DeCOP - Delineating the crossover control networks in plants

Summary General part Meiosis is a specialized type of cell division required for sexual reproduction. It ensures the reduction of the genome and the recombination of maternal and paternal chromosomal segments prior to the formation of generative cells. The process of meiotic recombination is initiated by programmed DNA double-strand breaks (DSBs), introduced by the conserved Spo11 protein. Ultimately, the positions of the DSBs define loci of mutual genetic exchange. However, in a single meiotic cell only a small sub-set of DSBs are destined to form genetic crossovers (COs), while the remainder are repaired via non-CO pathways. CO formation itself is subject to stringent control, which ensures that each homologue pair receives at least one obligate CO. A phenomenon known as CO interference then ensures that most (~85%) additional COs do not occur in an adjacent chromosomal region. As a result multiple COs are spaced well apart along the homologues. Understanding the factors that control DSB formation and processing to form COs is of fundamental scientific interest, moreover this knowledge will have important implications for manipulating meiotic recombination in crop plants. In recent years meiosis research in plants has largely focussed on the identification of meiotic genes/proteins involved in recombination pathways or the organization of the chromosome axes and synaptonemal complex. Although these studies clearly demonstrate the importance of these proteins, it remained mostly enigmatic how their activities are coordinated to ensure the controlled formation of COs. Hence this collaborative project (DeCOP) seeks to shift emphasis to focus on how recombination, chromosome organisation and remodelling are orchestrated to control the frequency and distribution of COs. Specifically, we seek to identify the protein networks that determine the fate of individual DSBs and establish when CO interference is established. We propose to 1) perform an innovative screen to identify novel factors that modulate CO formation and interference, 2) investigate the role of chromosome axis-associated proteins in CO maturation and interference, 3) determine the role of (ATM/ATR mediated) phosphorylation in coordinating meiotic DNA repair and CO formation and 4) to identify proteins involved in the final step of CO formation. The factors and processes studied in the DeCOP project will significantly enhance our understanding of the networks that govern crossover formation in plants. We therefore anticipate that our findings will strongly stimulate future crop breeding programmes. Summary Schlögelhofer project part Peter Schlögelhofer (project partner 1) will coordinate the DeCOP project and he will be involved in all four tasks outlined in the proposal. He will take the lead of "Task 3", dedicated to the analysis of the molecular role of ATM/ATR dependent phosphorylation of key proteins involved in DNA repair and CO formation. First, building on previous findings, a genetic suppressor screen will be performed, aiming at identifying targets of the ATR kinase, that suppress DMC1 function. Second, following a proteome-wide approach, in collaboration with project partner 2 (Karl Mechtler), we aim at identifying targets of the putative plant CHK2 kinase homologue. We identified the putative plant CHK2 kinase in a previous project as target of ATM/ATR. CHK2 represents a prominent target in other organisms and the here proposed project aims at delineating the signalling pathway downstream of ATM/ATR in plants. Furthermore, Peter Schlögelhofer, as all other partners, will be involved in the detailed analysis of candidates identified in the different genetic screens performed in the course of the project.

The collaborative ERA-CAPS DeCOP (Delineating the crossover control networks in plants) project has been a joint effort by six European research groups (P. Schlögelhofer MFPL, Univ. of Vienna, AUT, coordinator; K. Mechtler IMP, Vienna, AUT; I. Henderson Univ. of Cambridge, UK; H. Puchta Univ. of Karlsruhe, GER; E. Sanchez-Moran and Ch. Franklin both Univ. of Birmingham, UK). It focused on how meiotic recombination, chromosome organisation and remodelling are orchestrated to control the frequency and distribution of exchange of genetic information during meiosis in plants. Meiosis is a specialized type of cell division required for sexual reproduction. It ensures the reduction of the genome and the recombination of maternal and paternal chromosomal segments prior to the formation of generative cells. The process of meiotic recombination is initiated by programmed DNA double-strand breaks (DSBs). Ultimately, the positions of the DSBs define loci of mutual genetic exchange. However, in a single meiotic cell only a small subset of DSBs are destined to form genetic crossovers (COs), while the remainder are repaired via non-CO pathways. In this collaborative project we 1) identified novel factors that modulate CO formation and interference, 2) investigated the role of chromosome axis-associated proteins in CO maturation and interference, 3) determined the role of (ATM/ATR/CK2 mediated) phosphorylation in coordinating DNA repair and 4) identified proteins involved in the final step of meiotic CO formation. Some of the research work has already been published some other aspects are still under investigations. We especially highlight the findings that the protein ASY4 has been identified as a novel meiotic axis protein (Chambon et al., 2018), that PCH2 has been identified and described as a meiotic axis re-modelling factor in plants (Lambing et al., 2015), that cross- linking mass spectrometry techniques have been established and employed to characterize a meiotic protein complex (Rampler et al., 2016; Orban-Nemeth et al., 2018), that novel phosphorylation sites on meiotic axis proteins (Osman et al., 2018) have been identified, that in-depth phospho-proteomic studies established the kinase CK2 as a key mediator of DNA damage response (Schropp et al., in preparation), that local zygosity modulates meiotic interference (Ziolkowski et al., 2015), that HEI10 dosage positively correlates with CO frequency (Ziolkowski et al., 2017) and that DNA methylation and nucleosome density, identity and modifications define meiotic DSB and hence CO frequency (Choi et al, 2018; Underwood et al, 2018; Yelina et al, 2015). In summary, this European research consortium contributed considerably to the understanding of meiotic DSB formation, recombination and cross-over formation in plants. The coordination of research avoided competition and redundancy and, importantly, the regular exchange of ideas, know-how and research materials and the regular meetings of involved researches stimulated new research directions and collaborations.