The protein-protein interaction network of KNL2 in plants

着丝粒是有丝分裂和减数分裂中着丝粒复合物聚集在一起将染色体连接到纺锤体纤维上的特定染色体区域，在真核生物细胞分裂过程中负责染色体的精确分离。着丝粒功能和着丝点的丧失会导致染色体错误分离、基因组不稳定、非整倍体和细胞死亡。着丝粒的同一性在表观遗传上是由组蛋白H3变体cenH3的存在决定的，它触发功能性着丝点的组装。着丝点复合体由100多种蛋白质组成。KINETOCHORE NULL2 （KNL2）在新的cenH3沉积中起关键作用，并且是其他着丝粒蛋白募集到着丝粒所必需的。之前我们已经鉴定并鉴定了植物的KNL2同源物。AtKNL2的敲除突变体显示着丝粒上的cenH3水平降低。knl2突变体与野生型植物杂交产生单倍体植株。我们的研究还发现了一个在KNL2的c端靶向CENPC-k结构域的着丝粒。在这个项目中，我们的目标是鉴定和表征直接与KNL2相互作用或属于同一蛋白质复合物的蛋白质。为此，对Y2H文库进行了IP-MS分析和筛选。我们选择了与KNL2沉淀的分数较高的MOS1、NUF2、NDC80和DOT2蛋白进行进一步分析，这些蛋白已知参与细胞分裂的调控。为了测试所选候选蛋白是否直接与KNL2相互作用，将采用BiFC、Y2H和Co-IP方法。所选候选蛋白将通过分析其亚细胞定位及其对着丝粒组装、细胞分裂、植物生长和发育的失调表达的影响来进行功能表征。这些知识对于理解着丝点组装和功能的机制是必不可少的。此外，在申请人课题组中，它将用于优化基于knl2的单倍体在拟南芥和不同作物物种中的诱导。

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.