title: An inducible RNA interference system in Physcomitrella patens reveals a dominant role of augmin in phragmoplast microtubule generation.
abstract: Mitosis is a fundamental process of eukaryotic cell proliferation. However, the molecular mechanisms underlying mitosis remain poorly understood in plants partly because of the lack of an appropriate model cell system in which loss-of-function analyses can be easily combined with high-resolution microscopy. Here, we developed an inducible RNA interference (RNAi) system and three-dimensional time-lapse confocal microscopy in the moss Physcomitrella patens that allowed in-depth phenotype characterization of the moss genes essential for cell division. We applied this technique to two microtubule regulators, augmin and γ-tubulin complexes, whose mitotic roles remain obscure in plant cells. Live imaging of caulonemal cells showed that they proceed through mitosis with continual generation and self-organization of acentrosomal microtubules. We demonstrated that augmin plays an important role in γ-tubulin localization and microtubule generation from prometaphase to cytokinesis. Most evidently, microtubule formation in phragmoplasts was severely compromised after RNAi knockdown of an augmin subunit, leading to incomplete expansion of phragmoplasts and cytokinesis failure. Knockdown of the γ-tubulin complex affected microtubule formation throughout mitosis. We conclude that postanaphase microtubule generation is predominantly stimulated by the augmin/γ-tubulin machinery in moss and further propose that this RNAi system serves as a powerful tool to dissect the molecular mechanisms underlying mitosis in land plants.
authors.affiliation: Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Nakaoka
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Miki
authors.forename: Ryuta
authors.initials: R
authors.lastname: Fujioka
authors.forename: Ryota
authors.initials: R
authors.lastname: Uehara
authors.forename: Akiko
authors.initials: A
authors.lastname: Tomioka
authors.forename: Chikashi
authors.initials: C
authors.lastname: Obuse
authors.forename: Minoru
authors.initials: M
authors.lastname: Kubo
authors.forename: Yuji
authors.initials: Y
authors.lastname: Hiwatashi
authors.forename: Gohta
authors.initials: G
authors.lastname: Goshima
mesh_terms: D000705:Anaphase; D019068:Bryopsida / Q000166:cytology / Q000235:genetics* / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D017343:Genes, Plant / Q000235:genetics; D006801:Humans; D008870:Microtubules / Q000378:metabolism*; D010641:Phenotype; D010940:Plant Proteins / Q000378:metabolism*; D030821:Plants, Genetically Modified; D021381:Protein Transport; D034622:RNA Interference; D015203:Reproducibility of Results; D008941:Spindle Apparatus / Q000378:metabolism*; D014404:Tubulin / Q000378:metabolism
chemical_list: D010940:Plant Proteins; D014404:Tubulin