Research

The Arabidopsis RAD51 paralogs RAD51B, RAD51D and XRCC2 play partially redundant roles in somatic DNA repair and regulation

Yingxiang Wang1, Rong Xiao2*, Haifeng Wang1,3*, Zhihao Cheng1, Wuxing Li2, Genfeng Zhu1, Ying Wang1 and Hong Ma1,2,3 1State Key Laboratory of Genetic Engineering and Institute of Genetics, Institute of Plant Biology, Center for Evolutionary Biology, School of Life Sciences, Fudan University, Shanghai 200433, China; 2Department of Biology, Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA 16802, USA; 3Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China

Summary Author for correspondence: The eukaryotic RAD51 gene family has seven ancient paralogs conserved between plants  Hong Ma and animals. Among these, RAD51, DMC1, RAD51C and XRCC3 are important for homolo- Tel: +86 21 55665569 gous recombination and/or DNA repair, whereas single mutants in RAD51B, RAD51D or Email: [email protected] XRCC2 show normal meiosis, and the lineages they represent diverged from each other Received: 12 May 2013 evolutionarily later than the other four paralogs, suggesting possible functional redundancy. Accepted: 14 August 2013 The function of Arabidopsis RAD51B, RAD51D and XRCC2 in mitotic DNA repair  and meiosis was analyzed using molecular genetic, cytological and transcriptomic approaches. New Phytologist (2014) 201: 292–304 The relevant double and triple mutants displayed normal vegetative and reproductive  doi: 10.1111/nph.12498 growth. However, the triple mutant showed greater sensitivity than single or double mutants to DNA damage by bleomycin. RNA-Seq transcriptome analysis supported the idea that the Key words: Arabidopsis, DNA repair, gene triple mutant showed DNA damage similar to that caused by bleomycin. On bleomycin treat- regulation, homologous recombination, ment, many genes were altered in the wild-type but not in the triple mutant, suggesting that molecular evolution, RAD51 paralogs. the RAD51 paralogs have roles in the regulation of gene transcription, providing an explana- tion for the hypersensitive phenotype of the triple mutant to bleomycin. Our results provide strong evidence that Arabidopsis XRCC2, RAD51B and RAD51D have  complex functions in somatic DNA repair and gene regulation, arguing for further studies of these ancient genes that have been maintained in both plants and animals during their long evolutionary history.

correct genetic information in the repair process (West , Introduction et al. 2004; Bleuyard et al., 2006). In addition to its function in somatic Genome stability is important for cellular homeostasis and an DNA repair, HR is also required for normal meiosis to maintain organism must be able to repair DNA damage. Among a variety the association of homologous and contributes to of DNA damage, double-strand DNA breaks (DSBs) are caused the redistribution of genetic diversity among progeny. by ionizing radiation, genotoxic chemicals or errors in DNA repli- The genes involved in HR were first identified in budding cation (Kuzminov, 2001; Tonami et al., 2005). Failure to correctly yeast and mainly belong to the RAD52 epistasis group, including repair DSBs can cause genome instability, mutations, cell cycle RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55, arrest and even cell death (Glazer & Glazunov, 1999; Mills et al., RAD57, RAD59, MRE11 and XRS2 (Paques & Haber, 1999; 2003; Dudasova et al., 2004; Sasaki et al., 2004). DSBs are known Krogh & Symington, 2004). Further identification of their to be repaired by two major pathways: homologous recombination homologs in animals and plants suggests that the HR repair path- (HR) and non-homologous end-joining (NHEJ). The NHEJ way is highly conserved (Krogh & Symington, 2004; Bleuyard pathway involves the rejoining of two broken DNA ends without et al., 2006). Among them, members of the RAD51 family, a template of similar sequence, often resulting in deletions or inser- including DMC1, RAD51 and five RAD51 paralogs (RAD51B, tions. By contrast, HR is a relatively accurate pathway that RAD51C, RAD51D, XRCC2 and XRCC3) have crucial roles in depends on the homologous DNA sequence, thereby retaining the HR or DNA repair in mammals. Mutations in several of these genes lead not only to elevated sensitivity to DNA damaging *These authors contributed equally to this work. agents, but also to embryonic lethality (Tsuzuki et al., 1996; Shu

292 New Phytologist (2014) 201: 292–304 Ó 2013 The Authors www.newphytologist.com New Phytologist Ó 2013 New Phytologist Trust New Phytologist Research 293 et al., 1999; Deans et al., 2000; Pittman & Schimenti, 2000), Characterization of the double and triple mutants suggesting that they are important for DNA repair during the mitotic cell cycle. F1 double heterozygous plants were generated by crosses between Homologs of DMC1 and RAD51 have been studied in many , rad51b and rad51d homozygous single mutant plants. The eukaryotes, including fungi, invertebrate animals and plants triple heterozygous F1 plants were generated by crossing the (Bishop et al., 1992; Habu et al., 1996; Klimyuk & Jones, 1997; rad51b xrcc2 double homozygous mutant with rad51d. The F2 Couteau et al., 1999). In Arabidopsis thaliana, they function in or F3 progeny plants were genotyped with each of the gene- DNA repair via HR in somatic or meiotic cells (Couteau et al., specific primers for RAD51B and XRCC2, combining with the 1999; Bleuyard & White, 2004; Li et al., 2004, 2005; Abe et al., T-DNA left board primer (Supporting Information Table S1). 2005; Bleuyard et al., 2005; Osakabe et al., 2005). For simplicity, To genotype rad51d, PCR products were digested with SphI to unless otherwise noted, the RAD51 paralogous genes and produce two fragments of 233 and 86 bp for the wild-type and mutants refer to those of Arabidopsis. Both and one fragment of 319 bp for the mutant allele. knockout mutants are hypersensitive to DNA damaging agents and sterile with striking meiotic fragmentation, sug- Light microscopy gesting that RAD51C and XRCC3 are involved in mitotic DNA repair by somatic and meiotic recombination (Bleuyard & Photographs of plants were taken with a Sony digital camera White, 2004; Abe et al., 2005; Bleuyard et al., 2005; Li et al., DSC-707 (Tokyo, Japan). The viability of mature pollen grains 2005). By contrast, the rad51b, rad51d and xrcc2 mutants show was examined after staining with Alexander’s solution (Alexander, normal fertility without detectable meiotic defects, but are sensi- 1969). Mitosis was examined using root tips of 1-wk-old seed- tive to various DNA damaging agents (Bleuyard et al., 2005), lings, as described previously (Li et al., 2004). Male meiosis was and RAD51B and XRCC2 seem to have a role in the suppression examined using chromosome spreading with 4′,6-diamidino- of meiotic recombination (Ines et al., 2013), suggesting that 2-phenylindole (DAPI) staining, as described previously (Ross RAD51B, RAD51D and XRCC2 are involved in somatic and mei- et al., 1996). Both pollen and meiotic cells were photographed otic HR. Furthermore, the Arabidopsis RAD51B, RAD51C and using a Nikon dissecting microscope (Tokyo, Japan) with an RAD51 also interact in yeast two-hybrid systems, similar Optronics digital camera (Goleta, CA, USA). to their mammalian counterparts, suggesting that they have conserved functions (Osakabe et al., 2005). Treatment with DNA damaging agents In mammals, the embryonic lethality of mutations in RAD51 and its paralogs makes it difficult to analyze their function The eight genotypes of Col (wild-type), rad51b, rad51d (ssn1), in vivo. By contrast, none of the Arabidopsis RAD51 paralogs is xrcc2, rad51b rad51d, rad51d xrcc2, rad51b xrcc2 and rad51b required for survival in individual single mutants. It has been rad51d xrcc2 were treated with either of two types of DNA dam- reported that the RAD51B, RAD51D and XRCC2 homologs aging agent: the cross-linking agents cisplatin (Sigma P4394), form the three groups that occupy the last three branches in the methyl methanesulfonate (MMS; Sigma M4016) and mitomy- RAD51 family tree (Lin et al., 2006). Therefore, these genes cin-C (MMC; Sigma M4287); the DSB-inducing agent bleomy- might have overlapping/redundant functions in plant mitotic cell cin (Sigma B5507). Seeds were surface sterilized with 10% cycle or meiotic cells. It is also possible that RAD51B, RAD51D NaClO for 5 min and 75% ethanol for 5 min, and then sown on and XRCC2 are not required for normal meiosis, even when their Murashige and Skoog (MS) plates containing different concen- functions are lost simultaneously. Nevertheless, the Arabidopsis trations of MMC, bleomycin, cisplatin or MMS, as indicated in RAD51B, RAD51D and XRCC2 proteins might form a the text. The plates were placed at 4°C for 3 d, and then trans- complex that interacts with RAD51C in the process of DNA ferred to a growth chamber. The resistance or sensitivity was esti- repair by HR. To date, the genetic relationship among RAD51B, mated by the average fresh weight of four plants after growth for RAD51D and XRCC2 in Arabidopsis has not been studied. The 3 wk. study of their genetic interactions should provide clues to the understanding of the function and relationship of these genes The comet assay during their long evolutionary history. Fourteen-day-old plants grown on half-strength MS plates under 1 normal conditions were incubated in 2 glÀ of bleomycin for Materials and Methods l 6 h and then harvested in liquid nitrogen. Comet assay for DNA damage was performed according to a previously described proto- Plant materials and growth conditions col (Menke et al., 2001; Zhu et al., 2006) with minor modifica- The xrcc2 (SALK_029106) and rad51b (SALK_024755) T-DNA tions. Comet slides were prepared and subjected to 1 insertional lines have been characterized previously by Bleuyard electrophoresis on ice for 2 min (2 V cmÀ , 11 mA). Images of et al. (2005). rad51d (also named ssn1) was obtained from Profes- comets were captured under a Zeiss Axio Imager A2 fluorescence sor Xinnian Dong’s laboratory (Durrant et al., 2007). All plants microscope with a high-resolution microscopy camera AxioCam were grown in growth chambers under a 16-h light : 8-h dark MRc Rev. 3 FireWire (D). The comet data analysis was per- photoperiod at 22°C : 18°C, unless otherwise indicated. formed using CometScore software (http://autocomet.com). The

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DNA fragments in comet tails (% tail-DNA) were used to esti- enrichment analysis employing the online tools AgriGO with mate the extent of DNA damage. Fisher’s exact test and false discovery rate (FDR) correction (http://bioinfo.cau.edu.cn/agriGO/analysis.php). Transcription factor (TF) family annotations were downloaded from the Plant- RNA extraction and real-time PCR TFDB v2.0 database, containing 2023 TFs in 58 families for Three-week-old young plants grown without or with DNA dam- Arabidopsis thaliana (http://planttfdb.cbi.edu.cn/index.php; 1 aging agent at a concentration of 23.5 lg mlÀ (in the same man- Zhang et al., 2011). The heat map of the expressed TFs was ner as for the phenotypic analyses) were collected and quickly implemented by the pheatmap (Pretty Heatmaps) function in the frozen in liquid nitrogen. Total RNA was extracted using an pheatmap package (R version, 2.15, pheatmap version, 0.6.1; R RNeasy Plant Kit (Qiagen, Valencia, CA, USA) and its concen- Core Team, Vienna, Austria). tration was estimated on an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). Approximately 1 g of l Results total RNA was used to synthesize cDNA according to the manu- facturer’s instruction (Promega, Madison, WI, USA). Real-time Expression patterns of the three RAD51 paralogs PCR was performed as described previously (Yang et al., 2011) in triplicate for each sample, using gene-specific primers (Table S1). Previous studies have shown that RAD51B is expressed widely and at a higher level in the floral buds than in other tissues (Osakabe et al., 2005). Similarly, RAD51D is expressed widely, RNA-seq library preparation and sequencing but at very low levels (Durrant et al., 2007). The expression of The ribosomal RNA was then removed by two purification steps XRCC2 has not been reported. To further examine the expression with a PolyATtract® mRNA Isolation System (Promega) and a of these three Arabidopsis RAD51 paralogs (referred to hereafter Poly (A) PuristTM Kit (Ambion, Austin, TX, USA), respectively. as the RAD51 paralogs for convenience), we searched our micro- The removal of ribosomal RNAs was confirmed on an Agilent array data and male meiocyte transcriptome by RNA-Seq (Zhang 2100 Bioanalyzer. Approximately 0.8 lg of mRNA was frag- et al., 2005; Yang et al., 2011). Consistent with previous reports, mented by RNase III at 37°C for 10 min and ligated with adap- our microarray data showed that RAD51B and RAD51D were tor Mix A for reverse transcription. The 100–200 nucleotides of widely expressed with relatively low levels, as was XRCC2 the first-strand cDNA were recovered by separation in 6% TBE (Fig. S1a). Unlike DMC1, RAD51 and RAD51C, which showed (Tris-borate-EDTA)-Urea Gel (Invitrogen, Carlsbad, CA, USA). the highest expression in stage-6 anthers containing meiocytes, The fractionated cDNAs were amplified with 11–15 cycles of RAD51B and RAD51D were expressed at lower levels in stage-6 PCR and then purified to yield the SOLiD Fragment Library; anthers than in other tissues (Fig. S1a). Their low-level expression 600 pg of the library was used for emulsion PCR; 50-base was further confirmed by our male meiocyte transcriptome data sequence reads were obtained using a SOLiD sequencer (ABI, (Yang et al., 2011; Fig. S1b). Foster City, CA, USA). Mutants defective in the RAD51 paralogs showed normal Estimation of expression level and differential gene vegetative and reproductive growth expression Previous studies have shown that the rad51b, rad51d and xrcc2 Reads from each sample were aligned to The Arabidopsis Infor- single mutants display normal vegetative and reproductive mation Resource (TAIR) 10 Arabidopsis reference genome growth (Bleuyard et al., 2005; Durrant et al., 2007). To test (http://www.arabidopsis.org) using SOLiDTM BioScopeTM Soft- whether these genes were redundant for normal development, we ware 1.3 (https://products.appliedbiosystems.com), a SOLiD generated the relevant double and triple mutants and found that data analysis package for transcriptome sequencing and other they all exhibited normal development of the vegetative and floral sequencing technologies. Afterwards, the aligned reads matching organs and produced seedpods with normal seed numbers the annotated genes were used to estimate gene expression levels (Fig. 1). Furthermore, pollen grains from all single and multiple and to identify differentially expressed genes between treatments mutants were viable and indistinguishable from those of the by Cufflinks (v1.2; Trapnell et al., 2010). To reduce the false- wild-type (Fig. 1). Because mutations in RAD51 paralogs in positive rate, a threshold for differential expression was set at a P mouse and chicken cause severe mitotic defects that are accompa- value of 0.05 or less in the Cufflinks output, with a further nied by chromosome fragmentation (Takata et al., 2000, 2001), requirement of a minimal gene expression level of at least 1.0 it is possible that there are minor defects in mitotic cell division FPKM (fragments per kilobase of transcripts per millions of not observed from gross examination of the vegetative and repro- mapped reads). ductive growth of the Arabidopsis mutants. Mitotic chromo- somes were further examined using DAPI staining of cells from 1-wk-old root tips of the eight genotypes. The results showed (GO) enrichment analysis that mitotic chromosome features at metaphase, anaphase and We used the GO terms defined by the TAIR 10 GO Slim data- telophase were indistinguishable among the eight phenotypes: base (ftp.arabidopsis.org:/Ontologies/Gene_Ontology) for GO wild-type (70 cells), rad51b (54 cells), rad51d (35 cells), xrcc2

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Fig. 1 Phenotypes of wild-type (WT) and the Arabidopsis rad51b, rad51d, xrcc2 single, double and triple mutants. The left columns show the flower phenotype for the eight genotypes (as indicated on the left), the middle columns display the features of siliques and the right columns present pollen grains stained with Alexander’s solution of the eight genotypes. No obvious differences were found among the eight genotypes.

Fig. 2 Mitotic chromosomes in 1-wk-old Arabidopsis root tips of wild-type (WT) and the rad51b, rad51d, xrcc2 single, double and triple mutants. Mitotic chromosomes were visualized with 4′,6-diamidino- 2-phenylindole (DAPI) at metaphase, anaphase and telophase stages during the mitotic cell cycle.

(60 cells), rad51b rad51d (27 cells), rad51b xrcc2 (52 cells), might still be present, but not sufficiently severe to affect fertil- rad51d xrcc2 (59 cells) and rad51b rad51d xrcc2 (74 cells; Fig. 2). ity, as was the case for the mus81 mutant (Berchowitz et al., Taken together, these results suggest that these three genes com- 2007). We found that male meiosis in all mutant genotypes bined are not required for plant vegetative and reproductive showed normal chromosome behavior, including typical diaki- development. nesis with five bivalents (Fig. 3). In addition, similar to the wild-type, the bivalents in all mutants were well aligned at the equatorial plane at metaphase I (Fig. 3) and then segregated to The single, double or triple mutants in the RAD51 paralogs form two groups of chromosomes at anaphase I. The two appeared normal in male meiosis groups of chromosomes further underwent decondensation and In Arabidopsis, both RAD51C and XRCC3 are required for recondensation, and were again aligned at two division planes meiosis and DNA repair (Bleuyard & White, 2004; Abe et al., at metaphase II (Fig. 3). After anaphase II and telophase II, 2005; Li et al., 2005). Even though the mutants in the three sister chromatids were separated to form four nuclei, which RAD51 paralogs showed normal pollen phenotypes, we could were packed into four microspores at the end of male meiosis not rule out the possibility that minor male meiotic defects (Fig. 3). We examined a total of > 300 meiocytes for the triple

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Fig. 3 The Arabidopsis male meiosis in the wild-type (WT) and rad51b, rad51d, xrcc2 single, double and triple mutants. Chromosome features were stained by 4′,6-diamidino-2-phenylindole (DAPI) in the WT and mutants at diakinesis, metaphase I, metaphase II and tetrad stages. No obvious differences were found among the eight genotypes at each stage.

mutant, but found no obvious meiotic defects. Therefore, we bleomycin strongly inhibited the growth of all plants, with more conclude that these three genes are not essential for male meio- dramatic effects on mutant genotypes, especially for the triple sis, because mutations in the three genes together did not mutants, with significant differences compared with all other result in detectable abnormality. genotypes (Fig. 4e,f). The hypersensitivity of the triple mutant to the high dose of bleomycin was different from its response to the cross-linking agents, such as MMC, suggesting that the three RAD51B, RAD51D and XRCC2 were partially redundant RAD51 paralogs have partially redundant functions or are for somatic DSB repair involved in different pathways for the repair of DNA damage Mutations in different RAD51 paralogs caused sensitivity to induced by bleomycin. DNA damaging agents, such as c-irradiation, MMC, cisplatin To further evaluate the levels of DNA damage in the double and bleomycin, in both animal cells and Arabidopsis (Liu et al., and triple mutants exposed to bleomycin, the comet assay experi- 1998; Takata et al., 2001; Abe et al., 2005; Osakabe et al., 2005), ment, which reveals damaged DNA in a tail resembling that of a suggesting that the functions of the RAD51 paralogs in DNA comet after electrophoresis, was performed to estimate the damage repair are conserved between vertebrates and plants. To amount of DNA damage in 2-wk-old seedlings of the eight geno- investigate the relationship between RAD51B, RAD51D and types induced by bleomycin. The results showed that, under nor- XRCC2 in DNA damage repair, we first examined whether the mal growth conditions, wild-type and triple mutant plants double and triple mutant plants conferred greater sensitivity than showed no significant difference in the levels of DNA damage 1 the single mutants to MMC, a DNA cross-linking agent (Warren (Fig. 5a,b). By contrast, with 2 lg mlÀ bleomycin induction for et al., 1998). As shown in Fig. 4(c,d), the growth of the eight 6 h, the accumulation of DNA in the comet tail in the xrcc2 sin- genotypes was indistinguishable at a low dose of MMC gle mutants and all double and triple mutants was significantly 1 1 (30 lg mlÀ ). At 60 lg mlÀ or higher concentrations, the growth higher than that in rad51b, rad51d and the wild-type (Fig. 5a,b). of all mutants was inhibited significantly compared with that of It is especially worth noting that the triple mutants showed the the wild-type, to similar extents among the single, double and highest level of DNA damage with 92.22% of the nuclear DNA triple mutants (Fig. 4c,d). In a parallel experiment, the single, in the tail, consistent with the observation that the triple mutant double and triple mutants exhibited slight or mild sensitivity in a displayed the highest sensitivity to bleomycin. These results fur- similar manner to other DNA cross-linking agents, such as MMS ther support the proposal that these three genes have partially and cisplatin (data not shown). These results indicate that these redundant roles in DNA repair. three genes play similar, but not redundant, roles in the repair of damage caused by DNA cross-linking agents. Expression of DNA repair genes was induced in the triple Previous studies have demonstrated that RAD51B, RAD51D mutant or by bleomycin and XRCC2 have a role in DSB repair (Bleuyard et al., 2005; Osakabe et al., 2005; Durrant et al., 2007), but their genetic rela- Previous studies have revealed that mutants, such as and tionship in DSB repair is still lacking. Thus, we evaluated the rad51d, which are defective in mitotic HR or DNA repair, are sensitivity of the single, double and triple mutants to bleomycin, also affected in gene expression (Durrant et al., 2007; Tuteja which causes DSBs in DNA (Favaudon, 1982). As shown in et al., 2009; Wang et al., 2010; Liu & Gong, 2011). To investi- Fig. 4(e,f), all mutants grew normally on medium supplemented gate whether the hypersensitive phenotype of the triple mutant to 1 1 with 7.05 lg mlÀ of bleomycin. At 14.1 lg mlÀ , all single, bleomycin is related to the expression of HR or other double and triple mutants were more severely affected than the DNA repair genes, we examined several representative genes in wild-type, with the triple mutants being slightly more sensitive 3-wk-old seedlings with or without bleomycin treatment. In 1 than the double mutants (Fig. 4e,f). The dose of 23.5 lg mlÀ mammalian cells, RAD51B, RAD51D and XRCC2 form a

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(a) (b)

(c) (d)

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Fig. 4 Mitomycin-C (MMC) and bleomycin sensitivity assay of wild-type (WT) and rad51b, rad51d, xrcc2 single, double and triple mutants. The Arabidopsis plants were grown on half-strength Murashige and Skoog (MS) medium without or with different doses of methanesulfonate (MMS) or bleomycin for 3 wk, with 16 plants for each genotype in each replicate. The growth phenotypes were for normal conditions (a) for the eight genotypes 1 1 arranged as indicated in (b), for 60 and 120 lg mlÀ of MMC (c) and for 14.1 and 23.5 lg mlÀ of bleomycin (e); genotypes for plants shown in (c) and (e) are also the same as indicated in (b). Fresh weight was determined for groups of four plants, and the average weight for 16 plants was calculated. Shown are the average fresh weights from three independent tests for each genotype under a series of MMC treatments (d) and bleomycin treatments (f). Bars show standard error (**, P < 0.01 for comparison between each mutant and WT, as well as between the single or double mutants and the triple 1 mutant at 23.5 lg mlÀ in (d) and (f)). CK, control. complex with RAD51C (the BCDX2 complex), and RAD51C wild-type and triple mutant seedlings by the SOLiD 3 platform, with XRCC3 (the CX3 complex; Wiese et al., 2002); furthermore, as described previously (Yang et al., 2011). We obtained a total some of the plant members can also interact physically in a yeast of c. 378 million single-end reads of 50 bp (Table S2). Approxi- two-hybrid assay (Osakabe et al., 2005). Therefore, we first exam- mately 65.9% of reads were mapped to the Arabidopsis reference ined the expression of RAD51 paralogs, including RAD51 and genome (TAIR 10), representing c. 70.8% of the annotated genes RAD51C. As shown in Fig. 6, RAD51 and RAD51C expression in TAIR 10 (Table S3) and providing high-quality data to showed no significant difference between the wild-type and triple explore the transcriptome. mutants with or without bleomycin treatment (Fig. 6). By con- We first identified genes with altered expression in the triple trast, the expression level of GAMMA RESPONSE1 (GR1) was mutant compared with the wild-type (Fig. 7a), and found that increased dramatically by either the triple mutations or bleomycin 2111 genes were differentially expressed (FPKM ≥ 1 and induction (Fig. 6). We then examined the expression of BRCA1, P ≤ 0.05), including 1450 up-regulated (Table S4) and 661 which is important for HR and DNA repair in plants (Bundock & down-regulated (Table S5) genes, although many of these genes Hooykaas, 2002; Block-Schmidt et al., 2011), and found that might not be regulated directly by the RAD51 paralogs. The GO BRCA1 expression in the wild-type and triple mutants resembled categorization for the 1450 up-regulated genes showed that that of RAD51 under normal conditions (Fig. 6). By contrast, molecular functions of DNA repair, transcriptional regulator BRCA1 expression was sharply increased in the triple mutant com- activity, DNA binding, enzyme activity and developmental regu- 4 pared with the wild-type under bleomycin treatment (Fig. 6). It is lation were over-represented (P < 10À , Fig. 7b). Specifically, possible that DNA damage induced by bleomycin was repaired less among these were over 45 genes with known functions in somatic effectively in the triple mutant, as suggested by its greater sensitiv- DNA repair or meiotic recombination, such as COM1/GR1, ity to bleomycin relative to the single or double mutants, thereby MND1 and RPA1A, as well as RAD3, RAD54, MutS homolog 2, triggering the elevated expression of some of the DNA repair 4, 7 and DNA damage repair 1 (DRT101; Table S6), suggesting genes. that the triple mutant had more DNA damage even without bleomycin treatment, although this was not obvious using the comet assay. RAD51B, RAD51D and XRCC2 affected normal gene To investigate possible effects of the DNA damaging agent expression bleomycin on gene expression in the wild-type, we compared the To identify additional genes with altered expression in the triple wild-type transcriptome with or without bleomycin treatment, mutant, RNA-Seq was performed using mRNA isolated from and found that 4311 genes were differentially expressed

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(a)

Fig. 6 Analysis of the expression of RAD51, RAD51C, GR1 and BRCA1 genes in wild-type (Wt) and triple mutants using quantitative real-time PCR. The 3-wk bleomycin-treated and untreated Arabidopsis seedlings in wild-type and mutants were the same as in Fig. 4. Data are means SD of Æ three technical replicates (**, P < 0.01 for comparison with wild-type). Similar patterns were obtained from two biological replicates. CK, control.

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Fig. 5 Evaluation of DNA damage in wild-type (WT) and rad51b, rad51d, xrcc2 single, double and triple mutants by the comet assay. Samples for comet assay were prepared from untreated and bleomycin-treated 14-d-old Arabidopsis WT and mutant seedlings, as described in the Materials and Methods section. (a) Representative comet images are shown for the untreated WT and the triple mutant, as well as the eight treated genotypes. The red color shows the DNA stained by propidium iodide (PI). (b) The fraction of DNA from untreated and treated samples was separated in the comet tails by electrophoresis, which is defined as an indicator for double-strand DNA break (DSB) repair, and was quantified using a computerized CCD camera digital image analysis system (Tritek CometScore). The values shown are averages from two to four technical replicates, each with three slides, and from each slide 25 nuclei were scored. (*, P < 0.05; **, P < 0.01; ***, P < 0.001, for comparison between ▲▲▲ each mutant and WT; , P < 0.001 for comparison between the double Fig. 7 Comparisons of the differentially expressed genes affected by the mutants and the triple mutant). triple mutations and/or bleomycin treatment in Arabidopsis. (a) A comparison of the differentially expressed genes caused by the triple (FPKM ≥ 1and ≤ 0.05), with 2835 genes up-regulated mutations and bleomycin treatment (in the wild-type (Wt)). (b) Gene P ontology (GO) annotation of three sets of up-regulated genes from (a). (Table S7) and 1476 genes down-regulated (Table S8) in bleo- DEF_707, DEF_739 and DEF_2047 represent the differentially expressed mycin-treated seedlings. The GO categorization indicated that up-regulated genes in the triple mutant, in both triple mutant and DNA repair (53 genes; Table S6), response to stimulus, immune bleomycin-induced wild-type and in the bleomycin-induced wild-type, response, DNA binding and several kinds of enzymes were most respectively. (c) A comparison of the differentially expressed genes caused enriched in the up-regulated genes (Fig. 7b). In the down-regu- by the triple mutations and bleomycin treatment in the triple mutant. CK, control. lated genes, the most enriched categories were the same as those in the up-regulated genes of the triple mutant, such as transcrip- The fact that several known genes involved in DNA repair tional regulation, DNA binding and enzyme activity (Table S9), were induced in both the triple mutant and by bleomycin treat- but the specific genes did not overlap between these sets. ment suggested that the respective sets of differentially expressed

New Phytologist (2014) 201: 292–304 Ó 2013 The Authors www.newphytologist.com New Phytologist Ó 2013 New Phytologist Trust New Phytologist Research 299 genes might be similar when compared with the untreated Although the expression of similar numbers of genes was wild-type. To test this possibility, we compared the two sets of affected by bleomycin in both the wild-type and the triple differentially expressed genes, and found that the number of mutant, the fact that the triple mutant was hypersensitive to bleo- differentially expressed genes in the bleomycin-treated wild-type mycin suggested that distinct sets of genes might be affected in was more than double the number of genes differentially these two genotypes. Indeed, 1428 of the genes up-regulated in expressed in the triple mutant, suggesting that bleomycin has the bleomycin-treated wild-type were not induced in the triple more severe effects than the three mutations. In addition, among mutant by bleomycin, and 1001 of the genes induced in the tri- the up-regulated genes in the triple mutant, 739 genes (> 50%) ple mutant were not up-regulated in the treated wild-type were also found in the up-regulated genes caused by bleomycin (Fig. 8a, Table S18). Likewise, 624 of the genes that were down- treatment (Fig. 7a, Table S10), suggesting that a large number of regulated in the bleomycin-treated wild-type were not repressed genes were induced in the triple mutant probably as a result of by bleomycin in the triple mutant, whereas 1224 of the genes the accumulation of DNA damage when the repair functions were reduced. Furthermore, functional annotation of these genes showed that the main molecular functions were related to DNA (a) (b) repair, chromatin structure and stimulus response (Fig. 7b), including 30 genes related to DNA repair as mentioned already, such as COM/GR1, MND1, RPA1A, DRT101 and MSH7. These results support the idea that bleomycin and mutations in the three RAD51 paralogs both cause the accumulation of DNA damage. Moreover, transcriptional regulation was also most enriched in a set of 704 genes induced in the triple mutant, but not by bleomycin (Fig. 7b), whereas the categories of stimulus and immune response, enzyme activity and cell death were most enriched in the 2047 genes induced by bleomycin, but unaffected in the triple mutant (Fig. 7b), indicating that bleomycin treat- ment and the triple mutations also induced distinct changes in gene expression. (c) (d)

The triple mutant exhibited a strong transcriptomic response to bleomycin Although the triple mutant showed altered expression relative to the wild-type for many genes, it still responded to bleomy- cin in gene regulation. We therefore compared the transcripto- (e) mes between bleomycin-treated and untreated triple mutant seedlings, and found 2408 and 2076 genes to be up-regulated and down-regulated, respectively, in treated triple mutant seed- lings (Fig. 7c, Tables S11, S12). GO annotation showed that most enriched categories in the up-regulated genes were the same as those in the bleomycin-treated wild-type (Table S13), whereas transcriptional regulation, DNA binding and oxidore- ductase activity were enriched in down-regulated genes (f) (Table S14). We also found that bleomycin affected very different sets of genes relative to the triple mutations, as supported by the rela- tively large number of genes showing opposite effects for the tri- ple mutations and bleomycin: 270 were expressed at higher levels in the wild-type than in the triple mutant, but were repressed by Fig. 8 Additional comparisons of the differentially expressed genes caused by mutations in the RAD51 paralogs and/or bleomycin in Arabidopsis. (a) bleomycin, whereas 352 showed lower levels of expression in the A comparison of the genes differentially expressed by bleomycin in either wild-type than in the triple mutant, but were induced by bleomy- the wild-type (Wt) or triple mutant. (b) A comparison of the genes cin (Fig. 7c, Tables S15, S16). GO annotation analysis revealed differentially expressed by the triple mutations in either the absence or that TF and regulation activity, DNA binding and enzyme inhib- presence of bleomycin. (c) Hierarchical clustering of the 84 differentially itor activity were enriched in the 352 genes induced by bleomy- expressed genes encoding transcription factors in the four samples. (d–f) Heat-map of bHLH, ERF and MYB genes with putative or known functions cin in the triple mutant (Table S17), suggesting that the in development or responses to stress. Red represents genes with a higher up-regulatory genes are important for the response to stress by expression level in the mutants, and blue indicates reduced expression. CK, the triple mutant. control.

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repressed by bleomycin in the triple mutant did not decrease in these genes, making them more easily induced by bleomycin the bleomycin-treated wild-type (Fig. 8a). It is possible that the (Fig. 8c). Expression of the group III genes was reduced dramati- genes affected by bleomycin in the wild-type, but not in the triple cally in the wild-type by bleomycin, but the difference in the tri- mutant, require the function of the RAD51 paralogs for increased ple mutant, with or without bleomycin treatment, was not expression by bleomycin. Among the 1001 genes specifically dramatic (Fig. 8c), again suggesting a role of the RAD51 paralogs induced by bleomycin in the triple mutant are those related to in the altered expression caused by bleomycin. The group IV defense and immune responses, apoptosis, cell death and enzyme genes were negatively regulated by the RAD51 paralogs, but bleo- activities (Table S19). These genes might be sensitized by the mycin canceled the effect of the triple mutations (Fig. 8c). The defects of the RAD51 paralogs and might be more easily induced group V genes were slightly repressed by bleomycin in the wild- by bleomycin in the mutant compared with the wild-type. type, and even more repressed by bleomycin in the triple mutant, To further investigate whether the hypersensitivity of the triple suggesting an additive effect (Fig. 8c). Moreover, TF genes of the mutant to bleomycin is related to the expression of specific genes, same family also showed distinct expression patterns in the four we compared the expression of genes with known DNA repair samples. For example, among five bHLH genes examined here, functions, and found that most genes examined showed dramati- the expression pattern of At5G51780 and At1G74500 was similar cally lower levels of expression in the bleomycin-treated triple to that of the group I genes, At5G15160 was similar to the group mutant than in the bleomycin-treated wild-type (Table S6), simi- III genes, whereas At3G59060 and At5G56960 were similar to lar to the above real-time PCR results (Fig. 6). The low-level the group V genes (Fig. 8d). Similar phenomena were also found expression of these DNA repair genes, in addition to the triple for MYB and ERF genes (Fig. 8e,f). The variety of expression pat- mutations, suggested that the DNA repair capacity was low and terns of the TF genes caused by the triple mutation or bleomycin DNA damage probably accumulated abnormally in the triple treatment suggest that plants use many different regulators to mutant when treated with bleomycin. achieve a comprehensive response to DNA damage from differ- To identify additional differentially affected genes, we com- ent causes. pared the transcriptomes between the triple mutant and wild- type, both treated with bleomycin, and found 562 differentially Discussion expressed genes, with 238 up-regulated and 324 down-regulated in the triple mutant compared with the wild-type (FPKM ≥ 1 The role of RAD51B, RAD51D and XRCC2 in somatic DNA and P ≤ 0.05; Fig. 8b). Most of these genes (428) did not overlap repair with those differentially expressed between the triple mutant and wild-type when both were untreated with bleomycin (Fig. 8b), Similar to vertebrates, the Arabidopsis genome also contains suggesting an interaction between the triple mutations and bleo- seven RAD51 homologs, which can be divided into two ancient mycin. In other words, these 428 genes required the presence of groups, the RADa and RADb subfamilies (Lin et al., 2006). The bleomycin for differential expression by the triple mutations, RADa subfamily includes both RAD51 and DMC1, whereas the with 189 and 239 genes up-regulated and down-regulated, RADb subfamily includes RAD51B, RAD51C, RAD51D, XRCC2 respectively (Fig. 8b, Tables S20, S21). GO analysis of the 428 and XRCC3, which are also known as the five Arabidopsis genes revealed gene function categories for metabolism, signaling, RAD51 paralogs. Among the seven genes, RAD51, DMC1, stresses, catalytic activity and transcriptional regulation (Fig. S2). RAD51C and XRCC3 have non-redundant roles in meiotic HR A group for response to stimulus included 20 crucial genes for and are required for normal fertility (Li & Ma, 2006). Except for disease response, such as PR1, PRB1 and JAZ4 (Table S22), con- the meiosis-specific DMC1, the other three genes also function in sistent with the previous finding that RAD51 and RAD51D regu- somatic DNA repair (Bray & West, 2005). By contrast, single late directly defense-related genes (Durrant et al., 2007; Wang mutants defective in any of the RAD51B, RAD51D and XRCC2 et al., 2010). genes exhibit increased sensitivity to DNA damaging agents, sug- To further examine the genes affected by the triple mutations gesting that they have a role in HR and/or DNA repair, but they in the presence of bleomycin, we focused on 84 TF genes belong- show normal vegetative growth and fertility. The normal mor- ing to 10 families (21 up-regulated and 63 down-regulated), and phological phenotypes of these mutants are in dramatic contrast divided into five groups by hierarchical clustering, with addi- with the cellular phenotype and embryo lethality caused by the tional expression data from the triple mutant and wild-type with- mutations in their corresponding homologs in humans and mice out bleomycin (Fig. 8c). The expression patterns of these genes (Silva et al., 2010). In addition, yeast two-hybrid and immuno- suggested that transcriptional regulation was dramatically altered precipitation studies have shown that animal XRCC2, RAD51B in the triple mutant, even when its morphology was normal. The and RAD51D form a complex with RAD51C (the BCDX2 com- expression of group I genes was induced by bleomycin in the plex) and function as a complex in homologous recombinational wild-type, but generally similar in the triple mutant with or with- DNA repair (Dosanjh et al., 1998; Liu et al., 1998, 2002; Schild out bleomycin, indicating that their induction by bleomycin was et al., 2000; Masson et al., 2001; Wiese et al., 2002). This com- dependent on the RAD51B–RAD51D–XRCC2 functions plex may facilitate the formation of RAD51 foci important for (Fig. 8c). By contrast, the expression of group II genes was HR (Takata et al., 2000, 2001). Indeed, the survival of cell lines induced by bleomycin in the triple mutant, but not in the wild- carrying mutations in the RAD51 paralogs is reduced signifi- type, suggesting that these triple mutations might have sensitized cantly in response to c-irradiation treatment (Takata et al., 2000,

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2001). Recent studies have shown that the BCDX2 and CX3 supported by the comet assay (Fig. 5), consistent with the similar complexes act upstream and downstream, respectively, of RAD51 levels of RAD51 expression between the wild-type and triple recruitment on DNA damage in human cells (Chun et al., 2013). mutant (Fig. 6) and the indistinguishable patterns of radiation- A recent study has reported that Arabidopsis single mutants in induced RAD51 foci between each single mutant and the wild- the three genes do not affect the formation of radiation-induced type (Ines et al., 2013), as well as the normal growth of the rad51 RAD51 foci (Ines et al., 2013). Our study has demonstrated that mutant (Li et al., 2004). the triple mutant exhibits greater sensitivity to bleomycin than Bleomycin causes DNA DSBs (Favaudon, 1982) and inhibits the single and double mutants (Fig. 4), indicating that their func- the growth of both animal and plant cells, probably as a result of tions are partially redundant. For example, they might interact the accumulation of unrepaired DSBs. However, the molecular with proteins in parallel pathways that have overlapping func- basis of the bleomycin-induced growth phenotype in plants is tions in somatic DNA repair. This idea is supported by recent not clear. We found that of the > 4000 differentially expressed high-throughput proteomic analyses of protein complexes con- genes in the wild-type treated by bleomycin, over 58% of genes taining mouse RAD51C, RAD51D and XRCC2, which identi- were also found to be altered in the same direction in the triple fied > 100 candidates for interaction with each protein (Rajesh mutant, including 30 DNA repair genes (Table S6), suggesting et al., 2009). More than 60% of these proteins were involved in that bleomycin and the triple mutations have similar effects on a DNA/RNA modification or metabolism, including DNA mis- large number of genes, including those for DNA repair. match repair protein MSH2, DNA replication-licensing factor Although this result further supports the hypothesis that the MCM2, SFPQ and NONO. Further studies have demonstrated main functions of the three RAD51 paralogs are in DNA repair, that SFPQ–NONO form a heteromeric complex to repair DSB the fact that both bleomycin and the triple mutations also have by rejoining DSB ends (Rajesh et al., 2009, 2010). In addition, specific sets of differentially expressed genes suggests that each plant cells may require different recombination factors in differ- also has a distinct role in gene regulation. The existence of genes ent DNA repair pathways; for example, mutations in the plant that are specifically altered in the wild-type by bleomycin MRE11 and COM1 homologs do not affect either synthesis- suggests that bleomycin might cause more severe damage or dependent strand annealing (SDSA) or single-strand annealing different type (s) of damage than that found in the triple mutant. (SSA), whereas mutations in RAD51, RAD51C and XRCC3, as However, the presence of genes specifically induced/repressed by well as RAD54, affect SDSA but not SSA (Roth et al., 2012). The the triple mutations raises the possibility that some genes are idea that the three RAD51 paralogs together are involved in regulated by the RAD51 paralogs, but not by DNA damage, multiple pathways is supported by our transcriptomic results, perhaps in a manner similar to the regulation of pathogen-related which show that the expression of genes coding for factors genes by RAD51 and RAD51D. The RAD51 paralogs are involved in both SDSA and SSA pathways is affected in the triple thought to be ATPases that associate with RAD51 and chroma- mutant. It is also quite striking that the Arabidopsis RAD51B, tin (Li & Ma, 2006; Lin et al., 2006); it is possible that, in addi- RAD51D and XRCC2 genes have partially redundant functions tion to DNA repair, they affect chromatin structure and gene because they diverged at least 1 billion yr ago. expression. Although the triple mutant was clearly hypersensitive to bleo- mycin and probably deficient in DNA repair, > 4000 genes were Regulation of transcriptome by RAD51B, RAD51D and still differentially expressed in the triple mutant in response to XRCC2 bleomycin (Fig. 8a), consistent with the above discussion that Previous studies have shown that RAD51D and RAD51 regulate there is a substantial difference between the effects of mutations pathogen-related genes on salicylic acid induction (Durrant et al., and bleomycin treatment. Over 60% of the genes altered in the 2007; Wang et al., 2010), suggesting that other RAD51 paralogs triple mutant by bleomycin overlapped with those in the wild- might also have a role in transcriptional regulation. Our tran- type treated with bleomycin, indicating that these genes scriptomic analysis detected 2111 differentially expressed genes responded to bleomycin independent of the functions of the in the triple mutant compared with the wild-type (Fig. 7a). In RAD51 paralogs. Nevertheless, the hypersensitivity of the triple addition to several pathogen-related genes, other genes important mutant to bleomycin (Fig. 8a) can be explained by the observa- for DNA repair, abiotic stress and transcriptional regulation were tion that nearly one-half of the bleomycin-induced genes in the also enriched, suggesting that RAD51 paralogs have additional wild-type were not induced in the triple mutant, including some roles in gene regulation. It is possible that some of these genes genes crucial for the repair of damage caused by bleomycin, such might be regulated directly by the RAD51 paralogs, but many as COM1/GR1, MND1, RPA1A, RAD54, DRT101, ERCC1 and could be affected by the accumulation of DNA damage caused MSH2/7. We noted that a number of genes encoding TFs by the mutations in the RAD51 paralogs. However, this regula- (groups I and III, Fig. 8c) were induced/repressed by bleomycin tion does not seem to be vital for plant development under nor- in the wild-type but not in the triple mutant; some of these TFs mal conditions, as the triple mutant showed normal vegetative might be responsible for bleomycin-induced alteration in gene and reproductive growth (Figs 1, 2). Genetic studies support a expression in the wild-type, and the failure of the triple mutant major function of these genes in somatic DNA repair. The nor- to regulate the expression of these TF genes provides an explanation mal development of the mutants suggests that little DNA dam- for the lack of differential expression of many of the bleomycin- age, including DSBs, occurs under normal conditions, as induced genes.

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The other 40% of differentially regulated genes caused by bleo- homologous recombination in Arabidopsis thaliana. Nucleic Acids Research 39: mycin only in the mutant but not in the wild-type could be the 146–154. result of regulatory pathways affected by the RAD51 paralogs. In Bray CM, West CE. 2005. DNA repair mechanisms in plants: crucial sensors and effectors for the maintenance of genome integrity. New Phytologist 168: particular, a number of TF genes (groups II and IV, Fig. 8c) were 511–528. induced/repressed by bleomycin in the mutant but not in the Bundock P, Hooykaas P. 2002. Severe developmental defects, hypersensitivity to wild-type; these could provide the needed regulatory function for DNA-damaging agents, and lengthened telomeres in Arabidopsis mre11 mutant-specific changes in gene expression caused by bleomycin. mutants. Plant Cell 14: 2451–2462. Some of the genes might be important for DNA repair, and low- Chun J, Buechelmaier ES, Powell SN. 2013. Rad51 paralog complexes BCDX2 and CX3 act at different stages in the BRCA1–BRCA2-dependent homologous level accumulation of DNA damage in the triple mutant might recombination pathway. Molecular and Cellular Biology 33: 387–395. have sensitized the plant cells for response to bleomycin. Another Couteau F, Belzile F, Horlow C, Grandjean O, Vezon D, Doutriaux MP. 1999. perspective is that the combination of the triple mutations and Random chromosome segregation without meiotic arrest in both male and bleomycin might have a much greater effect (possible synergism) female meiocytes of a dmc1 mutant of Arabidopsis. Plant Cell 11: 1623–1634. than either alone. In any case, the comparison of transcriptomes Deans B, Griffin CS, Maconochie M, Thacker J. 2000. Xrcc2 is required for genetic stability, embryonic neurogenesis and viability in mice. EMBO Journal in both the triple mutant and wild-type, with or without bleomy- 19: 6675–6685. cin treatment, has revealed that there is an interaction between Dosanjh MK, Collins DW, Fan W, Lennon GG, Albala JS, Shen Z, Schild D. the triple mutations and bleomycin, suggestive of a complex func- 1998. Isolation and characterization of RAD51C, a new human member of the tion for the three RAD51 paralogs that was previously unappreci- RAD51 family of related genes. Nucleic Acids Research 26: 1179–1184. ated. Our results support multiple hypotheses and highlight the Dudasova Z, Dudas A, Chovanec M. 2004. Non-homologous end-joining factors of Saccharomyces cerevisiae. FEMS Microbiology Reviews 28: 581–601. importance of further studies with regard to the functions of these Durrant WE, Wang S, Dong XN. 2007. Arabidopsis SNI1 and RAD51D regulate ancient genes that have been maintained in both animals and both gene transcription and DNA recombination during the defense response. plants over the long history of eukaryotic evolution. Proceedings of the National Academy of Sciences, USA 104: 4223–4227. Favaudon V. 1982. On the mechanism of reductive activation in the mode of action of some anticancer drugs. Biochimie 64: 457–475. Acknowledgements Glazer VM, Glazunov AV. 1999. Molecular-genetic analysis of dual-stranded DNA break repair in Saccharomyces yeasts. Genetika 35: 1449–1469. We thank X. N. Dong (Duke University, Durham, NC, USA) Habu T, Taki T, West A, Nishimune Y, Morita T. 1996. The mouse and for providing the rad51d (ssn1) mutant, A. W. Dong’s laboratory human homologs of DMC1, the yeast meiosis-specific homologous (Fudan University, Shanghai, China) for comet assay assistance recombination gene, have a common unique form of exon-skipped transcript in – and the Ohio State University Arabidopsis Stock Center for meiosis. Nucleic Acids Research 24: 470 477. Ines OD, Degroote F, Amiard S, Goubely C, Gallego ME, White CI. 2013. providing the SALK lines. This work was supported by grants Effects of XRCC2 and RAD51B mutations on somatic and meiotic from the Ministry of Sciences and Technology of China recombination in Arabidopsis thaliana. Plant Journal 74: 959–970. (2011CB944603), the National Natural Science Foundation of Klimyuk VI, Jones JD. 1997. AtDMC1, the Arabidopsis homologue of the yeast China (91131007), Rijk Zwaan, Fudan University (to H.M.) DMC1 gene: characterization, transposon-induced allelic variation and meiosis- – and Zhuoxue Plan of Fudan University, and the Shanghai Com- associated expression. Plant Journal 11:1 14. Krogh BO, Symington LS. 2004. Recombination proteins in yeast. Annual mittee of Science and Technology Fund for 2013 Qimingxing Review of Genetics 38: 233–271. Project (13QA1400200) (to Y.W.). Kuzminov A. 2001. DNA replication meets genetic exchange: chromosomal damage and its repair by homologous recombination. Proceedings of the National Academy of Sciences, USA 98: 8461–8468. References Li W, Ma H. 2006. Double-stranded DNA breaks and gene functions in recombination and meiosis. Cell Research 16: 402–412. Abe K, Osakabe K, Nakayama S, Endo M, Tagiri A, Todoriki S, Ichikawa H, Li W, Yang X, Lin Z, Timofejeva L, Xiao R, Makaroff CA, Ma H. 2005. The Toki S. 2005. Arabidopsis RAD51C gene is important for homologous AtRAD51C gene is required for the chromosome synapsis and double-stranded recombination in meiosis and mitosis. Plant Physiology 139: 896–908. break repair in Arabidopsis. Plant Physiology 138: 965–976. Alexander MP. 1969. Differential staining of aborted and nonaborted pollen. Li WX, Chen CB, Markmann-Mulisch U, Timofejeva L, Schmelzer E, Ma H, Stain Technology 44: 117–122. Reiss B. 2004. The Arabidopsis AtRAD51 gene is dispensable for vegetative Berchowitz LE, Francis KE, Bey AL, Copenhaver GP. 2007. The role of development but required for meiosis. Proceedings of the National Academy of AtMUS81 in interference-insensitive crossovers in A. thaliana. PLoS Genetics 3: Sciences, USA 101: 10596–10601. e132. Lin ZG, Kong HZ, Nei M, Ma H. 2006. Origins and evolution of the recA/ Bishop DK, Park D, Xu L, Kleckner N. 1992. DMC1: a meiosis-specific yeast RAD51 gene family: evidence for ancient gene duplication and endosymbiotic homolog of E. coli recA required for recombination, synaptonemal complex gene transfer. Proceedings of the National Academy of Sciences, USA 103: formation, and cell cycle progression. Cell 69: 439–456. 10328–10333. Bleuyard JY, Gallego ME, Savigny F, White CI. 2005. Differing requirements Liu N, Lamerdin JE, Tebbs RS, Schild D, Tucker JD, Shen MR, Brookman for the Arabidopsis Rad51 paralogs in meiosis and DNA repair. Plant Journal KW, Siciliano MJ, Walter CA, Fan W et al. 1998. XRCC2 and XRCC3, new 41: 533–545. human Rad51-family members, promote chromosome stability and protect Bleuyard JY, Gallego ME, White CI. 2006. Recent advances in understanding against DNA cross-links and other damages. Molecular Cell 1: 783–793. of the DNA double-strand break repair machinery of plants. DNA Repair 5: Liu N, Schild D, Thelen MP, Thompson LH. 2002. Involvement of Rad51C in 1–12. two distinct protein complexes of Rad51 paralogs in human cells. Nucleic Acids Bleuyard JY, White CI. 2004. The Arabidopsis homologue of Xrcc3 plays an Research 30: 1009–1015. essential role in meiosis. EMBO Journal 23: 439–449. Liu Q, Gong Z. 2011. The coupling of epigenome replication with DNA Block-Schmidt AS, Dukowic-Schulze S, Wanieck K, Reidt W, Puchta H. 2011. replication. Current Opinion in Plant Biology 14: 187–194. BRCC36A is epistatic to BRCA1 in DNA crosslink repair and

New Phytologist (2014) 201: 292–304 Ó 2013 The Authors www.newphytologist.com New Phytologist Ó 2013 New Phytologist Trust New Phytologist Research 303

Masson JY, Stasiak AZ, Stasiak A, Benson FE, West SC. 2001. Complex transcription during plant immune responses. Proceedings of the National formation by the human RAD51C and XRCC3 recombination repair proteins. Academy of Sciences, USA 107: 22716–22721. Proceedings of the National Academy of Sciences, USA 98: 8440–8446. Warren AJ, Ihnat MA, Ogdon SE, Rowel EE, Hamilton JW. 1998. Binding of Menke M, Chen I, Angelis KJ, Schubert I. 2001. DNA damage and repair in nuclear proteins associated with mammalian DNA repair to the mitomycin Arabidopsis thaliana as measured by the comet assay after treatment with C-DNA interstrand crosslink. Environmental Molecular Mutagenesis 31: 70–81. different classes of genotoxins. Mutation Research 493: 87–93. West CE, Waterworth WM, Sunderland PA, Bray CM. 2004. Arabidopsis DNA Mills KD, Ferguson DO, Alt FW. 2003. The role of DNA breaks in genomic double-strand break repair pathways. Biochemical Society Transactions 32:964–966. instability and tumorigenesis. Immunological Reviews 194: 77–95. Wiese C, Collins DW, Albala JS, Thompson LH, Kronenberg A, Schild D. Osakabe K, Abe K, Yamanouchi H, Takyuu T, Yoshioka T, Ito Y, Kato T, 2002. Interactions involving the RAD51 paralogs RAD51C and XRCC3 in Tabata S, Kurei S, Yoshioka Y et al. 2005. Arabidopsis Rad51B is important for human cells. Nucleic Acids Research 30: 1001–1008. double-strand DNA breaks repair in somatic cells. Plant Molecular Biology 57: Yang HX, Lu PL, Wang YX, Ma H. 2011. The transcriptome landscape of 819–833. Arabidopsis male meiocytes from high-throughput sequencing: the complexity Paques F, Haber JE. 1999. Multiple pathways of recombination induced by and evolution of the meiotic process. Plant Journal 65: 503–516. double-strand breaks in Saccharomyces cerevisiae. Microbiology and Molecular Zhang H, Jin J, Tang L, Zhao Y, Gu X, Gao G, Luo J. 2011. PlantTFDB 2.0: Biology Reviews 63: 349–404. update and improvement of the comprehensive plant transcription factor Pittman DL, Schimenti JC. 2000. Midgestation lethality in mice deficient for the database. Nucleic Acids Research 39: D1114–D1117. RecA-related gene, Rad51d/Rad51 l3. Genesis 26: 167–173. Zhang XH, Feng BM, Zhang Q, Zhang DY, Altman N, Ma H. 2005. Genome- Rajesh C, Gruver AM, Basrur V, Pittman DL. 2009. The interaction profile of wide expression profiling and identification of gene activities during early homologous recombination repair proteins RAD51C, RAD51D and XRCC2 flower development in Arabidopsis. Plant Molecular Biology 58: 401–419. as determined by proteomic analysis. Proteomics 9: 4071–4086. Zhu Y, Dong A, Meyer D, Pichon O, Renou JP, Cao K, Shen WH. 2006. Rajesh P, Rajesh C, Wyatt MD, Pittman DL. 2010. RAD51D protects against Arabidopsis NRP1 and NRP2 encode histone chaperones and are required MLH1-dependent cytotoxic responses to O-6-methylguanine. DNA Repair 9: for maintaining postembryonic root growth. Plant Cell 18: 2879–2892. 458–467. Ross KJ, Fransz P, Jones GH. 1996. A light microscopic atlas of meiosis in Arabidopsis thaliana. Chromosome Research 4: 507–516. Roth N, Klimesch J, Dukowic-Schulze S, Pacher M, Mannuss A, Puchta H. Supporting Information 2012. The requirement for recombination factors differs considerably between Additional supporting information may be found in the online different pathways of homologous double-strand break repair in somatic plant cells. Plant Journal 72: 781–790. version of this article. Sasaki MS, Takata M, Sonoda E, Tachibana A, Takeda S. 2004. Recombination repair pathway in the maintenance of chromosomal integrity against DNA Fig. S1 Expression patterns of members of the RAD51 family. interstrand crosslinks. Cytogenetic and Genome Research 104: 28–34. Schild D, Lio YC, Collins DW, Tsomondo T, Chen DJ. 2000. Evidence for Fig. S2 Gene ontology (GO) annotation of 562 differentially simultaneous protein interactions between human Rad51 paralogs. Journal of Biological Chemistry 275: 16443–16449. expressed genes. Shu Z, Smith S, Wang L, Rice MC, Kmiec EB. 1999. Disruption of muREC2/ RAD51L1 in mice results in early embryonic lethality which can be partially Table S1 List of primers used in this study rescued in a p53(–/–) background. Molecular and Cellular Biology 19: – 8686 8693. Table S2 Summary of mapped reads Silva SN, Tomar M, Paulo C, Gomes BC, Azevedo AP, Teixeira V, Pina JE, Rueff J, Gaspar JF. 2010. Breast cancer risk and common single nucleotide polymorphisms in homologous recombination DNA repair pathway genes Table S3 Summary of transcript types detected by short reads XRCC2, XRCC3, NBS1 and RAD51. Cancer Epidemiology 34: 85–92. Takata M, Sasaki MS, Sonoda E, Fukushima T, Morrison C, Albala JS, Table S4 List of up-regulated genes in the triple mutant and Swagemakers SM, Kanaar R, Thompson LH, Takeda S. 2000. The Rad51 wild-type paralog Rad51B promotes homologous recombinational repair. Molecular and Cellular Biology. 20: 6476–6482. Takata M, Sasaki MS, Tachiiri S, Fukushima T, Sonoda E, Schild D, Table S5 List of down-regulated genes in the triple mutant and Thompson LH, Takeda S. 2001. Chromosome instability and defective wild-type recombinational repair in knockout mutants of the five Rad51 paralogs. – Molecular and Cellular Biology 21: 2858 2866. Table S6 List of differentially expressed genes related to DNA Tonami Y, Murakami H, Shirahige K, Nakanishi M. 2005. A checkpoint control linking meiotic S phase and recombination initiation in fission yeast. repair or meiotic recombination Proceedings of the National Academy of Sciences, USA 102: 5797–5801. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Table S7 List of the bleomycin-induced genes in the wild-type Salzberg SL, Wold BJ, Pachter L. 2010. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform Table S8 List of the bleomycin-repressed genes in the wild-type switching during cell differentiation. Nature Biotechnology 28: 511–515. Tsuzuki T, Fujii Y, Sakumi K, Tominaga Y, Nakao K, Sekiguchi M, Matsushiro A, Yoshimura Y, Morita T. 1996. Targeted disruption of the Rad51 gene leads Table S9 Gene ontology (GO) annotation of the bleomycin- to lethality in embryonic mice. Proceedings of the National Academy of Sciences, repressed genes in the wild-type USA 93: 6236–6240. Tuteja N, Ahmad P, Panda BB, Tuteja R. 2009. Genotoxic stress in plants: Table S10 List of the 739 overlapping genes shedding light on DNA damage, repair and DNA repair helicases. Mutation Research 681: 134–149. Wang S, Durrant WE, Song JQ, Spivey NW, Dong XN. 2010. Arabidopsis Table S11 List of the bleomycin-induced genes in the triple BRCA2 and RAD51 proteins are specifically involved in defense gene mutant

Ó 2013 The Authors New Phytologist (2014) 201: 292–304 New Phytologist Ó 2013 New Phytologist Trust www.newphytologist.com New 304 Research Phytologist

Table S12 List of the bleomycin-repressed genes in the triple Table S18 List of the 1001 genes induced in the triple mutant mutant that were not up-regulated in the bleomycin-treated wild-type

Table S13 Gene ontology (GO) annotation of the bleomycin- Table S19 Gene ontology (GO) annotation of the 1001 genes induced genes in the triple mutant Table S20 List of the 189 up-regulated genes in the wild-type Table S14 Gene ontology (GO) annotation of the bleomycin- and triple mutant, both treated with bleomycin repressed genes in the triple mutant Table S21 List of the 238 down-regulated genes in the wild-type Table S15 List of the 352 overlapping genes in the up-regulated and triple mutant, both treated with bleomycin set Table S22 List of the 20 crucial pathogen-related genes Table S16 List of the 270 overlapping genes in the down-regu- lated set Please note: Wiley Blackwell are not responsible for the content or functionality of any supporting information supplied by the Table S17 Gene ontology (GO) annotation of the 352 overlap- authors. Any queries (other than missing material) should be ping genes in the up-regulated set directed to the New Phytologist Central Office.

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