HEIP1 regulates crossover formation during meiosisin riceYafei Lia,b,1, Baoxiang Qina,c,1, Yi Shena, Fanfan Zhanga, Changzhen Liua, Hanli Youa, Guijie Dua, Ding Tanga,and Zhukuan Chenga,b,2

aState Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy ofSciences, 100101 Beijing, China; bJiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, 225009 Yangzhou,China; and cState Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Agriculture, Guangxi University, 530005Nanning, China

Edited by David C. Baulcombe, University of Cambridge, Cambridge, United Kingdom, and approved September 6, 2018 (received for review May 7, 2018)

During meiosis, the number of double-strand breaks (DSBs) farexceeds the final number of crossovers (COs). Therefore, toidentify proteins involved in determining which of these DSBsrepaired into COs is critical in understanding the mechanism of COcontrol. Across species, HEI10-related proteins play important rolesin CO formation. Here, through screening for HEI10-interactingproteins via a yeast two-hybrid system, we identify a CO proteinHEI10 Interaction Protein 1 (HEIP1) in rice. HEIP1 colocalizes withHEI10 in a dynamic fashion along the meiotic chromosomes andspecially localizes onto crossover sites from late pachytene to dip-lotene. Between these two proteins, HEI10 is required for theloading of HEIP1, but not vice versa. Moreover, mutations of theHEIP1 gene cause the severe reduction of chiasma frequency,whereas early homologous recombination processes are not dis-turbed and synapsis proceeds normally. HEIP1 interacts directlywith ZIP4 and MSH5. In addition, the loading of HEIP1 dependson ZIP4, but not on MER3, MSH4, or MSH5. Together, our resultssuggest that HEIP1 may be a member of the ZMM group and actsas a key element regulating CO formation.

rice | meiosis | crossover formation | HEIP1

Meiosis is a reductional type of cell division during which asingle round of DNA replication is followed by two rounds

of cell division, thus halving ploidy levels to produce haploidgametes. Accurate segregation of homologous chromosomes inmeiosis I is dependent on the formation of crossovers (COs). Atleast two types of COs coexist in most eukaryotes. Class I COsare sensitive to interference, a mechanism which ensures COsare more evenly spaced along a homologous pair than would beexpected by chance, and accounts for most COs. Class II COs donot exhibit interference (1). Mammals, budding yeast (Saccha-romyces cerevisiae), and plants all have both types of COs (2), butsome organisms have only one or the other (3, 4). According tothe double-strand break (DSB) repair model in S. cerevisiae (5),the formation of class I COs is dependent on a set of meiosis-specific proteins, collectively referred to as ZMM proteins(ZIP1, ZIP2, ZIP3, ZIP4, MER3, MSH4, and MSH5). ZMMproteins promote crossovers in the same recombination pathwayas shown by the similarity in phenotypes among zmm mutantsand colocalization of ZMM proteins (6). Several presumed ho-mologs of ZMM have been identified in plants, mice, and otherorganisms, suggesting these proteins are conserved. Additionalproteins involved in class I CO events include parting dancers(PTD) in Arabidopsis (7) and crossover-associated 1 (COSA-1)in Caenorhabditis elegans (8).Among ZMM proteins, ZIP1 is a central element (CE) com-

ponent of the synaptonemal complex (SC) (9), a meiosis-specifictripartite structure. MER3 is a DNA helicase protein that un-winds duplex DNA of various lengths in the 3′-to-5′ direction asan ATP-dependent process and extends the DNA joint moleculemade by RAD51 to facilitate the formation of double-Hollidayjunctions during meiotic homologous recombination (10). MSH4

and MSH5 proteins form a heterodimer complex, which binds tosingle-Holliday junctions or three-armed progenitor Holliday junc-tions as a sliding clamp, embracing the homologous chromosomesand promoting the formation of COs (11). ZIP2 is related toWD40-like repeat protein which appears to function in the promotion/stabilization of single-end invasion, while ZIP4 is a tetratricopeptiderepeat protein, which may facilitate specific interactions with apartner protein(s) (12). Yeast ZIP3 homologs have been identifiedin many other model organisms, and the ZIP3 family members aredivided into two subgroups: the ZIP3/RNF212 and HEI10 (13, 14),based on their sequence similarity network. The two subgroupsdisplay distinct differences in enzymatic activity (15–17), althoughthey share similarities in protein structure and dynamic localizationpatterns (18), and both are required for the formation of COs. TheZIP3/RNF212 group members appear to act solely as SUMO E3ligases (15), whereas the HEI10 group members appear to exhibitmore than just ubiquitin E3 ligase activity (16, 17).Here we report the identification of a protein, HEI10 Interaction

Protein 1 (HEIP1), which interacts directly with ZMM proteinsHEI10, ZIP4, and MSH5, and is essential for normal meiotic COformation in rice. Loss of HEIP1 results in decreased crossoverfrequency during meiosis. We also show that HEIP1 displays adynamic localization pattern during meiosis, and only a few brightfoci are retained on the meiotic chromosomes from late pachyteneto diplotene, which completely colocalize with HEI10. HEIP1 maybe a type of CO protein involved in the process of CO formation.

ResultsIsolation and Characterization of HEIP1. During meiosis, HEI10marks the sites of crossovers in mice, rice, and Arabidopsis (13,

Significance

Crossovers (COs) ensure the accurate segregation of homolo-gous chromosomes during meiosis. Failure to create the rightnumber of crossovers may lead to unequal distribution of ge-netic materials to daughter cells and sterility. CO proteins,which specially localize to crossover sites during meiosis, playcritical roles in CO formation. Here, we identify a CO protein inrice, named HEI10 Interaction Protein 1 (HEIP1), which is con-served in higher plants. Our results reveal its possible molec-ular mechanism for CO control in meiosis.

Author contributions: Y.L., Y.S., D.T., and Z.C. designed research; Y.L., B.Q., Y.S., F.Z., C.L.,H.Y., G.D., and D.T. performed research; and Y.L., B.Q., and Z.C. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1Y.L. and B.Q. contributed equally to this work.2To whom correspondence should be addressed. Email: zkcheng@genetics.ac.cn.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1807871115/-/DCSupplemental.

Published online October 1, 2018.

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16, 19). To gain more insight into the CO maturation, wescreened for HEI10-interacting proteins via a two-hybrid systemin yeast. A GAL4 activation domain (AD) fusion cDNA librarywas prepared from young rice panicles and positive clones wereselected for sequencing. Among them, one reproducible, positiveclone was identified, belonging to the Os01g0167700 gene. Thisgene was named HEI10 Interaction Protein 1 (HEIP1). The full-length cDNA sequence of HEIP1 was isolated by RT-PCR andRACE. The HEIP1 cDNA was 3,476 nucleotides long and wascomposed of a 2,808-bp ORF, a 470-bp 5′-noncoding region, anda 198-bp 3′-noncoding region (SI Appendix, Fig. S1). Sequencecomparison between genomic DNA and cDNA revealed that theHEIP1 gene was composed of 19 exons and 18 introns (Fig. 1A).To verify the interaction between HEI10 and HEIP1, we clonedthe full-length coding sequences encoding HEI10 and HEIP1into pGBKT7 and pGADT7 expression vectors, respectively.The cotransformed yeast cells grew on both double dropoutmedium (DDO, SD/-Leu/-Trp) and quadruple dropout medium(QDO, SD/-Ade/-His/-Leu/-Trp) supplemented with X-α-Galand aureobasidin A, suggesting that HEI10 interacts with HEIP1(Fig. 1B). Furthermore, the interaction between HEI10 andHEIP1 was also validated by a bimolecular fluorescence comple-mentation (BiFC) assay using rice protoplasts. Cyan fluorescent

protein (CFP) signals were detected in cells coexpressing HEI10-CFPN and HEIP1-CFPC as well as HEIP1-CFPN and HEI10-CFPC (Fig. 1C). The in vivo Co-IP assays were also conducted.As a result, HEIP1-Flag fusion proteins were immunoprecipitatedwith HEI10-Myc when they were transiently coexpressed in riceprotoplasts (Fig. 1D).

HEIP1 Is the Founding Member of a Gene Family Conserved in HigherPlants. A 2,808-bp HEIP1 ORF is predicted to encode a 935-aaprotein. Searches of public databases using the Position-SpecificIterative Basic Local Alignment Search Tool (PSI-BLAST) fromthe National Center for Biotechnology Information (NCBI)website were conducted to identify HEIP1 orthologs. Proteinswith sequence similarity to this gene were identified in multiplespecies, including LOC100835007 in Brachypodium distachyon(64% identity and 75% similarity over a 968-aa region),LOC103634850 in Zea mays (57% identity and 69% similarityover a 944-aa region), and AT2G30480 in Arabidopsis thaliana(28% identity and 45% similarity over a 475-aa region). We didnot identify candidate orthologs of HEIP1 outside the plantkingdom. Furthermore, additional NCBI DELTA-BLAST, Uni-port BLAST (https://www.uniprot.org/), and EMBL-EBI HMMERBLAST (https://www.ebi.ac.uk/Tools/hmmer/) searches were con-ducted; we could not identify the likely ortholog of HEIP1 beyondthe plant kingdom either, suggesting HEIP1 likely arose in plants.We aligned the HEIP1 amino acid sequence with representativeprotein sequences and constructed a neighbor-joining tree forHEIP1 homologs in plants (Fig. 1E and SI Appendix, Fig. S2). Wealso performed a search with the full-length protein sequences ofHEIP1 against motif libraries (https://www.genome.jp/tools/motif/).A potential GCK domain (E value 0.94) was detected at positions438–488 of HEIP1 from the NCBI’s conserved domain database(SI Appendix, Fig. S2), which might be involved in intracellularsignaling pathways or mediate heterodimerization according to theannotation. This domain is found only in plant proteins (https://www.ncbi.nlm.nih.gov/cdd/).

Mutation of HEIP1 Causes a Sterile Phenotype in Rice. To investigatethe function of HEIP1 in detail, we first attempted to screen oursterile mutant library, by using 60Co γ-rays as a mutagen to ir-radiate rice variety Zhongxian 3037, and isolated mutant lineswith allelic disruption in Os01g0167700. Using a map-basedscreening approach, we identified two mutants, heip1-1 and heip1-2. The heip1-1 mutant contained a single C-to-T nucleotidesubstitution in the sixth exon, changing arginine (CGA) at positionamino acid 184 to a stop codon (TGA), which led to prematuretermination of the protein. In heip1-2, a 1-bp deletion in the thirdexon led to frameshift and the formation of a premature stopcodon. We generated two additional mutants designated heip1-3and heip1-4 by CRISPR-Cas9 targeting (SI Appendix, Fig. S3).We then investigated the fertility in all heip1 mutants and

found that mature pollen grains were empty and shrunken in allmutants. Moreover, the heip1 plant did not set seed when pol-linated with wild-type pollen, indicating that micro- and mega-gametogenesis in heip1 were both affected. Aside from completesterility, the heip1 mutants exhibited normal vegetative growth(SI Appendix, Fig. S3).

Meiosis Is Disrupted in heip1. To determine the cause of sterility inheip1 mutants, we studied meiotic chromosomes of pollen mothercells (PMCs) at different meiotic stages in both wild type and theheip1-1 mutant. In the wild type, chromosomes were condensed toform long thin threads at leptotene. During zygotene and pachy-tene, homologous chromosomes paired up and synapsed. Fromdiplotene to diakinesis, SCs were disassembled, COs betweenhomologous chromosomes matured into visible chiasmata, chro-mosomes condensed further, and 12 bivalents were clearly ob-served. The bivalents aligned on the equatorial plate in an ordered

Fig. 1. Isolation and characterization of HEIP1. (A) Gene structure of HEIP1and mutation information of heip1, including mutation sites of heip1-1,heip1-2, heip1-3, and heip1-4. Black blocks represent exons, and un-translated regions are shown as gray boxes. (B) HEIP1 interacts with HEI10 inyeast two-hybrid assays. AgT and murine p53 were used as positive control.AD, prey vector pGADT7; BD, bait vector pGBKT7. (C) BiFC assays showingthe interaction between HEI10 and HEIP1 in rice protoplasts. (D) Coimmu-noprecipitation assays of Flag-HEIP1 and Myc-Hei10. IB, immunoblot; IP,immunoprecipitation. (E) Phylogenetic tree derived from the full-lengthamino acid sequences of HEIP1 and its homologs from nine plant species.(F) Meiotic chromosome behaviors in the heip1-1 mutant. (Scale bars: 5 μm.)

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pattern at metaphase I, and then, homologous chromosomesseparated and migrated in opposite directions at anaphase I.During the second meiotic division, the sister chromatids of eachchromosome separated, resulting in the formation of four daughtercells with 12 chromosomes each (SI Appendix, Fig. S4).In the heip1-1 mutant, meiotic chromosome behavior was al-

most identical to that of wild type from leptotene to zygotene.During pachytene, fully aligned chromosomes were detectedalong the whole chromosome (Fig. 1F). However, at diakinesis,bivalents were found to coexist with univalents in the samemutant PMC. At metaphase I, the bivalents aligned at theequator of the spindle, whereas the univalents were distributedrandomly. During anaphase I, the bivalents separated normallybut the univalents segregated randomly, resulting in an unequaldistribution of chromosomes in the two daughter cells. Thesecond meiotic division subsequently occurred. A tetrad withuneven chromosomes was detected. Similar defects were ob-served during male meiosis in heip1-2, heip1-3, and heip1-4 mu-tants (SI Appendix, Fig. S5 A–C), further indicating that themutant phenotype was caused by the disruption of HEIP1.

CO Levels Are Deficient in heip1.We further quantified the chiasmafrequency at metaphase I in both wild type and the heip1-1mutant using previously described criteria (20). Rod- and ring-shaped bivalents were treated as having one and two chiasmata,respectively. In wild type, the mean chiasma frequency per cellwas 20.85 ± 1.25 (n = 46), while in the heip1-1 mutant, thechiasma frequency was 2.89 ± 1.50 per cell which corresponds to2.56 ± 1.21 bivalents per PMC (n = 54). Thus, the mutation ofheip1-1 led to a significant reduction in both chiasma frequency(unpaired t test, P < 0.01) and the number of bivalents (unpairedt test, P < 0.01). The distribution of the remaining chiasmata wasalso analyzed in the heip1-1 mutant and compared with that inthe wild type. Statistical analysis showed that the residual chi-asmata distribution per cell in heip1-1 was consistent with apredicted Poisson distribution [SPSS Kolmogorov–Smirnov (K-S) test, P > 0.05], while the distribution of chiasmata in wild

type deviated significantly from a Poisson distribution amongdifferent PMCs (SPSS K-S test, P < 0.01). These results sug-gested the majority of residual chiasmata in heip1-1 are distrib-uted randomly (SI Appendix, Fig. S6).To further determine the role of HEIP1 in CO formation, we

generated a double mutant between hei10 and heip1 and ana-lyzed its chiasma number. The mean chiasma number per cellwas 2.64 ± 1.45 (n = 50) in hei10 heip1 (SI Appendix, Fig. S5D),which was very close to that in the heip1 single mutant, and theremaining chiasmata followed a Poisson distribution (SPSS K-Stest, P > 0.05) (SI Appendix, Fig. S6).

γH2AX, COM1, and DMC1 Localize Normally in heip1. To examinewhether CO deficiency is due to an early recombination defect,we detected γH2AX, COM1, and DMC1, three marker proteinspresent in early meiotic progress, in heip1-1 mutants by immu-nostaining assays. γH2AX, the histone H2AX variant, is con-sidered a reliable marker for DSBs. COM1, the homolog ofbudding yeast COM1/SAE2, participates in DSB end resection,whereas DMC1, the meiotic recombinase, mediates single-strandinvasion. In wild type, both of these proteins were observed aspunctuate foci on meiotic chromosomes. Similar levels wereobserved in the heip1-1 mutant. The number of γH2AX foci wasnot significantly different (unpaired t test, P = 0.848) betweenmeiocytes of heip1-1 (213 ± 57, n = 6) and the wild type (218 ±27, n = 9) (Fig. 2A). The number of COM1 foci in heip1-1 (257 ±32, n = 8) did not significantly differ (unpaired t test, P = 0.307)from that in wild type (278 ± 51, n = 10) (Fig. 2B). In wild type,the mean number of DMC1 foci at zygotene was 227 ± 25 (n =26). Similarly, the mean number of DMC1 foci in heip1-1 was219 ± 29 (n = 24) at the same stage, which did not differ from thewild type (unpaired t test, P = 0.338) (Fig. 2C). Two additionalmembers of the ZMM family, MER3 and ZIP4, are required forCO formation, and determining their localization may also in-dicate potential defects at early steps in crossover formation. Inheip1-1, both MER3 and ZIP4 appeared normal (SI Appendix,

Fig. 2. HEIP1 is not required for the localization of γH2AX, COM1, DMC1, PAIR2, PAIR3, and ZEP1 onto chromosomes. (A) Dual immunolocalization of REC8(green) and γH2AX (red) in the wild type and heip1. (B) Dual immunolocalization of REC8 (red) and COM1 (green) in the wild type and heip1. (C) Dualimmunolocalization of REC8 (red) and DMC1 (green) in the wild type and heip1. (D) Triple immunolocalization of PAIR2 (red), PAIR3 (green), and ZEP1 (blue)in the heip1 meiocytes. (Left) Merge results of PAIR2 and PAIR3. (Right) Merge results of PAIR2, PAIR3, and ZEP1. (Scale bars: 5 μm.)

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Fig. S7). These results suggest that the early homologous re-combination could be implemented in heip1.

Full Synapsis of Homologous Chromosomes Is Achieved in heip1.Synaptonemal complexes connect homologs along their lengthsduring the pachytene stage. An SC is a tripartite protein struc-ture consisting of two parallel axial elements (AEs), a structurethat forms between sister chromatids before synapsis, also calledlateral elements (LEs) after synapsis, and a CE. To explore therole of HEIP1 in synapsis, dual immunodetection was carried outin the heip1-1 mutant with antibodies related to both AEs/LEsand CEs. PAIR2 is only associated with unsynapsed AEs atleptotene and zygotene, and PAIR3 is associated with bothunsynapsed AEs and LEs of SC during prophase I, whereas thedistribution of ZEP1 signals indicates the extent of synapsis.Detailed analysis of PAIR2, PAIR3, and ZEP1 progression inwild-type meiotic prophase I was described by Wang et al. (21).In heip1-1, the localization of LEs and CEs from leptotene toearly pachytene was indistinguishable from that of wild type (Fig.2D). Therefore, we determined that HEIP1 may not be requiredfor synapsis to proceed. Furthermore, the synapsis initiation siteswere quantified by counting the number of ZEP1 stretches inearly to midzygotene according to criteria previously described(22). We found that the mean number of synapsis sites per cellwas not significantly different between heip1-1 and the wild type(unpaired t test, P > 0.05). In heip1-1, the mean number of ZEP1stretches was nearly 19 (n = 16, range 8–27), whereas it wasabout 18 (n = 19, range 9–25) for the wild type.

HEIP1 Proteins Are Present as Punctuate Foci and Colocalize withHEI10. To monitor the spatial and temporal location of HEIP1during rice meiosis, coimmunolocalization was performed withantibodies against HEIP1 and REC8 (a component of the mei-otic cohesin complex). HEIP1 was first visible as numerouspunctuated foci at early leptotene and the mean number ofHEIP1 foci increased rapidly, reaching its peak at late leptotene/early zygotene (324 ± 46, n = 12) (Fig. 3 A and B). With theproceeding of homologous pairing in zygotene, the foci of HEIP1decreased quickly and only 90 ± 29 foci (n = 12) remained atpachytene (Fig. 3C). However, with the further progression ofmeiosis, only a few bright foci were retained from late pachyteneto diplotene (Fig. 3 D and E). The mean number of HEIP1 fociper cell at this stage was 25 ± 3 (n = 20). They disappeared atdiakinesis and could be detected thereafter.A previous study showed that punctuate foci of HEI10 is also

retained at a limited number of sites during late prophase I (19).To establish the relationship between HEI10 and HEIP1, weperformed dual immunostaining experiments using antibodiesagainst HEIP1 and HEI10. In the leptotene stage, discrete fociof HEIP1 almost completely colocalized with foci of HEI10(97.80% of HEI10 foci contained HEIP1, 98.23% of HEIP1 focicontained HEI10; n = 5) (SI Appendix, Fig. S8 A–D). From zy-gotene to pachytene, HEI10 began to elongate along chromo-somes and still showed high colocalization with HEIP1 foci.Almost all HEI10 short stretches had at least one HEIP1 focus,and most HEIP1 foci localized on the linear HEI10 signals (SIAppendix, Fig. S8 E–H). Interestingly, during diplotene, HEI10foci and HEIP1 foci completely colocalized in all wild-typePMCs observed (n = 20) (SI Appendix, Fig. S8 I–K). At diaki-nesis, prominent HEI10 foci appeared and persisted to laterstages, whereas HEIP1 foci were no longer visible.Furthermore, the colocalization pattern between HEIP1 and

HEI10 was investigated in zep1. Although linear signal formationof HEI10 was severely disrupted in zep1, prominent fociappeared normal on chromosome axes and also showed highcolocalization with HEIP1 foci (SI Appendix, Fig. S9).

HEI10 Is Required for Loading of HEIP1, but Not Vice Versa.Consideringthe close localization pattern between HEIP1 and HEI10, dualimmunolocalization experiments were performed to investigatethe mutual dependency in the loading of HEI10 and HEIP1 ontochromosomes. In hei10 meiocytes, no obvious HEIP1 signals weredetected (SI Appendix, Fig. S10), suggesting that the proper lo-calization of HEIP1 relies on the presence of HEI10. In heip1-1,the loading of HEI10 was indistinguishable from that of wild typewhen small dot signals gradually developed into linear signal fromleptotene to early pachytene. However, at late pachytene, the di-vergence was apparent since the prominent foci did not developnormally (Fig. 4A). As a result, heip1-1 meiocytes had only 9 ± 2prominent foci, compared to 25 ± 3 in wild type (n = 21) (Fig. 4B).It seems that HEI10 loaded normally onto chromosomes untilearly pachytene in heip1; however, the formation of prominent fociat late pachytene was severely disrupted. Western blotting was alsoperformed to test the HEIP1 levels in both wild type and hei10,and vice versa. Our results showed that HEI10 levels in the wildtype and heip1-1 were comparable; nevertheless, almost no HEIP1was detected in hei10 (SI Appendix, Fig. S10).

The Interplay of HEIP1 and ZMM Proteins. MER3, ZIP4, MSH4, andMSH5 are members of the ZMM gene family, whose deletion or

Fig. 3. The localization pattern of HEIP1 in wild type. HEIP1 (green) presentsas punctuate foci on chromosomes (REC8 labeled red). (A) Leptotene. (B) Zy-gotene. (C) Pachytene. (D) Late pachytene. (E) Diplotene. (Scale bars: 5 μm.)

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mutation results in a severe reduction in crossover number. Inrice, ZIP4, MER3, MSH4, and MSH5 have been well character-ized (23–26). To further characterize the effect of ZMM proteinson HEIP1 localization, fluorescence immunolocalization studieswere performed using antibodies against REC8 and HEIP1 onmeiocytes of rice zmm mutants. In zip4, HEIP1 was not loadedonto meiotic chromosomes. Almost no immunostaining of HEIP1was detected in zip4meiotic chromosomes, suggesting that normalloading of HEIP1 is dependent on ZIP4. However, the localiza-tion of HEIP1 in mer3, msh4, and msh5 differed from that in zip4.In mer3, msh4, and msh5, HEIP1 foci appeared normally at zy-gotene (SI Appendix, Fig. S11). In addition, we performed dualimmunostaining experiments using antibodies against HEIP1 andMER3, raised in mice and rabbits, respectively. As shown in SIAppendix, Fig. S12, discrete foci of HEIP1 almost completelycolocalized with MER3 at zygotene.The similar function of these proteins in CO formation also

prompted us to further investigate possible interactions betweenHEIP1 and ZMM proteins. We cloned ORFs of HEIP1 andZMM proteins into the binding domain (BD) vector pGBKT7and the AD vector pGADT7, which cotransformed into the Y2HGold yeast strain, triggering their coexpression. We confirmed adirect interaction between the full-length HEIP1 and ZIP4proteins, whereas the BD vector containing full-length HEIP1 orMSH5 may autonomously activate the reporter genes in theabsence of interacting partners (25). The interaction betweenHEIP1 and MSH5 was validated by the yeast Y2H assay usingtruncated HEIP1 protein with no self-activation activity and Co-IP assays in rice protoplasts (Fig. 4 C–E). However, no in-teraction was detected between HEIP1 and the other ZMMproteins (MER3 and MSH4) (Fig. 4D). We propose that HEIP1may interact with ZIP4 and MSH5 to promote CO formation.

DiscussionHEIP1 Is Required for Class I CO Formation. By screening for HEI10-interacting proteins via a yeast two-hybrid system, we identifiedthe HEIP1 protein. In heip1, the chiasma frequency was severelyreduced compared with that in the wild type. Roughly 14% ofchiasmata remained in heip1, which is similar to that in msh5 (26).As previously shown in budding yeast, Arabidopsis, and rice, thereare two classes of CO occurring: one appears to be sensitive tointerference, while the other is not. First, our investigations into

heip1 mutants showed a reduced number of chiasmata withremaining chiasmata distributed randomly among cells. Similarly,residual chiasmata in hei10 heip1 mutants also displayed a randomdistribution. Second, the HEI10 prominent foci correspond to theclass I CO sites in rice. In heip1, the formation of prominentHEI10 foci was severely disrupted at late pachytene, and none ofthem was located on the remaining bivalents at diakinesis. Third,we revealed that HEIP1 interacted directly with HEI10, ZIP4, andMSH5. Thus, we suspect HEIP1 and ZMM may act in the sameCO formation pathway, which is likely interference sensitive.

HEIP1 Marks Putative CO Sites from Late Pachytene to Diplotene. Weshowed that the mean number of HEIP1 foci per cell from latepachytene to diplotene was ∼24 foci, similar to that of HEI10.Previous studies revealed that HEI10 prominent foci appear duringlate prophase I, where they marked the class I CO site (19). Fromlate pachytene to diplotene, HEI10 and HEIP1 foci almost com-pletely colocalized in all wild-type nuclei observed. Furthermore,previous studies showed that in zep1 mutants, HEI10 foci (class ICOs) increased ∼1.5-fold compared with that in wild type, which wasfurther confirmed by genetic analysis (27). We found that HEI10prominent foci always colocalized with HEIP1 in zep1 mutants.Across species, there appears to be four groups of proteins

localized at putative CO sites at late prophase I: DNA mismatchrepair protein MutL (MLH1–MLH3) (28–30), ZIP3/ZHP-3/Vilya/RNF212 SUMO E3 ligases (14, 18), HEI10 ubiquitin li-gases (13, 31), and the cyclic domain-containing proteins COSA-1/CNTD1, which are conserved only in metazoans (8, 32). Giventhe localization of HEIP1 from late pachytene to diplotene, itmight also belong to a group of CO proteins.

How Might HEIP1 Act in Meiosis? Recombination is initiated at alarge number of DSBs generated by Spo11 transesterase (33).These DSBs are then processed to generate free 3′ single-stranded overhangs which are rapidly coated with recombi-nases (34). In most species, the number of recombination sitesfar exceeds the number of CO events (34, 35). Recombinationnodules were originally identified as small electron-dense ovoidstructures appearing on the SCs (36). Recombination noduleshave been mainly classified into two classes: early nodule (EN)and late nodule (LN) (37). ENs present during zygotene–earlypachytene and have been postulated to correspond to initialrecombination sites, whereas LNs are believed to indicate COs

Fig. 4. The loading pattern of HEI10 in heip1 andinteractions between HEIP1 and ZMM proteins. (A)The immunodetection of HEI10 (green) in wild typeand heip1 during prophase I. (Scale bars: 5 μm.) (B)Number of the HEI10 foci on chromosomes at latepachytene in wild type and heip1. Each symbol rep-resents a single nucleus. ***P < 0.001. (C) Schematicdiagram of full-length and four truncated proteins,A–D, of HEIP1 used in yeast two-hybrid assays. (D)HEIP1 interacts with ZIP4 and MSH5. AD, prey vectorpGADT7; BD, bait vector pGBKT7. (E) Coimmuno-precipitation assays of Myc-HEIP1 and Flag-MSH5. IB,immunoblot; IP, immunoprecipitation.

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that are assumed to mature into chiasmata during mid-to-latepachytene (37, 38). Previous studies in mice suggest that mostearly RAD51–DMC1 complexes (300 ENs) acquire recombina-tion factors (39), including the meiosis-specific MutSγ complexMSH4–MSH5, whereas the RAD51–DMC1 component is lostfrom such “transition nodules” (100–200 TNs) (40). Only about25 TNs acquire the MLH1 protein that marks the sites of chi-asmata (LNs) (18, 41). The dynamic localization pattern ofHEIP1 during meiosis resembles LNs identified in other or-ganisms. Thus, we propose that HEIP1 plays an important role inmeiotic crossover maturation.In our investigation of HEIP1 and ZMM protein interactions,

we found that HEIP1 interacted directly with HEI10, ZIP4, andMSH5, which further supports the hypothesis that HEIP1 acts asthe backbone component in crossover formation. In hei10 and zip4,no obvious HEIP1 signals were detected, suggesting that HEIP1may act downstream of HEI10 and ZIP4. However, inmer3,msh4,and msh5, the HEIP1 foci appeared normal on chromosomes atzygotene, indicating that the localization of HEIP1 is independentof MER3, MSH4, and MSH5. Furthermore, prominent axisstaining of ubiquitin was detected on rice meiotic chromosomes(SI Appendix, Fig. S13), indicating that ubiquitination is also in-volved in meiosis of plants, which has been studied in yeast andmouse (42, 43). Almost no HEIP1 was detected in hei10 mutant,

suggesting that HEI10 may play a role in the stabilization ofHEIP1. In Arabidopsis, the exquisite dosage sensitivity of HEI10for crossover formation was well shown (44, 45). Considering theclose functional relationship between HEI10 and HEIP1, the ef-fect of increasing HEI10 and/or HEIP1 dosage on crossovers alsodeserves further study in rice. In addition, the lack of conservationsuggests HEIP1 may possibly play a structural role in a similarmanner to the axis-associated proteins and the central element ofthe synaptonemal complex (36). These proteins are also essentialfor crossover formation yet exhibit considerable sequence di-vergence between species.

Materials and MethodsThe heip1-1 and heip1-2 were isolated from an indica rice variety Zhongxian3037, and the heip1-3 and heip1-4were generated in a japonica variety Yandao8 by CRISPR-Cas9 targeting. Details on the following are available in SI Ap-pendix, SI Materials and Methods: plant materials, full-length cDNA cloning ofHEIP1, multiple alignments and phylogenetic tree construction, library screen-ing and Y2H assay, BiFC assay, coimmunoprecipitation assay, meiotic chromo-some preparation, antibody production, immunofluorescence, and Westernblot assay. The primers used in this study are listed in SI Appendix, Table S1.

ACKNOWLEDGMENTS. This work was supported by grants from the NationalKey Research and Development Program of China (2016YFD0100901) andthe National Natural Science Foundation of China (31460278 and 31771363).

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