Genome-Scale Sequence Disruption Following Biolistic Transformation in Rice and Maize.

Biolistic transformation delivers nucleic acids into plant cells by bombarding the cells with microprojectiles, which are micron-scale, typically gold particles. Despite the wide use of this technique, little is known about its effect on the cell's genome. We biolistically transformed linear 48-kb phage lambda and two different circular plasmids into rice (Oryza sativa) and maize (Zea mays) and analyzed the results by whole genome sequencing and optical mapping. Although some transgenic events showed simple insertions, others showed extreme genome damage in the form of chromosome truncations, large deletions, partial trisomy, and evidence of chromothripsis and breakage-fusion bridge cycling. Several transgenic events contained megabase-scale arrays of introduced DNA mixed with genomic fragments assembled by nonhomologous or microhomology-mediated joining. Damaged regions of the genome, assayed by the presence of small fragments displaced elsewhere, were often repaired without a trace, presumably by homology-dependent repair (HDR). The results suggest a model whereby successful biolistic transformation relies on a combination of end joining to insert foreign DNA and HDR to repair collateral damage caused by the microprojectiles. The differing levels of genome damage observed among transgenic events may reflect the stage of the cell cycle and the availability of templates for HDR.

Frequent Spindle Assembly Errors Require Structural Rearrangement to Complete Meiosis in Zea mays.

The success of an organism is contingent upon its ability to faithfully pass on its genetic material. In the meiosis of many species, the process of chromosome segregation requires that bipolar spindles be formed without the aid of dedicated microtubule organizing centers, such as centrosomes. Here, we describe detailed analyses of acentrosomal spindle assembly and disassembly in time-lapse images, from live meiotic cells of Zea mays. Microtubules organized on the nuclear envelope with a perinuclear ring structure until nuclear envelope breakdown, at which point microtubules began bundling into a bipolar form. However, the process and timing of spindle assembly was highly variable, with frequent assembly errors in both meiosis I and II. Approximately 61% of cells formed incorrect spindle morphologies, with the most prevalent being tripolar spindles. The erroneous spindles were actively rearranged to bipolar through a coalescence of poles before proceeding to anaphase. Spindle disassembly occurred as a two-state process with a slow depolymerization, followed by a quick collapse. The results demonstrate that maize meiosis I and II spindle assembly is remarkably fluid in the early assembly stages, but otherwise proceeds through a predictable series of events.

Aurora B Tension Sensing Mechanisms in the Kinetochore Ensure Accurate Chromosome Segregation.

The accurate segregation of chromosomes is essential for the survival of organisms and cells. Mistakes can lead to aneuploidy, tumorigenesis and congenital birth defects. The spindle assembly checkpoint ensures that chromosomes properly align on the spindle, with sister chromatids attached to microtubules from opposite poles. Here, we review how tension is used to identify and selectively destabilize incorrect attachments, and thus serves as a trigger of the spindle assembly checkpoint to ensure fidelity in chromosome segregation. Tension is generated on properly attached chromosomes as sister chromatids are pulled in opposing directions but resisted by centromeric cohesin. We discuss the role of the Aurora B kinase in tension-sensing and explore the current models for translating mechanical force into Aurora B-mediated biochemical signals that regulate correction of chromosome attachments to the spindle.

Anaphase asymmetry and dynamic repositioning of the division plane during maize meiosis.

The success of an organism is contingent upon its ability to transmit genetic material through meiotic cell division. In plant meiosis I, the process begins in a large spherical cell without physical cues to guide the process. Yet, two microtubule-based structures, the spindle and phragmoplast, divide the chromosomes and the cell with extraordinary accuracy. Using a live-cell system and fluorescently labeled spindles and chromosomes, we found that the process self- corrects as meiosis proceeds. Metaphase spindles frequently initiate division off-center, and in these cases anaphase progression is asymmetric with the two masses of chromosomes traveling unequal distances on the spindle. The asymmetry is compensatory, such that the chromosomes on the side of the spindle that is farthest from the cell cortex travel a longer distance at a faster rate. The phragmoplast forms at an equidistant point between the telophase nuclei rather than at the original spindle mid-zone. This asymmetry in chromosome movement implies a structural difference between the two halves of a bipolar spindle and could allow meiotic cells to dynamically adapt to errors in metaphase and accurately divide the cell volume.

Tethering sister centromeres to each other suggests the spindle checkpoint detects stretch within the kinetochore.

The spindle checkpoint ensures that newly born cells receive one copy of each chromosome by preventing chromosomes from segregating until they are all correctly attached to the spindle. The checkpoint monitors tension to distinguish between correctly aligned chromosomes and those with both sisters attached to the same spindle pole. Tension arises when sister kinetochores attach to and are pulled toward opposite poles, stretching the chromatin around centromeres and elongating kinetochores. We distinguished between two hypotheses for where the checkpoint monitors tension: between the kinetochores, by detecting alterations in the distance between them, or by responding to changes in the structure of the kinetochore itself. To distinguish these models, we inhibited chromatin stretch by tethering sister chromatids together by binding a tetrameric form of the Lac repressor to arrays of the Lac operator located on either side of a centromere. Inhibiting chromatin stretch did not activate the spindle checkpoint; these cells entered anaphase at the same time as control cells that express a dimeric version of the Lac repressor, which cannot cross link chromatids, and cells whose checkpoint has been inactivated. There is no dominant checkpoint inhibition when sister kinetochores are held together: cells expressing the tetrameric Lac repressor still arrest in response to microtubule-depolymerizing drugs. Tethering chromatids together does not disrupt kinetochore function; chromosomes are successfully segregated to opposite poles of the spindle. Our results indicate that the spindle checkpoint does not monitor inter-kinetochore separation, thus supporting the hypothesis that tension is measured within the kinetochore.

Beyond editing, CRISPR/Cas9 for protein localization: an educational primer for use with "A dCas9-based system identifies a central role for Ctf19 in kinetochore-derived suppression of meiotic recombination".

CRISPR/Cas9 has dramatically changed how we conduct genetic research, providing a tool for precise sequence editing. However, new applications of CRISPR/Cas9 have emerged that do not involve nuclease activity. In the accompanying article "A dCas9-based system identifies a central role for Ctf19 in kinetochore-derived suppression of meiotic recombination," Kuhl et al. utilize a catalytically dead Cas9 to localize proteins at specific genomic locations. The authors seek to understand the role of kinetochore proteins in the suppression of meiotic recombination, a phenomenon that has been observed in centromere regions. By harnessing the power of CRISPR/Cas9 to bind specific genomic sequences, Kuhl et al. localized individual kinetochore proteins to areas of high meiotic recombination and assessed their role in suppression. This primer article provides undergraduate students with background information on chromosomes, meiosis, recombination and CRISPR/Cas9 to support their reading of the Kuhl et al. study. This primer is intended to help students and instructors navigate the study's experimental design, interpret the results, and appreciate the broader scope of meiotic recombination and CRISPR/Cas9. Questions are included to facilitate discussion of the study.

Live-Cell Imaging of Meiotic Spindle and Chromosome Dynamics in Maize (Zea mays).

Live-cell imaging is a powerful tool that allows investigators to directly observe the dynamics of cellular processes. Live imaging has proven particularly useful in studying mitotic and meiotic chromosome segregation, where the assembly of spindles and movement of chromosomes can be quantified in ways not possible with fixed cells. This protocol describes how to image live meiosis in the agriculturally important plant, maize. The creation of fluorescently tagged tubulin allows visualization of maize spindles, and nucleic acid dyestain chromosomes. This protocol describes all steps required for live imaging, including how to grow plants, screen for relevant genotypes, harvest meiotic cells, and collect live movies of meiosis. While this protocol was developed for imaging fluorescently tagged tubulin, it can be easily modified to observe the meiotic dynamics of any fluorescently labeled protein of interest. © 2016 by John Wiley & Sons, Inc.

The Maize Divergent spindle-1 (dv1) Gene Encodes a Kinesin-14A Motor Protein Required for Meiotic Spindle Pole Organization.

The classic maize mutant divergent spindle-1 (dv1) causes failures in meiotic spindle assembly and a decrease in pollen viability. By analyzing two independent dv1 alleles we demonstrate that this phenotype is caused by mutations in a member of the kinesin-14A subfamily, a class of C-terminal, minus-end directed microtubule motors. Further analysis demonstrates that defects in early spindle assembly are rare, but that later stages of spindle organization promoting the formation of finely focused spindle poles are strongly dependent on Dv1. Anaphase is error-prone in dv1 lines but not severely so, and the majority of cells show normal chromosome segregation. Live-cell imaging of wild type and mutant plants carrying CFP-tagged ß-tubulin confirm that meiosis in dv1 lines fails primarily at the pole-sharpening phase of spindle assembly. These data indicate that plant kinesin-14A proteins help to enforce bipolarity by focusing spindle poles and that this stage of spindle assembly is not required for transition through the spindle checkpoint but improves the accuracy of chromosome segregation.

Genetic and genomic toolbox of Zea mays.

Maize has a long history of genetic and genomic tool development and is considered one of the most accessible higher plant systems. With a fully sequenced genome, a suite of cytogenetic tools, methods for both forward and reverse genetics, and characterized phenotype markers, maize is amenable to studying questions beyond plant biology. Major discoveries in the areas of transposons, imprinting, and chromosome biology came from work in maize. Moving forward in the post-genomic era, this classic model system will continue to be at the forefront of basic biological study. In this review, we outline the basics of working with maize and describe its rich genetic toolbox.

Chromosomal attachments set length and microtubule number in the Saccharomyces cerevisiae mitotic spindle.

The length of the mitotic spindle varies among different cell types. A simple model for spindle length regulation requires balancing two forces: pulling, due to micro-tubules that attach to the chromosomes at their kinetochores, and pushing, due to interactions between microtubules that emanate from opposite spindle poles. In the budding yeast Saccharomyces cerevisiae, we show that spindle length scales with kinetochore number, increasing when kinetochores are inactivated and shortening on addition of synthetic or natural kinetochores, showing that kinetochore-microtubule interactions generate an inward force to balance forces that elongate the spindle. Electron microscopy shows that manipulating kinetochore number alters the number of spindle microtubules: adding extra kinetochores increases the number of spindle microtubules, suggesting kinetochore-based regulation of microtubule number.