Live Cell Microscopy-Based RNAi Screening in the Moss Physcomitrella patens

2019

eLife

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Lagging chromosome is a hallmark of aneuploidy arising from errors in the kinetochore–spindle attachment in animal cells. However, kinetochore components and cellular phenotypes associated with kinetochore dysfunction are much less explored in plants. Here, we carried out a comprehensive characterization of conserved kinetochore components in the moss Physcomitrella patens and uncovered a distinct scenario in plant cells regarding both the localization and cellular impact of the kinetochore proteins. Most surprisingly, knock-down of several kinetochore proteins led to polyploidy, not aneuploidy, through cytokinesis failure in >90% of the cells that exhibited lagging chromosomes for several minutes or longer. The resultant cells, containing two or more nuclei, proceeded to the next cell cycle and eventually developed into polyploid plants. As lagging chromosomes have been observed in various plant species in the wild, our observation raised a possibility that they could be one of the natural pathways to polyploidy in plants.

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Kinetochore protein depletion underlies cytokinesis failure and somatic polyploidization in the moss Physcomitrella patens

Kozgunova

Nishina

2019

eLife

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“…Circular Cas9 and sgRNA plasmids were cotransformed with oligonucleotide templates into the wild-type strains carrying a Lifeact-mCherry marker or histone H2B-RFP and GFP-tubulin markers (Miki et al, 2016). In general, 10 μg Cas9 plasmid, 10 μg sgRNA plasmid and 10 μL oligonucleotide templates were used appropriately for each transformation.…”

Section: Methodsmentioning

confidence: 99%

Fast, Efficient, and Precise Gene Editing in the MossPhyscomitrella patens

2019

Preprint

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12Recent years, the bryophyte moss Physcomitrella patens has become an emerging 13 model organism for studying conserved signaling pathways and developmental 14 processes during plant evolution. Its short life cycle, ease of cultivation, and high rate of 15 homologous recombination have made it an ideal system for genetic analysis. However, 16 the presence of highly redundant genes and the difficulty of isolating hypomorphic 17 mutants have limited its broader use. Here we developed a simple, fast, and efficient 18 method to generate customized mutants in P. patens. We show that transient 19 cotransformation of CRISPR/Cas9 and oligonucleotide templates enables microindel 20 knock-in with high efficiency and accuracy. Using this method, we generated strains 21 carrying various types of mutations, including amino acid substitution, out-of-frame 22 deletion/insertion, splice site alteration, and small tag integration. We also demonstrate 23 that multiplex gene editing can be efficiently achieved to generate putative null and 24 hypomorphic mutants of redundant genes in one step. Thus our method will not only 25 simplify multiple-gene knockout, but also allows the generation of hypomorphic mutants 26 of genes of interest, especially those that are essential for viability. 27 28 Keywords 29 Bryophyte, Physcomitrella patens, CRISPR/Cas9, Oligonucleotide, Knock-in 30 4 2017a, 2017b). By forming a complex, its components, the endonuclease Cas9 and the 62 single guide RNA (sgRNA), are capable of introducing double-strand breaks (DSBs) at 63 predicted genomic loci once the sgRNA is customized with a targeting sequence (Jiang 64 and Doudna, 2017). These DSBs are repaired through the non-homologous end joining 65 or homology directed repair pathways, which leads to subsequent nucleotide changes 66 (Jiang and Doudna, 2017). In P. patens, CRISPR/Cas9 has been exploited to generate 67 heritable mutants (Nomura et al., 2016; Lopez-Obando et al., 2016; Collonnier et al., 68 2017a). However, it is unclear whether precise mutation knock-in can be achieved to 69 generate hypomorphic mutants. In addition, as microhomology-mediated end joining 70 (MMEJ) pathway is active in P. patens (Kamisugi et al., 2006; Collonnier et al., 2017a), 71 template-free editing can generate specific deletions at a high frequency, which may not 72 necessarily introduce out-of-frame mutations. For instance, a previous study reported 73 that 74% of the mutations generated by one CRISPR target are identical in-frame 74 deletions (Collonnier et al., 2017a).75 76Here we report a fast and efficient method to generate customized mutants in P. patens. 77 We show that transient cotransformation of CRISPR/Cas9 and oligonucleotide 78 templates induces microindel knock-in at a high efficiency and accuracy. This allows us 79 to introduce various types of mutations into the genome including amino acid 80 substitution, out-of-frame insertion/deletion, splice site alteration, or small tag integration. 81 We also demonstrate that multiplex gene editing can be effici...

“…The Kinesin-13 mutant plasmids (Kinesin-13b RIG -Cerulean, Kinesin-13b KVD/KEC -Cerulean, Kinesin-13b Loop12 -Cerulean) were generated by mutagenesis with the full-length Kinesin-13 plasmid and primers listed in Table S2, S3. The cloned Kinesin-13 sequences were ligated into pMN603 vector by a Gateway LR reaction, which contains EF1α promoter, Cerulean gene, nourseothricin-resistance cassette, PTA1 sequences designated for homologous recombination mediated integration (Miki et al, 2016; Yoshida et al, 2019).…”

Section: Methodsmentioning

confidence: 99%

Novel roles of Kinesin-13 and Kinesin-8 during cell growth and division in the mossPhyscomitrella patens

Edzuka

et al. 2019

Preprint

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Kinesin-13 and -8 are well-known microtubule (MT) depolymerases that regulate MT length and chromosome movement in animal mitosis. While much is unknown about plant Kinesin-8, Arabidopsis and rice Kinesin-13 have been shown to depolymerise MTs in vitro. However, mitotic function of both kinesins has yet to be understood in plants. Here, we generated the complete null mutants in plants of Kinesin-13 and -8 in the moss Physcomitrella patens. Both kinesins were found to be non-essential for viability, but the Kinesin-13 knockout (KO) line had increased mitotic duration and reduced spindle length, whereas the Kinesin-8 KO line did not display obvious mitotic defects. Surprisingly, spindle MT poleward flux, for which Kinesin-13 is responsible for in animals, was retained in the absence of Kinesin-13. Concurrently, MT depolymerase activity of either moss kinesins could not be observed, with MT catastrophe inducing (Kinesin-13) or MT gliding (Kinesin-8) activity observed in vitro. Interestingly, both KO lines showed waviness in their protonema filaments, which correlated with positional instability of the MT foci in their tip cells. Taken together, the results suggest that plant Kinesin-13 and -8 have diverged in both mitotic function and molecular activity, acquiring new roles in regulating MT foci positioning for directed tip-growth.One sentence summaryThis study uncovered the roles of Kinesin-13 and Kinesin-8 in regulating microtubule dynamics for mitotic spindle formation and straight tip cell growth in the moss Physcomitrella patens