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

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.Marie Curie International Training Network COMREC European Research Council (ERC) National Research Foundation of Korea Suh Kyungbae Foundatio

High-Throughput Fluorescent Pollen Tetrad Analysis Using DeepTetrad

Meiotic recombination initiates from ~100–200 s of programmed DNA double stranded breaks (DSBs) in plants. Meiotic DSBs can be repaired using homologous chromosomes to generate a crossover. Meiotic crossover is critical for chromosomal segregation and increasing genetic variation. The number of crossovers is limited to one and three per chromosome pair in most plant species. Genetic, epigenetic, and environmental factors control crossover frequency and distribution. Due to the limited number of crossovers it is challenging to measure crossover frequency along chromosomes. We adapted fluorescence-tagged lines (FTLs) that contain quartet1 mutations and linked transgenes expressing dsRed, eYFP, and eCFP in pollen tetrads into the deep learning-based image analysis tool, DeepTetrad. DeepTetrad enables the measurement of crossover frequency and interference by classifying 12 types of tetrads from three-color FTLs in a high-throughput manner, using conventional microscope instruments and a Linux machine. Here, we provide detailed procedures for preparing tetrad samples, tetrad imaging, running DeepTetrad, and analysis of DeepTetrad outputs. DeepTetrad-based measurements of crossover frequency and interference ratio will accelerate the genetic dissection of meiotic crossover control. © 2022, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.11Nscopu

Activation tagging screening of Arabidopsis dosage-dependent crossover rate modulators

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HEAT SHOCK FACTOR BINDING PROTEIN limits Meiotic Recombination by repressing HEI10 transcription in Arabidopsis

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HEAT SHOCK FACTOR BINDING PROTEIN limits Meiotic crossovers by repressing HEI10 transcription in Arabidopsis

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HIGH CROSSOVER RATE3 encoding J3 chaperone limits meiotic crossovers in Arabidopsis

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Activation tagging screening of Arabidopsis dosage-dependent crossover rate modulators

During meiosis, homologous chromosomes (homologs) pair and undergo reciprocal exchange of non-sister chromosomes, called crossover. Crossover is essential for segregation of homologs and provides genetic diversity. Number of crossovers tends to one per homolog pair in plants, indicating that anti-crossover factors restrict crossovers. Here, we performed genetic screening to identify novel crossover rate mutants using 420 fluorescent crossover reporter and activation tagging. Approximately 1,000 T1 individuals were measured for crossover frequency, which isolated 8 high crossover rate dominant (hcr-D) mutants and 1 low crossover rate dominant (lcr-D) mutant. T-DNA insertion sites were mapped via long read sequencing and adapter-ligation PCR, and overexpression of casual genes were examined by RT-qPCR. Using a genomic set of fluorescent seed reporter lines we revealed that hcr10-D showed increased crossover frequency in chromosomal arm but decreased crossover frequency around centromeres. Future studies will investigate the role of HCR10-D in meiotic recombination.2

DeepTetrad: high-throughput image analysis of meiotic tetrads by deep learning in Arabidopsis thaliana

Meiotic crossovers facilitate chromosome segregation and create new combinations of alleles in gametes. Crossover frequency varies along chromosomes and crossover interference limits the coincidence of closely spaced crossovers. Crossovers can be measured by observing the inheritance of linked transgenes expressing different colors of fluorescent protein in Arabidopsis pollen tetrads. Here we establish DeepTetrad, a deep learning-based image recognition package for pollen tetrad analysis that enables high-throughput measurements of crossover frequency and interference in individual plants. DeepTetrad will accelerate genetic dissection of mechanisms that control meiotic recombination.1

Control of meiotic crossover interference by a proteolytic chaperone network

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.ER

Arabidopsis HEAT SHOCK FACTOR BINDING PROTEIN is required to limit meiotic crossovers and HEI10 transcription.

The number of meiotic crossovers is tightly controlled and most depend on pro-crossover ZMM proteins, such as the E3 ligase HEI10. Despite the importance of HEI10 dosage for crossover formation, how HEI10 transcription is controlled remains unexplored. In a forward genetic screen using a fluorescent crossover reporter in Arabidopsis thaliana, we identify heat shock factor binding protein (HSBP) as a repressor of HEI10 transcription and crossover numbers. Using genome-wide crossover mapping and cytogenetics, we show that hsbp mutations or meiotic HSBP knockdowns increase ZMM-dependent crossovers toward the telomeres, mirroring the effects of HEI10 overexpression. Through RNA sequencing, DNA methylome, and chromatin immunoprecipitation analysis, we reveal that HSBP is required to repress HEI10 transcription by binding with heat shock factors (HSFs) at the HEI10 promoter and maintaining DNA methylation over the HEI10 5' untranslated region. Our findings provide insights into how the temperature response regulator HSBP restricts meiotic HEI10 transcription and crossover number by attenuating HSF activity.BBSRC grants BB/S006842/1, BB/S020012/1 and BB/V003984/1, European Research Council Consolidator Award ERC-2015-CoG-681987 ‘SynthHotSpot’ and Marie Curie International Training Network ‘COMREC’