Identification and characterization of EMB1674, as a novel KNL2 variant : revealing its centromere targeting mechanism and critical role in plant-specific kinetochore assembly

Abstract

The kinetochore ensures accurate chromosome segregation, initiated by the deposition of CENH3. KNL2 recognizes centromeres and facilitates CENH3 loading. Phylogenetic analysis revealed three independent KNL2 duplications in ferns, grasses, and eudicots. We classified previously uncharacterized KNL2 genes as αKNL2 and βKNL2 in eudicots, γKNL2 and δKNL2 in grasses. All KNL2 variants encode the conserved SANTA domain; however, only αKNL2 and γKNL2 possess the CENPC-k motif. In contrast, βKNL2 and δKNL2 lack this motif. Despite this, βKNL2 localizes to centromeres and is essential for CENH3 deposition. Homozygous βknl2 mutants exhibit severe developmental defects and do not survive on soil. Efficient centromeric targeting of βKNL2 requires both its SANTA domain and C-terminal motifs. Structural predictions and experimental data show that βKNL2 forms homodimers, binds centromeric DNA, and interacts with αKNL2, which facilitates its localization in a tissue-specific manner. This study reveals the functional divergence of duplicated KNL2 genes and highlights βKNL2’s unique role in plant-specific kinetochore assembly and genome stability.