant also had a significant reduction, with only 6.9±1.0 (n = 15; P = 2.6E-10) chiasmata per meiocyte formed (Fig 7E and 7K). Furthermore, the chiasmata numbers per meiocyte of atrad51c-/+ atxrcc3-/+ double heterozygote (7.1; P values = 2.0E-03 and 1.1E-02, respectively) and the triple heterozygote (6.9, P values = 1.7E-03 and 8.8E-03, respectively) were significantly lower than those of the other two double heterozygotes.

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Fig 7. Number of chiasmata and AtMLH1 foci in double and triple heterozygous mutants.

(A-E) Centromere-fluorescence in situ hybridization showing wild type (WT) and atrad51-/+ atrad51c-/+, atrad51-/+ atxrcc3-/+, atrad51c-/+ atxrcc3-/+ and atrad51-/+ atrad51c-/+ atxrcc3-/+ double and triple heterozygous mutant chromosome morphologies at diakinesis. Red dots indicate centromere signals. (F-J) Immunostaining of AtMLH1 foci (red dots) on WT, atrad51-/+ atrad51c-/+, atrad51-/+ atxrcc3-/+, atrad51c-/+ atxrcc3-/+ and atrad51-/+ atrad51c-/+ atxrcc3-/+ diakinesis chromosomes. (K) Number of chiasmata in WT, double and triple mutant chromosomes. (L) Number of AtMLH1 foci in WT, double and triple mutants. Scale bar: 5μm. * p<0.05, ** p<0.01 (one-tailed Student’s t-test).

https://doi.org/10.1371/journal.pgen.1006827.g007

Arabidopsis forms two types of COs: interference-sensitive Type I COs that require ZMM proteins like MSH4 and MLH1 [57–59], and interference-insensitive class II COs that are MUS81-dependent [60,61]. To assess the impact of RAD51 and its paralogs on Type I COs, we used an AtMLH1 antibody to visualize AtMLH1 foci, in WT, atrad51-/+ atrad51c-/+, atrad51-/+ atxrcc3-/+, atrad51c-/+ atxrcc3-/+and atrad51-/+ atrad51c-/+ atxrcc3-/+ meiocytes at diakinesis [59]. On average, WT meiocytes had 9.0±1.2 foci (n = 61, Fig 7F), whereas at similar stages, atrad51-/+ atrad51c-/+, atrad51-/+ atxrcc3-/+, atrad51c-/+ atxrcc3-/+ and atrad51-/+ atrad51c-/+ atxrcc3-/+ mutants had 7.9±1.4 (n = 40; P = 5.8E-05), 7.7±1.6 (n = 25; P = 5.4E-04), 6.4±1.3 (n = 39; P = 5.9E-16) and 5.9±1.0 (n = 16; P = 5.5E-12) foci, respectively (Fig 7G–7J and 7L). The reduction of AtMLH1 foci in the mutants is consistent with the observed reduction in chiasmata, and supports the idea that Type-I COs are reduced in the mutants.

Although the CO number was obviously reduced by ~30% in the atrad51-/+ atrad51c-/+ atxrcc3-/+ triple heterozygote, no univalents were observed, consistent with a mechanism that ensures at least one CO per chromosome [62]. If the COs were distributed among the 5 Arabidopsis bivalents randomly, they would follow the Poisson function P (k COs per bivalent) = (λke-λ)/k! where λ is the mean number of COs per bivalent. Using this function, from the analyses of 52 WT and 15 atrad51-/+ atrad51c-/+ atxrcc3-/+ triple heterozygote meiocytes, we would expect to find 36 and 19 univalents in WT and the triple mutant, respectively, but none were observed (Table 2).

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Table 2. Observed and expected nonexchange chromosomes.

https://doi.org/10.1371/journal.pgen.1006827.t002

To further quantify the remaining COs in atrad51-/+ atrad51c-/+ atxrcc3-/+, we used a flow cytometry-based assay that measures the segregation of transgenes encoding fluorescent marker proteins expressed using a pollen-specific LAT52 promoter (FTL markers) [63,64]. The number of viable pollen grains is dramatically reduced in atrad51-/+ atrad51c-/+ atxrcc3-/+, but it was still feasible to measure CO frequencies using this assay. We crossed atrad51-/+ atrad51c-/+ atxrcc3-/+ with line I2b, which carries two FTL markers (YFP and DsRed) on chromosome 2 (Fig 8A). Pollen grains which express both fluorescent proteins have not experienced a crossover between the markers, while those that express only one or the other have. The relative abundance of these two classes can be used to calculate the genetic distance between the two markers [65]. We scored 10,092 WT pollen grains and 15,460 pollen grains from the triple heterozygote (Fig 8B and 8C). The I2b map distance was 5.28±0.58 cM in WT and 2.87±0.33 cM in the triple heterozygote (Fig 8D–8G). The genetic distance between the two fluorescent markers was significantly reduced in atrad51-/+ atrad51c-/+ atxrcc3-/+ (Z score = 185.4, P value << 0.01) (Fig 8G), consistent with the reduction in chiasmata described above.

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Fig 8. Meiotic crossover frequency of I2b in WT and triple heterozygote via flow cytometry.

(A) Pollen grains from plants that are hemizygous for the DsRed2 (red grains) and eYFP (green pollen grains) markers flanking the I2b interval visualized using bright field (BF) microscopy or epi-fluorescence (R+G). Pollen grains expressing both markers are yellow. (B) Side scatter (SSC)/ forward scatter (FSC) plot of eYFP-DsRed/-- pollen grains (R1 gate) in WT with the number of pollen grains counted. Events in the R1 gate are represented as green points. (C) Side scatter (SSC)/ forward scatter (FSC) plot of eYFP-DsRed/-- pollen grains (R1 gate) in triple heterozygote with the number of pollen grains counted. Events in the R1 gate are represented as green points. (D) The R2 gate is comprised of yellow-only pollen grains; the R3 gate is comprised of the yellow+red pollen grains; the R4 gate R4 is comprised of non-fluorescent pollen grains; the R5 gate is comprised of red-only pollen grains. (E) DsRed / eYFP plot of eYFP-DsRed/-- pollen grains in WT with R2-R5 gates with the percentage of events in each quadrant is shown in each gate. (F) DsRed / eYFP plot of eYFP-DsRed/-- pollen grains in triple heterozygote with R2-R5 gates with the percentage of events in each quadrant is shown in each gate. (G) Formula to calculate the genetic distance of an interval flanked by the two fluorescent markers with results in WT and triple heterozygote. Standard deviations are shown in parentheses. Z score is 185.4: P value << 0.01.

https://doi.org/10.1371/journal.pgen.1006827.g008

Discussion

Formation of protein complexes between RAD51 and its paralogs is highly conserved

RAD51 family members are conserved across species, from yeast to humans [20]. The budding yeast S. cerevisiae has four RAD51 paralogs (Rad51, Dmc1, Rad55 and Rad57) [10], whereas humans have seven paralogs (RAD51, DMC1, RAD51B, RAD51C, RAD51D, XRCC2, XRCC3) [20]. In yeast, Rad55 interacts with Rad57 to form a stable heterodimer [10]. Similarly, in humans, two complexes are formed by the RAD51 paralogs: the BCDX2 and the CX3 complexes [36,37,39]. Moreover, a recent study in Caenorhabditis elegans showed that the RAD51 paralogs, RFS-1 and RIP-1, also exist as a heterodimer and interact with RAD51 [66].

Arabidopsis XRCC3 has been shown to interact with both RAD51 and RAD51C using a yeast two-hybrid assay [41]. We confirmed the yeast two-hybrid result (Fig 3A) and demonstrated that the AtRAD51C-AtXRCC3 interaction occurs in planta by using pull-down and BiFC assays (Fig 3B and 3C). It is noteworthy that both pull-down and BiFC assays support an interaction between AtRAD51C with AtRAD51 and AtXRCC3. Our results strongly support the idea that AtRAD51C is a central factor in complex formation, and is associated with AtRAD51 and AtXRCC3. These findings are consistent with previous results in human cells that show AtRAD51C associates with two protein complexes [36,37,39]. Our study is also the first time to show that RAD51 paralogs form a protein complex with RAD51 in plants, supporting the hypothesis that formation of RAD51-paralogs associated protein complexes is highly conserved across eukaryotes, including yeast, humans and plants.

Formation of a RAD51 paralog complex is required to facilitate RAD51 in HR

Previous studies in yeast showed that RAD51 paralogs are unable to form filaments with ssDNA and do not have a direct role in homology search or single strand invasion [10,23]. Nevertheless, studies in different organisms have reported that RAD51 paralogs play important roles in promoting RAD51 function in both mitotic and meiotic HR [46,67–69]. For example, the yeast Rad55-Rad57 complex has a role in RAD51-dependent HR [10,46]. Similar roles have been found for the C. elegans heterodimer of RAD51 paralogs RFS-1/RIP-1 [66] and the human CX3 complex [36,37,39].

Due to the lack of direct biochemical data, the role of RAD51 paralogs in meiotic HR in planta is unclear. Studies in the monocot model plant, Oryza sativa (rice), showed that the RAD51 paralogs OsRAD51C and OsXRCC3 are required for meiotic DSB repair and mutations in either result in sterility, chromosome entanglement and fragmentation [70,71]. These results are consistent with similar findings in Arabidopsis [13,14,32]. Immunostaining showed that OsXRCC3 is required for OsRAD51C localization on chromosomes [70], suggesting the existence of a potential OsRAD51C-OsXRCC3 complex in rice. Additionally, the single-end processing proteins OsCOM1 and OsDMC1 no longer associate with DSB sites in rice osxrcc3, which suggests that OsXRCC3, and by extension OsRAD51C, might function upstream of OsRAD51 [70,72]. Nevertheless, the relationship between OsRAD51 and its paralogs OsRAD51C and OsXRCC3 remains unclear, because a RAD51 antibody is currently unavailable in rice. In the present study, we showed that Arabidopsis RAD51 foci were obviously reduced in atrad51c and atxrcc3 mutants, consistent to the discovery in rice. Together, these studies, in both rice and Arabidopsis, strengthen the idea that AtRAD51 depends on its paralogs for normal function and that this relationship is highly conserved in eukaryotes.

RAD51 paralogs have a role in meiotic CO formation

Previous studies showed that RAD51 paralogs have a later role in processing meiotic recombination intermediates [40]. Direct evidence to support the RAD51C-XRCC3 complex having a role in the later meiotic recombination process come from the observation that the RAD51C-XRCC3 complex is associated with HJ resolvase activity. Moreover, RAD51C- and XRCC3-defective hamster cells have reduced resolvase activity and HJ progression [40,73]. Similarly, the Arabidopsis RAD51 paralogs AtRAD51B and AtXRCC2 were also reported to affect meiotic recombination in terms of CO number [74]. However, mutations in these paralogs show an increase in meiotic recombination frequency [74], suggesting that they have roles in meiotic CO formation. In the present study, we found that atrad51c atxrcc3 double heterozygous mutant and the atrad51 atrad51c atxrcc3 triple heterozygous mutant have significantly fewer COs (Fig 7D, 7E and 7I–7L), compared with WT. Given that the reduced number of AtRAD51 foci observed in the double and triple heterozygous mutants, we propose that a diminished capacity to form wild type level of RAD51 foci results in fewer COs in the mutants. The previous finding further supports this idea that a weaker atrad51 allele had fewer chromosome fragments and some univalents, and also formed bivalents between homologs and non-homologs [42]. Therefore, we speculate that AtRAD51 could function in two manners, both dependent on the AtRAD51 paralogs AtRAD51C and AtXRCC3. Most AtRAD51 foci are required for DNA repair using either homologs or sister chromatids as templates without CO formation, while a small number of AtRAD51 foci might play a role in normal CO formation dependent also on AtDMC1. Therefore, the AtRAD51C-AtXRCC3 is critical for ensuring wild type number of AtRAD51 foci and COs and facilitating proper homolog recombination and association.

A model for the role of RAD51 paralogs in meiotic recombination

Based on our results and previous studies, we propose a model for how AtRAD51C and AtXRCC3 function in conjunction with AtRAD51 in meiotic HR (Fig 9). Meiotic recombination is initiated by programmed DSBs that are catalyzed by AtSPO11-1 and other proteins. The broken ends are further processed by the MRX protein complex to produce ssDNA tails [2,75–77]. In WT, interaction between the AtRAD51C-AtXRCC3 complex and AtRAD51 is proposed to alter the latter’s configuration and facilitates its binding with the ssDNA tails, thus resulting in single end invasion. Consequently, repair of the DSBs yields either COs or NCOs. In the heterozygous mutants, the reduced AtRAD51 level is likely insufficient for supporting the AtDMC1 function, consistent with previous studies in both Arabidopsis and yeast showing that normal DMC1 function in meiosis requires RAD51 [6,78]. Thus, with reduced amounts of RAD51 proteins, single end invasion is possibly more promiscuous and targets both homologous and non-homologous templates, resulting in multivalent formation. This aspect of the model is supported by the observation that the triple heterozygous mutant and the weak atrad51 mutant had non-homologous associations and reduced COs. In the homozygous mutants, when AtRAD51 is either completely absent or reduced below a threshold, most or all DSBs are unrepaired, leading to severe chromosome fragmentation and chromosome entanglements. Further investigations are needed to establish the precise AtRAD51 thresholds and how the AtRAD51 paralogs maintain the necessary level of AtRAD51 during the single-end invasion process.

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Fig 9. Model of the role of RAD51C- and XRCC3-mediated meiotic recombination

(A) The number of RAD51 foci observed in different genotypes at zygotene. (B) Meiotic recombination is initiated by the formation of double-strand breaks (DSBs), catalyzed by SPO11. (C) DSB ends are processed by the MRE11- RAD50-NBS1 (MRN) protein complexes to generate 3′ ssDNA tails [2,3]. (D) In wild type, with normal RAD51 function, interactions between the RAD51C-XRCC3 complex and RAD51 may alter the configuration of RAD51 proteins and facilitate its binding to the single-strand DNA tails (E), and formation of nucleoprotein filaments (F). (G) The RAD51-mediated single end invasion results in formation of a recombination intermediate called a D-loop. (H) In the heterozygous mutant backgrounds, insufficient levels of RAD51C and XRCC3 lead to reduced RAD51 binding, leading to homolog invasion when RAD51 binds (G) or non-homolog invasion (J) in the absence of RAD51. (I-J) In the homozygous mutants, due to the absence, or severe reduction of RAD51, the single-ended DNA may form joint molecules with homologs, non-homologs or sister chromatids. The weight of the arrows indicates the proportional balance of the various intermediate products.

https://doi.org/10.1371/journal.pgen.1006827.g009

In summary, meiotic DSB repair is essential for sexual reproduction in eukaryotes including budding yeast, animals and flowering plants. RAD51 paralogs facilitate the establishment of RAD51 at DSBs and mediate and single end invasion. These functions are also highly conserved in eukaryotes. We propose that facilitation of normal RAD51 function by its paralogs, such as RAD51C and XRCC3, may be a general mechanism for meiotic DSB repair.

Materials and methods

Plant material and genotyping

The mutants atrad51-3 (SAIL_873_C08) [42], atrad51c (SALK_021960) [13], atxrcc3 (SALK_045564) [14] used in this study were shown previously to be null mutants in the Columbia (Col-0) background. atrad51-/+ atrad51c-/+ and atrad51-/- atrad51c-/- mutants were crossed by atrad51-/+ (male parent) and atrad51c-/+ (female parent), atrad51-/+ xrcc3-/+ and atrad51-/- xrcc3-/- mutants were crossed by atrad51-/+ (male parent) and xrcc3-/+ (female parent), atrad51c-/+ xrcc3-/+ and atrad51c-/- xrcc3-/- mutants were crossed by atrad51c-/+ (male parent) and xrcc3-/+ (female parent). Triple heterozygous mutants were crossed by atrad51c-/+ (male parent) and atrad51-/+ atxrcc3-/+ (female parent). Plants were grown at 21°C with 16 h light and 8 h dark. Mutant genotypes were confirmed by PCR using the primers described in S2 Table.

Phenotype analysis

A minimum of 10 plants were characterized for each mutant. Chromosome spreads were stained with DAPI and centromere FISH, and immuno-localization experiments were carried out as described previously [79]. Rabbit polyclonal AtRAD51 and γ-H2AX antibodies were used at 1:200 fold dilutions and Alexa Fluor 488 Goat Anti-Rat IgG (H+L) secondary antibody (A-21428, Invitrogen, Carlsbad, CA, USA) was used at a 1:1000 fold dilution [80]. Chiasmata distribution statistics were performed following the protocol of Sanchez et al. [81]. BAC DNA extraction (F19K16) and probe labeling were described previously [43]. Images of chromosome spreads were obtained using an Axio Imager A2 microscope (Zeiss, Heidelberg, Germany) equipped with a digital camera (Canon, Tokyo, Japan), and processed using Photoshop CS (Adobe Systems, Mountain View, CA). Images were initially captured in black & white and, if necessary, globally false-colored post-capture for visual contrast. AtRAD51 and γ-H2AX foci in WT and mutant lines were counted and statistically analyzed using ImageTool version 3.0 software (University of Texas Health Science Center, San Antonio, USA).

In mutants that lacked synapsis, we distinguished zygotene from pachytene chromosomes by their relative condensation, with pachytene being more condensed than zygotene chromosomes.

Constructs

To construct the vectors for yeast two-hybrid, pull-down and BiFC assays, full-length AtRAD51, AtRAD51C and AtXRCC3 cDNA were PCR-amplified using Phanta Super-Fidelity DNA polymerase (Vazyme Biotech Co., Ltd, China) and appropriate primers (S2 Table).

For the Y2H assay, full-length AtRAD51 and AtXRCC3 cDNA were purified and ligated into pGADT7 pGBKT7 by NdeI and BamHI double-enzyme digestion, and full-length AtRAD51C cDNA was purified and ligated into pGADT7 and pGBKT7 by NdeI and EcoRI double-enzyme digestion.

For the BiFC assay, full-length AtRAD51 and AtXRCC3 cDNA was purified and ligated into pXY103, pXY104, pXY105 and pXY106 by BamHI and SalI double-enzyme digestion, and full-length AtRAD51C cDNA was purified and ligated into pXY103, pXY104, pXY105 and pXY106 by XbaI and SalI double-enzyme digestion.

For the pull down assay, full-length AtRAD51 and AtXRCC3 cDNA was purified and ligated into pET32a and pGEX-6P-1 by BamHI and SalI double-enzyme digestion and full-length AtRAD51C cDNA was purified and ligated into pET32a and pGEX-6P-1 by EcoRI and SalI double-enzyme digestion. All constructs were verified by DNA sequencing.

Yeast two-hybrid assay

Plasmid vectors were transformed into the Y2H gold yeast strain (pGBKT7 constructs) or the Y187 yeast strain (pGADT7 constructs) using the LiAc/PEG method. Transformants were mated on YPDA medium for 48 h, and selected on SD/–Trp–Leu plates for 36 h. Transformants were then selected on SD/–His–Ade–Trp–Leu with X-α-Gal and AbA plates to test for positive interactions [82].

Pull-down assay

AtRAD51, AtRAD51C and AtXRCC3 were expressed in E. coli using the pGEX6P-1 and pET32a plasmids. The tagged proteins were mixed and incubated for 2 h at 4°C, then pulled down by GST beads for 1 h at 4°C. The protein mixture was confirmed by western blotting with a GST antibody (AG768, Beyotime Co. Ltd, China) or a His-tag antibody (AH367, Beyotime Co. Ltd, China) at 1:100 dilutions, followed by application of an horseradish peroxidase (HRP) goat anti-mouse IgG (H+L) secondary antibody (A0216, Beyotime Co. Ltd, China) at a 1:2000 dilution.

BiFC assay

BiFC plasmids (pXY103/104/105/106-RAD51, pXY103/104/105/106-RAD51C, pXY103/104/105/106-XRCC3 and pXY103/104/105/106) were transformed into Agrobacterium GV3101 cells. Transformants were harvested once the OD600 reached 2.0, and resuspended in MES/MgCl2/acetosyringone solution to a final OD600 of 1.0. Cell suspensions were mixed in 1:1 ratios of various combinations, and young Nicotiana benthamiana leaves were infiltrated. Leaves were excised and visualized using a LSM-710 confocal microscope (Zeiss) following 36 h incubation [83].

Flow cytometry

Open flowers from WT plants or atrad51-/+ atrad51c-/+ atxrcc3-/+plants that were hemizygous for the fluorescent-tagged line (FTL) interval I2b and either QRT+/+ or qrt-/+ were collected [64]. The flowers (50 or more) were mixed with 1 mL PBS buffer (10 mM CaCl2, 1 mM KCl, 2 mM MES, 5% w/v sucrose, pH 6.5) supplemented with 0.01% Triton X-100 in a 1.5-mL microcentrifuge tube. The mixture was vortexed at maximum speed for 2–3 min and the solution filtered through a 70-μm Falcon® cell strainer (352350, Corning Life Sciences, Tewksbury, MA, USA) at 450 ×g for 2 min at 4°C. The flow-through was resuspended in a fresh tube with 1 mL PBS buffer at 4°C. Flow cytometry analysis was performed using a Gallios flow cytometer (Beckman Coulter, Inc.). Statistical analysis was performed using Kaluza Analysis 1.3 software (Beckman Coulter, Inc.) using the two-color analysis methods described previously [65,84].

Statistical methods

Excel 2016 (Microsoft, USA) was used to calculate the mean and standard error of the AtRAD51 foci, γ-H2AX foci, MLH1 foci and the chiasmata numbers of WT and mutants. Data was compared using Student’s t-tests and P values were reported as either exact values or Gaussian approximations.

Supporting information

S1 Fig. Chromosome morphologies in atrad51-/- atrad51c-/-, atrad51-/- atxrcc3-/- and atrad51c-/- atxrcc3-/- double homozygous mutants.

(A-T) Wild-type (WT), atrad51-/- atrad51c-/-, atrad51-/- atxrcc3-/- and atrad51c-/- atxrcc3-/- mutant chromosome morphologies at pachytene, diakinesis, anaphase I, anaphase II and tetrad formation. In comparison with single homozygotes, atrad51-/- atrad51c-/-, atrad51-/- atxrcc3-/- and atrad51c-/- atxrcc3-/- mutants had similar chromosome phenotypes. Scale bar: 5 μm.

https://doi.org/10.1371/journal.pgen.1006827.s001

(PDF)

S2 Fig. Immunofluorescence of ASY1 at zygotene and ZYP1 at pachytene in wild type, atrad51, atrad51c and atxrcc3 mutants.

(A) Localization of ASY1 on wild-type (WT) chromosomes at zygotene. (B-D) Immunofluorescence of ASY1 at zygotene in atrad51, atrad51c and atxrcc3 mutants. (E) Localization of ZYP1 on wild-type (WT) chromosomes at pachytene. (F-H) Localization of ZYP1 at pachytene in atrad51, atrad51c and atxrcc3 mutants. Left panels show the chromosome morphology following staining with 6-diamidino-2-phenylindole (DAPI), middle panels show ASY1 signal (red line) or ZYP1 signal (red point/line), and right panels merge the DAPI-stained images with the ASY1/ZYP1 signal images. Scale bar: 5 μm.

https://doi.org/10.1371/journal.pgen.1006827.s002

(PDF)

S3 Fig. Fluorescence in situ hybridization analysis of chromosome behavior in atrad51-/+, atrad51c-/+ and atxrcc3-/+ single heterozygous mutants.

(A-P) Wild type (WT), atrad51-/+, atrad51c-/+ and atxrcc3-/+ mutant chromosome morphologies and centromere signals (shown as red dots) at pachytene, diakinesis, metaphase I and tetrad formation. In comparison with WT, atrad51-/+, atrad51c-/+ and atxrcc3-/+ mutants had similar chromosome phenotypes. Scale bar: 5 μm.

https://doi.org/10.1371/journal.pgen.1006827.s003

(PDF)

S4 Fig. Immunostaining of DMC1 signals in single, double and triple heterozygous mutant chromosomes at zygotene and pachytene stages.

The distribution of DMC1 among the eight genotypes examined shows no obvious differences on zygotene (A-H) and pachytene (I-P) chromosomes. Left panels show the chromosome morphology following staining with 6-diamidino-2-phenylindole (DAPI), middle panels show DMC1 foci (red dots), and right panels merge the DAPI-stained images with the DMC1 foci images. (Q-R) The number of DMC1 foci in chromosomes from the eight genotypes at zygotene and pachytene in A-P. Scale bar: 5 μm.

https://doi.org/10.1371/journal.pgen.1006827.s004

(PDF)

S1 Table. P-values of numbers of γ-H2AX foci for all comparisons at zygotene and pachytene.

https://doi.org/10.1371/journal.pgen.1006827.s005

(PDF)

S2 Table. Primers used in this study.

https://doi.org/10.1371/journal.pgen.1006827.s006

(XLSX)

Acknowledgments

We greatly appreciate the kind gift of AtMLH1 antibody from Prof. Raphaël Mercier at INRA, Centre de Versailles-Grignon (France). We also thank the Arabidopsis Biological Resource Center at Ohio State University for the Arabidopsis mutant seeds.

Author Contributions

  1. Conceptualization: YW HM.
  2. Data curation: HS ZC.
  3. Formal analysis: HS ZC GPC.
  4. Funding acquisition: YW HM.
  5. Investigation: YW HM HS ZC.
  6. Project administration: YW HM JL.
  7. Resources: YW HM.
  8. Software: GPC JH.
  9. Supervision: YW HM GPC.
  10. Validation: HM GPC.
  11. Visualization: YW HM HS ZC GPC JH.
  12. Writing – original draft: YW HS ZC.
  13. Writing – review & editing: YW HS ZC HM GPC.

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