Meiotic Chromosome Association 1 Interacts with TOP3a andRegulates Meiotic Recombination in RiceOPEN

QingHu,a,b,1 Yafei Li,a,b,1 HongjunWang,a,b Yi Shen,aChaoZhang,a,bGuijieDu,aDingTang,a andZhukuanChenga,b,2

a State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing100101, ChinabUniversity of Chinese Academy of Sciences, Beijing 100049, China

ORCID IDs: 0000-0002-3349-0056 (Q.H.); 0000-0002-9702-3000 (H.W.); 0000-0001-8428-8010 (Z.C.)

Homologous recombination plays a central role in guaranteeing chromosome segregation during meiosis. The preciseregulation of the resolution of recombination intermediates is critical for the success of meiosis. Many proteins, including theRECQ DNA helicases (Sgs1/BLM) and Topoisomerase 3a (TOP3a), have essential functions in managing recombinationintermediates. However, many other factors involved in this process remain to be defined. Here, we report the isolation ofmeiotic chromosome association 1 (MEICA1), a novel protein participating in meiotic recombination in rice (Oryza sativa).Loss of MEICA1 leads to nonhomologous chromosome association, the formation of massive chromosome bridges, andfragmentation. MEICA1 interacts with MSH7, suggesting its role in preventing nonallelic recombination. In addition, MEICA1has an anticrossover activity revealed by suppressing the defects of crossover formation in msh5 meica1 compared with thatin msh5, showing the similar function with its interacted protein TOP3a. Thus, our data establish two pivotal roles for MEICA1in meiosis: preventing aberrant meiotic recombination and regulating crossover formation.

INTRODUCTION

Meiosis is a specialized form of cell division producing haploidgerm cells from diploid progenitors and is essential for sexualreproduction in eukaryotes. During meiosis, crossovers (COs)formed between homologous chromosome pairs and ensureaccurate chromosome segregation. Homologous recombination(HR) is key to the generation of COs.Meiotic recombination startswith the introduction of programmed double-strand breaks(DSBs) mediated by SPO11, the ortholog of subunit A of an ar-chaealDNA topoisomerase (TopoVIA) (Keeneyet al., 1997;Robertet al., 2016). The DSB ends are subsequently resected to yieldextended 39-single-stranded DNA overhangs that are stabilizedby replication protein A (RPA) (Cao et al., 1990; Sugiyama et al.,1997; Mimitou and Symington, 2009). Next, RAD51 and DMC1,along with the accessory factors, displace RPA to promote ho-mology search, resulting in strandexchange and formation of jointmolecule (JM) intermediates (Shinohara et al., 1992; Cloud et al.,2012; Su et al., 2017). Ultimately, these events give rise to eitherCOs or noncrossovers (Börner et al., 2004; Osman et al., 2011).

During meiosis, HR normally occurs between allelic DNAsegments on homologous chromosomes. HR may also occurbetween nonallelic DNA sequences that share high sequencesimilarity (Stankiewicz and Lupski, 2002; Sasaki et al., 2010). Thisnonallelic HR (NAHR) is referred to as ectopic recombination.Although NAHR is less efficient than HR, they are likely to share

common mechanisms (Nag and Petes, 1990; Allers and Lichten,2001). Prevention of meiotic NAHR is critical to maintaining ge-nome stability during gametogenesis in eukaryotes. Becauseeukaryotic genomes contain numerous repetitive sequences,meiotic DSB repair through NAHR will lead to genomic rearrange-ment, resulting in genomic disorders in the germ line (Gu et al., 2008;Kim et al., 2016). Multiple strategies have evolved to prevent NAHRduring meiosis. Suppressing DSB formation within or near DNArepeats is themost straightforwardway to obviate NAHR (Kim et al.,2016). In Saccharomyces cerevisiae, the histone deacetylase Sir2serves as a key regulator of Spo11-dependent DSB formation(Mieczkowski et al., 2007). In mammals, PRDF1-RIZ homologydomain 9 (PRDM9) is a key determinant of DSB hotspot sites duringmeiosis (Baudat et al., 2010). Thus, it is likely that in meiotic cellsNAHR is inhibited through the regulation of DSB formation factorssuch as Sir2 and PRDM9. To avoid deleterious genome re-arrangements caused by NAHR in repetitive DNA regions (Parketet al., 1995), organisms also have mechanisms that constrain thechoice of HR template. One such mechanism is homologouspairing,whichhasbeenshown to restrictNAHR in yeast (Goldmanand Lichten, 2000). Additionally, the outcomes of NAHR eventscan be channeled into noncrossovers through mismatch repair(MMR) proteins and/or several helicases (Kim et al., 2016).MMR is closely linked to the success of meiotic recombination

(Kolas and Cohen, 2004). First, MMR proteins (including MSH4,MSH5, MLH1, and MLH3) promote CO formation during meiosis(Manhart and Alani, 2016). In addition, MMR is required for re-pairingmismatched nucleotides in the heteroduplex DNA (Bishopet al., 1987; Alani et al., 1994). This is necessary because meioticDSBs are preferentially repaired using homologous chromo-somes as templates, which contain nonidentical sequences.MMR aborts the strand exchange if excessive mismatches aredetected in the heteroduplex DNA (Datta et al., 1997; Spies andFishel, 2015). This process is known as heteroduplex rejection.

1 These authors contributed equally to this work.2 Address correspondence to zkcheng@genetics.ac.cn.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Zhukuan Cheng(zkcheng@genetics.ac.cn).OPENArticles can be viewed without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.17.00241

The Plant Cell, Vol. 29: 1697–1708, July 2017, www.plantcell.org ã 2017 ASPB.

Nonallelic DNA sequences are more divergent compared withallelic sequences, thus making it possible for MMR proteins tosuppress recombination between nonallelic sequences throughheteroduplex rejection (Datta et al., 1996; Li et al., 2006; Thamet al., 2016). In eukaryotes, two heterodimer complexes (MSH2-MSH6 and MSH2-MSH3) are responsible for the recognition ofmismatches (Alani, 1996; Gradia et al., 1997). MSH7, an MSH6-like protein unique to plants, forms a heterodimer with MSH2 inArabidopsis thaliana (Adé et al., 1999; Culligan and Hays, 2000).AtMSH2-AtMSH7 has an affinity for (T/G) mispairs in vitro, sug-gesting that this heterodimer hasa role in recognizingmismatches(Culligan and Hays, 2000).

The antirecombinational activities of several helicases are sug-gested to be involved in heteroduplex rejection (Spies and Fishel,2015).S.cerevisiaeSgs1 is aRecQhelicase,which is thecounterpartof BLM in human andRECQ4 in plants. Sgs1 can antagonize single-strand annealing by unwinding heteroduplex DNA (Sugawara et al.,2004). In eukaryotes, the RecQ helicase can interact with a type IAtopoisomerase (TOP3a) and an OB-fold protein (RMI1) to forma conserved complex (Gangloff et al., 1994; Chang et al., 2005). Thiscomplex, Sgs1-Top3-Rmi1 (STR) in yeast and BLM-TOP3a-RMI1/2in human, has the best-known anti-CO activity, catalyzing the dis-assembly of early D-loop intermediates without CO formation (DeMuyt et al., 2012; Zakharyevich et al., 2012; Séguéla-Arnaud et al.,2015). In addition, the STR members are required for processingabnormal JMs (Ohet al., 2007, 2008; JessopandLichten, 2008;Kauret al., 2015; Tang et al., 2015). Disruption of STR members in Ara-bidopsis leads todefects inmeiotic chromosomesegregationaswellasan increase inCOformation(Chelyshevaetal.,2008;Hartungetal.,2008; Higgins et al., 2011; Séguéla-Arnaud et al., 2015).

Here, we report a novel protein, MEICA1 (meiotic chromosomeassociation1), that isessential fornormalmeiotic recombination inrice (Oryza sativa). MEICA1 contains a conserved DUF4487 do-main. Loss of MEICA1 results in nonhomologous chromosomeassociations and chromosome fragmentations during meiosis.These chromosome defects are generated bymeiotic DSB repair,which depends on strand exchange directed by DMC1. Usingyeast two-hybrid assays, we demonstrate that MEICA1 interactswithMSH7andTOP3a. In addition, genetic analysis suggests thatMEICA1 may act as an anti-CO factor in the regulation of meioticrecombination. Thus, our data establish two pivotal roles forMEICA1 in meiosis: preventing aberrant meiotic recombinationand decreasing CO formation.

RESULTS

MEICA1 Is a Novel Protein Conserved in Higher Eukaryotes

To identify proteins involved in meiosis, we initiated a geneticscreen for sterile phenotypes, from which a mutant was identifiedand designatedmeica1. Themutant line produced progenies witha segregation ratio of 3:1 (fertile:sterile), indicating a single re-cessive mutation. We adopted a map-based cloning approachto identify the meica1 mutation. The target gene was mappedto an interval between markers Q2 and Q3 on chromosome3 (Supplemental Figure 1A). We sequenced PCR fragmentsamplified from the genomic DNA corresponding to this region in

the meica1 mutant and identified a mutation in the gene LOC_Os03g05040 (Figure 1A).To verify that LOC_Os03g05040 corresponds to the mutant

locus, a complementation test was conducted. Transformation ofa plasmid that contained the full-length genomic sequence ofLOC_Os03g05040 into meica1 mutant plants succeeded in res-cuing the fertility (SupplementalFigures1B to1Dand1Fto1H).Wealso generated MEICA1-RNA interference plants, most of whichshowed a phenotype similar tomeica1 (Supplemental Figures 1Eand 1I). These results indicate that the meiotic defects ofmeica1plants are indeed caused by mutation in the LOC_Os03g05040gene. Therefore, we defined the LOC_Os03g05040 gene asMEICA1 and the corresponding protein as MEICA1.According to the Rice Genome Annotation Project database

(http://rice.plantbiology.msu.edu/cgi-bin/gbrowse/rice), MEICA1 isaconservedgenewithunknownfunction.Weobtainedthefull-lengthcDNA ofMEICA1 by performing RT-PCR and rapid amplification ofcDNA ends (RACE).MEICA1 has seven exons and six introns, witha 3138-bp open reading frame (Figure 1A). Using 1045 amino acidsdeduced from the open reading frame sequence as a query, weperformed a search in Pfam (http://pfam.xfam.org/) and detectedaconservedDUF4487domain (Figure1C).Additionally,weexecutedPSI-BLAST (position-specific iterative BLAST) searches from theMax Planck Institute toolkit (http://toolkit.tuebingen.mpg.de/) toidentify orthologs of MEICA1 in other species. Multiple sequencealignment revealed that these proteins are highly conserved withinthe DUF4487 domain, especially the WCF sequence motif (Figure1D;Supplemental Figure2). Toanalyze thephylogenetic relationshipof the proteins containing the DUF4487 domain, a phylogenetictree was constructed with the full-length amino acid sequences ofthese proteins (Figure 1B). These results further confirmed that theMEICA1 protein is conserved throughout the eukaryotes.

MEICA1 Is Required for Accurate MeioticChromosome Segregation

The meica1 mutant exhibits normal vegetative growth but is al-most sterile, with only 0.95% seed-setting rate (SupplementalFigure 1C).We therefore examined the viability of mature pollen inmeica1 by staining with Alexander red and found that most pollengrains of meica1 are inviable (Supplemental Figure 1G).To investigate the reason for sterility in themeica1mutants, we

investigated male meiosis by staining pollen mother cells (PMCs)of both the wild type and meica1 with 49,6-diamidino-2-phenyl-indole (DAPI). In the wild type, meiotic chromosomes condensedto thin threadsat leptotene.The thin threads thenbegan topair andsynapse at zygotene. At pachytene, the paired chromosomescompleted synapsis and presented as thick threads (Figure 2A).After further condensation, 12 bivalents became clearly visible atdiakinesis and aligned on the equatorial plate at metaphase I(Figures 2B and 2C). During anaphase I, homologous chromo-somes separate from each other by the pulling of microtubules(Figure2D). Inmeiosis II, sister chromatidsseparated,giving rise tothe tetrad (Figures 2E and 2F).In the early stages of meiosis, the chromosomal behavior of

meica1 could not be distinguished from the wild type. Themeioticdefects of meica1 became evident at pachytene, when aber-rant associations could be detected among nonhomologous

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chromosomes as well as within the same chromosome (Figures2G,2M,and2N).Among thepachytenePMCssurveyed inmeica1,the average number of the distinguishable chromosome aber-rant association is 5.6 (n = 95, range 1–13; Supplemental Figure3). At diakinesis, unlike the 12 intact condensing bivalentsobserved in the wild type, associations were observed amongseveral chromosomes in meica1 (Figure 2H). These abnormalchromosome interactions becamemore obvious atmetaphase I(Figure 2I). At this stage, the chromosomes appeared as severalentangled masses, even bivalent-like structures still existed.During anaphase I, extensive chromosome bridges and frag-ments were observed in the plate (Figure 2J). The number ofbridges per cell was range from 1 to 11 (5.8 6 2.5, mean 6 SD;n = 25). After meiosis II, tetrads with unequal chromosomedistribution and micronuclei were formed (Figures 2K and 2L).Similar defects were observed during male meiosis inMEICA1-RNAi plants (Supplemental Figure 4). These data showedthat MEICA1 is essential for normal chromosomal segregationduring meiosis.

MEICA1 Is Not Necessary for HomologousBivalent Formation

Based on DAPI staining, synapsis of homologous chromosomeswas roughly normal in meica1. To confirm that MEICA1 is notinvolved in synapsis, we performed immunolocalization studieswith anti-ZEP1 (a transverse filament protein of synaptonemalcomplexes) andanti-REC8 (ameiotic-specificcohesionprotein) in

meica1 PMCs. ZEP1 displayed similar linear localization patternson pachytene chromosomes of meica1 and the wild type (Figure3A), suggesting that synapsis was complete in meica1.To further verify that the bivalent-like structures in meica1 are

pairs of homologous chromosomes, we performed fluorescentin situ hybridization (FISH) analyses on chromosome spreads ofPMCs using a 5S rDNA-specific probe, which clearly identifieschromosome11of rice. Inmeica1, twobright side-by-side signalsrepresenting the paired 5S rDNA loci were observed at thepachytene stage (Figure 3B). Two individual 5S rDNA signalsdistributed on the bivalent-like structure at metaphase I and mi-grated to the opposite poles at anaphase I (Figure 3B). These 5SrDNA distribution patterns were similar to those observed in thewild type, indicating that homologous paring and bivalent for-mation are completed in meica1.

MEICA1 Functions in a DMC1-Dependent Pathway

The aberrant chromosome interactions prevalent in meica1may arise through unusual meiotic recombination or abnormalmitoticDNAmetabolismevents.Todeterminewhether themeica1phenotype is dependent uponmeiotic DSB formation, the spo11-2meica1doublemutantsweregeneratedandanalyzed.Thespo11-2mutantproducesnovisiblemeioticDSBsandexhibits24univalentsthat are randomly distributed during metaphase I. The spo11-2meica1 double mutant has the same phenotype seen in spo11-2(Figure 4A). Therefore, chromosome associations in meica1 aredependent on meiotic DSB formation.

Figure 1. Characterization of MEICA1.

(A) Gene structure and mutation of MEICA1. Coding regions are shown as boxes, and untranslated regions are shown as lines.(B) Phylogenetic tree derived from the full-length amino acid sequences of proteins containing the DUF4487 domain. The scale bar represents 0.2substitutions per site.(C) Schematic representation of MEICA1.(D) Multiple sequence alignment of DUF4487 proteins. Only part of alignment is displayed here. The WCF motif is underlined. Os, Oryza sativa; At,Arabidopsis thaliana; Hs, Homo sapiens.

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Duringmeiosis, repair ofDSBsusing theclassical nonhomologousend joining (C-NHEJ) is a cause of ectopic associations for chro-mosomes (Lemmens et al., 2013). KU70 is essential for the C-NHEJpathway(DerianoandRoth,2013).ToinvestigatetheroleofC-NHEJinthe formation of chromosome associations inmeica1, we generatedthe ku70 meica1 double mutant. The meiotic chromosome behaviorof theku70meica1doublemutant issimilar to thatof themeica1singlemutant (Figure 4B), suggesting that aberrant associations in meica1are formed independent of KU70. Thus, the ectopic chromosomeinteractions in meica1 do not result from the C-NHEJ pathway, andthey are very likely to arise from another repair pathway.

Given previous evidence that meica1 defects are due to thefailure in meiotic DSB repair, we next analyzed the relationshipbetweenMEICA1 and DMC1, which is an essential component ofthe meiotic recombination machinery. The phenotype of dmc1meica1 double mutant resembles that of the dmc1 single mutant,displaying nearly 24 univalents at metaphase I (Figure 4A), in-dicating that MEICA1 acts downstream of DMC1. If so, MEICA1should not be required for localization of DMC1 or other factorsupstream of DMC1, such as COM1. We performed immunoloc-alization to detect the nuclear distribution of DMC1 and COM1.Both DMC1 and COM1 foci were similar in the wild type and themeica1mutant at zygotene (Figure4C), suggesting thatMEICA1 isnot necessary for the localization of these factors. These datarevealed that MEICA1 participates in meiotic recombination afterstrand invasion.

MEICA1 Is a Chromatin-Associated Protein with a DynamicLocalization Pattern

To define the spatial and temporal distribution of MEICA1 duringmeiosis in rice, we conducted dual immunolocalization experi-ments using polyclonal antibodies against the REC8 andMEICA1proteins. MEICA1 signals were absent at leptotene and first be-came detectable on zygotene chromosomes (Figure 5A). Atpachytene,MEICA1 could be detected as linear-like signals alongthe entire chromosome axes. Unlike REC8, MEICA1 signals werenarrow and discontinuous. MEICA1 signals disappeared at dip-lotene and the stages thereafter. To further reveal the localizationpattern of MEICA1, we conducted immunolocalization studiesusing structured illumination microscope. MEICA1 signals werelocated between the REC8 signals (Figure 5B), indicating thatMEICA1 localizes to the chromosome cores of PMCs. Thedistribution of MEICA1 on meiotic chromosomes implies thatMEICA1 may act on DNA structures formed in the process ofmeiotic recombination. To test the specificity of anti-MEICA1,immunolocalization studies were performed on PMCs ofmeica1,with noMEICA1 signal detected (Figure 5C).We further tested thelocalization of MEICA1 in several meiotic mutants, includingmtopVIB,dmc1, and zep1.MTOPVIB is essential formeioticDSBsformation in rice (Xueetal., 2016).MEICA1signalwasnotdetectedin mtopVIB (Figure 5D), indicating that MEICA1 functions in re-sponse to meiotic DSBs. DMC1 is required for strand-exchange

Figure 2. Loss of MEICA1 Results in Aberrant Chromosome Associations and Bridges during Meiosis.

(A) to (F) Meiotic chromosomes in the wild type.(G) to (N) Meiotic chromosomes in the meica1 mutant.(A) and (G)Pachytene, (B) and (H)Diakinesis, (C) and (I)Metaphase I, (D) and (J)Anaphase I, (E) and (K)Anaphase II, (F) and (L)Tetrad, and (M) and (N) Latepachytene. Aberrant associations are indicated in orange and blue frames. Bars = 5 mm.

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process of meiotic recombination (Cloud et al., 2012). FewMEICA1signals in dmc1 suggests that MEICA1 functions after DMC1 inrecombination (Figure 5D). No MEICA1 signal was detected inzep1 (Figure 5D), implying an essential role for ZEP1 in the lo-calization ofMEICA1. All these data demonstrated thatMEICA1 isa chromatin-associated protein, which is located to chromosomecores dependently of meiotic recombination as well as synapto-nemal complex assembly.

MEICA1 Interacts with MSH7 and TOP3a

To identify potential MEICA1-interacting proteins, we performedayeast two-hybrid screenusing the full-lengthMEICA1as thebait.From this screen, we identifiedMSH7 and TOP3a. To verify theseinteractions, the full-length coding sequences of MSH7 andTOP3awerecloned intopGBKT7, and the full-lengthMEICA1wascloned into pGADT7. The yeast cells were then cotransformedwith MEICA1-AD and MSH7-BD or TOP3a-BD. TransformantswithMEICA1-AD andMSH7-BDor TOP3a-BDgrewon bothDDOand QDO/X/A media, suggesting that MEICA1 interacts withMSH7andTOP3a (Figure6A).Wealsoconstructedthesplit-luciferasevectors containing the full-lengthMEICA1,MSH7,andTOP3a codingsequences and performed split-luciferase complementation assays,

which confirmed that both pairs of proteinswere able to interact inNicotiana benthamiana (Figure 6C). In yeast and human cells,Sgs1/BLM, TOP3a, and Rmi1/BLAP75 were demonstrated toform a conserved complex. We therefore investigated whetherMEICA1 interacted with RECQ4A and RMI1 using yeast two-hybrid assay. However, we detected no interaction between full-lengthMEICA1andRECQ4A,andno interactionbetweenMEICA1and RMI1 (Supplemental Figure 5).MSH7 was reported to be required for suppressing homoeolo-

gous recombination in plants (Dong et al., 2002; Tam et al., 2011).The fact thatMEICA1 interactswithMSH7suggests that they sharesimilar functions. Therefore, it is very likely thatMEICA1 localizes tomeiotic chromosomes to monitor or remove the abnormal het-eroduplex intermediates between nonallelic DNA sequences thatare responsible for nonhomologous chromosome associations.

Mutation of MEICA1 Increases Bivalent Formation inzmm Mutants

Previous studies revealed an important role for TOP3a in regu-lating theoutcomesofHR.ZMMs (Zip1-4,Mer3,Msh4, andMsh5)are proteins necessary for CO formation duringmeiosis. The zmmmutants display a great deal of univalents at metaphase I. InArabidopsis, a specific TOP3a mutant allele restores bivalentformation in zmmmutants, suggesting that TOP3ahasananti-COactivity in plants (Séguéla-Arnaud et al., 2015).Moreover, the anti-CO activity of TOP3a has been proven to be related to itsC-terminal domains (Séguéla-Arnaud et al., 2017). In order todissect the nature of the interaction betweenMEICA1andTOP3a,we generated a series of constructs with different domains ofMEICA1 and TOP3a (Figure 6B). We found that the interactionbetween themwasmediated by theC terminus (amino acids 550–928) of TOP3a (Figure 6A). The coding sequence of MEICA1 wasalso divided into three parts, including the N terminus (1–280), theDUF4487 domain (270–875), and the C terminus (865–1045)(Figure6B). The results showthat the interactionbetweenMEICA1and TOP3awasmediated by both the N terminus (1–280) and theC terminus (865–1045) of MEICA1 (Figure 6A).The interaction between MEICA1 and TOP3a prompted us to

analyze the meiotic chromosome behavior in the msh5 meica1doublemutant.MSH5 is aZMMprotein and is required for bivalentformation in rice.We found that thechromosomebridgesbetweenchromosomes inmeica1 arise independently from MSH5 (Figure7A;Supplemental Figure 6A).However,most of thePMCs inmsh5meica1 exhibited more bivalent-like structures than the msh5single mutant (Figure 7A). To determine the nature of thesebivalent-like structures, we performed FISH assays using the 5SrDNA probe and found out that 5S rDNA signals were located onthe bivalent-like structure in most of msh5 meica1 PMCs (Figure7A), indicating that the bivalent-like structures inmsh5meica1 arecomposed of homologous chromosome pairs. Inmsh5 mutants,only 6.5% (2 out of 31) PMCs exhibited coupled 5S rDNA signalson bivalent. While in msh5 meica1, 5S rDNA signals on bivalentweredetected inmost of PMCs (79.1%,53out of 67). Thus, lossofMEICA1 was able to increase bivalent formation in the msh5mutant background. We also generated the hei10 meica1 doublemutant and found similar results from cytological analysis(Supplemental Figure 6B). In addition, we analyzed the extent of

Figure 3. MEICA1 Is Dispensable for Synapsis and Bivalent Formation.

(A) Immunolocalization of ZEP1 (green) in thewild type andmeica1mutant.(B)FISH analysis using 5S rDNAprobes (green) in thewild type andmeica1mutant. Chromosomes stained with DAPI (blue). Bars = 5 mm.

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chromosome entanglements inmeica1,msh5 meica1, and hei10meica1 by quantifying the metaphase I aberrant aggregatedstructures (Supplemental Figure 6C). Statistically, the averagenumber of aggregated chromosomes in meica1 was 2.1 6 1.0(mean6 SD; n=74) and that inmsh5meica1was2.461.0 (n=70).No significant reduction of chromosome entanglements wasdetected in themsh5meica1doublemutant (P = 0.0671, unpairedt test). The extent of chromosome entanglements in hei10meica1(1.7 6 0.7; n = 43) was also similar to that in meica1 (P = 0.0551,unpaired t test). These data indicate that MEICA1 acts to preventabnormal chromosome interactions caused independently of theZMM pathway. Meanwhile, similar to TOP3a, MEICA1 may haveanti-CO activity during early meiosis.

meica1 Exhibits an Elevated Number of HEI10 Foci

In order to verify the frequency of CO formation inmeica1, we nextdetermined the localization of HEI10 bright foci at pachytene,which has been regarded as a good marker of the first class CO(CO I) (Chelysheva et al., 2012; Wang et al., 2012). The averagenumber of HEI10 foci in the wild type is 24.4 (n = 30, range 21–29),

while the average number inmeica1 is 29.7 (n = 29, range 20–38)(Figure 7B). The elevatednumberofHEI10 foci inmeica1 suggeststhat mutation ofMEICA1 leads to an increased frequency of CO.Thus, MEICA1 may possess the ability to abolish CO I formation.This hypothesis is further supported by the observation that thenumber of other ZMM foci, including MER3 and ZIP4, were in-creasedbymutation ofMEICA1 (Figure 7B). Our data showed thatthe localization of ZMM proteins, which are required for latemeiotic recombination events, was elevated by loss of MEICA1.Furthermore, the localization of early recombination factors, suchas COM1 and DMC1, were also investigated. The number ofCOM1 foci inmeica1 (2986 40, mean6 SD; n = 16) was similar tothat of the wild type (2896 38; n = 22; P = 0.4819, unpaired t test).There was also no significant difference (P = 0.1504, unpairedt test) in the number ofDMC1 foci betweenwild type (268633; n=18) andmeica1 (2886 45; n = 15). Accordingly, the localization ofCOM1 and DMC1 were not affected in meica1 mutants (Figure4C). It is very likely that MEICA1 functions to monitor or regulatethe process of heteroduplex intermediate formation mediated byDMC1. Therefore, mutation of MEICA1 leads to disorders in thisprocess, resulting in an elevated frequency of COs.

Figure 4. The Function of MEICA1 Is Dependent on Meiotic Recombination.

(A) Chromosome behavior of spo11-2, spo11-2 meica1, dmc1, and dmc1 meica1 at metaphase I meiocytes.(B) Chromosome associations and bridges existing in ku70 meica1 meiocytes.(C) Immunolocalization of COM1 and DMC1 in meica1 zygotene meiocytes. The numbers of COM1 and DMC1 foci are quantified. ns, no statisticaldifference; P > 0.05. Bars = 5 mm.

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DISSUSSION

Role of MEICA1 during Meiotic Recombination

To identify factors that function inmeiosis, we performed a screenfor rice mutants with meiotic defects. A sterile mutant line withaberrant chromosome interactions was obtained, and the cor-responding gene, MEICA1, was identified through map-basedcloning. The most remarkable defects observed in meica1 areectopic chromosome associations in metaphase I, extensivechromosome bridges, and fragmentation in anaphase I. First, werevealed that ectopic interactions in the absence of MEICA1depend on meiotic-specific DSB formation. Second, we provedthat MEICA1 is not necessary for homologous pairing and syn-apsis. Loss of MEICA1 does not disturb the localization of earlyrecombination factorsand the formationofhomologousbivalents,suggesting its dispensable role in the early steps of meiotic re-combination. Moreover, we demonstrated that the chromosome

defects in meica1 arise from DMC1-mediated recombinationevents. Third, the numbers of ZMM foci increased inmeica1, andmutation of MEICA1 restored CO formation in the zmm mutants,indicating a role for MEICA1 in managing the outcome of re-combination. Together, these results clearly demonstrate thatMEICA1 is required for the late stepsofmeiotic recombination andsuggest that MEICA1 may function to process recombinationintermediates.

MEICA1 May Act to Suppress NAHR

We detected associations between non-homologous chromo-somes in the meica1 mutant. There are several explanations forectopic associations during meiosis: C-NHEJ, alternative non-homologous end joining, single-strand annealing, and NAHR. Toverify the mechanism that leads to the formation of aberrant as-sociations, we conducted genetic analysis. KU70 is an essentialfactor of theC-NHEJpathway. The similar phenotypesof the ku70

Figure 5. MEICA1 Is a Meiotic Chromatin-Associated Protein.

(A) Dual immunolocalization of REC8 (red) and MEICA1 (green) on chromosome spread preparations from wild type.(B) A superresolution image is provided for better observation of MEICA1 staining. MEICA1 foci are located between the REC8 signals at pachytene.(C) Dual immunolocalization of REC8 and MEICA1 in meica1.(D) Immunolocalization of MEICA1 is disturbed in mtopVIB, dmc1, and zep1 mutants. Bars = 5 mm.

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meica1 doublemutant and themeica1 singlemutant suggest thataberrant associations inmeica1 result from repair pathways otherthanC-NHEJ.Thesimilarmeioticchromosomebehaviorsofdmc1anddmc1meica1 indicate thataberrant interactionsaredependent

on the function of DMC1. These genetic analyses demonstrate thattheectopic chromosomeassociations inmeica1 result fromerrors inDMC1-mediated homologous recombination, but not from theKU70-dependent C-NHEJ pathway.

Figure 6. MEICA1 Interacts with MSH7 and TOP3a.

(A)MEICA1 interactswithMSH7andTOP3a in yeast two-hybrid assays. SD/DDO (SD-Leu-Trp)was used to test the cotransformation efficiency. SD/QDO/X/A (SD-Leu-Trp-His-Ade+X-a-Gal+Aureobasidin A) was used as the selective medium to verify the interactions. AD, prey vector; BD, bait vector.(B) Schematic diagram of full-length and truncated proteins of MEICA1 and TOP3a used in this study.(C) MEICA1 interacts with MSH7 and TOP3a in split-luciferase complementation assays. The range of luminescence intensities are shown by pseu-docolored bars.

Figure 7. Relationship of MEICA1 and ZMM Proteins.

(A) Disruption of MEICA1 restores bivalent formation inmsh5 background. The bottom images show FISH analysis using 5S rDNA probes (green) inmsh5and msh5 meica1, respectively.(B) The numbers of ZMM foci are elevated in meica1 mutants. ****P < 0.0001; *P = 0.0257. Two-tailed Student’ s t tests. Bars = 5 mm.

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TheMMR system is closely linkedwith recombination. In yeast,MMR proteins play an important role in preventing recombinationbetween divergent sequences (Selva et al., 1995). The MMRsystem is also required for meiotic recombination (Alani et al.,1994; de Wind et al., 1995; Datta et al., 1996; Marsischky et al.,1999). These findings suggest that the suppression of homoe-ologous recombination mediated by MMR is responsible for in-hibiting nonallelic recombination during meiosis. As the MMRmembers, MSH2 to MSH6 heterodimers are responsible for therecognition of mismatched DNA base pairs (Alani, 1996). MSH7 isa plant-specific MutS protein sharing high similarity with MSH6(Adé et al., 1999). The Arabidopsis MSH2-MSH7 complex wasshown to have the capacity to recognize specific base-basemismatches (CulliganandHays, 2000;Wuetal., 2003;GómezandSpampinato, 2013). During meiosis, MSH7 is suggested to beassociated with homoeologous recombination (Dong et al., 2002;Tam et al., 2011). Loss of MEICA1 leads to nonhomologouschromosome associations, which have been proven to be ab-normal outcomesofmeiotic recombination. Although the functionof rice MSH7 is still elusive, it is very likely to have a similar an-tihomoeologous recombination activity just like its orthologs inother plants. The direct interaction between MEICA1 and MSH7implies that they may act in the same biological process. Hence,the aberrant associations in meica1 may be derived from ho-moeologous recombination between nonallelic sequences.

Relationship between MEICA1 and the STR Members

During recombination, the JM intermediates need to be carefullyprocessed to ensure accurate segregation of chromosomes. TheSTR members are implicated in processing meiotic JM inter-mediatesasacomplexorsubcomplex (Ohetal., 2007;JessopandLichten, 2008; Fasching et al., 2015; Kaur et al., 2015; Tang et al.,2015). Loss of Sgs1 leads to an increase in the number of COs andthe accumulation of aberrant JMs inmeiotic cells of yeast (Jessopand Lichten, 2008; Oh et al., 2008). The anti-CO activity of Sgs1relies on the formation of the STR complex (Fasching et al., 2015;Kaur et al., 2015; Tang et al., 2015). However, Top3 and Rmi1could form a subcomplex to resolve late meiotic recombinationintermediates independently of Sgs1 (Kaur et al., 2015; Tang et al.,2015). In Arabidopsis, the members of the STR complex are alsoessential for limiting CO frequency during meiosis (Séguéla-Arnaud et al., 2015, 2017). Moreover, TOP3a and RMI1 are in-volved in the resolution of meiotic recombination intermediates(Chelysheva et al., 2008; Hartung et al., 2008).

The most impressive defects in Arabidopsis top3a and rmi1mutants are entangled chromosomes, the formation of chromo-some bridges, and fragmentation during meiosis, indicating theaccumulation of recombination intermediates in the mutants(Chelysheva et al., 2008; Hartung et al., 2008). The meioticchromosome behaviors ofmeica1 are remarkably similar to thoseof Arabidopsis top3a and rmi1mutants, suggesting that MEICA1has a function in managing JM intermediates. To determinewhether MEICA1 has anti-CO activity similar to TOP3a, weconducted genetic analysis and found that bivalent formation inmsh5 can be partly restored by mutation of MEICA1. Thus, it istempting to speculate thatMEICA1may act to limit CO formation.The interaction betweenMEICA1 and TOP3a further supports the

hypothesis that MEICA1 acts a partner of TOP3a in processingmeiotic JM intermediates.To conclude, we have identified MEICA1 as a novel gene in-

volved inmeiotic recombination. We have provided evidence thatMEICA1 is essential for normal chromosome segregation. Moreimportantly, our data indicate a pivotal role forMEICA1 in regulatingof recombination intermediates during meiosis. First, MEICA1 isrequired for the suppression of NAHR. Second, MEICA1 may berequired to the resolution of JM intermediates.

METHODS

Plant Materials and Growth Conditions

MEICA1 was isolated from a sterile mutant induced by 60Co-g-ray irra-diation of rice (Oryza sativa). Other mutant alleles used in this study arespo11-2,mtopVIB-1,msh5-2, hei10-2, ku70-1, and dmc1a dmc1b (Honget al., 2010;Wang et al., 2012; Luo et al., 2013;Wang et al., 2016; Xue et al.,2016).Nipponbarewasusedas thewild type.All of theplantsweregrown inpaddy fields during the growing season.

Map-Based Cloning of MEICA1

In a screen for rice meiotic mutants, we identified a sterile mutant line fromYandao 8, a japonica rice variety. A map-based cloning approach wasadopted to isolate the target gene. To produce the mapping populations,we crossed meica1+ meica12 mutant plants with the indica rice varietyZhongxian 3037. Using 220 sterile plants identified in the F2 segregatingpopulation, the locus was first mapped to the long arm of chromosome 3.We then performed fine gene mapping with an additional 643 F3 segre-gates to pinpoint the target gene within a 50-kb region. All of the makersused formappingwere InDel (insertion-deletion)markers developedbasedon genomic sequence differences between indica variety 9311 and japonicavariety Nipponbare according to the data published at http://www.ncbi.nlm.nih.gov. Primer sequences are listed in Supplemental Table 1.

Complementation Test

A 9930-bp genomic DNA fragment containing 3068 bp of promoter regionand the entire MEICA1 gene region was inserted into the binary vectorpCAMBIA1300 to generate the plasmid for the complementation test. Theplasmid was transformed into Agrobacterium tumefaciens EHA105 byelectroporation and then into meica1+/2 embryonic calli.

Full-Length cDNA Cloning of MEICA1

Total RNA extraction was conducted using the TRIzol reagent (Invitrogen)from young rice panicles. Reverse transcription was performed with theSuperscript III RNaseH reverse transcriptase (Invitrogen) using primerAdaptor-T (18). For 59RACE and 39RACE, the SMARTer RACE cDNAamplification kit (Clontech) was used according to the manufacturer’sinstructions. Gene-specific primers RACE5-1 and RACE5-2, combinedwith the universal primers provided in the kit were used to perform 59RACEPCR. For 39RACE PCR, gene-specific primers RACE3-1 and RACE3-2were used along with the universal primers. The products of 59RACE-PCRand 39RACE-PCR were cloned into the PMD19-T vector (TaKaRa) andsequenced. The full-length coding sequence (CDS) of MEICA1 has beensubmitted to GenBank (https://www.ncbi.nlm.nih.gov/genbank/), and theaccession number is MF32161. To improve the expression of MEICA1 inyeast and Escherichia coli, we conducted codon optimization and genesynthesis at SangonBiotech. The optimizedCDShas also been submittedto the GenBank under accession number is MF32162. Primer sequencescan be found in the Supplemental Table 2.

MEICA1 in Rice Meiosis 1705

RNA Interference Experiment

A 243-bp region of the MEICA1 coding sequence was selected to makeRNAi constructs. The fragments for cloning were amplified using theprimers MEICA1-RNAi-F and MEICA1-RNAi-R (see Supplemental Table 3for the primer sequences) from wild-type Nipponbare panicle cDNA. ThePCR product was cloned into the BamHI-SalI and BglII-XhoI sites of thepUCCRNAi vector in an inverted repeat orientation. The stem-loop frag-ment was subcloned into the pCAMBIA 2300 vector, downstream of theActinpromoter. Theconstructwas introduced intoAgrobacteriumEHA105and transformed into embryonic calli of Yandao 8.

Yeast Two-Hybrid Assays

For yeast two-hybrid screening, the full-lengthCDSofMEICA1wasclonedinto the pGBKT7 vector using primers MEICA1-BD-F and MEICA1-BD-R.RNA from anthers of young rice panicles was used to construct the cDNAlibrary. Library construction and screening were performed with Match-maker library construction and screening kits (Clontech) according to themanufacturer’s instructions. The full-lengthCDSsof TOP3a,MSH7, RMI1,and RECQ4A were amplified using primers TOP3a-AD-F, TOP3a-AD-R,TOP3a-BD-F, TOP3a-BD-R, MSH73-AD-F, MSH7-AD-R, MSH7-BD-F,MSH7-BD-R, RMI1-AD-F, RMI1-AD-R, RMI1-BD-F, RMI1-BD-R, RECQ4A-AD-F, and RECQ4A-AD-R and cloned into the pGADT7 or PGBKT7 vectors.The bait and prey vectors were cotransformed into yeast Y2H Gold strainusing the Matchmaker Gold yeast two-hybrid system (Clontech). Co-transformantswere first cultured inDDO (SD/-Leu/-Trp)mediumat 30°C. Theactivation ability was assayed on QDO (SD/-Ade/-His/-Leu/-Trp) mediumcontaining with X-a-Gal and aureobasidin A. All primer pairs are listed inSupplemental Table 3.

Meiotic Chromosome Preparation

Young panicles of both wild-type and mutant lines were harvested andfixed in Carnoy’s solution (ethanol:glacial acetic, 3:1). Anthers at themeiotic stages were squashed in an acetocarmine solution on glass slidesand washed with 45% acetic acid. The slides were then frozen in liquidnitrogen. After removing the cover slips, the slides were dehydratedthrough an ethanol gradient (70, 90, and 100%). Chromosomes spreadswere counterstained with DAPI in an antifade solution (Vector Laborato-ries). Cytological analysiswasperformed under theZEISSA2 fluorescencemicroscope and images were captured with a micro CCD camera. Thesuperresolution images were captured under the DeltaVision microscope(GE Healthcare; OMXTMV4) and processed with SoftWoRx.

FISH

FISH analysis was conducted as described by Yang et al. (2016). ThepTa794 clone containing the coding sequences for the 5S rRNA genes ofwheat was used to identify rice chromosome 11.

Immunofluorescence

Fresh young panicles were fixed in 4% (w/v) paraformaldehyde for 10 to30min at room temperature. Anthers at the proper stagewere squashed inPBS solution on glass slides and covered with a cover slip. After removingcover slips, slides were dehydrated using an ethanol gradient (70, 90, and100%). Slideswere then incubated in a humid chamber at 37°C for 4 hwithdifferent combinations of antibodies, all diluted 1:500 in TNB buffer (0.1 MTris-HCl, pH 7.5, 0.15 M NaCl, and 0.5% blocking reagent). After threerounds of washing in PBS, Texas Red-conjugated goat anti-rabbit anti-body and fluorescein isothiocyanate-conjugated sheep anti-mouse anti-body (1:1000; SouthernBiotech) were added to the slides. After other threerounds of washing in PBS, chromosomes were counterstained with DAPI.

Antibody Production

Togenerate the antibodies againstMEICA1, a 453-bp fragment ofMEICA1cDNA (coding for 151 amino acids) was amplified from rice panicle cDNAwith primersMEICA1-AB-F andMEICA1-AB-R (sequences are listed in theSupplemental Table 3). This fragment was inserted into the expressionvector pET-30a(+) (Novagen). The His-fusion MEICA1 peptide was ex-pressed in BL21 (DE3) and purified with Ni-NTA agarose (Qiagen) ac-cording to the manufacturer’s instructions. The anti-MEICA1 polyclonalantibodywasobtainedby immunizingamousewith the fusionpeptide. Theantibodies against REC8, COM1, DMC1, ZIP4, MER3, ZEP1, and HEI10were previously generated in our lab.

Split-Luciferase Complementation Assay

The split-luciferase complementation assay was performed as previouslydescribed (Chen et al., 2008). Agrobacterium (GV3101) harboring theempty or recombinant vectors were infiltrated into expanded leaves ofNicotiana benthamiana and incubated in thegrowth room for 72 h. For LUCactivity measurement, 1 mM luciferin was sprayed onto the leaves. Afterincubation in thedark for 5min, a cooledCCD imaging apparatuswasusedto capture the LUC image. The primers used to construct plasmids arelisted in Supplemental Table 3.

Sequence Alignment and Statistical Analysis

The phylogenetic tree was constructed using MEGA version 6.0. Multiplealignmentswere conducted usingMAFFT (https://toolkit.tuebingen.mpg.de/mafft) and processed with ESPrint (http://espript.ibcp.fr/ESPript/ESPript/). Pollenmother cells used for quantitative analysiswere obtainedfrom at least three rice individuals at different times. Statistical analysiswas conducted using GraphPad Prism6.

Accession Numbers

Sequence data used in this article can be found in the NCBI databasesunder the following accession numbers: MEICA1 (full-length CDS), rice,MF32161; MEICA1 (codon-optimized CDS), rice, MF32162. Brachypodiumdistachyon, KQK23695; Zea mays, XP_008650960; Sorghum bicolor,XP_002465870; Aegilops tauschii, EMT10853; Vitis vinifera, XP_010650223;Glycine max, XP_006589330; Arabidopsis thaliana, NP_171959; Populustrichocarpa, XP_002304697; Amborella trichopoda, XP_006854270; Phys-comitrella patens, XP_001766106;Selaginellamoellendorffii, XP_002969503;Homo sapiens, NP_060656; Mus musculus, NP_958752; Danio rerio,XP_005160750; Xenopus tropicalis, NP_001120506; and Gallus gallus,XP_004943262.

Supplemental Data

Supplemental Figure 1. Map-based cloning of MEICA1 and pheno-types of wild-type, meica1 mutant, meica1COM, and MEICA1RNAi

plants.

Supplemental Figure 2. Alignment of full-length DUF4487 proteins.

Supplemental Figure 3. Distribution of the aberrant pachytenechromosome associations in meica1.

Supplemental Figure 4.Meiotic chromosome behaviors ofMEICA1RNAi

plants.

Supplemental Figure 5. Interactions were detected neither betweenfull-length MEICA1 and RMI1, nor between MEICA1 and RECQ4A inyeast two-hybrid assays.

Supplemental Figure 6. Chromosome associations and bridges weredetected in both msh5 meica1 and hei10 meica1 double mutants.

1706 The Plant Cell

Supplemental Table 1. Primers designed for map-based cloning.

Supplemental Table 2. Primers for RACE and genotyping.

Supplemental Table 3. Primers for plasmid construction.

Supplemental File 1. Text file of the alignment used to generate thephylogenetic tree in Figure 1B.

ACKNOWLEDGMENTS

This work was supported by grants from the National Key Research andDevelopment Program of China (2016YFD0100401), and the NationalNatural Science Foundation of China (31230038 and 31401357).

AUTHOR CONTRIBUTIONS

Z.C. and Q.H. conceived the original screening. Q.H., Y.L., and Y.S.designed the experiments and analyzed the data. Q.H., D.T., H.W., C.Z.,and G.D. performed most of the experiments. Q.H. wrote the article withcontributions of all the authors. Z.C. supervised and complemented thewriting.

Received March 27, 2017; revised June 14, 2017; accepted July 6, 2017;published July 10, 2017.

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1708 The Plant Cell