DeCOP - Delineating the crossover control networks 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), 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. In general, the Mechtler group will develop, optimize and apply mass spectrometry-based bioanalytical methods in the context of this proposal. There are two main areas relevant to the planned experimental strategy. First, using label-free approaches and cross-linking, we will elucidate the composition of involved protein complexes and their quantitative change over meiosis. Second, we will apply a combination of isotopic labeling, diverse phosphopeptide enrichment methods and two- dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS) to study the quantitative response of the phosphoproteome to loss-of-function mutations of several putative master regulator kinases involved in meiosis. This will allow us to identify crucial effector proteins downstream of these kinases.

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. This collaborative project (DeCOP) focused on how recombination, chromosome organisation and remodelling are orchestrated to control the frequency and distribution of COs. Specifically, we wanted to identify the protein networks that determine the fate of individual DSBs and establish when CO interference is established. We 1) performed an innovative screen to identify novel factors that modulate CO formation and interference, 2) investigated the role of chromosome axis-associated proteins in CO maturation and interference, 3) studied the role of (ATM/ATR/CK2 mediated) phosphorylation in coordinating meiotic DNA repair and CO formation and 4) identified proteins involved in the final step of CO formation. In the frame of the joint project, the group of Karl Mechtler based at the Research Institute of Molecular Pathology in Vienna analyzed meiotic protein complexes by mass spectrometry (MS) and identified protein interaction partners and modifications. Furthermore, they performed a proteome-wide MS-based screen to study phosphorylation events in dependency of the activity of the kinases ATM, ATR and CK2 in the DNA damage pathway. Finally, new MS methods -based on chemical cross-linking- were established which allow to study dynamic changes in the structure of protein complexes.