Journal of Experimental Botany Advance Access published June 1, 2015 Journal of Experimental Botany doi:10.1093/jxb/erv253
RESEARCH PAPER
XRCC3 is essential for proper double-strand break repair and homologous recombination in rice meiosis Bingwei Zhang1,*, Mo Wang2,*, Ding Tang2, Yafei Li2, Meng Xu1, Minghong Gu1, Zhukuan Cheng2,†, Hengxiu Yu1,† 1
* These authors contributed equally to this work. To whom correspondence should be addressed. E-mail: [email protected]; [email protected]
†
Received 16 November 2014; Revised 22 April 2015; Accepted 28 April 2015 Editor: James Murray
Abstract RAD51 paralogues play important roles in the assembly and stabilization of RAD51 nucleoprotein filaments, which promote homologous pairing and strand exchange reactions in organisms ranging from yeast to vertebrates. XRCC3, a RAD51 paralogue, has been characterized in budding yeast, mouse, and Arabidopsis. In the present study, XRCC3 in rice was identified and characterized. The rice xrcc3 mutant exhibited normal vegetative growth but complete male and female sterility. Cytological investigations revealed that homologous pairing and synapsis were severely disrupted in the mutant. Meiotic chromosomes were frequently entangled from diplotene to metaphase I, resulting in chromosome fragmentation at anaphase I. The immunostaining signals from γH2AX were regular, implying that double-strand break (DSB) formation was normal in xrcc3 meiocytes. However, COM1 was not detected on early prophase I chromosomes, suggesting that the DSB end-processing system was destroyed in the mutant. Moreover, abnormal chromosome localization of RAD51C, DMC1, ZEP1, ZIP4, and MER3 was observed in xrcc3. Taken together, the results suggest that XRCC3 plays critical roles in both DSB repair and homologous chromosome recombination during rice meiosis. Key words: DSB, homologous recombination, meiosis, rice, XRCC3.
Introduction Meiosis is a specialized cell division process. In meiosis, one round of DNA replication is followed by two sequential cell divisions, which leads to the formation of haploid cells. The chromosome number of the zygote, derived from the fusion of male and female gametes, is restored to that of the parent
generation. This process ensures the organisms that undergo sexual replication have a stable number of chromosomes. In addition, genetic information in homologous chromosomes is exchanged mutually during meiosis, which creates diversity in gamete formation and is an important source of variation
Abbreviations: AE, axial element; DSB, DNA double-strand break; FISH, fluorescent in situ hybridization; HR, homologous recombination; LE, lateral element; MMC, mitomycin C; MMEJ, microhomology-mediated end joining; NHEJ, non-homologous end joining; ORF, open reading frame; PMC, pollen mother cell; PBS, phosphate-buffered saline; RPA, replication protein A; RT-PCR, reverse-transcriptase PCR; SC, synaptonemal complex; ssDNA, single-stranded DNA; TF, transverse filament. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: [email protected]
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Key Laboratory of Plant Functional Genomics of Ministry of Education/Jiangsu Key Laboratory of Crop Genetics and Physiology/ Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, China 2 State Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
Page 2 of 13 | Zhang et al. For example, the loss of function of RAD51B, RAD51D or XRCC2 in Arabidopsis does not disturb mitosis or meiosis under normal growth conditions (Bleuyard et al., 2005), while it is not the case in vertebrates. The loss of function of Arabidopsis XRCC3 or RAD51C causes severe abnormalities in meiosis, including chromosome fragmentation and defective homologue pairing and synapsis. Further evidence suggests that the Arabidopsis RAD51C-XRCC3-associated Holliday junction resolvase complex plays essential roles in the resolution of recombination intermediates prior to chromosome segregation (Bleuyard et al., 2005). Rice (Oryza sativa) is one of the most important crops worldwide and is always used as a model monocot in molecular biological studies, especially since the rice genome has been completely sequenced and efficient Agrobacteriummediated transformation systems have been established. Among the five rice RAD51 paralogues, only RAD51C has been well characterized (Kou et al., 2012; Tang et al., 2014). Mutants lacking functional RAD51C exhibit increased sensitivity to various DNA-damaging agents. A complete SC cannot form at pachytene and massive chromosome fragments are observed beginning at metaphase I in their meiocytes. However, the function of the other rice RAD51 paralogues has not been well revealed (Luo et al., 2014). In the present study, the rice XRCC3 gene has been isolated using a mapbased cloning approach. Chromosome behaviour and immunostaining analyses of xrcc3 mutants demonstrated that XRCC3 is required for proper DSB repair, SC assembly, and homologous recombination in rice meiosis.
Materials and methods Plant materials The xrcc3-1 mutant was isolated from the offspring of a japonica rice variety, Wuxiangjing 9, which had undergone tissue culture. As xrcc3-1 is completely sterile for both male and female gametes, XRCC3-1+/– was crossed with an indica rice variety, 9311, to construct a mapping population. Completely sterile plants from the F2 or F3 generations were used for fine mapping of the target gene. The xrcc3-2 mutant was isolated from a japonica rice variety, Yandao 8, which had been treated with 60Coγ-ray. The XRCC3-2+/– plants were also crossed with 9311 to produce the mapping populations. All plant materials were grown in paddy fields under normal conditions. Molecular cloning of XRCC3 To map XRCC3, STS markers (S1–S9) were developed based on sequence differences between indica variety 9311 and japonica variety Nipponbare according to data published at http://www. ncbi.nlm.nih.gov. Primer pairs P1 and P2 were used to detect the genomic deletion in xrcc3-1 and xrcc3-2, respectively. All the primers mentioned above are listed in Supplementary Table S1 at JXB online. Complementation test A 6.91 kb genomic DNA fragment containing the entire XRCC3 coding region was inserted into binary vector pCAMBIA1300 to generate the transformation plasmid for the complementation test. The plasmid was transformed into Agrobacterium tumefaciens EHA105 by electroporation and was then transformed into calli derived from offspring seeds of XRCC3-1+/– plants.
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in eukaryotes (Zickler and Kleckner, 1999; Page and Hawley, 2003). In eukaryotes, homologous recombination (HR) depends on the formation of double-strand breaks (DSBs), a process that is mediated by the conserved topoisomerase-like enzyme SPO11 (Bergerat et al., 1997; Keeney et al., 1997; Murakami and Keeney, 2008). The SPO11 protein remains covalently linked to the 5′ termini at the incision sites of the DSB. For DSBs to be repaired by HR, DNA is nicked at a distance from the incision site of the single-stranded DNA (ssDNA), linked to SPO11 by a protein complex, MRX/N (Mre11– Rad50–Xrs2/Nbs1) and Sae2 (Com1)/CtIP/Ctp1, to yield 3′ ssDNA tails (Cao et al., 1990; Mimitou and Symington, 2008, 2009; Edlinger and Schlogelhofer, 2011). After proper end-processing, the ssDNA is bound by replication protein A (RPA). RPA is displaced by Rad51 and Dmc1 to form a nucleoprotein filament that can catalyse homologous pairing and strand invasion, eventually resulting in HR (Edlinger and Schlogelhofer, 2011). RAD51 and DMC1 are two members of the highly conserved RecA recombinase family (San Filippo et al., 2008). In yeast, Rad51 paralogues, including Rad55 and Rad57, are also involved in Rad51-dependent DSB repair and HR. RAD51 and its paralogues may be generated from different ancestors of eukaryotic RecA/RAD51like genes (Lin et al., 2006; San Filippo et al., 2008). These proteins share a highly conserved central RecA/RAD51 domain (Kans and Mortimer, 1991; Lovett, 1994; Lin et al., 2006). In yeast, Rad55 and Rad57 form a stable heterodimer to stimulate the Rad51-promoting strand exchange reaction (Sung, 1997; Fortin and Symington, 2002; Liu et al., 2011). Loss of function of any of the Rad51 paralogues (Rad55 or Rad57) leads to a loss of meiosis-specific RAD51 foci and a deficiency in meiotic recombination as well as reduced spore viability. Five RAD51 paralogues have been identified in vertebrates, including RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3 (Dosanjh et al., 1998; Liu et al., 1998, 2002). These RAD51 paralogues share 20–30% sequence identity with RAD51 and with each other. Two primary RAD51 paralogue complexes are thought to exist in vivo: one contains RAD51B, RAD51C, RAD51D, and XRCC2 (BCDX2 complex), the other comprises RAD51C and XRCC3 (CX3 complex) (Liu et al., 2002). In mouse, disruption of one of the RAD51 paralogues (RAD51B, RAD51BC, RAD51D, or XRCC2) leads to embryonic lethality (Shu et al., 1999; Deans et al., 2000; Pittman and Schimenti, 2000; Kuznetsov et al., 2007), indicating that each of the RAD51 paralogues is essential and non-redundant during mouse development. Moreover, RAD51C was also proved to be associated with the resolution of double Holliday junctions during meiosis by characterizing a hypomorphic allele in mouse (Kuznetsov et al., 2007). Plants also harbour five RAD51 paralogues, including RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3. In Arabidopsis, mutants of these RAD51 paralogues are hypersensitive to DNA-damaging agents (Bleuyard et al., 2005; Li et al., 2005; Vignard et al., 2007). Their functions have differentiated a great deal compared with other organisms.
The role of XRCC3 in rice meiosis | Page 3 of 13 Reverse-transcriptase PCR (RT-PCR) for transcript expression analysis Total RNA was extracted from the young rice panicles using TRIZOL reagent (Invitrogen) according to the manufacturer’s instructions. Isolated RNA (1 μg) was reverse-transcribed with the Oligo-dT(18) primer using M-MLV reverse transcriptase (Promega). The RT-PCR was carried out using XRCC3RT–F (5′-CCATCGCCT CCCTTTTCC-3′) and XRCC3RT–R (5′-CCGGGTGTTGACGCA GTT-3′) for XRCC3 and UBQF (5′-CAAGATGATCTGCCGC AAATGC-3′) and UBQR (5′-TTAACCAGTCCATGAACCCG-3′) for the Ubiquitin gene.
Immunofluorescence Fresh young panicles from xrcc3-1, xrcc3-2, and wild-type plants were sampled and fixed in 4% (w/v) paraformaldehyde at room temperature for 30 min. Anthers at the appropriate stages were separated, crushed on a slide with phosphate-buffered saline (PBS) solution, and covered with a cover slip. After soaking the slides in liquid nitrogen and removing the cover slip, the slides were dehydrated through an ethanol series (70%, 90%, and 100%) prior to immunostaining. Then, the slides were incubated in a humidity chamber at 37 °C for 4 h with different antibodies diluted (1:500) in TNB buffer (0.1 M TRIS–HCl, pH 7.5, 0.15 M NaCl, and 0.5% blocking reagent). After three rounds of washing in PBS, secondary detection antibodies, including Texas-red-anti-rabbit antibody and fluorescein isothiocyanate anti-mouse antibody (1:1000) were added to the slides, which were incubated in a humidity chamber at 37 °C for 30 min. After three rounds of washing in PBS, images were observed and captured with fluorescence microscopy. Mitomycin C (MMC) sensitivity test Husked seeds from the heterozygous XRCC3-1+/– and wild-type plants were surface-sterilized, inoculated on solid half-strength MS medium containing 0 or 20 μg ml–1 MMC (Biochempartner), and grown in a light incubator. Fourteen days later, the genotypes and phenotypes of the seedlings were assayed. Accession number Sequence data from this article can be found in GenBank (http:// www.ncbi.nlm.nih.gov/GeneBank) under accession number KM983395.
Results Characterization of a sterile mutant A tissue-culture-derived mutant was identified showing complete sterility from the progeny of a japonica rice variety,
Isolation of XRCC3 in rice A map-based cloning approach was used to clone the target gene. A mapping population was constructed by crossing heterozygous plants harbouring the mutant gene with an indica cultivar, 9311. A total of 982 F2 and F3 complete sterile plants were used for gene mapping. Linkage analysis mapped the target gene to the long arm of chromosome 2. The target gene was further delimited to a 130 kb region. PCR analysis using primers (see Supplementary Table S1 at JXB online) located in this region revealed that a 21 kb DNA fragment was deleted in the mutant. This 21 kb region contains four open reading frames (ORFs), i.e. Os02g35450, Os02g35460, Os02g35470, and Os02g35480, according to the Rice Genome Annotation Project Database (http://rice. plantbiology.msu.edu/cgi-bin/gbrowse/rice) (Fig. 1E). The protein encoded by Os02g35450 is predicted to be related to DNA repair and recombination and shows high similarity to XRCC3 in Arabidopsis (127/290, 43.8% residue identity; see Supplementary Fig. S1 at JXB online). The three other ORFs encode proteins of unknown function. RT-PCR analysis was then conducted using primers specific for XRCC3; no XRCC3 transcript was detected in the young panicles of the mutant (see Supplementary Fig. S2 at JXB online). To verify whether the loss of function of XRCC3 was responsible for the mutant phenotype, a complementation test was conducted using full-length XRCC3. The results show that full-length XRCC3 could successfully rescue the sterile phenotype of the mutant plants (Fig. 1). These data confirmed that the loss of function of XRCC3 was indeed responsible for the sterile phenotype of the mutant; this mutant was designated xrcc3-1. Another mutant, xrcc3-2, was also identified from a japonica rice variety Yandao 8, which exhibits the same plant sterile phenotype and chromosome behaviours as xrcc3-1 (see Supplementary Fig. S3 at JXB online). Map-based cloning and DNA sequencing revealed a 143 kb deletion in this mutant, which included XRCC3. This deletion led to the loss of XRCC3 function in xrcc3-2. The XRCC3 ORF, which was obtained from the wild-type Wuxiangjing 9 via RT-PCR, is 873 bp in length with no introns and encodes a protein of 290 amino acids, which is consistent with the results from Nipponbare (http://www.ncbi.nlm.nih.gov/).
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Meiotic chromosome preparation and fluorescent in situ hybridization (FISH) Young panicles of both wild-type and mutant plants were harvested and fixed in Carnoy’s solution (ethanol:glacial acetic acid, 3:1) and stored at –20 °C. Meiotic chromosomes were prepared as described by Wang et al. (2009). FISH analysis was conducted as described by Zhang et al. (2005). The pTa794 clone (containing the coding sequence for the 5S ribosomal RNA genes of wheat) was used as the FISH probe. The probe was detected using a fluorescein isothiocyanate conjugated anti-digoxigenin antibody (Vector Laboratories). The chromosomes were counterstained with 4′, 6′-diamidino-phenylindole (DAPI) in Vectashield antifading solution (Vector laboratories). Chromosome images were observed under an Olympus BX61 fluorescence microscope and captured with a DVC1412 CCD camera using IPLab software.
Wuxiangjing 9. This mutant closely resembles the wild type during the vegetative stage (Fig. 1A, B). In the segregation population, plants with a normal phenotype and sterile mutants were present in a 3:1 segregation ratio, suggesting that this mutant harbours a recessive mutation (95:27, χ2=0.58; P >0.05). To investigate the male fertility of this mutant, mutant and wild-type pollen grains were stained with an iodine–potassium iodide solution (I2–KI). All of the mature mutant pollen grains were empty and shrunken, while the wild-type pollen grains were dark and round (Fig. 1C, D), indicating that microspores of the mutant were aborted. The mutant failed to set seeds when pollinated with wildtype pollen grains, suggesting that megagametogenesis was also affected in this mutant.
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Fig. 1. Characterization of the xrcc3-1 plant and map-based cloning of XRCC3. (A) Comparison of a wild-type plant (left), an xrcc3-1 plant (middle), and a complementary plant (right). (B) Panicles of the wild-type (left), the xrcc3-1 (middle), and the complementary plant (right). (C) Normal pollen grains of the wild type. (D) Complete sterile pollen grains of the xrcc3-1 plant. (E) Map-based cloning of XRCC3. CEN, centromere; Rec, the number of recombinants between xrcc3-1 and the adjacent markers. Scale bars: 50 μm.
Abnormal meiotic chromosome behaviours in xrcc3 To clarify the cause of sterility of the xrcc3 mutant, the meiotic chromosome behaviour was carefully examined in pollen mother cells (PMCs) of both wild-type and xrcc3-1 plants (Fig. 2). In the wild type, the chromosomes appeared
as thin threads at leptotene (Fig. 2A), and homologous chromosomes exhibited partial pairing and synapsis at zygotene (Fig. 2B). During pachytene, since synapsis had been completed between homologous chromosomes, thick, thread-like chromosomes were observed (Fig. 2C). During diplotene and
The role of XRCC3 in rice meiosis | Page 5 of 13 chromosomes were deficient. During diplotene and diakinesis, irregularly shaped univalents were scattered throughout the nucleus, and univalents were frequently connected by filamentous DNA (Fig. 2M, N). Visible chromosome fragments were also present, as well as some loosely connected multivalents during diakinesis (Fig. 2N). These chromosomes aggregated into a block at metaphase I (Fig. 2O). During anaphase I, these entangled chromosomes separated from each other and moved randomly to the opposite poles. At the same time, chromosome bridges were always observed and chromosome fragments remained near the equatorial plate (Fig. 2P). Although most chromosomes had reached the two poles at telophase I, chromosome fragments still halted near the centre of the cell (Fig. 2Q). As a result, the two daughter cells often harboured some micronuclei. Although these
Fig. 2. Meiosis of male meiocytes in both wild type and xrcc3-1. (A–I) Meiosis in wild type. (A) Leptotene. (B) Zygotene. (C) Pachytene. (D) Diplotene. (E) Diakinesis. (F) Metaphase I. (G) Anaphase I. (H) Tlophase I. (I) Tetrads. (J–R) Meiosis in xrcc3-1. (J) Leptotene. (K) Zygotene. (L) Pachytene. (M) Diplotene. (N) Diakinesis. (O) Metaphase I. (P) Anaphase I. (Q) Telophase I. (R) Irregular tetrads. Scale bars: 5 μm.
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diakinesis, the chromosomes were highly condensed, and 12 short bivalents were distributed in the cells (Fig. 2D, E). At metaphase I, all 12 bivalents aligned on the equatorial plate (Fig. 2F), and homologous chromosomes subsequently separated and migrated to opposite poles at anaphase I (Fig. 2G). Finally, all chromosomes reached the two poles at telophase I (Fig. 2H). During the second meiotic division, sister chromatids segregated and moved to opposite poles, and regular tetrads formed at the end of meiosis II (Fig. 2I). In xrcc3-1 meiocytes, chromosomes were observed as thin threads during leptotene (Fig. 2J), which was also the case in the wild type. However, the chromosomes appeared as single threads and did not pair with their homologous chromosomes from zygotene to pachytene (Fig. 2K, L), which implies that paring and synapsis between homologous
Page 6 of 13 | Zhang et al. cells could subsequently undergo the second meiotic division, abnormal tetrads with some micronuclei were ultimately generated (Fig. 2R). The chromosome behaviours of xrcc3-2 were similar to those of xrcc3-1 (see Supplementary Fig. S3 at JXB online). These results indicate that the loss of function of XRCC3 profoundly disturbs the normal progression of meiosis, and the complete sterility of the xrcc3 mutant may be caused by deficient in homologous chromosome pairing, synapsis, and severe chromosomal fragmentation.
XRCC3 is dispensable for DSB formation
Homologous chromosome paring is deficient in xrcc3 To examine whether the deficiency of XRCC3 affected chromosome paring further, FISH was performed on both wildtype and xrcc3-1 meiotic chromosomes using 5S rDNA as a specific probe; 5S rDNA is a tandem repetitive sequence (represented by a single locus in the rice genome) located on the
Normal SC lateral element (LE) installation with deficient central element installation in xrcc3 To investigate whether localization of the LEs of the SC was normally installed in xrcc3-1, immunostaining was conducted using antibodies against rice REC8, PAIR2, and PAIR3. PAIR2, a homologue of S. cerevisiae Hop1 and Arabidopsis ASY1, is required for homologous chromosomes synapsis and always associates with unpaired chromosome axes during early meiosis I (Nonomura et al., 2006). Dualimmunostaining was conducted using two antibodies against rice REC8 and PAIR2, and more than 50 meiocytes were observed both for xrcc3-1 and the wild type. In xrcc3-1 meiocytes, PAIR2 loaded normally on to chromosomes. Strong PAIR2 signals along the chromosomes were detected in xrcc3-1 meiocytes during pachytene, which colocalized with REC8 (Fig. 5D) and did not show any difference from the wild type (Fig. 5A). PAIR3, a putative coiled-coil protein, is also an axis-associated member of the SC (Wang et al., 2011). To detect the localization of PAIR3 in xrcc3-1, dual-immunostaining was conducted using antibodies against REC8 and PAIR3. By observing about 50 meiocytes in both xrcc3-1 and the wild type, it was found that PAIR3 distribution on chromosomes in xrcc3-1 meiocytes was similar to that of the wild type (Fig. 5B, E). During pachytene, PAIR3 foci were observed along the entire chromosomes and colocalized with REC8 in xrcc3-1 (Fig. 5E). Therefore, the loss of function of XRCC3
Fig. 3. Dual immunolocalization of REC8 (red) and γH2Ax (green) in the wild type and xrcc3-1 PMCs. (A) Meiocyte of the wild type at the zygotene stage. (B) Meiocyte of xrcc3-1 at the zygotene stage. (C) Numbers of γH2AX foci in the meiocytes of wild type and xrcc3-1 durring the zygotene stage. Scale bars: 5 μm.
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The histone variant H2AX is rapidly phosphorylated to γH2AX at DSB sites. Then, γH2AX foci are considered to be reliable markers of DSBs (Löbrich et al., 2010; Lang et al., 2012). To investigate whether DSBs occurred normally in xrcc3-1, antibodies that recognize γH2AX and REC8 were used to perform immunostaining. REC8 is a parallel axial element of the SC (Shao et al., 2011). In xrcc3-1, REC8 localization was the same as that of the wild type at different stages of meiosis. At zygotene, REC8 foci were observed along all of the chromosomes in both the wild type and xrcc3-1 (Fig. 3). In wild-type PMCs, γH2AX foci were first detected as dot-like signals at leptotene. During zygotene, the number of γH2AX foci peaked at 186 ± 49 (n=24) (Fig. 3A, C). In xrcc3-1 PMCs, γH2AX signals showed similar patterns and the greatest accumulation of γH2AX was observed during the zygotene stage (181 ± 54, n=23) (Fig. 3B, C). There was no significant difference between the wild type and the xrcc3-1 mutant in terms of the number of γH2AX foci during the zygotene stage (P=0.697). Therefore, this suggests that XRCC3 is dispensable for the formation of DSBs.
short arm close to the centromere of chromosome 11. During leptotene, two distinct 5S rDNA signals were visible in the PMCs of xrcc3-1 (Fig. 4B), which resembled those in the wild type (Fig. 4A), indicating that homologous chromosomes did not pair in the xrcc3-1 mutant or the wild type. Only one 5S rDNA signal was detected, indicating that two homologous chromosomes had been paired during pachytene in the wild type (n=22) (Fig. 4C). However, paired 5S rDNA signals were never observed in xrcc3-1 meiocytes (n=20) (Fig. 4D). These results further confirm that homologous chromosomes paring is deficient in xrcc3-1.
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does not affect the correct chromosome loading of these three LEs. The transverse filament (TF) protein ZEP1, a homologue of Arabidopsis thaliana ZYP1, is a central element of the rice SC (Wang et al., 2010). To investigate whether loss of function of XRCC3 affects the localization of ZEP1 and the installation of the SC, a dual immunolocalization experiment was performed using antibodies against ZEP1 and REC8. More than 50 xrcc3-1 and wild-type meiocytes were checked. In wild-type meiocytes, ZEP1 first appeared as punctuate foci at early zygotene and then elongated quickly to form short linear signals along the chromosomes. At pachytene, ZEP1 signals were located along the entire chromosomes (Fig. 5C). In the meiocytes of xrcc3-1, the ZEP1 signals could not elongate to form continuous linear signals during prophase I. For example, only some dots or rod-like ZEP1 foci at pachytene were detected (Fig. 5F). These results suggest that SC formation was severely disrupted in xrcc3 meiocytes. Therefore, SC installation was deficient in xrcc3.
XRCC3 is required for proper localization of COM1, RAD51C, and DMC1 COM1, a homologue of Sae2 in S. cerevisiae, takes part in DSB end-processing (Ji et al., 2012). To verify the distribution of COM1 in xrcc3-1 meiocytes, dual immunolocalization was performed using antibodies against COM1 and REC8. More than 50 xrcc3-1 and wild-type meiocytes were carefully investigated. In wild-type meiocytes, the foci of COM1 first became visible during interphase, and the number of COM1 foci increased as prophase I progressed. The highest levels of punctate signals were found in zygotene (Fig. 6A). During early pachytene, COM1 foci began to diminish and shifted
from chromosomes into the nucleoplasm. After late pachytene, COM1 signals were no longer detectable. However, no COM1 signals were detected during the entire meiotic division process in xrcc3-1 meiocytes (Fig. 6D). These results imply that XRCC3 is required for chromosome localization of COM1 in rice meiocytes and that the DSB end-processing system was destroyed in xrcc3-1 meiocytes. RAD51C is another RAD51 paralogue that also plays an important role in rice DSB repair via homologous recombination (Tang et al., 2014). To investigate RAD51C localization in xrcc3-1, dual-immunostaining was performed using antibodies against rice REC8 and RAD51C and more than 50 xrcc3-1 and wild-type meiocytes were evaluated. In the wild type, the RAD51C signals were first observed as punctuate foci during leptotene. The quantity of RAD51C foci gradually increased and reached its maximal level at zygotene (Fig. 6B). RAD51C signals colocalized with REC8, indicating that RAD51C foci were localized on chromosomes. However, no RAD51C foci were detected in xrcc3-1 meiocytes (Fig. 6E). These results imply that functional XRCC3 is indispensable for the localization of RAD51C on to chromosomes during meiosis and that DSB repair via homologous recombination was blocked in xrcc3-1. DMC1, the rice homologue of the yeast Dmc1, is also crucial for homologous pairing (Deng and Wang, 2007). More than 50 xrcc3-1 and wild-type meiocytes immunostained with antibodies against REC8 and DMC1 were observed. In the wild type, the quantity of DMC1 foci reached its maximal level at zygotene (Fig. 6C). No DMC1 foci in xrcc3-1 meiocytes were detected (Fig. 6F). These results imply that XRCC3 is required for the proper localization of DMC1 on the chromosome and further verify a serious homologous pairing deficiency in xrcc3-1.
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Fig. 4. FISH using 5S rDNA probe on chromosomes in the wild-type and xrcc3-1 PMCs. (A, C) Meiocytes of the wild type at the leptotene and pachytene stages. (B, D) Meiocytes of xrcc3-1 at the leptotene and pachytene stages. Chromosomes are stained with DAPI. Scale bars: 5 μm.
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Fig. 5. Immunofluorescent localization of PAIR2, PAIR3, and ZEP1 antibodies in both wild type and xrcc3-1 PMCs. (A) PAIR2, (B) PAIR3, and (C) ZEP1 on prophase I chromosomes in the wild type. (D) PAIR2, (E) PAIR3, and (F) ZEP1 on prophase I chromosomes in xrcc3-1. An anti-REC8 antibody is used to indicate the chromosomes. Scale bars: 5 μm.
Correct loading of MER3 and ZIP4 depends on XRCC3 MER3, a homologue of yeast Mer3, is one of the rice ZMM proteins (ZIP1, ZIP2, ZIP3, ZIP4, MSH4, MSH5, and
MER3). MER3 can be used to mark the correct installation of recombination elements (Wang et al., 2009). To investigate whether loss of function of XRCC3 affects normal recombination, the distribution of MER3 by dual-immunostaining using antibodies against REC8 and MER3 was monitored.
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Fig. 6. Immunofluorescent localization of COM1, RAD51C, and DMC1 antibodies on meiotic chromosomes in the wild type and xrcc3-1 meiocytes. (A) COM1, (B) RAD51C, and (C) DMC1 on the chromosomes at the zygotene stages in the wild type. (D) COM1, (E) RAD51C, and (F) DMC1 on the chromosomes at the zygotene stages in xrcc3-1. An anti-REC8 antibody is used to indicate the chromosomes. Scale bars: 5 μm.
More than 50 xrcc3-1 and wild-type meiocytes were observed. In the wild type, numerous MER3 foci were observed in the meiocyte at zygotene (Fig. 7A). By contrast, no MER3 foci were detected in xrcc3-1 meiocytes (Fig. 7C), indicating the presence of a severe recombination defect in xrcc3-1. ZIP4, another ZMM protein, is important for loading of MER3 onto chromosomes and chiasma formation (Shen et al., 2012). To monitor the distribution of ZIP4 in xrcc3
meiocytes, dual immunolocalization was carried using antibodies against REC8 and ZIP4 and about 50 xrcc3-1 and wild-type meiocytes were checked. In the wild type, ZIP4 foci could be detected slightly later than REC8 signals at early leptotene, and most ZIP4 foci colocalized with REC8. The number of ZIP4 foci increased rapidly and reached its peak at late leptotene to early zygotene (Fig. 7B). Subsequently, the number of ZIP4 foci rapidly decreased, and only a few
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foci could be detected in late pachytene. The ZIP4 foci disappeared at diplotene and could not be detected thereafter. In addition, no ZIP4 signals could be observed during meiosis I in xrcc3-1, confirming the presence of a serious recombination deficiency (Fig. 7D).
self-fertilized heterozygous XRCC3-1+/– plants were sterilized and grown on medium with 0 or 20 μg ml–1 MMC for 14 d. The growth rate of xrcc3-1 seedlings on medium with 20 μg ml–1 MMC was significantly slower than that of the wild type (Fig. 8), implying that XRCC3 also plays an essential role in DNA repair of somatic cells.
The xrcc3 mutant is hypersensitive to DNA mutagens To investigate whether mutation of XRCC3 affects the cellular response to somatic DNA damage, the sensitivity of xrcc3-1 to the crosslinking reagent MMC was tested. MMC can induce inter-strand DNA cross-linking; this type of DNA lesion is exclusively repaired by HR (Sasaki et al., 2004). Therefore, this test can provide evidence for the role of XRCC3 in HR processes during mitosis. Husked seeds from
Discussion Functional conservation and divergence of XRCC3 in different organisms XRCC3 has been characterized in several organisms. Multiple protein alignment analysis suggests that vertebrate XRCC3 is most closely related to yeast Rad57 (Symington, 2002).
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Fig. 7. Immunofluorescent localization of MER3 and ZIP4 antibodies on meiotic chromosomes in the wild type and xrcc3-1 meiocytes. (A) MER3 and (B) ZIP4 on chromosomes at the zygotene stages in the wild type. (C) MER3 and (D) ZIP4 on chromosomes at the zygotene stages in xrcc3-1. An anti-REC8 antibody is used to indicate the chromosomes. Scale bars: 5 μm.
The role of XRCC3 in rice meiosis | Page 11 of 13
In S. cerevisiae, Rad57 combines with the other Rad51 paralogue protein, Rad55, to form a stable heterodimer that stimulates the Rad51-promoting strand exchange reaction (Sung, 1997; Fortin and Symington, 2002; Liu et al., 2011). A null mutation of Rad57 causes the loss of meiosis-specific Rad51 foci and defective meiotic recombination as well as reduced spore viability, sensitivity to DNA cross-linking agents, and cold-sensitive DNA repair (Hays et al., 1995; Johnson and Symington, 1995; Symington, 2002), suggesting that Rad57 takes part in DNA repair during both mitosis and meiosis in S. cerevisiae. Vertebrate XRCC3 interacts with RAD51C to form a heterodimer complex (CX3) (Liu et al., 2002). The hamster xrcc3 mutant, irs1SF, exhibits a highly significant increase in chromosome missegregation, and a 25-fold reduction in homology-directed repair of DSBs compared with the wild type (Griffin, 2002). Although the CX3 complex acts downstream of RAD51 recruitment in response to DNA damage (Chun et al., 2013), human HCT116 cells lacking XRCC3 exhibit a mild reduction in sister chromatid exchange, impaired RAD51 focus formation, and increased levels of chromosome aberrations; endoreduplication is increased 5–7-fold in these cells (Yoshihara et al., 2004). These observations indicate that XRCC3 plays a conserved role in maintaining genome stability in vertebrates during both meiosis and mitosis. However, human HCT116 and chicken B-lymphocyte DT40 cell lines, which lack XRCC3, exhibit milder phenotypes with respect to recombinational repair (2–3-fold sensitivity to MMC) compared with hamster irs1SF, which exhibits extreme sensitivity to MMC (60-fold). By contrast, the Drosophila xrcc3 mutant Spn-B is not hypersensitive to DSB-inducing agents (Abdu et al., 2003). So far, only Arabidopsis xrcc3 mutant has been characterized in plants. Several abnormal chromosome behaviours occur, including the lack of homologous chromosomes pairing, synapsis, chromosome entanglement, and chromosome fragmentation, demonstrating that XRCC3 is important for homologous recombination and for maintaining its genome integrity in Arabidopsis (Vignard et al., 2007). Severe
defects in homologue paring and synapsis, massive chromosome entanglement, and fragmentation in rice xrcc3 meiocytes were observed. Compared with the reduced number of DMC1 foci in Arabidopsis xrcc3, no DMC1 foci in rice xrcc3 were detected.
Abnormal chromosome entanglement and fragmentation in xrcc3 Organisms have evolved effective mechanisms for DSB repair including three major repair pathways: HR, non-homologous end-joining (NHEJ), and microhomology-mediated end-joining (MMEJ) (McVey and Lee, 2008; Symington and Gautier, 2011). HR ensures accurate repair of DSBs by using the intact sister chromatid or homologous chromosome as a template (Symington and Gautier, 2011). NHEJ is the simplest mechanism involved in DSB repair, ensuring chromosome integrity by direct ligation of broken DNA ends (Lieber, 2010; Symington and Gautier, 2011). In the MMEJ pathway, microhomologous sequences are annealed by the end-resection machinery to align ends prior to ligation (McVey and Lee, 2008). NHEJ and MMEJ are error-prone repair pathways that allow nucleotide alterations at the rejoining site, as they use no or limited sequence homology to rejoin ends of non-homologous chromosomes. The pathway used for DNA DSB repair depends on the occurrence of through end-processing. In eukaryotes, three DSB repair pathways function in a complementary, balanced manner during the cell cycle (Symington and Gautier, 2011). MRN/X and Com1/Sae2/CtIP are involved in DSB endprocessing, while the Com1/Sae2/CtIP homologue may help maintain the balance between the three DSB repair pathways, NHEJ, MMEJ, and HR (Rahal et al., 2010; Symington and Gautier, 2011). CtIP, a COM1 homologue in chicken DT40 cells, regulates the balance between error-prone DNA end-joining and error-free HR in the DSB repair pathway through its phosphorylation of serine 327 and the recruitment of BRCA1 (Yun and Hiom, 2009). A similar mechanism also occurs in C. elegans, as the HR pathway does not
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Fig. 8. Seedlings of the wild type and xrcc3-1 mutant were treated with MMC. (A) xrcc3-1 seedlings (right) exhibited more growth retardation than the wild-type seedlings (left) on half-strength MS medium with MMC. (B) Statistical analysis of the wild-type and xrcc3-1 seedling height.
Page 12 of 13 | Zhang et al. occur in COM-1-deficient germ cells, which are committed to meiotic DSB repair by NHEJ, resulting in crossover defects and extensive chromosomal aggregation (Lemmens et al., 2013). In the rice com1 mutant, non-homologous entanglements in meiotic cells lead to the formation of chromosome fragments with two telomeres produced via the interaction of non-homologous chromosomes through NEHJ (Ji et al., 2012). In the present study, no COM1 signals and massive chromosome entanglements were observed in xrcc3-1 meiocytes, which are similar to the com1 mutant, implying that DSB ends cannot be processed normally in rice xrcc3. It is suspected that DSBs might be repaired using another option, NHEJ, in xrcc3-1 meiocytes.
Supplementary data can be found at JXB online. Supplementary Table S1. Primers for XRCC3 gene cloning. Supplementary Fig. S1. Alignment of amino acid sequence encoded by rice and Arabidopsis XRCC3 genes. Supplementary Fig. S2. The lack of a transcript of the XRCC3 gene in the young panicles of the xrcc3-1 mutant. Supplementary Fig. S3. Meiosis of male meiocytes in xrcc3-2.
Acknowledgements This work was supported by grants from the Ministry of Sciences and Technology of China (2011CB944602 and 2012AA10A301) and the National Natural Science Foundation of China (31170288 and 31230038).
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RESEARCH PAPER
XRCC3 is essential for proper double-strand break repair and homologous recombination in rice meiosis Bingwei Zhang1,*, Mo Wang2,*, Ding Tang2, Yafei Li2, Meng Xu1, Minghong Gu1, Zhukuan Cheng2,†, Hengxiu Yu1,† 1
* These authors contributed equally to this work. To whom correspondence should be addressed. E-mail: [email protected]; [email protected]
†
Received 16 November 2014; Revised 22 April 2015; Accepted 28 April 2015 Editor: James Murray
Abstract RAD51 paralogues play important roles in the assembly and stabilization of RAD51 nucleoprotein filaments, which promote homologous pairing and strand exchange reactions in organisms ranging from yeast to vertebrates. XRCC3, a RAD51 paralogue, has been characterized in budding yeast, mouse, and Arabidopsis. In the present study, XRCC3 in rice was identified and characterized. The rice xrcc3 mutant exhibited normal vegetative growth but complete male and female sterility. Cytological investigations revealed that homologous pairing and synapsis were severely disrupted in the mutant. Meiotic chromosomes were frequently entangled from diplotene to metaphase I, resulting in chromosome fragmentation at anaphase I. The immunostaining signals from γH2AX were regular, implying that double-strand break (DSB) formation was normal in xrcc3 meiocytes. However, COM1 was not detected on early prophase I chromosomes, suggesting that the DSB end-processing system was destroyed in the mutant. Moreover, abnormal chromosome localization of RAD51C, DMC1, ZEP1, ZIP4, and MER3 was observed in xrcc3. Taken together, the results suggest that XRCC3 plays critical roles in both DSB repair and homologous chromosome recombination during rice meiosis. Key words: DSB, homologous recombination, meiosis, rice, XRCC3.
Introduction Meiosis is a specialized cell division process. In meiosis, one round of DNA replication is followed by two sequential cell divisions, which leads to the formation of haploid cells. The chromosome number of the zygote, derived from the fusion of male and female gametes, is restored to that of the parent
generation. This process ensures the organisms that undergo sexual replication have a stable number of chromosomes. In addition, genetic information in homologous chromosomes is exchanged mutually during meiosis, which creates diversity in gamete formation and is an important source of variation
Abbreviations: AE, axial element; DSB, DNA double-strand break; FISH, fluorescent in situ hybridization; HR, homologous recombination; LE, lateral element; MMC, mitomycin C; MMEJ, microhomology-mediated end joining; NHEJ, non-homologous end joining; ORF, open reading frame; PMC, pollen mother cell; PBS, phosphate-buffered saline; RPA, replication protein A; RT-PCR, reverse-transcriptase PCR; SC, synaptonemal complex; ssDNA, single-stranded DNA; TF, transverse filament. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: [email protected]
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Key Laboratory of Plant Functional Genomics of Ministry of Education/Jiangsu Key Laboratory of Crop Genetics and Physiology/ Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, China 2 State Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
Page 2 of 13 | Zhang et al. For example, the loss of function of RAD51B, RAD51D or XRCC2 in Arabidopsis does not disturb mitosis or meiosis under normal growth conditions (Bleuyard et al., 2005), while it is not the case in vertebrates. The loss of function of Arabidopsis XRCC3 or RAD51C causes severe abnormalities in meiosis, including chromosome fragmentation and defective homologue pairing and synapsis. Further evidence suggests that the Arabidopsis RAD51C-XRCC3-associated Holliday junction resolvase complex plays essential roles in the resolution of recombination intermediates prior to chromosome segregation (Bleuyard et al., 2005). Rice (Oryza sativa) is one of the most important crops worldwide and is always used as a model monocot in molecular biological studies, especially since the rice genome has been completely sequenced and efficient Agrobacteriummediated transformation systems have been established. Among the five rice RAD51 paralogues, only RAD51C has been well characterized (Kou et al., 2012; Tang et al., 2014). Mutants lacking functional RAD51C exhibit increased sensitivity to various DNA-damaging agents. A complete SC cannot form at pachytene and massive chromosome fragments are observed beginning at metaphase I in their meiocytes. However, the function of the other rice RAD51 paralogues has not been well revealed (Luo et al., 2014). In the present study, the rice XRCC3 gene has been isolated using a mapbased cloning approach. Chromosome behaviour and immunostaining analyses of xrcc3 mutants demonstrated that XRCC3 is required for proper DSB repair, SC assembly, and homologous recombination in rice meiosis.
Materials and methods Plant materials The xrcc3-1 mutant was isolated from the offspring of a japonica rice variety, Wuxiangjing 9, which had undergone tissue culture. As xrcc3-1 is completely sterile for both male and female gametes, XRCC3-1+/– was crossed with an indica rice variety, 9311, to construct a mapping population. Completely sterile plants from the F2 or F3 generations were used for fine mapping of the target gene. The xrcc3-2 mutant was isolated from a japonica rice variety, Yandao 8, which had been treated with 60Coγ-ray. The XRCC3-2+/– plants were also crossed with 9311 to produce the mapping populations. All plant materials were grown in paddy fields under normal conditions. Molecular cloning of XRCC3 To map XRCC3, STS markers (S1–S9) were developed based on sequence differences between indica variety 9311 and japonica variety Nipponbare according to data published at http://www. ncbi.nlm.nih.gov. Primer pairs P1 and P2 were used to detect the genomic deletion in xrcc3-1 and xrcc3-2, respectively. All the primers mentioned above are listed in Supplementary Table S1 at JXB online. Complementation test A 6.91 kb genomic DNA fragment containing the entire XRCC3 coding region was inserted into binary vector pCAMBIA1300 to generate the transformation plasmid for the complementation test. The plasmid was transformed into Agrobacterium tumefaciens EHA105 by electroporation and was then transformed into calli derived from offspring seeds of XRCC3-1+/– plants.
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in eukaryotes (Zickler and Kleckner, 1999; Page and Hawley, 2003). In eukaryotes, homologous recombination (HR) depends on the formation of double-strand breaks (DSBs), a process that is mediated by the conserved topoisomerase-like enzyme SPO11 (Bergerat et al., 1997; Keeney et al., 1997; Murakami and Keeney, 2008). The SPO11 protein remains covalently linked to the 5′ termini at the incision sites of the DSB. For DSBs to be repaired by HR, DNA is nicked at a distance from the incision site of the single-stranded DNA (ssDNA), linked to SPO11 by a protein complex, MRX/N (Mre11– Rad50–Xrs2/Nbs1) and Sae2 (Com1)/CtIP/Ctp1, to yield 3′ ssDNA tails (Cao et al., 1990; Mimitou and Symington, 2008, 2009; Edlinger and Schlogelhofer, 2011). After proper end-processing, the ssDNA is bound by replication protein A (RPA). RPA is displaced by Rad51 and Dmc1 to form a nucleoprotein filament that can catalyse homologous pairing and strand invasion, eventually resulting in HR (Edlinger and Schlogelhofer, 2011). RAD51 and DMC1 are two members of the highly conserved RecA recombinase family (San Filippo et al., 2008). In yeast, Rad51 paralogues, including Rad55 and Rad57, are also involved in Rad51-dependent DSB repair and HR. RAD51 and its paralogues may be generated from different ancestors of eukaryotic RecA/RAD51like genes (Lin et al., 2006; San Filippo et al., 2008). These proteins share a highly conserved central RecA/RAD51 domain (Kans and Mortimer, 1991; Lovett, 1994; Lin et al., 2006). In yeast, Rad55 and Rad57 form a stable heterodimer to stimulate the Rad51-promoting strand exchange reaction (Sung, 1997; Fortin and Symington, 2002; Liu et al., 2011). Loss of function of any of the Rad51 paralogues (Rad55 or Rad57) leads to a loss of meiosis-specific RAD51 foci and a deficiency in meiotic recombination as well as reduced spore viability. Five RAD51 paralogues have been identified in vertebrates, including RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3 (Dosanjh et al., 1998; Liu et al., 1998, 2002). These RAD51 paralogues share 20–30% sequence identity with RAD51 and with each other. Two primary RAD51 paralogue complexes are thought to exist in vivo: one contains RAD51B, RAD51C, RAD51D, and XRCC2 (BCDX2 complex), the other comprises RAD51C and XRCC3 (CX3 complex) (Liu et al., 2002). In mouse, disruption of one of the RAD51 paralogues (RAD51B, RAD51BC, RAD51D, or XRCC2) leads to embryonic lethality (Shu et al., 1999; Deans et al., 2000; Pittman and Schimenti, 2000; Kuznetsov et al., 2007), indicating that each of the RAD51 paralogues is essential and non-redundant during mouse development. Moreover, RAD51C was also proved to be associated with the resolution of double Holliday junctions during meiosis by characterizing a hypomorphic allele in mouse (Kuznetsov et al., 2007). Plants also harbour five RAD51 paralogues, including RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3. In Arabidopsis, mutants of these RAD51 paralogues are hypersensitive to DNA-damaging agents (Bleuyard et al., 2005; Li et al., 2005; Vignard et al., 2007). Their functions have differentiated a great deal compared with other organisms.
The role of XRCC3 in rice meiosis | Page 3 of 13 Reverse-transcriptase PCR (RT-PCR) for transcript expression analysis Total RNA was extracted from the young rice panicles using TRIZOL reagent (Invitrogen) according to the manufacturer’s instructions. Isolated RNA (1 μg) was reverse-transcribed with the Oligo-dT(18) primer using M-MLV reverse transcriptase (Promega). The RT-PCR was carried out using XRCC3RT–F (5′-CCATCGCCT CCCTTTTCC-3′) and XRCC3RT–R (5′-CCGGGTGTTGACGCA GTT-3′) for XRCC3 and UBQF (5′-CAAGATGATCTGCCGC AAATGC-3′) and UBQR (5′-TTAACCAGTCCATGAACCCG-3′) for the Ubiquitin gene.
Immunofluorescence Fresh young panicles from xrcc3-1, xrcc3-2, and wild-type plants were sampled and fixed in 4% (w/v) paraformaldehyde at room temperature for 30 min. Anthers at the appropriate stages were separated, crushed on a slide with phosphate-buffered saline (PBS) solution, and covered with a cover slip. After soaking the slides in liquid nitrogen and removing the cover slip, the slides were dehydrated through an ethanol series (70%, 90%, and 100%) prior to immunostaining. Then, the slides were incubated in a humidity chamber at 37 °C for 4 h with different antibodies diluted (1:500) in TNB buffer (0.1 M TRIS–HCl, pH 7.5, 0.15 M NaCl, and 0.5% blocking reagent). After three rounds of washing in PBS, secondary detection antibodies, including Texas-red-anti-rabbit antibody and fluorescein isothiocyanate anti-mouse antibody (1:1000) were added to the slides, which were incubated in a humidity chamber at 37 °C for 30 min. After three rounds of washing in PBS, images were observed and captured with fluorescence microscopy. Mitomycin C (MMC) sensitivity test Husked seeds from the heterozygous XRCC3-1+/– and wild-type plants were surface-sterilized, inoculated on solid half-strength MS medium containing 0 or 20 μg ml–1 MMC (Biochempartner), and grown in a light incubator. Fourteen days later, the genotypes and phenotypes of the seedlings were assayed. Accession number Sequence data from this article can be found in GenBank (http:// www.ncbi.nlm.nih.gov/GeneBank) under accession number KM983395.
Results Characterization of a sterile mutant A tissue-culture-derived mutant was identified showing complete sterility from the progeny of a japonica rice variety,
Isolation of XRCC3 in rice A map-based cloning approach was used to clone the target gene. A mapping population was constructed by crossing heterozygous plants harbouring the mutant gene with an indica cultivar, 9311. A total of 982 F2 and F3 complete sterile plants were used for gene mapping. Linkage analysis mapped the target gene to the long arm of chromosome 2. The target gene was further delimited to a 130 kb region. PCR analysis using primers (see Supplementary Table S1 at JXB online) located in this region revealed that a 21 kb DNA fragment was deleted in the mutant. This 21 kb region contains four open reading frames (ORFs), i.e. Os02g35450, Os02g35460, Os02g35470, and Os02g35480, according to the Rice Genome Annotation Project Database (http://rice. plantbiology.msu.edu/cgi-bin/gbrowse/rice) (Fig. 1E). The protein encoded by Os02g35450 is predicted to be related to DNA repair and recombination and shows high similarity to XRCC3 in Arabidopsis (127/290, 43.8% residue identity; see Supplementary Fig. S1 at JXB online). The three other ORFs encode proteins of unknown function. RT-PCR analysis was then conducted using primers specific for XRCC3; no XRCC3 transcript was detected in the young panicles of the mutant (see Supplementary Fig. S2 at JXB online). To verify whether the loss of function of XRCC3 was responsible for the mutant phenotype, a complementation test was conducted using full-length XRCC3. The results show that full-length XRCC3 could successfully rescue the sterile phenotype of the mutant plants (Fig. 1). These data confirmed that the loss of function of XRCC3 was indeed responsible for the sterile phenotype of the mutant; this mutant was designated xrcc3-1. Another mutant, xrcc3-2, was also identified from a japonica rice variety Yandao 8, which exhibits the same plant sterile phenotype and chromosome behaviours as xrcc3-1 (see Supplementary Fig. S3 at JXB online). Map-based cloning and DNA sequencing revealed a 143 kb deletion in this mutant, which included XRCC3. This deletion led to the loss of XRCC3 function in xrcc3-2. The XRCC3 ORF, which was obtained from the wild-type Wuxiangjing 9 via RT-PCR, is 873 bp in length with no introns and encodes a protein of 290 amino acids, which is consistent with the results from Nipponbare (http://www.ncbi.nlm.nih.gov/).
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Meiotic chromosome preparation and fluorescent in situ hybridization (FISH) Young panicles of both wild-type and mutant plants were harvested and fixed in Carnoy’s solution (ethanol:glacial acetic acid, 3:1) and stored at –20 °C. Meiotic chromosomes were prepared as described by Wang et al. (2009). FISH analysis was conducted as described by Zhang et al. (2005). The pTa794 clone (containing the coding sequence for the 5S ribosomal RNA genes of wheat) was used as the FISH probe. The probe was detected using a fluorescein isothiocyanate conjugated anti-digoxigenin antibody (Vector Laboratories). The chromosomes were counterstained with 4′, 6′-diamidino-phenylindole (DAPI) in Vectashield antifading solution (Vector laboratories). Chromosome images were observed under an Olympus BX61 fluorescence microscope and captured with a DVC1412 CCD camera using IPLab software.
Wuxiangjing 9. This mutant closely resembles the wild type during the vegetative stage (Fig. 1A, B). In the segregation population, plants with a normal phenotype and sterile mutants were present in a 3:1 segregation ratio, suggesting that this mutant harbours a recessive mutation (95:27, χ2=0.58; P >0.05). To investigate the male fertility of this mutant, mutant and wild-type pollen grains were stained with an iodine–potassium iodide solution (I2–KI). All of the mature mutant pollen grains were empty and shrunken, while the wild-type pollen grains were dark and round (Fig. 1C, D), indicating that microspores of the mutant were aborted. The mutant failed to set seeds when pollinated with wildtype pollen grains, suggesting that megagametogenesis was also affected in this mutant.
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Fig. 1. Characterization of the xrcc3-1 plant and map-based cloning of XRCC3. (A) Comparison of a wild-type plant (left), an xrcc3-1 plant (middle), and a complementary plant (right). (B) Panicles of the wild-type (left), the xrcc3-1 (middle), and the complementary plant (right). (C) Normal pollen grains of the wild type. (D) Complete sterile pollen grains of the xrcc3-1 plant. (E) Map-based cloning of XRCC3. CEN, centromere; Rec, the number of recombinants between xrcc3-1 and the adjacent markers. Scale bars: 50 μm.
Abnormal meiotic chromosome behaviours in xrcc3 To clarify the cause of sterility of the xrcc3 mutant, the meiotic chromosome behaviour was carefully examined in pollen mother cells (PMCs) of both wild-type and xrcc3-1 plants (Fig. 2). In the wild type, the chromosomes appeared
as thin threads at leptotene (Fig. 2A), and homologous chromosomes exhibited partial pairing and synapsis at zygotene (Fig. 2B). During pachytene, since synapsis had been completed between homologous chromosomes, thick, thread-like chromosomes were observed (Fig. 2C). During diplotene and
The role of XRCC3 in rice meiosis | Page 5 of 13 chromosomes were deficient. During diplotene and diakinesis, irregularly shaped univalents were scattered throughout the nucleus, and univalents were frequently connected by filamentous DNA (Fig. 2M, N). Visible chromosome fragments were also present, as well as some loosely connected multivalents during diakinesis (Fig. 2N). These chromosomes aggregated into a block at metaphase I (Fig. 2O). During anaphase I, these entangled chromosomes separated from each other and moved randomly to the opposite poles. At the same time, chromosome bridges were always observed and chromosome fragments remained near the equatorial plate (Fig. 2P). Although most chromosomes had reached the two poles at telophase I, chromosome fragments still halted near the centre of the cell (Fig. 2Q). As a result, the two daughter cells often harboured some micronuclei. Although these
Fig. 2. Meiosis of male meiocytes in both wild type and xrcc3-1. (A–I) Meiosis in wild type. (A) Leptotene. (B) Zygotene. (C) Pachytene. (D) Diplotene. (E) Diakinesis. (F) Metaphase I. (G) Anaphase I. (H) Tlophase I. (I) Tetrads. (J–R) Meiosis in xrcc3-1. (J) Leptotene. (K) Zygotene. (L) Pachytene. (M) Diplotene. (N) Diakinesis. (O) Metaphase I. (P) Anaphase I. (Q) Telophase I. (R) Irregular tetrads. Scale bars: 5 μm.
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diakinesis, the chromosomes were highly condensed, and 12 short bivalents were distributed in the cells (Fig. 2D, E). At metaphase I, all 12 bivalents aligned on the equatorial plate (Fig. 2F), and homologous chromosomes subsequently separated and migrated to opposite poles at anaphase I (Fig. 2G). Finally, all chromosomes reached the two poles at telophase I (Fig. 2H). During the second meiotic division, sister chromatids segregated and moved to opposite poles, and regular tetrads formed at the end of meiosis II (Fig. 2I). In xrcc3-1 meiocytes, chromosomes were observed as thin threads during leptotene (Fig. 2J), which was also the case in the wild type. However, the chromosomes appeared as single threads and did not pair with their homologous chromosomes from zygotene to pachytene (Fig. 2K, L), which implies that paring and synapsis between homologous
Page 6 of 13 | Zhang et al. cells could subsequently undergo the second meiotic division, abnormal tetrads with some micronuclei were ultimately generated (Fig. 2R). The chromosome behaviours of xrcc3-2 were similar to those of xrcc3-1 (see Supplementary Fig. S3 at JXB online). These results indicate that the loss of function of XRCC3 profoundly disturbs the normal progression of meiosis, and the complete sterility of the xrcc3 mutant may be caused by deficient in homologous chromosome pairing, synapsis, and severe chromosomal fragmentation.
XRCC3 is dispensable for DSB formation
Homologous chromosome paring is deficient in xrcc3 To examine whether the deficiency of XRCC3 affected chromosome paring further, FISH was performed on both wildtype and xrcc3-1 meiotic chromosomes using 5S rDNA as a specific probe; 5S rDNA is a tandem repetitive sequence (represented by a single locus in the rice genome) located on the
Normal SC lateral element (LE) installation with deficient central element installation in xrcc3 To investigate whether localization of the LEs of the SC was normally installed in xrcc3-1, immunostaining was conducted using antibodies against rice REC8, PAIR2, and PAIR3. PAIR2, a homologue of S. cerevisiae Hop1 and Arabidopsis ASY1, is required for homologous chromosomes synapsis and always associates with unpaired chromosome axes during early meiosis I (Nonomura et al., 2006). Dualimmunostaining was conducted using two antibodies against rice REC8 and PAIR2, and more than 50 meiocytes were observed both for xrcc3-1 and the wild type. In xrcc3-1 meiocytes, PAIR2 loaded normally on to chromosomes. Strong PAIR2 signals along the chromosomes were detected in xrcc3-1 meiocytes during pachytene, which colocalized with REC8 (Fig. 5D) and did not show any difference from the wild type (Fig. 5A). PAIR3, a putative coiled-coil protein, is also an axis-associated member of the SC (Wang et al., 2011). To detect the localization of PAIR3 in xrcc3-1, dual-immunostaining was conducted using antibodies against REC8 and PAIR3. By observing about 50 meiocytes in both xrcc3-1 and the wild type, it was found that PAIR3 distribution on chromosomes in xrcc3-1 meiocytes was similar to that of the wild type (Fig. 5B, E). During pachytene, PAIR3 foci were observed along the entire chromosomes and colocalized with REC8 in xrcc3-1 (Fig. 5E). Therefore, the loss of function of XRCC3
Fig. 3. Dual immunolocalization of REC8 (red) and γH2Ax (green) in the wild type and xrcc3-1 PMCs. (A) Meiocyte of the wild type at the zygotene stage. (B) Meiocyte of xrcc3-1 at the zygotene stage. (C) Numbers of γH2AX foci in the meiocytes of wild type and xrcc3-1 durring the zygotene stage. Scale bars: 5 μm.
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The histone variant H2AX is rapidly phosphorylated to γH2AX at DSB sites. Then, γH2AX foci are considered to be reliable markers of DSBs (Löbrich et al., 2010; Lang et al., 2012). To investigate whether DSBs occurred normally in xrcc3-1, antibodies that recognize γH2AX and REC8 were used to perform immunostaining. REC8 is a parallel axial element of the SC (Shao et al., 2011). In xrcc3-1, REC8 localization was the same as that of the wild type at different stages of meiosis. At zygotene, REC8 foci were observed along all of the chromosomes in both the wild type and xrcc3-1 (Fig. 3). In wild-type PMCs, γH2AX foci were first detected as dot-like signals at leptotene. During zygotene, the number of γH2AX foci peaked at 186 ± 49 (n=24) (Fig. 3A, C). In xrcc3-1 PMCs, γH2AX signals showed similar patterns and the greatest accumulation of γH2AX was observed during the zygotene stage (181 ± 54, n=23) (Fig. 3B, C). There was no significant difference between the wild type and the xrcc3-1 mutant in terms of the number of γH2AX foci during the zygotene stage (P=0.697). Therefore, this suggests that XRCC3 is dispensable for the formation of DSBs.
short arm close to the centromere of chromosome 11. During leptotene, two distinct 5S rDNA signals were visible in the PMCs of xrcc3-1 (Fig. 4B), which resembled those in the wild type (Fig. 4A), indicating that homologous chromosomes did not pair in the xrcc3-1 mutant or the wild type. Only one 5S rDNA signal was detected, indicating that two homologous chromosomes had been paired during pachytene in the wild type (n=22) (Fig. 4C). However, paired 5S rDNA signals were never observed in xrcc3-1 meiocytes (n=20) (Fig. 4D). These results further confirm that homologous chromosomes paring is deficient in xrcc3-1.
The role of XRCC3 in rice meiosis | Page 7 of 13
does not affect the correct chromosome loading of these three LEs. The transverse filament (TF) protein ZEP1, a homologue of Arabidopsis thaliana ZYP1, is a central element of the rice SC (Wang et al., 2010). To investigate whether loss of function of XRCC3 affects the localization of ZEP1 and the installation of the SC, a dual immunolocalization experiment was performed using antibodies against ZEP1 and REC8. More than 50 xrcc3-1 and wild-type meiocytes were checked. In wild-type meiocytes, ZEP1 first appeared as punctuate foci at early zygotene and then elongated quickly to form short linear signals along the chromosomes. At pachytene, ZEP1 signals were located along the entire chromosomes (Fig. 5C). In the meiocytes of xrcc3-1, the ZEP1 signals could not elongate to form continuous linear signals during prophase I. For example, only some dots or rod-like ZEP1 foci at pachytene were detected (Fig. 5F). These results suggest that SC formation was severely disrupted in xrcc3 meiocytes. Therefore, SC installation was deficient in xrcc3.
XRCC3 is required for proper localization of COM1, RAD51C, and DMC1 COM1, a homologue of Sae2 in S. cerevisiae, takes part in DSB end-processing (Ji et al., 2012). To verify the distribution of COM1 in xrcc3-1 meiocytes, dual immunolocalization was performed using antibodies against COM1 and REC8. More than 50 xrcc3-1 and wild-type meiocytes were carefully investigated. In wild-type meiocytes, the foci of COM1 first became visible during interphase, and the number of COM1 foci increased as prophase I progressed. The highest levels of punctate signals were found in zygotene (Fig. 6A). During early pachytene, COM1 foci began to diminish and shifted
from chromosomes into the nucleoplasm. After late pachytene, COM1 signals were no longer detectable. However, no COM1 signals were detected during the entire meiotic division process in xrcc3-1 meiocytes (Fig. 6D). These results imply that XRCC3 is required for chromosome localization of COM1 in rice meiocytes and that the DSB end-processing system was destroyed in xrcc3-1 meiocytes. RAD51C is another RAD51 paralogue that also plays an important role in rice DSB repair via homologous recombination (Tang et al., 2014). To investigate RAD51C localization in xrcc3-1, dual-immunostaining was performed using antibodies against rice REC8 and RAD51C and more than 50 xrcc3-1 and wild-type meiocytes were evaluated. In the wild type, the RAD51C signals were first observed as punctuate foci during leptotene. The quantity of RAD51C foci gradually increased and reached its maximal level at zygotene (Fig. 6B). RAD51C signals colocalized with REC8, indicating that RAD51C foci were localized on chromosomes. However, no RAD51C foci were detected in xrcc3-1 meiocytes (Fig. 6E). These results imply that functional XRCC3 is indispensable for the localization of RAD51C on to chromosomes during meiosis and that DSB repair via homologous recombination was blocked in xrcc3-1. DMC1, the rice homologue of the yeast Dmc1, is also crucial for homologous pairing (Deng and Wang, 2007). More than 50 xrcc3-1 and wild-type meiocytes immunostained with antibodies against REC8 and DMC1 were observed. In the wild type, the quantity of DMC1 foci reached its maximal level at zygotene (Fig. 6C). No DMC1 foci in xrcc3-1 meiocytes were detected (Fig. 6F). These results imply that XRCC3 is required for the proper localization of DMC1 on the chromosome and further verify a serious homologous pairing deficiency in xrcc3-1.
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Fig. 4. FISH using 5S rDNA probe on chromosomes in the wild-type and xrcc3-1 PMCs. (A, C) Meiocytes of the wild type at the leptotene and pachytene stages. (B, D) Meiocytes of xrcc3-1 at the leptotene and pachytene stages. Chromosomes are stained with DAPI. Scale bars: 5 μm.
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Fig. 5. Immunofluorescent localization of PAIR2, PAIR3, and ZEP1 antibodies in both wild type and xrcc3-1 PMCs. (A) PAIR2, (B) PAIR3, and (C) ZEP1 on prophase I chromosomes in the wild type. (D) PAIR2, (E) PAIR3, and (F) ZEP1 on prophase I chromosomes in xrcc3-1. An anti-REC8 antibody is used to indicate the chromosomes. Scale bars: 5 μm.
Correct loading of MER3 and ZIP4 depends on XRCC3 MER3, a homologue of yeast Mer3, is one of the rice ZMM proteins (ZIP1, ZIP2, ZIP3, ZIP4, MSH4, MSH5, and
MER3). MER3 can be used to mark the correct installation of recombination elements (Wang et al., 2009). To investigate whether loss of function of XRCC3 affects normal recombination, the distribution of MER3 by dual-immunostaining using antibodies against REC8 and MER3 was monitored.
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Fig. 6. Immunofluorescent localization of COM1, RAD51C, and DMC1 antibodies on meiotic chromosomes in the wild type and xrcc3-1 meiocytes. (A) COM1, (B) RAD51C, and (C) DMC1 on the chromosomes at the zygotene stages in the wild type. (D) COM1, (E) RAD51C, and (F) DMC1 on the chromosomes at the zygotene stages in xrcc3-1. An anti-REC8 antibody is used to indicate the chromosomes. Scale bars: 5 μm.
More than 50 xrcc3-1 and wild-type meiocytes were observed. In the wild type, numerous MER3 foci were observed in the meiocyte at zygotene (Fig. 7A). By contrast, no MER3 foci were detected in xrcc3-1 meiocytes (Fig. 7C), indicating the presence of a severe recombination defect in xrcc3-1. ZIP4, another ZMM protein, is important for loading of MER3 onto chromosomes and chiasma formation (Shen et al., 2012). To monitor the distribution of ZIP4 in xrcc3
meiocytes, dual immunolocalization was carried using antibodies against REC8 and ZIP4 and about 50 xrcc3-1 and wild-type meiocytes were checked. In the wild type, ZIP4 foci could be detected slightly later than REC8 signals at early leptotene, and most ZIP4 foci colocalized with REC8. The number of ZIP4 foci increased rapidly and reached its peak at late leptotene to early zygotene (Fig. 7B). Subsequently, the number of ZIP4 foci rapidly decreased, and only a few
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foci could be detected in late pachytene. The ZIP4 foci disappeared at diplotene and could not be detected thereafter. In addition, no ZIP4 signals could be observed during meiosis I in xrcc3-1, confirming the presence of a serious recombination deficiency (Fig. 7D).
self-fertilized heterozygous XRCC3-1+/– plants were sterilized and grown on medium with 0 or 20 μg ml–1 MMC for 14 d. The growth rate of xrcc3-1 seedlings on medium with 20 μg ml–1 MMC was significantly slower than that of the wild type (Fig. 8), implying that XRCC3 also plays an essential role in DNA repair of somatic cells.
The xrcc3 mutant is hypersensitive to DNA mutagens To investigate whether mutation of XRCC3 affects the cellular response to somatic DNA damage, the sensitivity of xrcc3-1 to the crosslinking reagent MMC was tested. MMC can induce inter-strand DNA cross-linking; this type of DNA lesion is exclusively repaired by HR (Sasaki et al., 2004). Therefore, this test can provide evidence for the role of XRCC3 in HR processes during mitosis. Husked seeds from
Discussion Functional conservation and divergence of XRCC3 in different organisms XRCC3 has been characterized in several organisms. Multiple protein alignment analysis suggests that vertebrate XRCC3 is most closely related to yeast Rad57 (Symington, 2002).
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Fig. 7. Immunofluorescent localization of MER3 and ZIP4 antibodies on meiotic chromosomes in the wild type and xrcc3-1 meiocytes. (A) MER3 and (B) ZIP4 on chromosomes at the zygotene stages in the wild type. (C) MER3 and (D) ZIP4 on chromosomes at the zygotene stages in xrcc3-1. An anti-REC8 antibody is used to indicate the chromosomes. Scale bars: 5 μm.
The role of XRCC3 in rice meiosis | Page 11 of 13
In S. cerevisiae, Rad57 combines with the other Rad51 paralogue protein, Rad55, to form a stable heterodimer that stimulates the Rad51-promoting strand exchange reaction (Sung, 1997; Fortin and Symington, 2002; Liu et al., 2011). A null mutation of Rad57 causes the loss of meiosis-specific Rad51 foci and defective meiotic recombination as well as reduced spore viability, sensitivity to DNA cross-linking agents, and cold-sensitive DNA repair (Hays et al., 1995; Johnson and Symington, 1995; Symington, 2002), suggesting that Rad57 takes part in DNA repair during both mitosis and meiosis in S. cerevisiae. Vertebrate XRCC3 interacts with RAD51C to form a heterodimer complex (CX3) (Liu et al., 2002). The hamster xrcc3 mutant, irs1SF, exhibits a highly significant increase in chromosome missegregation, and a 25-fold reduction in homology-directed repair of DSBs compared with the wild type (Griffin, 2002). Although the CX3 complex acts downstream of RAD51 recruitment in response to DNA damage (Chun et al., 2013), human HCT116 cells lacking XRCC3 exhibit a mild reduction in sister chromatid exchange, impaired RAD51 focus formation, and increased levels of chromosome aberrations; endoreduplication is increased 5–7-fold in these cells (Yoshihara et al., 2004). These observations indicate that XRCC3 plays a conserved role in maintaining genome stability in vertebrates during both meiosis and mitosis. However, human HCT116 and chicken B-lymphocyte DT40 cell lines, which lack XRCC3, exhibit milder phenotypes with respect to recombinational repair (2–3-fold sensitivity to MMC) compared with hamster irs1SF, which exhibits extreme sensitivity to MMC (60-fold). By contrast, the Drosophila xrcc3 mutant Spn-B is not hypersensitive to DSB-inducing agents (Abdu et al., 2003). So far, only Arabidopsis xrcc3 mutant has been characterized in plants. Several abnormal chromosome behaviours occur, including the lack of homologous chromosomes pairing, synapsis, chromosome entanglement, and chromosome fragmentation, demonstrating that XRCC3 is important for homologous recombination and for maintaining its genome integrity in Arabidopsis (Vignard et al., 2007). Severe
defects in homologue paring and synapsis, massive chromosome entanglement, and fragmentation in rice xrcc3 meiocytes were observed. Compared with the reduced number of DMC1 foci in Arabidopsis xrcc3, no DMC1 foci in rice xrcc3 were detected.
Abnormal chromosome entanglement and fragmentation in xrcc3 Organisms have evolved effective mechanisms for DSB repair including three major repair pathways: HR, non-homologous end-joining (NHEJ), and microhomology-mediated end-joining (MMEJ) (McVey and Lee, 2008; Symington and Gautier, 2011). HR ensures accurate repair of DSBs by using the intact sister chromatid or homologous chromosome as a template (Symington and Gautier, 2011). NHEJ is the simplest mechanism involved in DSB repair, ensuring chromosome integrity by direct ligation of broken DNA ends (Lieber, 2010; Symington and Gautier, 2011). In the MMEJ pathway, microhomologous sequences are annealed by the end-resection machinery to align ends prior to ligation (McVey and Lee, 2008). NHEJ and MMEJ are error-prone repair pathways that allow nucleotide alterations at the rejoining site, as they use no or limited sequence homology to rejoin ends of non-homologous chromosomes. The pathway used for DNA DSB repair depends on the occurrence of through end-processing. In eukaryotes, three DSB repair pathways function in a complementary, balanced manner during the cell cycle (Symington and Gautier, 2011). MRN/X and Com1/Sae2/CtIP are involved in DSB endprocessing, while the Com1/Sae2/CtIP homologue may help maintain the balance between the three DSB repair pathways, NHEJ, MMEJ, and HR (Rahal et al., 2010; Symington and Gautier, 2011). CtIP, a COM1 homologue in chicken DT40 cells, regulates the balance between error-prone DNA end-joining and error-free HR in the DSB repair pathway through its phosphorylation of serine 327 and the recruitment of BRCA1 (Yun and Hiom, 2009). A similar mechanism also occurs in C. elegans, as the HR pathway does not
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Fig. 8. Seedlings of the wild type and xrcc3-1 mutant were treated with MMC. (A) xrcc3-1 seedlings (right) exhibited more growth retardation than the wild-type seedlings (left) on half-strength MS medium with MMC. (B) Statistical analysis of the wild-type and xrcc3-1 seedling height.
Page 12 of 13 | Zhang et al. occur in COM-1-deficient germ cells, which are committed to meiotic DSB repair by NHEJ, resulting in crossover defects and extensive chromosomal aggregation (Lemmens et al., 2013). In the rice com1 mutant, non-homologous entanglements in meiotic cells lead to the formation of chromosome fragments with two telomeres produced via the interaction of non-homologous chromosomes through NEHJ (Ji et al., 2012). In the present study, no COM1 signals and massive chromosome entanglements were observed in xrcc3-1 meiocytes, which are similar to the com1 mutant, implying that DSB ends cannot be processed normally in rice xrcc3. It is suspected that DSBs might be repaired using another option, NHEJ, in xrcc3-1 meiocytes.
Supplementary data can be found at JXB online. Supplementary Table S1. Primers for XRCC3 gene cloning. Supplementary Fig. S1. Alignment of amino acid sequence encoded by rice and Arabidopsis XRCC3 genes. Supplementary Fig. S2. The lack of a transcript of the XRCC3 gene in the young panicles of the xrcc3-1 mutant. Supplementary Fig. S3. Meiosis of male meiocytes in xrcc3-2.
Acknowledgements This work was supported by grants from the Ministry of Sciences and Technology of China (2011CB944602 and 2012AA10A301) and the National Natural Science Foundation of China (31170288 and 31230038).
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Supplementary data
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