The protein-protein interaction network of KNL2 in plants

着丝粒是在有丝分裂和减数分裂中组装动粒复合体以将染色体附着到纺锤体纤维的特定染色体区域，并且负责真核生物细胞分裂期间染色体的精确分离。着丝粒功能和动粒的丧失导致染色体错误分离、基因组不稳定、非整倍性和细胞死亡。着丝粒的身份是由称为cenH 3的组蛋白H3变体的存在而表观遗传地指定的，其触发功能性着丝粒的组装。动粒复合物由100多种蛋白质形成。动粒NULL 2（KNL 2）在新的cenH 3沉积中起关键作用，并且是将其他动粒蛋白募集到着丝粒所需的。以前，我们确定和表征的KNL 2同源植物。AtKNL 2的敲除突变体显示着丝粒处cenH 3水平降低。knl 2突变体与野生型植物的杂交导致产生单倍体植物。我们的研究还揭示了在KNL 2的C-末端处靶向CENPC-k结构域的着丝粒。在这个项目中，我们的目标是识别和表征直接与KNL 2相互作用或属于同一蛋白质复合物的蛋白质。为此，进行Y2 H文库的IP-MS分析和筛选。已选择用KNL 2沉淀的具有高评分并且已知参与细胞分裂调节的MOS 1、NUF 2、NDC 80和DOT 2蛋白用于进一步分析。为了测试所选择的候选蛋白是否与KNL 2直接相互作用，将应用BiFC、Y2 H和Co-IP方法。所选择的候选蛋白质将通过分析它们的亚细胞定位和它们的失调表达对动粒组装、细胞分裂、植物生长和发育的影响来功能性地表征。这些知识对于理解动粒组装和功能的机制是必不可少的。此外，在申请人的研究小组中，它将用于优化拟南芥和不同作物物种中基于KNL 2的单倍体诱导。

Centromeres are specific chromosomal regions where kinetochore complexes assemble in mitosis and meiosis to attach chromosomes to the spindle fibers, and are responsible for accurate segregation of chromosomes during cell division in eukaryotes. Loss of the centromeres function and kinetochores causes chromosome mis-segregation, genome instability, aneuploidy, and cell death. Centromere identity is specified epigenetically by the presence of the histone H3 variant termed cenH3 which triggers the assembly of functional kinetochores. The kinetochore complexes are formed by more than 100 proteins. KINETOCHORE NULL2 (KNL2) plays the key role in new cenH3 deposition and is required for the recruitment of other kinetochore proteins to the centromere. Previously we identified and characterized the KNL2 homologue of plants. A knock-out mutant for AtKNL2 showed a reduced level of cenH3 at centromeres. Crossing of the knl2 mutant with a wild-type plant resulted in the generation of haploid plants. Our studies revealed also a centromere targeting CENPC-k domain at the C-terminus of KNL2. In this project, we aim to identify and characterize proteins that directly interact with KNL2 or belong to the same protein complex. For this purpose, IP-MS analysis and screening of the Y2H library were performed. The MOS1, NUF2, NDC80 and DOT2 proteins precipitated with KNL2 with a high score and known to be involved in the regulation of cell divisions have been selected for further analysis. To test whether selected candidate proteins interact with KNL2 directly, BiFC, Y2H and Co-IP methods will be applied. The selected candidate proteins will be functionally characterized by analysing their subcellular localization and effects of their deregulated expression on kinetochore assembly, cell division, plant growth, and development. Such knowledge is essential to understand the mechanism of kinetochore assembly and function. Moreover, in the applicant's research group, it will be used to optimize KNL2-based haploid induction in Arabidopsis and different crop species.