A coadapted KNL1 and spindle assembly checkpoint axis orchestrates precise mitosis in Arabidopsis

Abstract

The kinetochore scaffold 1 (KNL1) protein recruits spindle assembly checkpoint (SAC) proteins to ensure accurate chromosome segregation during mitosis. Despite such a conserved function among eukaryotic organisms, its molecular architectures have rapidly evolved so that the functional mode of plant KNL1 is largely unknown. To understand how SAC signaling is regulated at kinetochores, we characterized the function of the KNL1 gene in Arabidopsis thaliana. The KNL1 protein was detected at kinetochores throughout the mitotic cell cycle, and null knl1 mutants were viable and fertile but exhibited severe vegetative and reproductive defects. The mutant cells showed serious impairments of chromosome congression and segregation, that resulted in the formation of micronuclei. In the absence of KNL1, core SAC proteins were no longer detected at the kinetochores, and the SAC was not activated by unattached or misaligned chromosomes. Arabidopsis KNL1 interacted with SAC essential proteins BUB3.3 and BMF3 through specific regions that were not found in known KNL1 proteins of other species, and recruited them independently to kinetochores. Furthermore, we demonstrated that upon ectopic expression, the KNL1 homolog from the dicot tomato was able to functionally substitute KNL1 in A. thaliana, while others from the monocot rice or moss associated with kinetochores but were not functional, as reflected by sequence variations of the kinetochore proteins in different plant lineages. Our results brought insights into understanding the rapid evolution and lineage-specific connection between KNL1 and the SAC signaling molecules.

Keywords: Arabidopsis; KNL1; SAC; kinetochore.

Fig. 1.

KNL1 plays a critical role in vegetative growth and reproduction in Arabidopsis. (A) The diagram illustrates the gene structure of KNL1 (At2g04235) with exons in boxes and introns in lines. The position of T-DNA insertion and gene editing site are indicated in the diagram. The sgRNA target sequence and changes in the knl1-2^cr and knl1-3^cr sequences are highlighted in red. Mutant transcripts are predicted to introduce premature stop codons (*) resulted from frameshifts. (B) Comparison of the 3-wk-old plants of wild-type (WT) plant (1), knl1-1 mutant (2), knl1-2^cr mutant (3), knl1-3^cr mutant (4), and knl1-1 plant expressing the KNL1-GFP fusion protein (5). (C) Three knl1 mutant alleles show serious growth reduction when compared to the WT and rescued plants grown for 6 wk. (D) The knl1 mutants have frequently aborted seeds in the opened siliques. (E) Quantification of seed production in WT (n = 486), knl1 mutants (n = 512, 534, 508), and rescued plants (n = 492). (F) The knl1 mutants frequently produce 4-pronged trichome while the WT and rescued plants have primarily 3-pronged trichomes on the leaf surface.

Fig. 2.

Localization of KNL1-GFP during mitosis in Arabidopsis. (A) At interphase, KNL1 is concentrated at discrete foci in the nucleoplasm. (B) Paired KNL1 signal can be detected in cells bearing the preprophase band microtubule array. (C) At late prophase when a spindle microtubule array is detected, KNL1 appears exclusive in paired kinetochores. (D and E) The KNL1 pairs associate with chromosomes following nuclear envelope breakdown, and later exhibit biorientation at the metaphase plate and are connected to paired kinetochore fiber microtubules. (F) At anaphase, the KNL1 signal highlights kinetochores of the separated sister chromatids. (G and H) After arriving at spindle poles at telophase, KNL1 foci later become suspended in the nucleus when daughter nuclei are formed during cytokinesis. The merged images have KNL1-GFP detected by the anti-GFP antibody in green, microtubules in red, and DNA in blue. Micrographs are representative of 100% mitotic cells (n = 180) from three independent transgenic lines (N = 3). (Scale bars, 5 μm.)

Fig. 3.

KNL1 plays a critical role in chromosome congression and segregation. (A) Comparative views of chromosome alignment in WT and knl1-1 mutant cells at metaphase. Misaligned chromosomes are indicated by white arrowheads in the knl1-1 cells. (B) Comparative views of cytokinetic cells in WT and knl1-1 plants. One or more micronuclei caused by KNL1 depletion are indicated by white arrowheads. Merged images have microtubules in green and DNA in magenta. (C) Quantitative assessment of cells exhibiting misaligned chromosomes at metaphase and cells producing micronuclei following cytokinesis in WT (n = 150) and knl1-1 plants (n = 165). (D) Live-cell imaging of WT cells expressing GFP-TUB6 and Histone-RFP. Representative snapshot images are acquired from Movie S1. (E) Live-cell imaging of knl1-1 cells expressing GFP-TUB6 and Histone-RFP. Images are acquired from Movie S2. Misaligned chromosomes and lagging chromosomes are indicated by arrowheads. (F) Representative time-lapse images of chromosome migration in knl1-1 cells. Chromosome centroids are plotted distant from the initial position over time, and the movement of individual chromosomes is tracked by lines using different colors in the Last panel. Arrows show the direction of chromosome migration; arrowheads point at chromosome bridges. Live-cell images are representative of 16 mitotic videos from WT (n = 16) and 20 mitotic videos from knl1-1 (n = 20) plants. (Scale bars, 5 μm.)

Fig. 4.

KNL1 is essential for kinetochore localization of core SAC proteins in A. thaliana. (A) Comparison of seedlings of the WT, knl1-1 mutant, and knl1-1 mutant expressing KNL1-GFP with and without 100 nM oryzalin treatment after grown for 10 d. (B) Quantification of root lengths in the seedlings in (A) with and without oryzalin treatment. Data are means ± SD measured from three independent experiments (N = 3) each containing six individual measurements (n = 6). ** indicates significance (P < 0.01, pairwise comparison using one-way ANOVA analysis). (C and D) GFP-BUB3.3 localized to kinetochores upon expression in the bub3.3 mutant (C) but becomes diffuse in the cytoplasm in the knl1-1 mutant cells (D) at prometaphase (Top) and anaphase (Bottom). (E and F) BMF1-GFP is detected at kinetochores upon expression in the bmf1 mutant (E) and in knl1-1 mutant cells (F) in representative cells at prophase (Top row) and anaphase (Bottom row). (G and H) BMF3-GFP localization is shown at kinetochore when expressed in the bmf3 mutant (G) and becomes diffuse in the knl1-1 mutant cells (H). (I and J) GFP-MAD1 localizes to kinetochores upon expression in the mad1 mutant (I) and becomes diffuse in the cytoplasm in the knl1-1 mutant cells (J). Merged images have GFP-tagged proteins in green, microtubules in red, and DNA in blue. Micrographs are representative of more than 100 cells from three independent lines (n ≥ 100, N = 3) with similar results. (Bars, 5 μm.)

Fig. 5.

KNL1 interacts with BUB3.3 and BMF3. (A) Assessment of interactions between KNL1 and SAC components by Y2H assays. The empty vector is used as a negative control (Ø). The yeast cultures were spotted on vector-selective (-L/-W, Left column) and interaction-selective (-L/-W/-H/-A, Right column) media and photographed after incubation at 30 °C for 2 d. (B) Bimolecular fluorescence complementation (BIFC) assay examining interactions of KNL1 (fused with the N-terminal fragment of YFP) and SAC proteins (fused with the C-terminal fragment of YFP) in Nicotiana benthamiana. (C) Schematic representation of full-length and truncated versions of KNL1 used to map BUB3.3 and BMF3 binding domains. (D) Y2H interactions between truncated KNL1 variants and BMF3/BUB3.3. (E) BIFC assay to examine interactions between BUB3.3/BMF3 and truncated KNL1 variants. (F) In vitro pull-down assays of recombinant GST fusions of KNL1 variants with His-BMF3/BUB3.3 immobilized beads. BIFC experiments were repeated three times (N = 3) with similar results. (Scale bars, 25 μm.)

Fig. 6.

KNL1 deploys a eudicot-specific domain to recruit BUB3.3 and BMF3 to kinetochores. (A) Growth phenotypes of 3-week-old plants of WT, knl1-1, and mutant plants expressing KNL1 and KNL1^ΔESD. (B) Representative images of chromosome segregation in knl1-1 mutant cells expressing KNL1-FLAG (Top rows) and KNL1^ΔESD-FLAG (Bottom rows), misaligned chromosomes are indicated by white arrowheads. Merged images have microtubules in red and DNA in green. (C and D) Localization of GFP-BUB3.3 (C) and BMF3-GFP (D) in knl1-1 mutant expressing KNL1-FLAG or KNL1^ΔESD-FLAG. While both KNL1-FLAG and KNL1^ΔESD-FLAG are detected at kinetochores, GFP-BUB3.3 and BMF3-GFP colocalize with KNL1-FLAG but not KNL1^ΔESD-FLAG. The merged images have FLAG-tagging proteins detected by the FLAG antibody in red, GFP-tagging proteins detected by the GFP antibody in green, and DNA stained by DAPI in blue. Micrographs are representative of more than 100 cells from three independent transgenic lines with similar results (n ≥ 100, N = 3). (Scale bars, 5 μm.)

Fig. 7.

KNL1 of a eudicot but not monocot or bryophyte origin captures the function in A. thaliana. (A) Growth phenotypes of 5-week-old plants of WT, knl1-1, and mutant plants expressing KNL1 orthologs from P. patens, S. lycopersicum, and O. sativa. AtKNL1 is used as the positive control. (B and C) Assessment of the kinetochore localization of GFP-BUB3.3 (B) and BMF3-GFP (C) in knl1-1 mutant cells expressing FLAG-tagged PpKNL1, SlKNL1, or OsKNL1 detected by the anti-FLAG antibody. The merged images have FLAG-tagging proteins in red, GFP-tagging proteins in green, and DAPI-stained DNA in blue. Micrographs are representative of more than 100 cells from three independent transgenic lines (n ≥ 100, N = 3) with similar results. (Scale bars, 5 μm.)