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Control of meiotic crossover interference by a proteolytic chaperone network

Nature Plants volume 10, pages 453–468 (2024)Cite this article

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Abstract

Meiosis is a specialized eukaryotic division that produces genetically diverse gametes for sexual reproduction. During meiosis, homologous chromosomes pair and undergo reciprocal exchanges, called crossovers, which recombine genetic variation. Meiotic crossovers are stringently controlled with at least one obligate exchange forming per chromosome pair, while closely spaced crossovers are inhibited by interference. In Arabidopsis, crossover positions can be explained by a diffusion-mediated coarsening model, in which large, approximately evenly spaced foci of the pro-crossover E3 ligase HEI10 grow at the expense of smaller, closely spaced clusters. However, the mechanisms that control HEI10 dynamics during meiosis remain unclear. Here, through a forward genetic screen in Arabidopsis, we identified high crossover rate3 (hcr3), a dominant-negative mutant that reduces crossover interference and increases crossovers genome-wide. HCR3 encodes J3, a co-chaperone related to HSP40, which acts to target protein aggregates and biomolecular condensates to the disassembly chaperone HSP70, thereby promoting proteasomal degradation. Consistently, we show that a network of HCR3 and HSP70 chaperones facilitates proteolysis of HEI10, thereby regulating interference and the recombination landscape. These results reveal a new role for the HSP40/J3-HSP70 chaperones in regulating chromosome-wide dynamics of recombination via control of HEI10 proteolysis.

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Fig. 1: Isolation of hcr3 as a dominant-negative j3 mutant.
Fig. 2: Increased crossover frequencies in chromosome arms in hcr3 and J3::J3G155R plants.
Fig. 3: J3G155R expression increases crossovers in a hybrid context.
Fig. 4: hcr3 increases interfering crossovers and reduces interference.
Fig. 5: hcr3 elevates interfering crossovers and closely spaced crossovers per bivalent.
Fig. 6: J3 interacts with ZMMs and co-localizes with HEI10.
Fig. 7: HCR3 mediates proteolysis of HEI10.

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Data availability

Seeds of Arabidopsis transgenic plants used for this study are freely available on request. GBS data in this study are available in the ArrayExpress database at EMBL-EBL (http://www.ebi.ac.uk/arrayexpress) under the accession numbers E-MTAB-10168, E-MTAB-11586, E-MTAB-12663, E-MTAB-12692, E-MTAB-12694, E-MTAB-12695, E-MTAB-12696, E-MTAB-12697, E-MTAB-12726 and E-MTAB-13412–13415. RNA-seq data have been deposited at EMBL-EBI under accession numbers E-MTAB-12699 and E-MTAB-13417. Source data are provided with this paper.

Code availability

All custom codes involved in the paper are available at https://github.com/KyuhaChoi-Lab/HCR3.

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Acknowledgements

We thank G. Copenhaver (University of North Carolina at Chapel Hill), A. Levy (The Weizmann Institute of Science) and S. Poethig (University of Pennsylvania) for providing CTLs/FTLs. We thank C. Franklin (University of Birmingham) for providing ASY1 and RAD51 antibodies. We appreciate the support of IJPB plant observatory technological platforms. This research was supported by Suh Kyungbae Foundation SUHF-17020079, Samsung Science and Technology Foundation SSTF-BA2202-09, Next-Generation BioGreen 21 Program PJ01337001 and National Research Foundation of Korea NRF-2020R1A2C2007763 to K.C.; National Research Foundation of Korea NRF-2021R1A6A3A01087206 to J.K.; Agence Nationale de la Recherche CO-PATT-ANR-20-CE12-0006 and Saclay Plant Sciences-SPS ANR-17-EUR-0007 to M.G.; BBSRC grants BB/S006842/1, BB/S020012/1 and BB/V003984/1 to I.R.H.; European Research Council Consolidator Award ERC-2015-CoG-681987 to C.L. and I.R.H.; BBSRC grant BB/X011003/1 to C.L.; BBSRC Discovery Fellowship BB/V005774/1 to C.M.; BBSRC Institute Strategic Programme GEN BB/P013511/1 to M.H.

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Author notes

  1. These authors contributed equally: Heejin Kim, Jaeil Kim.

Authors and Affiliations

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

    Heejin Kim, Jaeil Kim, Namil Son, Dohwan Byun, Jihye Park, Youngkyung Lee, Yeong Mi Park, Ildoo Hwang & Kyuha Choi

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

    Pallas Kuo, Christophe Lambing & Ian R. Henderson

  3. Rothamsted Research, Harpenden, UK

    Pallas Kuo & Christophe Lambing

  4. John Innes Centre, Norwich Research Park, Norwich, UK

    Chris Morgan, John A. Fozard & Martin Howard

  5. Institut Jean-Pierre Bourgin (IJPB), Université Paris-Saclay, INRAE, AgroParisTech, Versailles, France

    Aurélie Chambon, Julie Guérin, Aurélie Hurel & Mathilde Grelon

  6. Department of Chromosome Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany

    Raphael Mercier

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Contributions

H.K., J.K., C.M., I.R.H. and K.C. designed the study. H.K., J.K., N.S., P.K., C.M., A.C., D.B., J.P., Y.L., Y.M.P., J.G., A.H., C.L. and K.C. performed experiments. H.K., J.K., D.B, J.P., Y.L., Y.M.P., I.H., R.M., I.R.H. and K.C. contributed to genetics, genomics and biochemical results. H.K., P.K., C.M., A.C., J.G., A.H., C.L. and M.G. contributed to cytological data. C.M. generated the data of HEI10 foci. P.K., A.C., J.G., A.H., C.L. and M.G. contributed to quantification of MLH1 foci. H.K., J.K., N.S., P.K., C.M., A.C., D.B., J.P., Y.L., Y.M.P., J.A.F, C.L., M.H., M.G., I.R.H. and K.C. analysed the results. N.S. conducted analyses of GBS and RNA-seq data. J.A.F analysed the HEI10 coarsening model. C.L., M.H., M.G., I.H., I.R.H. and K.C. contributed to the supervision. H.K., J.K. and K.C. wrote and revised the paper. All authors commented, discussed and provided input on the paper.

Corresponding author

Correspondence to Kyuha Choi.

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Nature Plants thanks Zhukuan Cheng and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Characterization of j3 and j2 T-DNA insertion mutants.

a, Gene structures for J3 and J2. Positions of T-DNA insertion (black triangles), hcr3 mutation (red triangle) and RT–PCR primers (arrows) are shown. b, Agarose gels showing RT–PCR products of J3 and J2 in Col, hcr3, j3-1, j3-3 and j2-2. c, Plot showing normalized transcript reads for J3 and J2 in RNA-seq of Col seedlings (J3, n = 4, J2, n = 4) and buds (J3, n = 4, J2, n = 4). Data are presented as mean values ± s.d. values of normalized transcript reads. n = the number of biologically independent samples. d, Principle component analysis of three replicates of RNA-seq libraries in Col, hcr3, j3-1, j3-3 and j2-2 seedlings. e, As for (d) but showing integrative genomic viewer windows showing the transcript levels for J3 and J2. f, Immunoblot analysis of transiently expressed epitope Myc-tagged and endogenous J3 and J2 in Col protoplasts. IB, immunoblot. CB, Coomassie blue. Anti-J3 antibody detects both J3 and J2 proteins (J3/J2). g,h, Immunoblot (g) and quantification (h) analyses of J3/J2 using anti-J3 antibody in seedlings and buds of Col, hcr3, j3-1, j3-3 and j2-2. J3/J2, seedling Col (n = 5), hcr3 (n = 5), j3-1 (n = 5), j3-3 (n = 5), j2-2 (n = 5); bud Col (n = 3), hcr3 (n = 3), j3-1 (n = 3), j3-3 (n = 3), j2-2 (n = 3). Immunoblots of tubulin and Coomassie blue staining were used for loading control and quantification. Black dots represent the normalized intensities of immunoblot replicates. n = the number of independent experiments. Red dots and horizontal lines indicate mean ± s.d. of intensities of immunoblot replicates (two-sided Welch’s t-test). i,j, Representative images (i) and quantification (j) of flowering time in Col, hcr3, j3-1, j3-3 and j2-2. Black dots represent leaf numbers of individual plants and red dots and horizontal lines indicate mean ± s.d. values of leaf numbers from individual plants (two-sided Welch’s t-test).

Source data

Extended Data Fig. 2 Functional redundancy between J3 and J2 in meiotic crossover frequency.

a, Seed (triangles) and pollen (circles) FTLs across the Arabidopsis genome. Lines represent the interval positions. b,c, Crossover frequency (cM) of pollen (b) and seed (c) FTLs in Col, j3-1, j3-3 and j2-2. d, 420 crossover frequency (cM) in Col, j3-1, j3-1, j2-2/+ j2-2 and j3-1/+ j2-2. e, As for (d) but showing Col, hcr3, j3-1, j3-1 J3::HA-J2 (T1), j3-1 meiMIGS-J2 (T1) and transgenic plants (T1) expressing HA-J2G156R under the J3, RPS5A and SPO11-1 promoters. Coloured (b,c) or black (d,e) dots represent cM values of individual plants. n = the number of individual plants. Black (b,c) or red (d,e) dots and horizontal lines indicate mean ± s.d. of cM values from individual plants (two-sided Welch’s t-test). f, Yeast two-hybrid analysis showing protein interactions of J3 and J3G155R with themselves and J2. g,h, Co-immunoprecipitation analysis showing dimerization of J3 and J3G155R with themselves (g) and J2 (h) in Arabidopsis protoplasts.

Source data

Extended Data Fig. 3 Functional redundancy between J3 and J2 in pollen development, embryogenesis and meiotic chromosome segregation.

a,b, Anthers containing Alexander-stained pollen grains (a) and plot (b) showing viable pollen grains per anther in Col, hcr3, j3-1, j3-3, j2-2, j3-1 j2-2/+ and j3-1/+ j2-2. Scale bars, 100 μm. Black dot indicates viable pollens of individual anther. Red dot and horizontal lines indicate mean values ± s.d. values of viable pollens for genotype. n = the number of individual anthers. c,d, As for (a,b) but showing representative images (c) and plot (d) for number of seeds per silique. White asterisks indicate aborted seeds. Scale bars, 1 mm. Blue and red dots indicate numbers of plump and aborted seeds per silique, respectively. Black dot and horizontal lines indicate mean values ± s.d. values of seed numbers for genotype. n = the number of individual siliques. e, Table of genotyping results and representative images showing seedling lethality of j3-1 j2-2 from the progeny of self-fertilized j3-1 j2-2/+ and j3-1/+ j2-2. Scale bars, 1 mm (top), 1 cm (bottom). f, As for (e) but showing plot and representative DAPI-stained images of chromosomal abnormalities at metaphase I or anaphase I. Red arrows indicate chromosome fragmentation or interlock. Scale bars, 5 μm. (b,d) Data are presented as mean values ± s.d. (two-sided Welch’s t-test).

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Extended Data Fig. 4 Genomic analyses for crossover number and distribution in Col × Ler and J3::J3G155R Col × Ler.

a, Schematic of generation of J3::J3G155R Col × Ler hybrid plants and crossover map populations. b, 420 crossover frequencies in Col × Ler (n = 7) and J3::J3G155R Col × Ler (#4, n = 8, #6, n = 10, #8, n = 10,) F1 hybrid plants. Black dots indicate cM values of individual plants. Red dots and horizontal lines represent mean ± s.d. of cM values from individual plants (two-sided Welch’s t-test). n = the number of individual plants. c, Average crossover number per chromosome in Col × Ler and J3::J3G155R Col × Ler F2 individuals. d, As for (c) but showing normalized crossover frequencies (cM/Mb) along chromosome arms from the telomere (TEL) to the centromere (CEN). e,f, As for (c,d), but showing male and female meiosis. g,h, As for (c,d), but showing recq4a recq4b background.

Extended Data Fig. 5 Genomic crossover maps for F2 individuals from independent J3::J3G155R and SPO11-1::J3G155R Col × Ler plants.

a, CTL5.14 crossover frequencies in Col × Ler (n = 11) and two independent SPO11-1::J3G155R Col × Ler (#1, n = 11, #3, n = 12,) F1 hybrid plants. Black dots indicate cM values of individual plants. Red dots and horizontal lines represent mean ± s.d. of cM values from individual plants (two-sided Welch’s t-test). n = the number of individual plants. b, Histograms showing the ratio of F2 individuals containing different crossover numbers in each population. Vertical dotted red lines indicate the mean value. c, Average number of crossovers per chromosome in F2 individuals in each population. d,e, As for (c) but showing normalized crossover frequencies (cM/Mb) along chromosome arms from the telomere (TEL) to the centromere (CEN) (d) and across the genome (e).

Extended Data Fig. 6 Genome-wide crossover mapping of j3 knockout Col × Ler hybrids and quantification of MLH1 foci in j3 mutants.

a, Gene structure and nucleotide sequences of J3 with hcr3 mutation (red triangle), positions of CRISPR/Cas9 sgRNAs and Cas9-mediated mutations for the j3 null mutants in Ler. Nucleotide deletions (green), insertions (blue). b, Anthers containing Alexander-stained pollens of Ler, j3-5, j3-6 and j3-7. Scale bars, 100 μm. c, As for (b), but showing pollen viability in Col (n = 12), hcr3 (n = 13), j3-1 (n = 6), j3-3 (n = 6), j2-2 (n = 6), Ler (n = 6), j3-5 (n = 7), j3-6 (n = 8) and j3-7 (n = 9). Black dots indicate pollen viability per sample. n = the number of independent samples. d, Seed number per silique in Ler (n = 23) and j3-5 (n = 21). Coloured dots indicate the numbers of plump (blue) and aborted (red) seeds. n = the number of individual siliques. e, 420 crossover frequency in Col × Ler (n = 7), j3-1 × Ler (n = 6), Col × j3-5 (n = 6) and j3-1 × j3-5 (n = 7) F1 hybrids. n = the number of individual plants. f, Histogram of sex-averaged crossover number in Col/Ler (n = 240) and j3-1/j3-5 (n = 96) F2 individuals. g, As for (f), but showing average crossover number per chromosome. h,i, As for (f), but showing normalized crossover frequencies (cM/Mb) along chromosome arms from the telomere (TEL) to the centromere (CEN) (h) and across the genome (i). j, Quantification of immunostained MLH1 foci of meiotic cells at diplotene stage in Col (n = 63), j3-1 (n = 30) and j3-3 (n = 26). Coloured dots indicate numbers of MLH1 foci per cell. Black dots and horizontal lines indicate mean ± s.d. of values of MLH1 foci (two-sided Wilcoxon test, j3-1 P = 1.15 × 10−2, j3-3 P = 5.88 × 10−3). n = the number of independent cells. (c,d,e) Data were presented as mean ± s.d. of values and significance was tested by a two-sided Welch’s t-test.

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Extended Data Fig. 7 J3 and J3G155R co-localize with HEI10 in vivo.

a, Representative images of co-immunostained J3/J2 (white), ZYP1 (green) and HEI10-Myc (red) at late-pachytene stage in Col HEI10::HEI10-Myc and hcr3 HEI10::HEI10-Myc. b, As for (a) but showing at diplotene and diakinesis stage in Col HEI10::HEI10-Myc. c, As for (a) but showing immunostained HA-J3, HA-J3G155R and HEI10-Myc at late-pachytene stage in J3::HA-J3 HEI10::HEI10-Myc and at mid-pachytene stage in J3::HA-J3G155R HEI10::HEI10-Myc plants. Three yellow dotted line boxes (1, 2, 3) in the merged images of co-immunostained J3/J2 and HEI10 are enlarged and displayed. Scale bars, 5 μm. Experiments (a–c) were performed at least three times.

Extended Data Fig. 8 J3-HSP70 chaperone machinery restricts crossover frequency.

a, Co-immunoprecipitation analysis of J3 and HSP70 in Arabidopsis protoplasts. b, In vitro pull-down assay of J3 and HSP70-1. c, Co-immunoprecipitation analysis of J2 with HSP70 and HEI10 in Arabidopsis protoplasts. d, Co-localization of J3/J2 and HSP70 with HEI10, PTD and MSH5 in Arabidopsis protoplasts. YN indicates N-terminal 1-158 amino acid resides of yellow fluorescent protein (YFP) and YC, C-terminal 159-238 amino acid residues of YFP for BiFC assay. Scale bars, 20 μm. e, RT–qPCR analysis of HSP70-1-5 genes in unopen flower buds of Col and meiMIGS-HSP70-1 plants. Red and blue dots indicate two technical duplicates of three biological replicates (n = 6 for each genotype) for RT–qPCRs. Mean ± s.d. of values are shown by black dots and horizontal lines. f, Seed FTLs across genome. g, 420 crossover frequencies in meiMIGS-HSP70-1 and meiMIGS-HSP70-3 transgenic lines. 420 Col (n = 12), meiMIGS-HSP70-1 T1 (n = 8), meiMIGS-HSP70-1 T2#5 (n = 6), meiMIGS-HSP70-1 T2#7 (n = 4), meiMIGS-HSP70-2 T1 (n = 8), meiMIGS-HSP70-2 T2#1 (n = 8), meiMIGS-HSP70-2 T2#4 (n = 8), h, As for (g) but showing CTLs in meiMIGS-HSP70-1. CTL1.17 Col (n = 6), meiMIGS-HSP70-1 (n = 7); CTL1.22 Col (n = 5), meiMIGS-HSP70-1 (n = 5); CTL3.15 Col (n = 6), meiMIGS-HSP70-1 (n = 5); CTL4.1 Col (n = 6), meiMIGS-HSP70-1 (n = 5); CTL4.7 Col (n = 6), meiMIGS-HSP70-1 (n = 6). i, As for (g) but showing meiMIGS-HSP70/Ler hybrid plants. Col/Ler (n = 8), meiMIGS-HSP70-1/Ler (n = 8), meiMIGS-HSP70-2/Ler (n = 8). j, Histogram of crossover number in Col × Ler (blue, n = 240) and meiMIGS-HSP70-1 × Ler (red, n = 96) F2 individuals. Dotted blue and red lines indicate mean values. k, As for (j) but showing normalized crossover frequencies (cM/Mb) along chromosome arms from the telomere (TEL) to the centromere (CEN). Black (g,i) or coloured (h) dots indicate cM values of individual plants. Red (g,i) or black (h) dots and horizontal lines represent mean ± s.d. of cM values from individual plants (two-sided Welch’s t-test). (g,h,i) n = the number of biologically independent plants.

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Extended Data Fig. 9 Ubiquitination modification and proteasome-dependent degradation of HEI10 in Arabidopsis protoplasts.

a, Immunoprecipitation and immunoblot analysis of ubiquitin (Ub)-conjugated HEI10-HA in Arabidopsis protoplasts. The plasmid constructs for either FLAG-tagged ubiquitin (FLAG-Ub) or HEI10-HA, or both were co-transfected into protoplasts. IP, immunoprecipitation. IB, immunoblot. Coomassie blue (CB) stained membrane is shown as a loading control. Experiments were performed at least three times. b,c, Immunoblot analysis (b) and quantification plot (c) of HEI10-HA protein upon treatment of translation elongation (cycloheximide, CHX) and proteasome (MG132) inhibitors in Arabidopsis protoplasts. Coloured dots indicate the normalized intensities of immunoblots. Black dots and horizontal lines indicate mean ± s.d. of values of the normalized immunoblot intensities at 0h (CHX, n = 3, CHX+MG132, n = 3, P = 0.47), 0.5h (CHX, n = 3, CHX+MG132, n = 3, P = 4.5 × 10−2), 1h (CHX, n = 3, CHX+MG132, n = 3, P = 7.3 × 10−3) and 2h (CHX, n = 3, CHX+MG132, n = 3, P = 2.9 × 10−2). Significance between time points was examine using a two-sided Welch’s t-test. n = the number of independent experiments.

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Extended Data Fig. 10 HCR1 and HCR3 are required for restricting class I crossovers.

a, Crossover frequencies of CTL1.26 in Col (n = 8), hcr1 (n = 8), hcr3 (n = 7) and hcr1 hcr3 (n = 7) (Supplementary Table 27). Black dots indicate cM values of individual plants. Red dots and horizontal lines represent mean ± s.d. of cM values from individual plants. Significance between genotypes was tested by a two-sided Welch’s t-test (Col vs hcr1, P = 1.18 × 10−7, Col vs hcr3, P = 4.00 × 10−10, Col vs hcr1 hcr3, P = 2.73 × 10−7, hcr1 vs hcr1 hcr3, P = 6.28 × 10−6, hcr3 vs hcr1 hcr3, P = 1.39 × 10−3). n = the number of biologically independent plants. b, Immunoblot analysis of HEI10 in Col, hcr1, hcr3 and hcr1 hcr3. Coomassie blue-stained membrane and tubulin protein blot were shown as a loading control. Experiments were performed at least three times.

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Supplementary Figs. 1–6, Tables 1–28, references and source data for Supplementary Figs. 5 and 6.

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Kim, H., Kim, J., Son, N. et al. Control of meiotic crossover interference by a proteolytic chaperone network. Nat. Plants 10, 453–468 (2024). https://doi.org/10.1038/s41477-024-01633-y

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