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HIGH CROSSOVER RATE1 encodes PROTEIN PHOSPHATASE X1 and restricts meiotic crossovers in Arabidopsis

Nature Plants volume 7, pages 452–467 (2021)Cite this article

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Abstract

Meiotic crossovers are tightly restricted in most eukaryotes, despite an excess of initiating DNA double-strand breaks. The majority of plant crossovers are dependent on class I interfering repair, with a minority formed via the class II pathway. Class II repair is limited by anti-recombination pathways; however, similar pathways repressing class I crossovers have not been identified. Here, we performed a forward genetic screen in Arabidopsis using fluorescent crossover reporters to identify mutants with increased or decreased recombination frequency. We identified HIGH CROSSOVER RATE1 (HCR1) as repressing crossovers and encoding PROTEIN PHOSPHATASE X1. Genome-wide analysis showed that hcr1 crossovers are increased in the distal chromosome arms. MLH1 foci significantly increase in hcr1 and crossover interference decreases, demonstrating an effect on class I repair. Consistently, yeast two-hybrid and in planta assays show interaction between HCR1 and class I proteins, including HEI10, PTD, MSH5 and MLH1. We propose that HCR1 plays a major role in opposition to pro-recombination kinases to restrict crossovers in Arabidopsis.

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Fig. 1: A forward genetic screen for mutants with changed 420 crossover frequency.
Fig. 2: HIGH CROSSOVER RATE1 encodes PROTEIN PHOSPHATASE X1.
Fig. 3: Euchromatic crossover frequency increases and crossover interference decreases in hcr1 and meiMIGS-PPX1-PPX2.
Fig. 4: Genome-wide mapping of crossovers in meiMIGS-PPX1-PPX2.
Fig. 5: Meiotic MLH1 foci are elevated in hcr1 whereas RAD51, ASY1 and ZYP1 immunostaining are unchanged.
Fig. 6: HCR1 genetically and physically interacts with the class I crossover pathway.
Fig. 7: HCR1/PPX1–PP4 control of meiotic crossover recombination in Arabidopsis.

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Data availability

Genome sequencing data of F2 plants can be found at the ArrayExpress repository hosted by the European Bioinformatics Institute under accessions E-MTAB-9621 and E-MTAB-10168. Source data are provided with this paper.

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Acknowledgements

We thank G. Copenhaver, A. Levy and S. Poethig for FTLs/CTLs, R. Mercier for fancm-1, L. Ziolkowska and C. Underwood for helping grow the EMS population, M. Grelon for MLH1 antibodies, C. Franklin for ASY1, ZYP1 and RAD51 antibodies and the Gurdon Institute for access to microscopes. This work was funded by the Suh Kyungbae Foundation (Jaeil Kim, Juhyun Kim, J.P., E.-J.K., H.K., D.B., Y.M.P. and K.C.), Next-Generation BioGreen 21 Program PJ01337001 (Jaeil Kim, Juhyun Kim, J.P., E.-J.K., H.K., D.B., Y.M.P. and K.C.) and PJ01342301 (H.S.C., S.L. and I.H.), Rural Development Administration, Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education NRF-2020R1A2C2007763 (H.K., D.B. and K.C.), Marie Curie International Training Network ‘COMREC’ (DN), Biotechnology and Biological Sciences Research Council grant EpiSpiX BB/N007557/1 (X.Z. and I.H.), Biotechnology and Biological Sciences Research Council ERA-CAPs grant BB/M004937/1 (C.L. and I.H.) and European Research Council Consolidator Award ERC-2015-CoG-681987 ‘SynthHotSpot’ (C.L., A.J.T. and I.H.).

Author information

Author notes

  1. These authors contributed equally: Divyashree C. Nageswaran, Jaeil Kim.

Authors and Affiliations

  1. Department of Plant Sciences, University of Cambridge, Cambridge, UK

    Divyashree C. Nageswaran, Christophe Lambing, Pallas Kuo, Andrew J. Tock, Xiaohui Zhao, Kyuha Choi & Ian R. Henderson

  2. Department of Life Sciences, Pohang University of Science and Technology, Pohang, Republic of Korea

    Jaeil Kim, Juhyun Kim, Jihye Park, Eun-Jung Kim, Hyun Seob Cho, Heejin Kim, Dohwan Byun, Yeong Mi Park, Seungchul Lee, Ildoo Hwang & Kyuha Choi

Authors
  1. Divyashree C. Nageswaran
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  2. Jaeil Kim
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  3. Christophe Lambing
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  4. Juhyun Kim
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  5. Jihye Park
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  6. Eun-Jung Kim
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  7. Hyun Seob Cho
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  8. Heejin Kim
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  9. Dohwan Byun
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  10. Yeong Mi Park
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  11. Pallas Kuo
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  12. Seungchul Lee
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  13. Andrew J. Tock
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  14. Xiaohui Zhao
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  15. Ildoo Hwang
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  16. Kyuha Choi
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  17. Ian R. Henderson
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Contributions

Design and conception of experiments: D.C.N., Jaeil Kim, C.L., Juhyun Kim, J.P., E.-J.K., P.K., K.C. and I.H. Acquisition and analysis of data: D.C.N., Jaeil Kim, C.L., Juhyun Kim, J.P., H.S.C., H.K., D.B., Y.M.P., P.K., S.L., A.J.T., X.Z., I.H. and K.C. Writing of the manuscript: D.C.N., Jaeil Kim, C.L., Juhyun Kim, K.C. and I.H.

Corresponding authors

Correspondence to Kyuha Choi or Ian R. Henderson.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Plants thanks the anonymous reviewers for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 420 crossover frequency in wild type and M2 plants derived from the EMS population.

Box and whisker plot showing 420 crossover frequency (cM) for wild type (Col/Col) 420/++ plants (n=75) and EMS-treated M2 420/++ plants (n=1,217). Black dots indicate 420 crossover frequency in individual plants. Horizontal lines of black (wild type, Col) and red (EMS M2) box plots represent maximum, 3rd quartile, median, 1st quartile and minimum in 420 cM, respectively. In this study, wild type plants show a mean value of 19.5 cM (standard deviation=1.5) within 420, and the majority (81.4%, 991/1,217) of M2 plants display 420 crossover frequency within the range of 18-22 cM (Mean=21.4 cM, SD=1.5). 420 crossover frequency in M2 plants was significantly increased compared to wild type (one-sided Welch’s t-test P=2.2×10−16), which may have been caused by heterozygous EMS polymorphisms.

Extended Data Fig. 2 EMS mutations identified in FANCM (hcr4) and TAF4b (lcr1).

a, FANCM gene structure is shown, including the EMS mutation site in hcr4/fancm-11. The red arrow indicates the G to A substitution within exon 15, which causes a G to S amino acid substitution. Exons are shown as boxes (black=CDS, grey=UTR). Scale bar=0.5 kb. b, Multiple sequence alignment of the DEHDc (blue line) and HELICC (green line) domains of FANCM in different species. The mutation positions of the fancm-1 to fancm-10 alleles that were previously identified, and fancm-11 (hcr4), are shown. The fancm-11 mutation is located in a conserved motif within the SF2 helicase domain (bold arrow). c, Gene structure of TAF4b is shown with the location of the lcr1 (taf4b-3) mutation indicated in exon 3 (red arrow), which causes a premature stop codon.

Extended Data Fig. 3 T-DNA insertions in Arabidopsis PP4/PPX complex genes.

a, The gene structures of PPX1 (At4g26720), PPX2 (At5g55260) and PP4R2 (At5g17070) are shown. Exons are shown as boxes (black=CDS, grey=UTR). Scale bar=0.5 kb. The EMS induced hcr1-1 mutation is located at the splice donor site of the 3rd intron, shown by the asterisk. The red arrows indicate the location of primers for RT-qPCR in PPX1 and PPX2. The hcr1-2 T-DNA (GK_651B07) insertion position in the 5´-UTR is indicated. The position of the ppx2-1 (GK_488H09), ppx2-2 (SALK_049725), and pp4r2 (SALK_093051) T-DNA insertions are shown, which are located in the 4th intron, 8th exon and 7th intron, respectively. The arrows spanning the ppx2 and pp4r2 T-DNA insertions indicate primer positions used for RT-PCR. b, RT-PCR amplification and quantification for PPX1, PPX2 and PP4R2 mRNA expression in wild type Col, hcr1-1, ppx1-2, ppx2-1 and pp4r2. Floral cDNA from two biological replicates were evaluated by RT-PCR amplification for PPX1, PPX2, PP4R2 (shown in a) and GAPC expression. RT-PCR amplicon sizes for wild type, hcr1-1, ppx1-2, ppx2-1, pp4r2 cDNAs and wild type genomic DNA (positive/negative control) are shown. c, Plot showing RT-qPCR enrichment of PPX1 and PPX2 in hcr1-1 and ppx2-1. Relative transcript levels of PPX1 and PPX2 were measured in wild type, hcr1-1, and ppx2-1 using qRT-PCR. TUB2 was used for normalization. The y axis indicates fold-enrichment of PPX1 and PPX2 transcript levels, compared to PPX1 and PPX2 in wild type. RT-qPCR reactions of two technical replicates for each of four biological samples were shown as dots. Mean values are indicated by horizontal lines. Significance between wild type and mutants was assessed by one-sided Welch’s t-tests. The P values between Col and hcr1-1 for PPX1 was 0.186, for PPX2 was 3.49×10−5; between Col and ppx2-1 for PPX1 was 1.77×10−10 and for PPX2 was 3.64×10−9. Asterisks indicate P<0.001. d, Photograph showing developmental phenotypes of wild type, hcr1-2, hcr1-1, ppx2-1, hcr1-2 ppx2-1 and hcr1-1 ppx2-1 grown alongside one another. e, Photograph showing seeds of wild type and hcr1-1/+ ppx2-2/+ plants. Asterisks indicate defective seeds. f, Photograph showing F2 seedlings grown from self-fertilization of F1 hcr1-1/+ ppx2-2/+ plants, with asterisks indicating developmentally delayed seedlings.

Extended Data Fig. 4 Alignment of PP4 homolog protein sequences from diverse eukaryotes.

a, Amino acid sequence alignment of AtPPX1, the predicted hcr1-1 truncated protein, AtPPX2 and PP4 homologs from different eukaryotic species. The predicted hcr1-1 truncated protein consisting of 143 residues is shown. The underlined region indicates amino acids generated due to the retention of the 3rd intron. Hash symbols indicate the locations of conserved PP4 catalytic motifs (GDXHG, GDXVDRG and GNHE) and the histidine (H) residues required for metal binding in C-terminal region. b, As for a, but showing percent identity of amino acid sequence between PP4 homologs.

Extended Data Fig. 5 Meiosis-specific knockdown of PPX1 and PPX2 in meiMIGS transgenic plants.

a, qRT-PCR analysis of PPX1/HCR1 and PPX2 transcripts in floral buds of wild type and meiMIGS-PPX1, meiMIGS-PPX2 and meiMIGS-PPX1-PPX2 T2 transgenic lines. The y axis indicates fold-enrichment of PPX1 and PPX2 transcripts, compared to PPX1 in wild type. DMC1 was used as a meiotic gene for normalization. Replicate measurements are shown as dots and mean values shown by horizontal lines. b, Correlation between PPX1 and PPX2 transcript levels in wild type, meiMIGS-PPX1, meiMIGS-PPX2, and meiMIGS-PPX1-PPX2 lines. The x and y axis indicate relative PPX1 and PPX2 transcript levels in meiMIGS-PPX1 (blue), meiMIGS-PPX2 (red), and meiMIGS-PPX1-PPX2 (green) lines respectively, compared to PPX1 and PPX2 expressions in wild type Col plant. The correlation coefficient between PPX1 and PPX2 expression values across these samples was r=0.80, which was significantly different than expected if there were no relationship (P value=1.21×10−5).

Extended Data Fig. 6 Crossover frequency and interference measured in wild type and hcr1-1 using fluorescent pollen.

a, Crossover frequency measured using the pollen FTLs I1bc and I3bc from wild type and hcr1-1. Crossover frequency in each interval of the three-color FTLs was measured using the DeepTetrad pipeline3 (Supplementary Table 20). b, Crossover interference ratio measured using FTL pollen tetrads in wild type and hcr1-1. Crossover interference ratio (IFR) were calculated using the DeepTetrad pipeline. c, Plots showing the % of tetrads containing double crossovers, using data from the three-color FTL intervals in wild type and hcr1-1. d, As for c, but showing FTL data from the I1bc, I1fg, I3bc and I5ab intervals in wild type and meiMIGS-PPX1-PPX2. Tetrads were classified into 12 fluorescence classes (A-L) by DeepTetrad. Mean values are indicated by horizontal lines.

Source data

Extended Data Fig. 7 SPO11-1-oligonucleotides and nucleosome occupancy around wild type and meiMIGS-PPX1-PPX2 crossovers.

10 kb windows surrounding crossover midpoints identified from wild type or meiMIGS-PPX1-PPX2 plants, or the same number of randomly selected positions, were analysed for SPO11-1-oligos (log2(SPO11-1-oligos/gDNA), red) or nucleosome occupancy (log2(MNase-seq/gDNA), blue).

Extended Data Fig. 8 Yeast two hybrid assays showing interactions of HCR1/PPX1 with meiotic proteins.

a, Yeast two hybrid assays testing interaction between HCR1/PPX1 and Class I (ZMM) proteins. The yeast co-transformants were grown until OD600 = 1 and spotted on synthetic dropout media (SD) lacking leucine/tryptophan (-LT) and leucine/trptophan/histidine/adenine (-LTHA) for 3, 5 or 7 days. b, Yeast two hybrid assays of HCR1/PPX1 and meiotic proteins involved in axis formation, DSB formation and DNA repair. The yeast transformants were grown until OD600 = 1, then diluted 10-, 100- and 1,000-fold in water, and spotted on SD (-LT) and SD (-LTHA) plates to examine growth in 3, 5, or 7 days (Supplementary Table 23).

Extended Data Fig. 9 The EVH1 domain of Arabidopsis PP4R3A interacts with meiotic proteins.

a, Yeast two-hybrid assays testing interaction between the PP4R3A EVH1 domain and meiotic proteins. PP4R3A-N indicates the PP4R3A N-terminal region (1-166 aa) containing the EVH1 domain. The yeast co-transformants were grown until OD600 = 1 and spotted on synthetic dropout media (SD) lacking leucine/tryptophan (-LT) and leucine/trptophan/histidine/adenine (-LTHA) for 3 and 5 days. The yeast transformants were grown until OD600 = 1, then diluted 10-, 100- and 1,000-fold in water, and spotted on SD (-LT) and SD (-LTHA) plates to examine growth. b, Venn diagram summarizing yeast two hybrid assays of meiotic proteins that interact with HCR1/PPX1 and the PP4R3A EVH1 domain. c, A schematic model of Arabidopsis PP4 holoenzyme complex that recognizes target protein HEI10 for dephosphorylation via the PP4R3A EVH1 domain and PPX1.

Extended Data Fig. 10 Genome-wide prediction of PP4 complex target proteins during meiosis.

a, Protein domain (green) structure of Arabidopsis PP4 subunits PPX1, PPX2, PP4R2 and PP4R3. b, Amino acid alignment of the PP4R3A homolog EVH1 domain (red box). Hash symbols (#) indicate conserved tyrosine (Y) and tryptophan (W) residues. c, As for a, but showing the positions of FxxP motifs and phosphorylation consensus sites in PTD, HEI10, MSH5 and MLH1. d, Venn diagram showing overlap of meiotically expressed, nuclear proteins with FxxP motifs. e, Venn diagram showing overlap of candidate PP4 target proteins with CDK, DDK or ATM/ATR kinase consensus motifs, predicted using GPS 5.0. The location of HCR1 Y2H interactors are indicated within the Venn diagram. f, Histogram showing a significant enrichment of proteins containing phosphorylation sites in the predicted 1,367 PP4 targets, compared to 1,000 sets of randomly chosen genes (n=1,367). The vertical red line indicates observed predicted PP4 target proteins containing phosphorylation sites, compared to the random sets (black lines). g, Gene ontology (GO) enrichment analysis of the predicted PP4 targets, using PANTHER (http://pantherdb.org/). Benjamini-Hochberg False Discovery Rate (FDR) correction was used for enrichment test.

Supplementary information

Supplementary Information

Supplementary Tables 1–24.

Reporting Summary

Source data

Source Data Fig. 6

Unprocessed western blots for Fig. 6f.

Source Data Extended Data Fig. 3

Unprocessed DNA gels for Extended Data Fig. 3b.

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Nageswaran, D.C., Kim, J., Lambing, C. et al. HIGH CROSSOVER RATE1 encodes PROTEIN PHOSPHATASE X1 and restricts meiotic crossovers in Arabidopsis. Nat. Plants 7, 452–467 (2021). https://doi.org/10.1038/s41477-021-00889-y

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