OsHOP2 regulates the maturation of crossovers by

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DR ZHUKUAN

Article type

CHENG (Orcid ID : 0000-0001-8428-8010)

: Regular Article

OsHOP2 regulates the maturation of crossovers by promoting homologous pairing and synapsis in rice meiosis

Wenqing Shia*, Ding Tanga*, Yi Shena, Zhihui Xuea, Fanfan Zhanga, Chao Zhanga,b, Lijun Rena,b, Changzhen Liua,b, Guijie Dua, Yafei Lia, Changjie Yanc, Zhukuan Chenga,b a

State Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of

Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China. b c

University of Chinese Academy of Sciences, Beijing 100049, China.

Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou

University, 225009 Yangzhou, China *These authors contributed equally to this work.

Corresponding author: Zhukuan Cheng ([email protected]) Telephone: 0086-10-6480, 6551 Fax: 0086-10-6480, 6595

Received: 10 July 2018 Accepted: 7 December 2018

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/nph.15664 This article is protected by copyright. All rights reserved.

ORCID ID:

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Wenqing Shi, https://orcid.org/0000-0003-3820-9259 Ding Tang, https://orcid.org/0000-0003-2187-4180 Yi Shen, https://orcid.org/0000-0001-8403-9882

Chao Zhang, https://orcid.org/0000-0002-2064-5450 Lijun Ren, https://orcid.org/0000-0001-5957-1113 Guijie Du, https://orcid.org/0000-0002-8287-7102 Yafei Li, https://orcid.org/0000-0002-0010-5940 Zhukuan Cheng, https://orcid.org/0000-0001-8428-8010

Summary 

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Meiotic recombination is closely linked with homologous pairing and synapsis. Previous studies have shown that HOMOLOGOUS PAIRING PROTEIN2 (Hop2), plays an essential role in homologous pairing and synapsis. However, the mechanism by which HOP2 regulates crossover (CO) formation has not been elucidated. Here, we show that OsHOP2 mediates the maturation of COs by promoting homologous pairing and synapsis in rice meiosis. We used a combination of genetic analysis, immunolocalization and super-resolution imaging to analyze the function of OsHOP2 in rice meiosis. We showed that full-length pairing, synapsis and CO formation are disturbed in Oshop2 meiocytes. Moreover, structured illumination microscopy (SIM) showed that OsHOP2 localized to chromatin and displayed considerable co-localization with axial elements (AEs) and central elements (CEs). Importantly, the interaction between OsHOP2 and ZEP1, provided further evidence that OsHOP2 was involved in assembly or stabilization of the structure of the synaptonemal complex (SC). Although the initiation of recombination and CO designation occur normally in Oshop2 mutants, mature COs were severely reduced, and HEI10 foci were only present on the synapsed region.

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

Together, we speculate that OsHOP2 may serve as a global regulator to coordinate

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homologous pairing, synapsis and meiotic recombination in rice meiosis.

Key words: rice, meiosis, HOMOLOGOUS PAIRING PROTEIN2 (HOP2), synapsis, recombination

Introduction Meiosis is a specialized set of cell divisions that generates haploid gametes from diploid

parental cells during sexual reproduction. During meiosis, homologous recombination (HR) produces non-crossover (NCO) and crossover (CO) products, providing the basis for increasing genetic diversity and establishing physical links between homologous chromosomes. In addition to HR, homologous pairing and synapsis also play critical roles during this highly organized process. All of these events are spatially and temporally coordinated to promote faithful segregation of homologous chromosomes at the first meiotic division (Gerton & Hawley, 2005; Kleckner, 2006). During meiosis, the events described above occur in the context of highly organized

chromosomes. At early prophase I, each pair of sister chromatids are organized into two co-oriented linear arrays of chromatin loops connected by a shared axis. DSBs (double-strand breaks) occur in tethered loop-axis complexes (Blat et al., 2002; Panizza et al., 2011). After that, interactions between two individual DNA segments, mediate the spatial coalignment of entire homologous chromosomes during early- to mid-leptotene (Zickler & Kleckner, 2015). Concomitantly, the sites of these interactions are visualized as interaxis bridges that mature into structures known as axial associations (AAs). Formation of the synaptonemal complex (SC) appears to be initiated at AAs at early zygotene and continues extension along the chromosome until it is fully formed at pachytene. The AAs serve as sites for assembly of a proteinaceous core (including Zip1p, Zip2p, Zip3p, and Msh4p) that provides a link between synapsis and meiotic COs (Page and Hawley, 2004). Overall, the association of


recombination complexes with chromosome axes as well as highly dynamic organization of

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chromosome axes, play crucial roles in the coordination of inter-homolog (IH) recombination, pairing and synapsis.

Homologous recombination is initiated by formation of DNA DSBs, which are catalyzed

by the topoisomerase-like protein SPORULATION11 (SPO11) (Keeney et al., 1997). Incision sites are further processed by the Mre11-Rad50-Xrs2 (MRX) complex, to yield 3’ ssDNA tails (Mimitou & Symington, 2009). Then, the free ssDNA ends are coated by RAD51 and DISRUPTED MEIOTIC CDNA1 (DMC1), two homologs of the bacterial RecA protein, to promote strand invasion and formation of double-Holliday junction (dHJ) (Hunter & Kleckner, 2001). Finally, the resolution of the dHJ following cutting at alternative strands give rise either to CO or NCO (Allers & Lichten, 2001; Borner et al., 2004).

In many organisms, meiotic recombination is coupled with homologous pairing and

synapsis. The mutants involved in DSB formation or progression of meiotic recombination, are defective in homologous pairing, synapsis, and CO formation. In the absence of mouse SPO11 protein, pairing and SC formation are defective; synapsis can be restored by inducing DSBs in spo11 mutants (Romanienko & Camerini-Otero, 2000). In Arabidopsis dmc1, synapsis and IH COs are disturbed and meiotic DSBs are repaired using the sister chromatid as a template in a RAD51-dependent manner (Couteau et al., 1999; Vignard et al., 2007). In addition, the maturation of recombination intermediates into COs is dependent on the ZMM (for Zip1, Zip2, Zip3, Zip4, Msh4, Msh5 and Mer3) proteins that are essential for the formation of class I COs. In S. cerevisiae zmm mutants, homologous pairing and synapsis are affected (Lynn et al., 2007). However, in plant zmm mutants, there are defects in CO formation, but pairing and synapsis occurs normally (Mercier et al., 2005; Chelysheva et al., 2007; Higgins et al., 2008; Macaisne et al., 2008; Wang et al., 2012). This suggests that the functions of ZMM proteins for pairing and SC assembly are divergent in different organisms.


The SC is a meiosis-specific supramolecular structure that physically links homologous

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chromosomes and ensures the formation of meiotic COs. The SC is a tripartite protein structure with two parallel lateral elements (LEs), connected together by transverse filaments (TFs) (Page & Hawley, 2004). The axial/lateral elements provide IH recombination bias by interacting with recombination proteins, ensuring that COs occur between homologous chromosomes rather than sister chromatids (Brown & Bishop, 2015). TF proteins are thought to function in protein-protein interactions with other central element (CE) proteins via coiled-coil domains. These multiple interactions among SC proteins are required for the assembly or stabilization of SC structure (Cahoon & Hawley, 2016). In addition to being a central component of SC, TFs play a role in CO maturation (Page & Hawley, 2004). Zip1, a TF protein, is also required for processing of recombination intermediates (Storlazzi et al., 1996). The Drosophila C(3)G and mouse SYCP1 protein, two TF proteins, are required for the maturation of DSBs into CO (Page & Hawley, 2001; de Vries et al., 2005). In C. elegants, SYP-1 and SYP-2 (two TF proteins) display similar functions in CO formation (MacQueen et al., 2002; Colaiacovo et al., 2003). Overall, these findings suggest that the mature SC plays a key role in promoting the maturation of recombination intermediates into CO.

HOP2 proteins have been identified in yeast, mouse and Arabidopsis, with similar

functions in promoting homologous pairing and synapsis (Leu et al., 1998; Tsubouchi & Roeder, 2002; Petukhova et al., 2003; Uanschou et al., 2013). However, the role of HOP2 in regulation of CO maturation has not been reported. Here, we characterized the rice HOP2 homolog (OsHOP2) and explored its molecular mechanism in rice meiosis. Our studies show that OsHOP2 is crucial for CO maturation by promoting full-length pairing and synapsis.


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Materials and Methods Plant materials The Oshop2-1 mutant was isolated from the japonica rice variety Yandao 8 induced by

60

Co γ-ray irradiation. The Oshop2-2 was identified from Huanghuazhan (an indica rice

variety). The Oshop2-3 and Oshop2-4 mutants were generated using CRISPR-Cas9 targeting method in the japonica rice variety Yandao 8. The Oshop2-1 allele was used for data generation throughout our research. The Osspo11-1, pair1, Oscom1, Osrad51c, Oszip4 mutants used in this study were reported previously (Yu et al., 2010; Ji et al., 2012; Shen et al., 2012; Tang et al., 2014). All plant materials were grown in paddy fields.

Molecular cloning For map-based cloning of Oshop2, heterozygous mutant plants of Oshop2-1 were

crossed with the indica variety Zhongxian 3037, to generate the mapping populations. Markers were developed based on sequence differences between the japonica variety and the indica variety. The primer sequences of markers are listed in the Supplemental Table S1.

Cloning the full-length OsHOP2 cDNA Total RNA was extracted from rice young panicles (5-8 cm) using TRIZOL reagent

(Invitrogen). Reverse-transcribed into cDNA was used the SuperScript® III Reverse Transcriptase (Invitrogen, Cat #18080-044). The full-length OsHOP2 cDNA was confirmed using the 5’-Full RACE kit (TaKaRa, Cat #6107) and the 3’-Full RACE kit (TaKaRa, Cat #6106). The products of 5’ and 3’ RACE-PCR were cloned and sequenced.


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CRISPR-Cas9 targeting of OsHOP2 and RNAi analysis For CRISPR-Cas9 targeting of OsHOP2, the reported CRISPR-Cas9 binary vector

pC1300-cas9 and the intermediate vector SK-gRNA were used in this study. The target sequences of OsHOP2 were listed in the Supplemental Table S1. These two constructs were introduced into A. tumefaciens strain EHA105 and then independently transformed into the japonica variety Yandao 8. Two fragments of OsHOP2 cDNA sequence were selected for RNAi analysis. One is 353 bp, another is 252 bp. The primer is listed in the Supplemental Table S1.

Yeast two-hybrid assay The yeast two-hybrid assays were conducted using the MatchmakerTM Gold Yeast

Two-Hybrid system (Clontech No.630489). ORFs of OsHOP2 and ZEP1, were amplified with KOD-plus polymerase and cloned into pGADT7 and pGBKT7 to generate AD and BD recombinants. These plasmids were cotransformed into Y2Hgold strain in an AD-BD-coupled manner. Detailed procedures were described in the manufacturer's handbook (Yeast Protocols Handbook; PT3024-1; Clontech).

BiFC assay To conduct BiFC assays, OsHOP2 and ZEP1 were amplified by specific primers

(Supplemental Table S1) with KOD-plus polymerase and then ligated into BiFC vectors, including pSCYNE (SCN) and pSCYCE(R) (SCC) (Waadt et al., 2008). The generated plasmids were transformed into protoplasts by previous method (Bart et al., 2006). After incubation in the dark for 18 h at 28°C, the CFP signals were captured under a confocal laser scanning microscope at an excitation wavelength 405 nm (Leica TCS SP5).


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Antibodies The primary antibodies against OsREC8, PAIR3, γH2AX, OsCOM1, OsDMC1, OsZIP4,

OsMER3, ZEP1 and HEI10 were generated previously. To generate the antibody against OsHOP2, the full length of OsHOP2 ORF was amplified with primer OsHOP2-Ab (Supplemental Table S1) and ligated into the expression vector pET-30a (Novagen). The fusion peptide expression and purification were carried out as described previously (Wang et al., 2009). The polyclonal antibody was raised from mouse and guinea pig.

Meiotic chromosome preparation Fresh young panicles were fixed in Carnoy’s solution (ethanol:glacial acetic acid, 3:1).

Microsporocytes undergoing meiosis were squashed in an acetocarmine solution. Slides with chromosomes were frozen in liquid nitrogen and removed the coverslip rapidly. Then the slides were dehydrated through a graded ethanol series (70, 90 and 100%) for 5 min each and air-dried. Chromosomes were counterstained with 4',6-diamidino-2-phenylindole (DAPI) in an anti-fade solution (Vector Labora-tories, Burlingame, CA, USA). Images were captured under a Zeiss A2 fluorescence microscope with a micro-charge-coupled device camera.

Fluorescence in situ hybridization The FISH analysis was conducted as described previously (Zhang et al., 2005). The

pAtT4 clone contains telomere repeats and the pTa794 clone has 5S ribosomal RNA genes from wheat (Cuadrado & Jouve, 1994). The bulked oligonucleotide probes (11L and 11S) were newly developed based on the procedures of a recent report (Hou et al., 2018). Chromosome images were captured under the Olympus BX51 fluorescence microscope with a microCCD camera using software IPLab4.0.


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Immunofluorescence assay and image analysis For immunofluorescence assays, the young panicles were fixed with fresh 4% (w/v)

paraformaldehyde for 30 min. The procedure was performed as previously described (Wang et al., 2009). Images were captured under a Zeiss A2 fluorescence microscope with a micro-charge-coupled device camera. The super-resolution images were captured using a DeltaVision microscope (GE healthcare, OMXTM V4) and processed with SoftWoRx (Applied Precision) to generate projected images. Colocalization analysis was performed using

an

automated

graphic

plugins

running

with

ImageJ

1.37a

software

(http://wwwfacilities.uhnresearch.ca /wcif), according to the description reported by Li et al. (Li et al., 2004)

Western-Blot Assay Proteins were extracted from rice panicles with buffer solution containing 50 mM

Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP40, and 1% SDS, 5 Mm DTT, 1 mM PMSF and proteinase inhibitor cocktail (Roche LifeScie). Protein samples were separated by SDS-PAGE on a 12% polyacrylamide gel and electroblotted onto PVDF membranes (GE Healthcare). Western blots were performed with OsHOP2 antibodies diluted 1/5,000 and anti-mouse IgG antibodies conjugated to horseradish peroxidase (Abcam) diluted 1/10,000. Anti-guinea pig IgG conjugated to horseradish peroxidase (Jackson) diluted 1/10,000. HSP90 was detected by probing with an anti-HSP90 antibody (BGI).

Computational and database analysis The

multiple

sequence

alignment

(https://toolkit.tuebingen.mpg.de/#/tools/mafft)

was

conducted

using

MAFFT

and

colored

with

ESPript

(http://espript.ibcp.fr/ESPript/ESPript/). The protein domains were predicted by SMART (http://smart.embl-heidelberg.de/) and drawn by IBS software (http://ibs.biocuckoo.org/).


Results

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Map-based cloning and characterization of OsHOP2

by

A complete sterile mutant was identified from a japonica rice variety, Yandao 8, induced 60

Co γ-ray irradiation. The mutant plants showed normal vegetative growth but were

completely sterile. I2-KI staining showed that the pollen grains were empty and shrunken in the mutant (Fig. 1a). Moreover, the mutant did not set seeds when pollinated with the wild-type pollens, indicating that the mutant was both male and female sterile. The self-fertilization of heterozygous plants produced a 3:1 segregation ratio (fertile, 183; sterile, 57), which suggested that this mutant is a single recessive mutant (χ2=0.34; P >0.05).

To isolate the target gene, a mapping population was constructed by crossing

heterozygous plants with the indica rice variety Zhongxian 3037. The gene was first mapped on the long arm of chromosome 3, and was then further located to a physical region of approximately 150 kb by fine-mapping (Fig. S1a). According to the Rice Genome Annotation Project Database (http://rice.plantbiology.msu.edu/cgi-bin/gbrowse/ rice), in this region, Os03g50220 is annotated as homologous-pairing protein meu13, an ortholog of yeast HOP2 protein. Sequencing analysis revealed a single nucleotide (G) insertion in exon 1 (ORF 24) (Fig. 1b), resulting in the frameshift and premature termination codon at position 18 in the amino acid sequence (Fig. S1b). Thus, we deduced that the mutation of Os03g50220 was responsible for the sterile phenotype and named the mutant Oshop2-1. Another allele, Oshop2-2, was identified from Huanghuazhan (an indica variety), with a large fragment insertion in intron 5 of OsHOP2 (Fig. 1b). In order to confirm that meiotic defects were caused by the mutation of OsHOP2, we obtained two other alleles of OsHOP2 using CRISPR-Cas9 targeting, named Oshop2-3 and Oshop2-4 (Fig. 1b). Specifically, Oshop2-3 carried a single nucleotide (T) insertion in exon 4, and Oshop2-4 had a deletion (of AAGAC) in exon 5. Both alleles resulted in the frameshift and a new stop codon at position 127 and 192 in the amino acid sequence, respectively (Fig. S1b).


The full-length cDNA sequence of OsHOP2 was obtained using RT-PCR and RACE

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(rapid amplification of cDNA ends), and is consistent with the full-length cDNA (AK102972) of OsHOP2 (https://www.ncbi.nlm.nih.gov/nuccore/32988181). OsHOP2 is composed of six exons and five introns (Fig. 1b). It encodes a 228-amino acid (AAs) protein. According to the crystal structure (Kang et al., 2015), OsHOP2 contains an N-terminal winged-helix domain (WHD) and three leucine zippers: LZ1, LZ2, and LZ3 (Fig. 1c). Multiple alignment and phylogenetic analysis indicated that HOP2 is an evolutionarily conserved protein among eukaryotes (Fig. S2a and S2b). Furthermore, the results of RT-PCR showed that OsHOP2 was highly expressed in young panicles, and a slight and a severe reduction of transcript was detected in Oshop2-1 mutants and OsHOP2RNAi lines, respectively (Fig. S3a). Moreover, the expression upstream and downstream of the insertion in Oshop2-2 mutant was examined. The results showed that the expression of OsHOP2 after the insertion site was significantly reduced and had a reduction of about 30% before the insertion (Fig. S3b).

Chromosome behaviors in Oshop2 meiocytes To explore the reason for sterility of Oshop2 mutants, meiotic chromosome behaviors

were investigated in meiocytes of both wild-type and mutant plants. In wild type, the chromosomes were fully synapsed and thick threads were observed at pachytene. Twelve bivalents aligned on the equatorial plate at metaphase I. Homologous chromosomes subsequently separated and migrated to opposite poles at anaphase I. After the second meiotic division, the sister chromatids separated from each other and produced tetrad spores (Fig. 1d).

Compared with wild type, most chromosomes were widely dispersed as single threads at

pachytene in the Oshop2-1 mutant; some regions were synapsed and condensed into thick threads. When meiocytes reached metaphase I, few bivalents, but a majority of univalents were observed within the nucleus. During anaphase I, chromosomes were unequally separated, and lagging chromosomes were observed. At the end of meiosis, abnormal tetrads with unequal chromosome distribution and micronuclei were observed (Fig. 1d).


Further cytological observations revealed that the meiotic defects in Oshop2-2,

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Oshop2-3, and Oshop2-4 were similar to that in Oshop2-1 (Fig. S4b, S4c and S4d). In addition, the meiotic defects in sterile OsHOP2RNAi plants were consistent with that in Oshop2-1 plants (Fig. S4e and S4f). These results proved that the meiotic chromosome behavior of Oshop2-1 was induced by the deletion of OsHOP2, further leading to sterility. Thus, Oshop2-1 was selected for all subsequent studies.

OsHOP2 is required for full-length homologous pairing and synapsis Telomere bouquet clustering is essential for homologous pairing in early prophase I.

Fluorescence in situ hybridization (FISH) was performed to detect bouquet formation in both wild type and Oshop2 meiocytes, using a telomere-specific probe (pAtT4). The results showed that nearly all of the pAtT4 signals were clustered within a defined region at early zygotene in both wild type (n=56) and Oshop2 (n=80) meiocytes (Fig. S5a), indicating that OsHOP2 is not required for telomere bouquet formation. To further examine homologous pairing in the Oshop2 mutant, FISH was carried out on

meiotic chromosomes of both wild type and Oshop2 by chromosome painting. The painting probes, which were specific to the short (11S) and long arms (11L) of chromosome 11, were prepared and used in the FISH assay. In wild type, fully paired pachytene chromosomes were labeled by the red (11L) and green (11S) signal (n=35) (Fig. 2a and 2c), which indicated that homologous pairing was completed. In Oshop2 mutants, 26.3% of detected meiocytes showed paired or partially paired signals of two probes and 73.7% of meiocytes displayed unpaired signals (n=57) (Fig. 2a and 2c). 5S rDNA as a specific probe was also used to examine homologous pairing. 5S rDNA is a tandem repetitive sequence located close to the centromere of chromosome 11. In wild type, two paired 5S rDNA signals were visible at pachytene, indicating that homologous chromosomes had been paired (n=67). However, two apart 5S rDNA signals were observed at pachytene in Oshop2 (77.4%, n=93), and 22.6% of meiocytes showed paired 5S rDNA signals (Fig. S5b). Together, these results suggested that OsHOP2 is required for full-length homologous pairing.


In order to monitor the assembly of SCs in both wild type and Oshop2, we conducted

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immunostaining using an antibody against ZEP1. In wild type, ZEP1 signals were fully elongated along the entire chromosomes at pachytene. In Oshop2, meiocytes displayed considerable variation in SC length at pachytene (Fig. 2b and 2d). Then 165 pachytene meiocytes were measured to monitor the level of SCs. The results showed that 35.2% of meiocytes displayed punctuate or short liner ZEP1 signals. Among them, the SC level was less than 20%. 32.7% of meiocytes showed a few longer ZEP1 stretches and SC level was between 20% and 50%. In 27.3% of meiocytes, many long ZEP1 tracks were presented along the chromosomes and SC level reached 50% to 80%. Only 4.8% of meiocytes showed high level loading of ZEP1 signal and SC assembly was close to 80% to 100% (Fig. 2d).

Previous studies have reported that Arabidopsis hop2(ahp2) mutants have a prolonged

zygotene (Stronghill et al., 2010). The low degree of fully synapsed cells in Oshop2 mutants may cause by the delay of meiosis. Therefore, we examined the stages of meiosis in a set of primary inflorescences of wild type and Oshop2 by immunostaining using an antibody against ZEP1. Rice spikelets with a size distribution (from small to large) were selected to detect the level of SC in each spikelet (Fig. S5c). The results showed that the Oshop2 meiocytes still exhibited short linear ZEP1 signals when the SCs have been completed in wild type meiocytes (3.7-4.1 mm). The average SC level in Oshop2 was less than 20%. With the increase of the spikelet length, the SC assembly was gradually increasing. The average SC level reached a maximum of about 55% in the 5.1-5.3 mm long spikelets. However, different Oshop2 meiocytes showed considerable variation in SC assembly during this stage (the values of SC level ranged from 10% to 90%). Together, these results indicate that the lack of OsHOP2 protein delays as well as disrupts the full assembly of SC in rice.


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OsHOP2 is dynamically localized to chromatin during meiosis To investigate localization of the OsHOP2 protein in meiosis, we performed

immunostaining in wild-type meiocytes, using a polyclonal antibody against OsHOP2 raised from mouse. PAIR3, an axial protein, indicates the meiotic progression (Wang et al., 2011). OsHOP2 was first detected as punctuated foci at leptotene. At zygotene, with chromosome aggregation, OsHOP2 signals were present with a high signal intensity. At pachytene, the PAIR3 signal extended along the entire chromosomes and OsHOP2 was detected along the length of the chromosome. The OsHOP2 labeling was thicker than that of PAIR3. OsHOP2 signals gradually became weak, and were no longer detectable at diakinesis (Fig. 3a). In order to observe the localization of OsHOP2 on chromosome more clearly, immunocytochemistry combined with a super-resolution structured illumination microscope (SIM) was used. The high resolution of the SIM provides more details for protein localization (Lambing et al., 2015; Lee et al., 2015). Results showed that the dense OsHOP2 signals were localized to chromatin loop and some bright OsHOP2 foci exhibited colocalization with axes protein (Fig. 3b and 3c).

Previous studies found that OsMTOPVIB was also localized on chromosomes as

punctuate foci (Xue et al., 2016). Thus, dual-immunolocalization was detected between OsHOP2 and OsMTOPVIB, using a polyclonal antibody against OsHOP2 raised from guinea pig. At leptotene, both OsMTOPVIB and OsHOP2 appeared as punctuate foci on chromosomes (Fig. S6a). Although some OsMTOPVIB foci displayed random overlap with OsHOP2 signals, most signals did not colocalize with OsHOP2. At zygotene, both OsMTOPVIB and OsHOP2 signals were present with high signal intensity due to the chromosome aggregation (Fig. S6b). During pachytene, OsMTOPVIB and OsHOP2 were detected along chromosomes, and no obvious colocalization was observed (Fig. S6c and S6d).


We also detected the distribution of OsHOP2 in other mutants. We investigated OsHOP2

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loading in mutants with disrupted meiotic initiation of recombination (Ospo11-1), DSB processing (Oscom1), strand invasion and homology search (Osdmc1). The distribution of OsHOP2 was not affected in these mutants (Fig. S7a-7c), showing that localization of OsHOP2 is independent of the recombination processes. Moreover, OsHOP2 signal was undetectable in Oshop2 mutants in both western blot and cytology experiments, indicating the specificity of the OsHOP2 antibody (Fig. S8a-S8c).

OsHOP2 shows considerable co-localization with SC proteins and interacts with ZEP1 To explore the role of OsHOP2 in SC formation, dual-immunolocalization was detected

between OsHOP2 and SC proteins. Axial element formation of SC requires OsREC8, which is a component of the cohesion complex (Shao et al., 2011). ZEP1 is a central element that bridges two parallel lateral elements (LEs) (Wang et al., 2010). Using super-resolution SIM on wild-type meiocytes, the results showed that two parallel OsREC8 linear tracks were distributed along the entire length of the chromosomes at pachytene. OsHOP2 surrounded OsREC8 staining with high signal density and numerous signals displayed co-localization with OsREC8 as bright foci (Fig. 4a). Although the corresponding signals of OsHOP2 were located on the axis of the chromosome, some obvious OsHOP2 signals also appeared to be co-localized with ZEP1 linear signals, exhibiting bright yellow signal points (Fig. 4b). Furthermore, quantitative colocalization analysis was performed and several colocalization parameters were determined (Rr, R, and ICQ) (Fig. S9). The results showed that OsHOP2 had considerable co-localization with OsREC8 and ZEP1. Overall, the considerable co-localization between OsHOP2 and SC components provide direct evidence that OsHOP2 might promote SC assembly, or stabilize the structure of SCs.

In order to identify the mechanism of OsHOP2 in regulating the formation of SCs, we

performed yeast two-hybrid assays between OsHOP2 and ZEP1. Surprisingly, the Y2H experiments showed that OsHOP2 can interact with ZEP1. The central element was aligned in head-to-head, with its N terminus in the central region and C terminus positioned in the LE (de Boer & Heyting, 2006). We divided ZEP1 protein into segments with different sizes according to its domain. The results showed that 135-240 amino acid residues (a coiled-coil


motif) of ZEP1 protein were sufficient to establish the interaction with OsHOP2 (Fig. 4c). Also, these interactions were further confirmed in BiFC assays (Fig. 4d). Our data showed

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that the LZ1-LZ2 domains of OsHOP2 were essential for interaction with ZEP1 and itself (Fig. 4e and Fig. S10). Taken together, these results suggested that OsHOP2 can form a complex with ZEP1 to promote the completion of SCs, possibly by stabilizing the structure of SCs.

The function of OsHOP2 is dependent on early recombination events The univalents in Oshop2 led us suspect that DSB may be reduced. γH2AX is a reliable

DSB marker has been used to monitor the level of DSBs (Xue et al., 2016). OsREC8, as an axial element, is indicative of meiotic chromosomes (Shao et al., 2011). In Oshop2, the number of γH2AX foci (197 ± 7, n=15), were not significantly different from wild type (216 ± 8, n=15) at early zygotene (Fig. 5a and 5b). These results suggest that OsHOP2 is not required for DSB formation. Additionally, we also examined the signal distribution of meiotic recombination factors, including OsCOM1 and OsDMC1 in both wild type and mutants. OsCOM1 is involved in DSB end-processing and is essential for rice meiotic recombination (Ji et al., 2012). OsDMC1, an important recombinase, plays a central role in homologous recombination (Wang et al., 2016). Immunostaining results revealed that the loading and foci of these two factors were not significantly different between wild type and Oshop2 (Fig. 5a and 5b), suggesting that early recombination events occurred normally in Oshop2.

To explore the function of OsHOP2 in the meiotic recombination pathway, we generated

pair1 Oshop2 double mutants. Rice PAIR1 is thought to be the homolog of AtPRD3, which is involved in DSB formation in Arabidopsis (De Muyt et al., 2009). The double mutants showed a typical pair1 phenotype: asynaptic chromosomes were present at pachytene and 24 univalents were observed at metaphase I (Fig. 5c). In addition, we generated Oscom1 Oshop2 and Osrad51c Oshop2 double mutants. In Oscom1, both homologous pairing and synapsis were abolished at pachytene, and an entangled chromosome mass was detected at metaphase I. OsRAD51C is essential for meiotic DSB repair (Tang et al., 2014). In Osrad51c, homologous pairing and synapsis were defective at pachytene and extensive chromosome fragments were produced at metaphase I. The phenotypes of Oscom1 Oshop2 and Osrad51c This article is protected by copyright. All rights reserved.

Oshop2 double mutants were similar to that of the Oscom1 and Osrad51c single mutants (Fig.

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5c). These results suggested that the function of OsHOP2 is dependent on the presence of meiotic DSBs and functions after OsCOM1 and OsRAD51C in the DSB repair cascade.

CO maturation is abolished following loss of OsHOP2 To quantify the number of chiasmata in Oshop2 and wild type, we investigated the shape

of bivalents at diakinesis according to previously described criteria (Moran et al., 2001). Rod-shaped bivalents are considered to have one chiasma, while ring-shaped bivalents are treated as having two chiasmata. The chiasma frequency in wild type was 19.8±1.52 (n=33) per cell, while in Oshop2 mutant it was 3.21±1.29 (n=235) per cell, corresponding to 3.01 bivalents per cell (Fig. 7c). Previous study revealed that HEI10 could indicate the first class CO (CO I) (Wang et al., 2012). In wild type, bright HEI10 foci were observed (23.5 ± 3, n=33) at late pachytene (Fig. 6a). Compare to wild type, the appearance of the bright dot-like HEI10 signals was delayed in Oshop2 mutants. At late pachytene, immature HE10 foci seem to be clustered on the synapsed stretches. This may be caused by the delayed SC assembly. At diplotene, the prominent HEI10 foci were significantly decreased in Oshop2 (12.3±5.9, n=29) (Fig. 6a). Collectively, these results indicated that CO maturation was disturbed in Oshop2. To investigate whether COs occurred at sites where chromosome synapsed, we carried

out co-immunolocalization of ZEP1 and HEI10 in wild-type and Oshop2 meiocytes. In wild type, the HEI10 linear signals displayed extensive co-localization with ZEP1 at early pachytene. At pachytene, most HEI10 linear signals were split into linear arrays of dots. During late pachytene, the bright HEI10 foci were always present on the synapsed regions (Fig. 6b). In Oshop2 mutants, a number of HEI10 linear signals were present on the regions labeled by ZEP1 at early pachytene. At pachytene, the discontinuous punctate HEI10 signals were more apparent and were localized on ZEP1 linear signals. Finally, the bright HEI10 foci were mostly found on the stretches of SC at late pachytene (81.2%, n=29) (Fig. 6b). These results indicated that mature SC has a positive correlation with maturation of CO.


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OsHOP2 promotes CO maturation upstream of ZMM proteins The reduction of chiasmata in Oshop2 prompted us to explore the relationship between

OsHOP2 and ZMM proteins. OsZIP4 and OsMER3, two ZMM proteins, are involved in the formation of interference-sensitive COs (Wang et al., 2009; Shen et al., 2012). In Oshop2 mutants, the signal patterns and numbers of OsZIP4 and OsMER3 were not significantly different from wild type at zygotene (Fig. 7a and S11a). These results suggested that the early CO designation occur normally in Oshop2. In addition, we generated the Oszip4 Oshop2 double mutant and investigated its chromosome behaviors. In Oszip4, fully paired and synapsed chromosomes were detected during pachytene, and a mixture of univalents and bivalents were present at metaphase I (Fig. 7b). However, homologous pairing and synapsis were completely abolished at pachytene in the double mutant. During metaphase I, many univalents and a few bivalents were detected in Oszip4 Oshop2 (Fig. 7b). Compared with Oszip4 (5.97±1.61, n=36), the CO numbers were further reduced in Oszip4 Oshop2 (1.57± 1.14, n=35) (Fig. 7c). Based on these data, we speculated that OsHOP2 might also promote the formation of class II COs in Oszip4 background.

In order to determine whether the maturation of CO depend on the SC, we generated the

zep1 Oshop2 double mutant and investigated its chromosome behavior. In zep1, two threads of homologous chromosomes were always visible, whereas they could align along the entire chromosome at pachytene. And 12 bivalents aligned on the equatorial plate at metaphase I (Fig. 7b). In zep1 Oshop2, homologous pairing and synapsis were abolished at pachytene and many univalents were detected at metaphase I (Fig. 7b). The chromosome behavior in zep1 Oshop2 was similar to those in Oshop2 rather than in zep1. Compared with Oshop2 (3.21 per cell), the CO number were further reduced in the double mutant (1.35±1.12, n=88) (Fig 7c). These results suggested that OsHOP2 functions upstream of ZEP1 during CO formation.

The co-localization analysis of OsHOP2 with OsMER3, HEI10 and OsDMC1 showed

that no co-localization of OsHOP2 with OsDMC1 and OsMER3 was detected. Instead, they were always randomly overlapped (Fig. S11b and S11d). Moreover, the bright foci of HEI10


at late pachytene did not exhibit obvious co-localization with OsHOP2 signals (Fig. S11c).

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These results suggested that OsHOP2 does not associate specifically with the sites for meiotic recombination. This is consistent with the observation of MND1 in Arabidopsis and yeast (Zierhut et al., 2004; Vignard et al., 2007)

Discussion Functional conservation of HOP2 in meiosis Previous studies have shown that HOP2 plays an essential role in promoting

homologous pairing and synapsis in yeast and mouse. Synapsis takes place mostly between nonhomologous chromosomes in the absence of HOP2. It is difficult to observe the products of recombination, due to hop2 arrests at meiotic prophase I (Leu et al., 1998; Petukhova et al., 2003). However, in yeast spo11 hop2 double mutant, the meiotic arrest is bypassed, suggesting the delayed cell cycle is triggered by a defect in recombination and/or synapsis (Leu et al., 1998).

In Arabidopsis hop2, prolonged meiotic progress and a substantial amount of pairing

and synapsis were observed (Stronghill et al., 2010). Moreover, one Arabidopsis hop2 allele displays chromosome bridges and fragmentation, while the other allele exhibits intact chromosomes (Uanschou et al., 2013). HOP2 and MND1 can form a stable complex during meiosis (Tsubouchi & Roeder, 2002; Pezza et al., 2007). Homologous chromosome pairing and synapsis are disrupted and entangled mass of chromosomes are presented in Arabidopsis mnd1 (Vignard et al., 2007). These data suggest HOP2/MND1 function in promoting homologous DNA repair.

Here, we found that the chromosome pairing and SC assembly were delayed in Oshop2

mutants. The Oshop2 meiocytes showed a considerable variation in SC length and a few cells even reached the level of wild type. Compare to Arabidopsis, abnormal chromosomal associations were observed in very few cells and chromosome fragmentation was rarely detected. Although the initiation of recombination and CO designation occurred normally in Oshop2 mutants, mature COs were severely reduced. Our results also provided direct


evidence that OsHOP2 was involved in assembly or stabilization of the SC structure. Based on these data, we speculate that OsHOP2 do play a role in mediating the maturation of CO. It

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may facilitate or stabilize the interplay between recombination complex and SC elements to ensure CO maturation.

The interaction between OsHOP2 and ZEP1 indicates that OsHOP2 plays a key role in

promoting the full-length pairing and synapsis and regulating IH CO formation. In the absence of OsHOP2, homologous pairing and synapsis are disturbed and the HEI10 bright foci only localize on the synapsed region (Fig 8). We suspect that OsHOP2 acts as an important meiotic modulator to coordinate homologous pairing, synapsis, and recombination.

OsHOP2 provides a link between homologous recombination and SC formation Meiotic recombination and SC are closely linked. SC is preferentially nucleated at

CO-designated sites at early prophase I. And the CO sites are always distributed along the SC central components at late prophase I (Kleckner, 2006). However, the connection between synapsis and CO formation is not well understood mechanistically. The SC might monitor the interhomolog interactions, with its formation signal that recombination intermediates are properly formed. The SC may stabilize chromosome structure around sites of COs to regulate recombination (Zickler & Kleckner, 2015).

In Oshop2, the maturation of HEI10 bright foci is delayed, which might be tightly

coupled with delayed SC assembly. Similar observations were also observed in other meiotic mutants. In Arabidopsis pch2 and pss1 mutants, HEI10 or MLH1 foci (mark mature CO sites) are only associated with stretches of SC (Duroc et al., 2014; Lambing et al., 2015). Recently, it was reported that rice ZYGO1 promotes homologous pairing, synapsis and CO formation by mediating bouquet formation (Zhang et al., 2017). In these mutants, DSB formation and early recombination intermediates are successfully established, while the intermediates do not mature into COs. Although these mutants exhibit similar phenotypes to OsHOP2, they may regulate CO formation through different pathways.


Although the maturation of CO coupled with SC assembly, the function of SC on

CO

formation is limited. In yeast, zip1 shows modest defects in meiotic recombination but

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completely abolishes the CO interference (Sym & Roeder, 1994). Similarly, in rice zep1, the increased COs suggest that SC might function in inhibiting excessive CO formation (Wang et al., 2010). SC may prevent the inappropriate dismantlement of CO intermediates (Woglar & Villeneuve, 2018). Here, the chromosome behavior in zep1 Oshop2 suggests OsHOP2 functions upstream of ZEP1 and the interference of SC on CO maturation depends on the formation of stable recombinant intermediates. OsHOP2 does play a crucial role in maintaining the stabilization of recombinant interactions.

The interaction between OsHOP2 and ZEP1 suggests that OsHOP2 may be involved in

the assembly or stabilization of SCs. Alternatively, OsHOP2 may mediate the interaction of ZEP1 with other proteins, including SC elements and recombinant proteins. It is also possible that OsHOP2 may stable association of recombination complexes with chromosome axes or SCs, to promote pairing, synapsis, and recombination process.

OsHOP2 acts as a key regulator on meiotic chromatin loops In yeast, HOP2 localizes to meiotic chromatin as punctate foci and its localization are

not affected in spo11 mutant (Leu et al., 1998). HOP2 and MND1 can form a stable complex during meiosis (Tsubouchi & Roeder, 2002; Pezza et al., 2007). In yeast, MND1 localizes to chromatin throughout meiotic prophase I and its localization is also independent of SPO11. Moreover, MND1 staining is more diffuse and continuous, whereas RAD51 displays discrete foci (Tsubouchi & Roeder, 2002). In Arabidopsis, MND1 localized to chromatin and its loading is not affected in the absence of recombination, axis formation, or cohesion. And there is no obvious colocalization between MND1 and DMC1 foci (Vignard et al., 2007). In this study, we showed that OsHOP2 protein was extensively distributed on chromatin

as diffuse foci and its localization is independent of the recombination processes, suggesting that the function of HOP2 is evolutionarily conserved during meiosis. Moreover, lack of significant co-localization between OsHOP2 and other recombinant proteins

suggests that

OsHOP2 does not associate specifically with the sites for meiotic recombination. Previous


studies have shown that OsMTOPVIB and AtSPO11-1 display similar distribution of signal

Accepted Article

on chromatin loop during meiosis (Xue et al., 2016; Choi et al., 2018). The tethered loop-axis complex is important for controlling DSB formation (Panizza et al., 2011). The localization of them on chromatin might be closely related to their functions in DSB formation. No remarkable co-localization between OsMTOPVIB and OsHOP2 indicates that OsHOP2 might not be involved in the formation of DSB. In yeast, they favor a view that HOP2/MND1 may has a genome wide positive effect.

They may antagonize structural constraints of chromosomes and make the homolog accessible for DSB repair (Zierhut et al., 2004). Obviously, HOP2/MND1 is rich in leucine zipper motif, which is responsible for binding DNA. Biochemical evidence also shows that HOP2-MND1 have the ability to bind DNA (Pezza et al., 2007). Accordingly, HOP2/MND1 might regulate gene expression by binding to the regulatory region of genes. In general, the function of OsHOP2 on chromatin needs to be further studied to elucidate the role of OsHOP2 in coordinating homologous pairing, synapsis, and meiotic recombination in meiosis.

Acknowledgements This work was supported by grants from the Ministry of Sciences and Technology of China (2016YFD0100901), and the National Natural Science Foundation of China (31500255 and 31771363).

Author contributions Z.C. and W.S. designed the research project. D.T. and Y.L. supervised the experiments. W.S., D.T., Y.S., Z.X., F.Z., C.Z, L.R., C.L., C.Y. and G.D. performed most of the experiments. W.S. and D.T. analyzed the image data. W.S. wrote the paper. Z.C. supervised and complemented the writing. W.S. and D.T. contributed equally to this work.


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Figure legends

Fig. 1 Identification of rice (Oryza sativa) Oshop2 mutant and meiotic chromosome behaviors in Oshop2 meiocytes. (a) Comparison of a wild type (WT) plant (Left) and Oshop2 mutant plant (Center). Pollen grains stained in 1% I2-KI solution in a WT plant (Upper Right) and Oshop2 plant (Lower Right). Bars=50 μm. (b) Schematic representation of OsHOP2 gene and the mutation sites in OsHOP2, including Oshop2-1, Oshop2-2, Oshop2-3 and Oshop2-4. Coding regions are shown as black boxes, and untranslated regions are shown as gray boxes. (c) OsHOP2 contains an N-terminal winged-helix domain (WHD) (AAs 18-68) and three leucine zippers: LZ1 (AAs 77-114), LZ2 (AAs 118-148), and LZ3 (AAs 155-227). (d) Meiotic chromosome behaviors in both WT and Oshop2 mutant. Bars=5 μm.

Fig. 2 OsHOP2 is required for the full-length homologous pairing and synapsis in rice (Oryza sativa). (a) The chromosome pairing status in wild type (WT) and Oshop2. The painting probes labeled by red (11L) and green (11S) signal, to track the long and short arms of chromosome 11. Chromosomes were stained with 4',6-diamidino-2-phenylindole (DAPI, blue). Left panel, fully paired pachytene chromosomes in WT; middle panel, the Oshop2 meiocytes show partially paired signals of two probes; right panel, the Oshop2 meiocytes display unpaired signals. Bars=5 μm. (b) Immunostaining for ZEP1 (green) in WT and Oshop2 at pachytene. OsREC8 signals (red) indicate the meiocytes. Bars=5 μm. (c) Histogram of cells according to their pairing status. (d) Histogram of cells according to their proportion of synapsed regions.

Fig. 3 OsHOP2 is dynamically localized to chromatin during rice (Oryza sativa) meiosis. (a) Dual immunolocalization of PAIR3 (red, from guinea pig) and OsHOP2 (green, from mouse) in wild type (WT) meiocytes. PAIR3 is used to indicate the meiotic chromosomes. OsHOP2 proteins are present as punctuated foci at leptotene and display a high signal intensity region at zygotene. At pachytene, OsHOP2 signals are detected along the entire length of the chromosome and are no longer detectable at diakinesis. Bars=5 μm. (b) Structured illumination microscopy (SIM) is used to observe the distribution of OsHOP2 at pachytene. Chromosomes were stained with DAPI (blue). Bar=5 μm. (c) A single chromosome image shows that dense OsHOP2 signals localized to the chromatin loop and some bright OsHOP2 foci exhibit colocalization with axes protein. Bar=5 μm.

Fig. 4 OsHOP2 shows considerable co-localization with SC proteins and interacts with ZEP1 in rice (Oryza sativa). (a) Immunostaining of OsHOP2 (green, from mouse) and OsREC8 (red, from rabbit) in wild type (WT) meiocytes at pachytene. Numerous OsHOP2 signals display co-localization with OsREC8 as bright foci. Magnified images of the blocked regions are shown in the right panels with channels separated out (top, middle) and merged (bottom). Bar=5 μm. (b) Immunostaining of OsHOP2 (green, from mouse) and ZEP1 (red, from guinea pig) in WT meiocytes at pachytene. Massive OsHOP2 signals are clearly co-localized with ZEP1 linear signals, exhibiting bright yellow signal points. Magnified images of the blocked regions are shown in the right panels. Bar=5 μm. (c) OsHOP2 interacts with ZEP1 in Y2H assays. Interactions


Accepted Article

between bait and prey were examined on the control media 2 (SD/-Leu/-Trp) and selective media 4 (SD/-Ade/-His/-Leu/-Trp). AD, prey vector; BD, bait vector. (d) LZ1-LZ2 domains of OsHOP2 are essential for the interaction with ZEP1 in Y2H assays. (e) BiFC assays show the interaction between OsHOP2 and ZEP1 and OsHOP2 with itself in rice protoplasts. Bars=5 μm.

Fig. 5 The function of OsHOP2 is dependent on early recombination events in rice (Oryza sativa). (a) Immunolocalization of γH2AX (red), OsCOM1 (green), and OsDMC1 (green) at early prophase I stage in WT and Oshop2. Bars=5 μm. (b) Quantification of γH2AX, OsCOM1, and OsDMC1 foci per meiocyte in WT and Oshop2. Statistical analyses reveals no significant differences (NS) between the wild type (WT) (n values are 15, 12 and 16, respectively) and Oshop2 (n values are 12, 12 and 16, respectively). The P values are 0.1097, 0.6237, and 0.5986, respectively, from two-tailed Student’s t-tests. NS, not significant. (c) Genetic analysis of OsHOP2 with PAIR1, OsCOM1 and OsRAD51C. The pair1 Oshop2 double mutant shows a typical pair1 phenotype, indicating OsHOP2 is dependent on the presence of meiotic DSBs. The phenotype of Oscom1 Oshop2 and Osrad51c Oshop2 double mutants are similar with that of the Oscom1 and Osrad51c single mutant. Bars=5 μm.

Fig. 6 Crossover (CO) maturation is abolished in Oshop2 meiocytes in rice (Oryza sativa). (a) Immunolocalization of HEI10 is shown in wild type (WT) (left panel) and in Oshop2 (middle panel). Right panel, scatter plot of HEI10 in WT (n values are 33) and Oshop2 (n values are 29). ****P