Chromosome ends initiate homologous chromosome pairing during rice meiosis.

During meiotic prophase I, chromosomes undergo large-scale dynamics to allow homologous chromosome pairing, prior to which chromosome ends attach to the inner nuclear envelope and form a chromosomal bouquet. Chromosome pairing is crucial for homologous recombination and accurate chromosome segregation during meiosis. However, the specific mechanism by which homologous chromosomes recognize each other is poorly understood. Here, we investigated the process of homologous chromosome pairing during early prophase I of meiosis in rice (Oryza sativa) using pooled oligo probes specific to an entire chromosome or chromosome arm. We revealed that chromosome pairing begins from both ends and extends towards the center from early zygotene through late zygotene. Genetic analysis of both trisomy and autotetraploidy also showed that pairing initiation is induced by both ends of a chromosome. However, healed ends that lack the original terminal regions on telocentric and acrocentric chromosomes cannot initiate homologous chromosome pairing, even though they may still enter the telomere clustering region at the bouquet stage. Furthermore, a chromosome that lacks the distal parts on both sides loses the ability to pair with other intact chromosomes. Thus, the native ends of chromosomes play a crucial role in initiating homologous chromosome pairing during meiosis and likely have a substantial impact on genome differentiation.

De novo centromere formation in pericentromeric region of rice chromosome 8.

Neocentromeres develop when kinetochores assemble de novo at DNA loci that are not previously associated with CenH3 nucleosomes, and can rescue rearranged chromosomes that have lost a functional centromere. The molecular mechanisms associated with neocentromere formation in plants have been elusive. Here, we developed a Xian (indica) rice line with poor growth performance in the field due to approximately 272 kb deletion that spans centromeric DNA sequences, including the centromeric satellite repeat CentO, in the centromere of chromosome 8 (Cen8). The CENH3-binding domains were expanded downstream of the original CentO position in Cen8, which revealed a de novo centromere formation in rice. The neocentromere formation avoids chromosomal regions containing functional genes. Meanwhile, canonical histone H3 was replaced by CENH3 in the regions with low CENH3 levels, and the CenH3 nucleosomes in these regions became more periodic. In addition, we identified active genes in the deleted centromeric region, which are essential for chloroplast growth and development. In summary, our results provide valuable insights into neocentromere formation and show that functional genes exist in the centromeric regions of plant chromosomes.

Epigenomic features of DNA G-quadruplexes and their roles in regulating rice gene transcription.

A DNA G-quadruplex (G4) is a non-canonical four-stranded nucleic acid structure involved in many biological processes in mammals. The current knowledge on plant DNA G4s, however, is limited; whether and how DNA G4s impact gene expression in plants is still largely unknown. Here, we applied a protocol referred to as BG4-DNA-IP-seq followed by a comprehensive characterization of DNA G4s in rice (Oryza sativa L.); we next integrated dG4s (experimentally detectable G4s) with existing omics data and found that dG4s exhibited differential DNA methylation between transposable element (TE) and non-TE genes. dG4 regions displayed genic-dependent enrichment of epigenomic signatures; finally, we showed that these sites displayed a positive association with expression of DNA G4-containing genes when located at promoters, and a negative association when located in the gene body, suggesting localization-dependent promotional/repressive roles of DNA G4s in regulating gene transcription. This study reveals interrelations between DNA G4s and epigenomic signatures, as well as implicates DNA G4s in modulating gene transcription in rice. Our study provides valuable resources for the functional characterization or bioengineering of some of key DNA G4s in rice.

DEEP GREEN PANICLE1 suppresses GOLDEN2-LIKE activity to reduce chlorophyll synthesis in rice glumes.

Understanding the regulation mechanisms of photosynthesis is key to improving its efficiency and, ultimately, crop yield. In this study, we report that DEEP GREEN PANICLE1 (DGP1) is involved in photosynthesis regulation in rice (Oryza sativa L.). We identified the dgp1 mutant, which has increased chlorophyll content in glumes. The mutated gene was isolated by map-based cloning. Knockout plants, generated using a gene editing approach, mimic the phenotype of dgp1. Overexpression of DGP1 leads to chlorotic leaves and glumes. DGP1 is a plant-specific protein with a conserved TIGR01589 domain. The expression of DGP1 was detected in green tissues and is induced by light. Moreover, genes involved in key steps of chlorophyll synthesis are upregulated in the glumes of dgp1. Importantly, we found that DGP1 interacts with the rice proteins GOLDEN2-LIKE1 (OsGLK1) and GOLDEN2-LIKE2 (OsGLK2), the two transcription factors involved in the regulation of photosynthesis. Transactivation assays showed that DGP1 represses the activation activity of OsGLK1 on its target genes. Our results demonstrate that DGP1 is a repressor of OsGLK activity and thus photosynthesis in rice. Manipulation of this gene and its homologs in other crops may provide new approaches for high photosynthetic efficiency breeding.

OsRAD51 Plays a Vital Role in Promoting Homologous Recombination in Rice Meiosis.

Meiotic recombination plays a pivotal role in achieving accurate chromosomal segregation and increasing genetic diversity. In the homologous recombination pathway, the detailed mechanisms of how OsRAD51 and OsDMC1 work in rice meiosis remain to be explored. Here, we obtained different types of mutants for Osrad51a1, Osrad51a2, Osdmc1a, and Osdmc1b through CRISPR/Cas9. Both Osrad51a1 and Osrad51a2 exhibited normal vegetative growth and fertility. Osrad51 (Osrad51a1 Osrad51a2) mutant plants show normal vegetative growth but exhibit complete sterility, indicating that OsRAD51A1 and OsRAD51A2 are functionally redundant in rice fertility. In contrast to the wild type, Osrad51 chromosomes are not paired perfectly at pachytene and synaptonemal complex (SC) formation is deficient. Moreover, univalents and multivalent associations were observed at metaphase I, chromosome fragments presented at anaphase I, and crossover formation is basically suppressed in Osrad51 pollen mother cells (PMCs). OsRAD51 foci emerge at leptotene and disappear from late pachytene and chromosome localization of OsRAD51 depends on the formation of double-strand breaks (DSBs). Most OsRAD51 foci can co-localize with OsDMC1 signals. OsRAD51 is essential for the loading of OsDMC1 onto chromosomes, and vice versa. In addition, both OsRAD51 and OsDMC1 can interact with OsFIGL1 and OsBRCA2, two important components in rice meiosis. Moreover, the Osrad51 Osdmc1 (Osrad51a1 Osrad51a2 Osdmc1a Osdmc1b) quadruple mutant PMCs exhibited similar defective phenotypes as Osrad51 in homologous pairing, synapsis, and DSB repair. Taken together, our results suggest that the recombinases DMC1 and RAD51 may functionally depend on each other and play important roles in meiotic recombination during meiosis in rice.

COM1, a factor of alternative non-homologous end joining, lagging behind the classic non-homologous end joining pathway in rice somatic cells.

The role of rice (Oryza sativa) COM1 in meiotic homologous recombination (HR) is well understood, but its part in somatic double-stranded break (DSB) repair remains unclear. Here, we show that for rice plants COM1 conferred tolerance against DNA damage caused by the chemicals bleomycin and mitomycin C, while the COM1 mutation did not compromise HR efficiencies and HR factor (RAD51 and RAD51 paralogues) localization to irradiation-induced DSBs. Similar retarded growth at the post-germination stage was observed in the com1-2 mre11 double mutant and the mre11 single mutant, while combined mutations in COM1 with the HR pathway gene (RAD51C) or classic non-homologous end joining (NHEJ) pathway genes (KU70, KU80, and LIG4) caused more phenotypic defects. In response to γ-irradiation, COM1 was loaded normally onto DSBs in the ku70 mutant, but could not be properly loaded in the MRE11RNAi plant and in the wortmannin-treated wild-type plant. Under non-irradiated conditions, more DSB sites were occupied by factors (MRE11, COM1, and LIG4) than RAD51 paralogues (RAD51B, RAD51C, and XRCC3) in the nucleus of wild-type; protein loading of COM1 and XRCC3 was increased in the ku70 mutant. Therefore, quite differently to its role for HR in meiocytes, rice COM1 specifically acts in an alternative NHEJ pathway in somatic cells, based on the Mre11-Rad50-Nbs1 (MRN) complex and facilitated by PI3K-like kinases. NHEJ factors, not HR factors, preferentially load onto endogenous DSBs, with KU70 restricting DSB localization of COM1 and XRCC3 in plant somatic cells.

Dual-color oligo-FISH can reveal chromosomal variations and evolution in Oryza species.

Fluorescence in situ hybridization using probes based on oligonucleotides (oligo-FISH) is a useful tool for chromosome identification and karyotype analysis. Here we developed two oligo-FISH probes that allow the identification of each of the 12 pairs of chromosomes in rice (Oryza sativa). These two probes comprised 25 717 (green) and 25 215 (red) oligos (45 nucleotides), respectively, and generated 26 distinct FISH signals that can be used as a barcode to uniquely label each of the 12 pairs of rice chromosomes. Standard karyotypes of rice were established using this system on both mitotic and meiotic chromosomes. Moreover, dual-color oligo-FISH was used to characterize diverse chromosomal abnormalities. Oligo-FISH analyses using these probes in various wild Oryza species revealed that chromosomes from the AA, BB or CC genomes generated specific and intense signals similar to those in rice, while chromosomes with the EE genome generated less specific signals and the FF genome gave no signal. Together, the oligo-FISH probes we established will be a powerful tool for studying chromosome variations and evolution in the genus Oryza.

OsATM Safeguards Accurate Repair of Meiotic Double-Strand Breaks in Rice.

ATAXIA TELANGIECTASIA-MUTATED (ATM) protein has been well studied for its roles in the DNA damage response. However, its role in meiosis has not been fully explored. Here, we characterized the functions of the rice (Oryza sativa) ATM homolog during meiosis. Aberrant chromosome associations and DNA fragmentations were observed after the completion of homologous pairing and synapsis in Osatm pollen mother cells (PMCs). Aberrant chromosome associations disappeared in Osspo11-1 Osatm-1 double mutants and more severe defects were observed in Osdmc1 Osatm, suggesting that OsATM functions downstream of OsSPO11-1-catalyzed double-strand break formation and in parallel with OsDMC1-mediated homologous recombination. We further demonstrated that phosphorylation of H2AX in PMCs did not depend on OsATM, in contrast to the situation in somatic cells. Moreover, the removal of OsDMC1 from chromosomes in Osatm PMCs was delayed and the number of HEI10 foci (markers of interference-sensitive crossover intermediates) decreased. Together, these findings suggest that OsATM plays important roles in the accurate repair of meiotic double-strand breaks in rice.

The SUN Domain Proteins OsSUN1 and OsSUN2 Play Critical but Partially Redundant Roles in Meiosis.

During meiosis, Sad1/UNC-84 (SUN) domain proteins play conserved roles in promoting telomere bouquet formation and homologous pairing across species. Arabidopsis (Arabidopsis thaliana) AtSUN1 and AtSUN2 have been shown to have overlapping functions in meiosis. However, the role of SUN proteins in rice (Oryza sativa) meiosis and the extent of functional redundancy between them remain elusive. Here, we generated single and double mutants of OsSUN1 and OsSUN2 in rice using genome editing. The Ossun1 Ossun2 double mutant showed severe defects in telomere clustering, homologous pairing, and crossover formation, suggesting that OsSUN1 and OsSUN2 are essential for rice meiosis. When introducing a mutant allele of O. sativa SPORULATION11-1 (OsSPO11-1), which encodes a topoisomerase initiating homologous recombination, into the Ossun1 Ossun2 mutant, we observed a combined Osspo11-1- and Ossun1 Ossun2-like phenotype, demonstrating that OsSUN1 and OsSUN2 promote bouquet formation independent of OsSPO11-1 but regulate pairing and crossover formation downstream of OsSPO11-1. Importantly, the Ossun1 single mutant had a normal phenotype, but meiosis was disrupted in the Ossun2 mutant, indicating that OsSUN1 and OsSUN2 are not completely redundant in rice. Further analyses revealed a genetic dosage-dependent effect and an evolutionary differentiation between OsSUN1 and OsSUN2 These results suggested that OsSUN2 plays a more critical role than OsSUN1 in rice meiosis. Taken together, this work reveals the essential but partially redundant roles of OsSUN1 and OsSUN2 in rice meiosis and demonstrates that functional divergence of SUN proteins has taken place during evolution.

The OsRR24/LEPTO1 Type-B Response Regulator is Essential for the Organization of Leptotene Chromosomes in Rice Meiosis.

Response regulators play significant roles in controlling various biological processes; however, their roles in plant meiosis remain unclear. Here, we report the identification of OsRR24/LEPTOTENE1 (LEPTO1), a rice (Oryza sativa) type-B response regulator that participates in the establishment of key molecular and morphological features of chromosomes in leptotene, an early stage of prophase I in meiosis. Although meiosis initiates normally, as indicated by staining of the centromere-specific histone CENH3, the meiotic chromosomes in lepto1 mutant pollen mother cells fail to form the thin thread-like structures that are typical of leptotene chromosomes in wild-type pollen mother cells. Furthermore, lepto1 mutants fail to form chromosomal double-strand breaks, do not recruit meiosis-specific proteins to the meiotic chromosomes, and show disrupted callose deposition. LEPTO1 also is essential for programmed cell death in tapetal cells. LEPTO1 contains a conserved signal receiver domain (DDK) and a myb-like DNA binding domain at the N terminus. LEPTO1 interacts with two authentic histidine phosphotransfer (AHP) proteins, OsAHP1 and OsAHP2, via the DDK domain, and a phosphomimetic mutation of the DDK domain relieves its repression of LEPTO1 transactivation activity. Collectively, our results show that OsRR24/LEPTO1 plays a significant role in the leptotene phase of meiotic prophase I.

Deep Sequencing Discovery and Profiling of Known and Novel miRNAs Produced in Response to DNA Damage in Rice.

Under extreme environmental conditions such as ultraviolet and ionizing radiation, plants may suffer DNA damage. If these damages are not repaired accurately and rapidly, they may lead to chromosomal abnormalities or even cell death. Therefore, organisms have evolved various DNA repair mechanisms to cope with DNA damage which include gene transcription and post-translational regulation. MicroRNA (miRNA) is a type of non-coding single-stranded RNA molecule encoded by endogenous genes. They can promote DNA damage repair by regulating target gene transcription. Here, roots from seedlings of the japonica rice cultivar 'Yandao 8' that were treated with bleomycin were collected for transcriptome-level sequencing, using non-treated roots as controls. A total of 14,716,232 and 17,369,981 reads mapping to miRNAs were identified in bleomycin-treated and control groups, respectively, including 513 known and 72 novel miRNAs. Compared with the control group, 150 miRNAs showed differential expression levels. Target predictions of these differentially expressed miRNAs yielded 8731 potential gene targets. KEGG annotation and a gene ontology analysis indicated that the highest-ranked target genes were classified into metabolic processes, RNA degradation, DNA repair, and so on. Notably, the DNA repair process was significantly enriched in both analyses. Among these differentially expressed miRNAs, 58 miRNAs and 41 corresponding potential target genes were predicted to be related to DNA repair. RT-qPCR results confirmed that the expression patterns of 20 selected miRNAs were similar to those from the sequencing results, whereas four miRNAs gave opposite results. The opposing expression patterns of several miRNAs with regards to their target genes relating to the DNA repair process were also validated by RT-qPCR. These findings provide valuable information for further functional studies of miRNA involvement in DNA damage repair in rice.

OsPINOID Regulates Stigma and Ovule Initiation through Maintenance of the Floral Meristem by Auxin Signaling.

Stigma and ovule initiation is essential for sexual reproduction in flowering plants. However, the mechanism underlying the initiation of stigma and ovule primordia remains elusive. We identified a stigma-less mutant of rice (Oryza sativa) and revealed that it was caused by the mutation in the PINOID (OsPID) gene. Unlike the pid mutant that shows typical pin-like inflorescences in maize (Zea mays) and Arabidopsis (Arabidopsis thaliana), the ospid mutant does not display any defects in inflorescence development and flower initiation, and fails to develop normal ovules in most spikelets. The auxin activity in the young pistil of ospid was lower than that in the wild-type pistil. Furthermore, the expression of most auxin response factor genes was down-regulated, and OsETTIN1, OsETTIN2, and OsMONOPTEROS lost their rearrangements of expression patterns during pistil and stamen primordia development in ospid Moreover, the transcription of the floral meristem marker gene, OSH1, was down-regulated and FLORAL ORGAN NUMBER4, the putative ortholog of Arabidopsis CLAVATA3, was up-regulated in the pistil primordium of ospid These results suggested that the meristem proliferation in the pistil primordium might be arrested prematurely in ospid Based on these results, we propose that the OsPID-mediated auxin signaling pathway plays a crucial role in the regulation of rice stigma and ovule initiation by maintaining the floral meristem.

Rice RAD51 paralogs play essential roles in somatic homologous recombination for DNA repair.

Synthesis-dependent strand annealing (SDSA) and single-strand annealing (SSA) are the two main homologous recombination (HR) pathways in double-strand break (DSB) repair. The involvement of rice RAD51 paralogs in HR is well known in meiosis, although the molecular mechanism in somatic HR remains obscure. Loss-of-function mutants of rad51 paralogs show increased sensitivity to the DSB-inducer bleomycin, which results in greatly compromised somatic recombination efficiencies (xrcc3 in SDSA, rad51b and xrcc2 in SSA, rad51c and rad51d in both). Using immunostaining, we found that mutations in RAD51 paralogs (XRCC3, RAD51C, or RAD51D) lead to tremendous impairment in RAD51 focus formation at DSBs. Intriguingly, the RAD51C mutation has a strong effect on the protein loading of its partners (XRCC3 and RAD51B) at DSBs, which is similar to the phenomenon observed in the case of blocking PI3K-like kinases in wild-type plant. We conclude that the rice CDX3 complex acts in SDSA recombination while the BCDX2 complex acts in SSA recombination in somatic DSB repair. Importantly, RAD51C serves as a fulcrum for the local recruitment of its partners (XRCC3 for SDSA and RAD51B for SSA) and is positively modulated by PI3K-like kinases to facilitate both the SDSA and SSA pathways in RAD51 paralog-dependent somatic HR.

Chromosome painting and its applications in cultivated and wild rice.

BACKGROUND: The chromosome-specific probe is a fundamental tool of chromosome painting and has been commonly applied in mammalian species. The technology, however, has not been widely applied in plants due to a lack of methodologies for probe development. Identification and labeling of a large number of oligonucleotides (oligos) specific to a single chromosome offers us an opportunity to establish chromosome-specific probes in plants. However, never before has whole chromosome painting been performed in rice. RESULTS: We developed a pooled chromosome 9-specific probe in rice, which contains 25,000 oligos based on the genome sequence of a japonica rice (Oryza sativa L., AA, 2n = 2× = 24). Chromosome 9 was easily identified in both japonica and indica rice using this chromosome 9-painting probe. The probe was also successfully used to identify and characterize chromosome 9 in additional lines of O. sativa, a translocation line, two new aneuploids associated with chromosome 9 and a wild rice (Oryza eichingeri A. Peter, CC, 2n = 2× = 24). CONCLUSION: The study reveals that a pool of oligos specific to a chromosome is a useful tool for chromosome painting in rice.

Chromatin states responsible for the regulation of differentially expressed genes under 60Co~γ ray radiation in rice.

BACKGROUND: The role of histone modifications in the DNA damage response has been extensively studied in non-plant systems, including mammals and yeast. However, there is a lack of detailed evidence showing how chromatin dynamics, either an individual mark or combined chromatin states, participate in regulating differentially expressed genes in the plant DNA damage response. RESULTS: In this study, we used RNA-seq and ChIP-seq to show that differentially expressed genes (DEGs), in response to ionizing radiation (IR), might be involved in different pathways responsible for the DNA damage response. Moreover, chromatin structures associated with promoters, exons and intergenic regions are significantly affected by IR. Most importantly, either an individual mark or a certain chromatin state was found to be highly correlated with the expression of up-regulated genes. In contrast, only the chromatin states, as opposed to any individual marks tested, are related to the expression of the down-regulated genes. CONCLUSIONS: Our findings demonstrate that IR-related DEGs are modulated by distinct epigenetic mechanisms. Either chromatin states or distinct histone dynamics may act sequentially or in combination in regulating up-regulated genes, but the complex chromatin structure is mainly responsible for the expression of down-regulated genes. Thus, this study provides new insights into how up- and down-regulated genes are epigenetically regulated at the chromatin levels, thereby helping us to understand distinct epigenetic mechanisms that function in the plant DNA damage response.

RNAi-mediated resistance to rice black-streaked dwarf virus in transgenic rice.

Rice black-streaked dwarf virus (RBSDV), a member of the genus Fijivirus in the family Reoviridae, causes significant economic losses in rice production in China and many other Asian countries. Development of resistant varieties by using conventional breeding methods is limited, as germplasm with high level of resistance to RBSDV have not yet been found. One of the most promising methods to confer resistance against RBSDV is the use of RNA interference (RNAi) technology. RBSDV non-structural protein P7-2, encoded by S7-2 gene, is a potential F-box protein and involved in the plant-virus interaction through the ubiquitination pathway. P8, encoded by S8 gene, is the minor core protein that possesses potent active transcriptional repression activity. In this study, we transformed rice calli using a mini-twin T-DNA vector harboring RNAi constructs of the RBSDV genes S7-2 or S8, and obtained plants harboring the target gene constructs and the selectable marker gene, hygromycin phosphotransferase (HPT). From the offspring of these transgenic plants, we obtained selectable marker (HPT gene)-free plants. Homozygous T5 transgenic lines which harbored either S7-2-RNAi or S8-RNAi exhibited high level resistance against RBSDV under field infection pressure from indigenous viruliferous small brown planthoppers. Thus, our results showed that RNA interference with the expression of S7-2 or S8 genes seemed an effective way to induce high level resistance in rice against RBSD disease.

OsHUS1 facilitates accurate meiotic recombination in rice.

Meiotic recombination normally takes place between allelic sequences on homologs. This process can also occur between non-allelic homologous sequences. Such ectopic interaction events can lead to chromosome rearrangements and are normally avoided. However, much remains unknown about how these ectopic interaction events are sensed and eliminated. In this study, using a screen in rice, we characterized a homolog of HUS1 and explored its function in meiotic recombination. In Oshus1 mutants, in conjunction with nearly normal homologous pairing and synapsis, vigorous, aberrant ectopic interactions occurred between nonhomologous chromosomes, leading to multivalent formation and subsequent chromosome fragmentation. These ectopic interactions relied on programmed meiotic double strand breaks and were formed in a manner independent of the OsMER3-mediated interference-sensitive crossover pathway. Although early homologous recombination events occurred normally, the number of interference-sensitive crossovers was reduced in the absence of OsHUS1. Together, our results indicate that OsHUS1 might be involved in regulating ectopic interactions during meiosis, probably by forming the canonical RAD9-RAD1-HUS1 (9-1-1) complex.

Biofortification of rice with the essential amino acid lysine: molecular characterization, nutritional evaluation, and field performance.

Rice (Oryza sativa L.), a major staple crop worldwide, has limited levels of the essential amino acid lysine. We previously produced engineered rice with increased lysine content by expressing bacterial aspartate kinase and dihydrodipicolinate synthase and inhibiting rice lysine ketoglutarate reductase/saccharopine dehydrogenase activity. However, the grain quality, field performance, and integration patterns of the transgenes in these lysine-enriched lines remain unclear. In the present study, we selected several elite transgenic lines with endosperm-specific or constitutive regulation of the above key enzymes but lacking the selectable marker gene. All target transgenes were integrated into the intragenic region in the rice genome. Two pyramid transgenic lines (High Free Lysine; HFL1 and HFL2) with free lysine levels in seeds up to 25-fold that of wild type were obtained via a combination of the above two transgenic events. We observed a dramatic increase in total free amino acids and a slight increase in total protein content in both pyramid lines. Moreover, the general physicochemical properties were improved in pyramid transgenic rice, but the starch composition was not affected. Field trials indicated that the growth of HFL transgenic rice was normal, except for a slight difference in plant height and grain colour. Taken together, these findings will be useful for the potential commercialization of high-lysine transgenic rice.

Central region component1, a novel synaptonemal complex component, is essential for meiotic recombination initiation in rice.

In meiosis, homologous recombination entails programmed DNA double-strand break (DSB) formation and synaptonemal complex (SC) assembly coupled with the DSB repair. Although SCs display extensive structural conservation among species, their components identified are poorly conserved at the sequence level. Here, we identified a novel SC component, designated central region component1 (CRC1), in rice (Oryza sativa). CRC1 colocalizes with ZEP1, the rice SC transverse filament protein, to the central region of SCs in a mutually dependent fashion. Consistent with this colocalization, CRC1 interacts with ZEP1 in yeast two-hybrid assays. CRC1 is orthologous to Saccharomyces cerevisiae pachytene checkpoint2 (Pch2) and Mus musculus THYROID receptor-interacting protein13 (TRIP13) and may be a conserved SC component. Additionally, we provide evidence that CRC1 is essential for meiotic DSB formation. CRC1 interacts with homologous pairing aberration in rice meiosis1 (PAIR1) in vitro, suggesting that these proteins act as a complex to promote DSB formation. PAIR2, the rice ortholog of budding yeast homolog pairing1, is required for homologous chromosome pairing. We found that CRC1 is also essential for the recruitment of PAIR2 onto meiotic chromosomes. The roles of CRC1 identified here have not been reported for Pch2 or TRIP13.

XRCC3 is essential for proper double-strand break repair and homologous recombination in rice meiosis.

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.