The protein-protein interaction network of KNL2 in plants

• Applicant: Dr. Inna Lermontova
• Subject Area: Plant Genetics and Genomics ; Cell Biology
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 467635690

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.

• DFG Programme: Research Grants
• Co-Investigator: Dmitri Demidov, Ph.D.