Biology subjects

Kalidass, M.

Publications and source records attributed to Kalidass, M..

4 recordsLinked to original sources

bioRxiv2025

The C-terminal SUMOylation-dependent regulation of αKNL2 governs its centromere targeting and interaction with CENH3

The centromere is a specialized domain that facilitates chromosome segregation during mitosis and serves as the site for kinetochore formation. KINETOCHORE NULL2 (KNL2) is essential for the recognition and loading of the centromeric histone H3 variant, CENH3, to centromeres. A yeast two-hybrid screen for KNL2 interactors identified components of the SUMOylation pathway. However, the role of KNL2 SUMOylation in Arabidopsis has not yet been determined. In this study, we demonstrated that the C-terminal part of KNL2 interacts with SUMO3 and ULP1d, as shown by BiFC and co-immunoprecipitation assays. Bioinformatic and functional analysis identified three SUMOylation and two SUMO-interacting motif (SIM) sites in the C-terminal region of KNL2, which are critical for growth, fertility, and chromosome alignment. Of the three SUMOylation sites, Lys474 and Lys511 were the most critical for the centromeric localization of KNL2, underscoring the importance of KNL2 SUMOylation for its function. Additionally, both in vitro and in vivo assays showed that KNL2-C undergoes SUMOylation by SUMO1 or SUMO3. The SUMO protease mutant, ulp1d-2 led to the slight accumulation of SUMOylated KNL2 in Arabidopsis. We further showed that SUMOylation of KNL2 promotes its binding to CENH3 and controls protein stability. Our findings show that C-terminal SUMOylation of KNL2 is crucial for its centromeric localization, interaction with CENH3, and kinetochore assembly, emphasizing the significance of post-translational modifications in chromosome segregation and cell division in plants.

molecular biology↗

bioRxiv2024

Centromeric localization of KNL2 and CENP-C proteins in plants depends on their centromere-targeting domain and DNA-binding regions

In eukaryotic organisms, proper chromosome segregation during cell division depends on the centromeric histone H3 (CENH3) variant. Our previous studies identified a plant CENH3 assembly factor, Kinetochore Null2 (KNL2), that possesses a centromere-targeting motif, CENPC-k, similar to the CENPC motif in CENP-C. Additionally, we have demonstrated that KNL2 can bind DNA in vitro, independent of its CENPC-k motif. Thus, the mechanism underlying the binding of KNL2 to centromeric DNA remains elusive. Our study shows that the CENPC-k and CENPC motifs alone are not sufficient to target the centromere in N. benthamiana and A. thaliana. In-silico analysis revealed flanking DNA-binding regions near the CENPC-k and CENPC motifs, suggesting their importance in interacting with centromeric DNA. Fusion of protein fragments containing these motifs to EYFP facilitated targeting to the centromere. Deletion of DNA-binding domains reduced the centromeric localization of KNL2-C, whereas fusion of CENPC-k to the H-NS protein from E. coli targeted it to centromeres. We conclude that targeting of KNL2 and CENP-C proteins to centromeres is dependent on the CENPC-k/CENPC motifs, and their sequence-independent DNA-binding promotes anchoring at the centromere. Understanding the targeting mechanisms of KNL2 and CENP-C may help to engineer kinetochore structure by targeting chromatin modifying proteins to centromeres.

plant biology↗

bioRxiv2023

Heat stress impairs centromere structure and segregation of meiotic chromosomes in Arabidopsis

Heat stress is a major threat to global crop production, and understanding its impact on plant fertility is crucial for developing climate-resilient crops. Despite the known negative effects of heat stress on plant reproduction, the underlying molecular mechanisms remain poorly understood. Here, we investigated the impact of elevated temperature on centromere structure and chromosome segregation during meiosis in Arabidopsis thaliana. Consistent with previous studies, heat stress leads to a decline in fertility and micronuclei formation in pollen mother cells. Our results reveal that elevated temperature causes a decrease in the amount of centromeric histone and the kinetochore protein BMF1 at meiotic centromeres with increasing temperature. Furthermore, we show that heat stress increases the duration of meiotic divisions and prolongs the activity of the spindle assembly checkpoint during meiosis I, indicating an impaired efficiency of the kinetochore attachments to spindle microtubules. Our analysis of mutants with reduced levels of centromeric histone suggests that weakened centromeres sensitize plants to elevated temperature, resulting in meiotic defects and reduced fertility even at moderate temperatures. These results indicate that the structure and functionality of meiotic centromeres in Arabidopsis are highly sensitive to heat stress, and suggest that centromeres and kinetochores may represent a critical bottleneck in plant adaptation to increasing temperatures.

plant biology↗

bioRxiv2022

High temperature increases centromere-mediated genome elimination frequency in Arabidopsis deficient in cenH3 or its assembly factor KNL2

Double haploid production is the most effective way of creating true-breeding lines in a single generation. In Arabidopsis, haploid induction via mutation of the centromere-specific histone H3 (cenH3) has been shown when outcrossed to wild-type. Here we report that a mutant of the cenH3 assembly factor KNL2 can be used as a haploid inducer. We elucidated that short temperature stress of the knl2 mutant increased the efficiency of haploid induction from 1 to 10%. Moreover, we have demonstrated that a point mutation in the CENPC-k motif of KNL2 is sufficient to generate haploid inducing lines, suggesting that haploid inducing lines in crops can be identified in a naturally occurring or chemically induced mutant population, avoiding the GMO approach at any stage. In addition, we have shown that the cenh3-4 mutant, which does not induce haploids under standard growth conditions, functions as a haploid inducer after exposure to short temperature stress.

plant biology↗