Yafei Li

CENTRAL REGION COMPONENT1, a Novel Synaptonemal Complex Component, Is Essential for Meiotic Recombination Initiation in Rice

This work identifies CRC1 (for CENTRAL REGION COMPONENT1), a novel synaptonemal complex component that is essential for the initiation of homologous recombination in meiosis. CRC1 is the rice ortholog of budding yeast Pch2 and mouse TRIP13, but the roles of CRC1 identified here have not been reported for Pch2 or TRIP13. 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.

Ten Years of Gene Discovery for Meiotic Event Control in Rice

Meiosis is the crucial process by which sexually propagating eukaryotes give rise to haploid gametes from diploid cells. Several key processes, like homologous chromosomes pairing, synapsis, recombination, and segregation, sequentially take place in meiosis. Although these widely conserved events are under both genetic and epigenetic control, the accurate details of molecular mechanisms are continuing to investigate. Rice is a good model organism for exploring the molecular mechanisms of meiosis in higher plants. So far, 28 rice meiotic genes have been characterized. In this review, we give an overview of the discovery of rice meiotic genes in the last ten years, with a particular focus on their functions in meiosis.

The Role of OsMSH5 in Crossover Formation during Rice Meiosis

MSH5, a meiosis-specific member of the MutS-homolog family, is required for normal level of recombination in budding yeast, mice, Caenorhabditis elegans, and Arabidopsis. Here, we report the identification and characterization of its rice homolog, OsMSH5, and demonstrate its function in rice meiosis. Five independent Osmsh5 mutants exhibited normal vegetative growth and severe sterility. The synaptonemal complex is well installed in Osmsh5, while the chiasma frequency is greatly reduced to approximately 10% of that observed in the wild-type, leading to the homologous non-disjunction and complete sterile phenotype. OsMSH5 is predominantly expressed in panicles. Immunofluorescence studies indicate that OsMSH5 chromosomal localization is limited to the early meiotic prophase I. OsMSH5 can be loaded onto meiotic chromosomes in Oszip4, Osmer3, and hei10. However, those ZMM proteins cannot be localized normally in the absence of OsMSH5. Furthermore, the residual chiasmata were shown to be the least frequent among the zmm mutants, including Osmer3, Oszip4, hei10, and Osmsh5. Taken together, we propose that OsMSH5 functions upstream of OsZIP4, OsMER3, and HEI10 in class I crossover formation.

OsRAD51C is essential for double-strand break repair in rice meiosis

RAD51C is one of the RAD51 paralogs that plays an important role in DNA double-strand break repair by homologous recombination. Here, we identified and characterized OsRAD51C, the rice homolog of human RAD51C. The Osrad51c mutant plant is normal in vegetative growth but exhibits complete male and female sterility. Cytological investigation revealed that homologous pairing and synapsis were severely disrupted. Massive chromosome fragmentation occurred during metaphase I in Osrad51c meiocytes, and was fully suppressed by the CRC1 mutation. Immunofluorescence analysis showed that OsRAD51C localized onto the chromosomes from leptotene to early pachytene during prophase I, and that normal loading of OsRAD51C was dependent on OsREC8, PAIR2, and PAIR3. Additionally, ZEP1 did not localize properly in Osrad51c, indicating that OsRAD51C is required for synaptonemal complex assembly. Our study also provided evidence in support of a functional divergence in RAD51C among organisms.

OsDMC1 Is Not Required for Homologous Pairing in Rice Meiosis

OsDMC1, a conserved recombinase, maintains the stability of the single-end invasion process but is not required for homologous pairing in rice, which is quite different from the DMC1 homologs identified so far in other organisms. Meiotic homologous recombination is pivotal to sexual reproduction. DMC1, a conserved recombinase, is involved in directing single-end invasion between interhomologs during meiotic recombination. In this study, we identified OsDMC1A and OsDMC1B, two closely related proteins in rice (Oryza sativa) with high sequence similarity to DMC1 proteins from other species. Analysis of Osdmc1a and Osdmc1b Tos17 insertion mutants indicated that these genes are functionally redundant. Immunolocalization analysis revealed OsDMC1 foci occurred at leptotene, which disappeared from late pachytene chromosomes in wild-type meiocytes. According to cytological analyses, homologous pairing is accomplished in the Osdmc1a Osdmc1b double mutant, but synapsis is seriously disrupted. The reduced number of bivalents and abnormal OsHEI10 foci in Osdmc1a Osdmc1b establishes an essential role for OsDMC1 in crossover formation. In the absence of OsDMC1, early recombination events probably occur normally, leading to normal localization of γH2AX, PAIR3, OsMRE11, OsCOM1, and OsRAD51C. Moreover, OsDMC1 was not detected in pairing-defective mutants, such as pair2, pair3, Oscom1, and Osrad51c, while it was loaded onto meiotic chromosomes in zep1, Osmer3, Oszip4, and Oshei10. Taken together, these results suggest that during meiosis, OsDMC1 is dispensable for homologous pairing in rice, which is quite different from the DMC1 homologs identified so far in other organisms.

Semi-Rolled Leaf2 modulates rice leaf rolling by regulating abaxial side cell differentiation

Highlight Semi-Rolled Leaf2 encodes a novel plant-specific protein that modulates leaf rolling by regulating cell development in the abaxial sides of rice leaves.

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.

MRE11 is required for homologous synapsis and DSB processing in rice meiosis

Mre11, a conserved protein found in organisms ranging from yeast to multicellular organisms, is required for normal meiotic recombination. Mre11 interacts with Rad50 and Nbs1/Xrs2 to form a complex (MRN/X) that participates in double-strand break (DSB) ends processing. In this study, we silenced the MRE11 gene in rice and detailed its function using molecular and cytological methods. The OsMRE11-deficient plants exhibited normal vegetative growth but could not set seed. Cytological analysis indicated that in the OsMRE11-deficient plants, homologous pairing was totally inhibited, and the chromosomes were completely entangled as a formation of multivalents at metaphase I, leading to the consequence of serious chromosome fragmentation during anaphase I. Immunofluorescence studies further demonstrated that OsMRE11 is required for homologous synapsis and DSB processing but is dispensable for meiotic DSB formation. We found that OsMRE11 protein was located on meiotic chromosomes from interphase to late pachytene. This protein showed normal localization in zep1, Oscom1 and Osmer3, as well as in OsSPO11-1RNAi plants, but not in pair2 and pair3 mutants. Taken together, our results provide evidence that OsMRE11 performs a function essential for maintaining the normal HR process and inhibiting non-homologous recombination during meiosis.

Crossover Formation During Rice Meiosis Relies on Interaction of OsMSH4 and OsMSH5

MSH4 encodes a MutS protein that plays a specialized role in meiosis. In eukaryotic species, such as budding yeast, mice, Caenorhabditis elegans, and Arabidopsis, msh4 mutants display meiotic defects with a reduced number of chiasmata. Here, we characterized rice MSH4 by map-based cloning. In Osmsh4 mutants, the chiasma frequency was dramatically decreased to ∼10% of the wild type, but the synaptonemal complex was normally installed. The double mutant analysis showed that in the Osmsh4 Osmsh5 mutant, the reduction of chiasmata was greater than other zmm mutants. This was consistent with the absence of localization for OsZIP4 and OsMER3 in Osmsh4 and suggests an earlier role for OsMSH4 and OsMSH5 than other ZMM proteins where they may be required to stabilize progenitor Holliday junctions. Using yeast two-hybrid and pull-down assays, we verified the direct physical association between OsMSH4 and OsMSH5 and OsMSH5 and HEI10 in plants for the first time. The MSH4–MSH5 heterodimer has been demonstrated in mammals to stabilize the formation of progenitor and double Holliday junctions that may be resolved as crossovers (COs). We propose that OsMSH4 interacts with OsMSH5 to promote formation of the majority of COs in rice.

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 programed 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.