bioRxiv preprint doi: https://doi.org/10.1101/606285; this version posted April 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Patronus is the elusive plant securin, preventing chromosome separation by 2 antagonizing separase 3 Laurence Cromer1, Sylvie Jolivet1, Dipesh Kumar Singh1, Floriane Berthier1, Nancy 4 De Winne2, Geert De Jaeger2, Shinichiro Komaki3,4, Maria Ada Prusicki3, Arp 5 Schnittger3, Raphael Guérois5 and Raphael Mercier1* 6 7 1 Institut Jean-Pierre Bourgin, UMR1318 INRA-AgroParisTech, Université Paris- 8 Saclay, RD10, 78000 Versailles, France. 9 2 Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, 10 Belgium, VIB Center for Plant Systems Biology, Ghent, Belgium 11 3 Department of Developmental Biology, Institute for Plant Sciences and 12 Microbiology, University of Hamburg, 22609 Hamburg, Germany. 13 4 Present address : Nara Institute of Science and Technology, Graduate School of 14 Biological Sciences, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan 15 5 Institute for Integrative Biology of the Cell (I2BC), Commissariat à l’Energie 16 Atomique et aux Energies Alternatives (CEA), Centre National de la Recherche 17 Scientifique (CNRS), Université Paris-Sud, CEA-Saclay, Gif-sur-Yvette, France 18 * Corresponding author. [email protected] 19 1 bioRxiv preprint doi: https://doi.org/10.1101/606285; this version posted April 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 20 Abstract 21 22 Chromosome distribution at anaphase of mitosis and meiosis is triggered by 23 separase, an evolutionarily conserved protease. Separase must be tightly regulated 24 to prevent the untimely release of chromatid cohesion and disastrous chromosome 25 distribution defects. Securin is the key inhibitor of separase in animals and fungi, but 26 has not been identified in other eukaryotic lineages. Here, we identified PATRONUS1 27 and PATRONUS2 (PANS1 and PANS2) as the Arabidopsis homologues of securin. 28 Disruption of PANS1 is known to lead to the premature separation of chromosomes 29 at meiosis, and the simultaneous disruption of PANS1 and PANS2 is lethal. Here, we 30 show that PANS1 targeting by the anaphase-promoting-complex is required to trigger 31 chromosome separation, mirroring the regulation of securin. We showed that PANS1 32 acts independently from Shugosins. In a genetic screen for pans1 suppressors, we 33 identified SEPARASE mutants, showing that PANS1 and SEPARASE have 34 antagonistic functions in vivo. Finally, we showed that the PANS1 and PANS2 35 proteins interact directly with SEPARASE. Altogether, our results show that PANS1 36 and PANS2 act as a plant securin. Remote sequence similarity was identified 37 between the plant patronus family and animal securins, suggesting that they indeed 38 derive from a common ancestor. Identification of patronus as the elusive plant 39 securin illustrates the extreme sequence divergence of this central regulator of 40 mitosis and meiosis. 41 2 bioRxiv preprint doi: https://doi.org/10.1101/606285; this version posted April 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 42 Introduction 43 Balanced segregation of chromosomes at both mitosis and meiosis requires that 44 chromatid cohesion complexes be removed after proper chromosome alignment in 45 the spindle. Inaccuracies in this process cause chromosome mis-segregation and 46 aneuploidy, contributing to cancer and birth defects. Separase, which is conserved in 47 fungi, animals and plants, triggers cohesion release by cleaving the kleisin subunit of 48 the cohesin complex, opening the cohesin ring and allowing chromosome 49 segregation (1). Securin is the primary regulator of separase activity in animals and 50 fungi, forming a complex with separase and blocking substrate access to its active 51 site. Securin, which is defined by its functional and biochemical properties, is a 52 largely unstructured protein whose sequence is conserved only between closely 53 related species (2–6). At the onset of anaphase, the anaphase-promoting-complex 54 (also known as the cyclosome) (APC/C) triggers the degradation of securin, releasing 55 separase activity and allowing chromosome separation. In vertebrates, the Cdk1- 56 CyclinB1 complex is an additional regulator of separase activity (7). At meiosis I, the 57 cleavage of cohesins is also spatially controlled: peri-centromeric cohesins are 58 protected from separase cleavage by shugoshin-PP2A through dephosphorylation of 59 the kleisin subunit (8, 9). 60 In Arabidopsis, separase is essential for cohesion release and chromosome 61 segregation at both meiosis and mitosis (10, 11), APC/C regulates the progression of 62 division (12, 13) and the shugoshins SGO1 and SGO2 and PP2A are required for 63 cohesion protection at meiosis (14–16). However, securin is missing from the picture, 64 raising the intriguing possibility that securin has been lost in the green lineage and 65 that plant separase is regulated by a different mechanism. 3 bioRxiv preprint doi: https://doi.org/10.1101/606285; this version posted April 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 66 We previously characterised the two paralogues PATRONUS1 and PATRONUS2 in 67 Arabidopsis (PANS1, PANS2) (14). PANS1 is essential for the protection of sister 68 chromatid cohesion between the two meiotic divisions, through an unknown 69 mechanism. In the pans1 mutant, sister chromatid cohesion is lost before metaphase 70 II, leading to chromosome segregation defects at meiosis II (14, 17, 18). In addition, 71 the pans1 mutant also has a slight growth defect, which is exacerbated under stress 72 conditions, associated with a certain level of mitotic defects and aneuploidy in 73 somatic cells (14, 17, 19). The pans2 mutant is indistinguishable from the wild type, 74 but has synthetic lethal interaction with pans1, suggesting that PANS1 and PANS2 75 have an essential but redundant role at mitosis (14). PANS1 and PANS2 share 42% 76 identity and encode proteins of unknown function. PANS1 has been shown to interact 77 with APC/C through its D-box and KEN-box domains. Patronus proteins are well- 78 conserved in dicots, one of the two major clades of flowering plants, with most 79 species having one or two homologues (14). In monocots, the other major clade of 80 flowering plants, PANS proteins share limited similarity with RSS1 (RICE SALT 81 SENSITIVE 1, also known as Os02g39390), a protein that regulates cell cycle under 82 stress conditions in rice (20). Although both PANS1, PANS2 and RSS1 clearly play 83 important role in cell division, the molecular function of PANS1, PANS2 and RSS1 84 has remained elusive. 85 4 bioRxiv preprint doi: https://doi.org/10.1101/606285; this version posted April 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 86 Results 87 Expression of destruction-box-less PATRONUS1 prevents cohesion release 88 and chromosome segregation 89 The sequences of PANS proteins contain a conserved destruction-box (D-box) 90 domain (RxxLxxxN), recognised by the anaphase-promoting complex (APC/C), which 91 triggers the destruction of the targeted protein by the proteasome. A mutant version 92 of PANS1 mutated in its D-box (PANS1ΔD; RxxL LxxV) loses its capacity to 93 interact with the APC/C activator subunit CDC20 in yeast two-hybrid (Y2H) assays 94 (14). Expression of PANS1ΔD under its own promoter is lethal, suggesting that 95 PANS1 accumulation prevents plant development (14). To assess the function of the 96 PANS1 D-box at meiosis, we expressed PANS1ΔD under the meiosis-specific 97 promoter DMC1. Wild-type PANS1 expressed under the DMC1 promoter was able to 98 complement the meiotic defects of the pans1 mutant (n=2/4) (Figure S1). In contrast 99 pDMC1::PANS1ΔD caused full sterility when transformed in wild-type or pans1 plants 100 (n=15/15 and 2/2, respectively). In addition, pDMC1::PANS1ΔD plants showed a 101 variable growth defect, from barely developing plants to wild-type-like plants (Figure 102 S2), suggesting that the pDMC1 promoter can drive variable expression in somatic 103 tissues, but all plants showed complete sterility. In pDMC1::PANS1ΔD plants, meiotic 104 chromosome spreads did not reveal any defect in prophase or early metaphase I 105 (Figure 1). However, among the 119 post-prophase cells observed, none showed the 106 configuration typical of anaphase I, metaphase II, anaphase II or telophase II, 107 suggesting that the meiotic cells do not progress beyond metaphase I. We observed 108 normal metaphase I with five bivalents aligned on the metaphase plate (compare 109 Figure 1F with Figure 1A), but also metaphase I with five over-stretched bivalents 110 (Figure 1G). We also observed configurations resembling 5 bioRxiv preprint doi: https://doi.org/10.1101/606285; this version posted April 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.