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 g-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 g-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 g-tubulin complex affected microtubule formation throughout mitosis. We conclude that postanaphase microtubule generation is predominantly stimulated by the augmin/g-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.

The rise and fall of the phragmoplast microtubule array

“…In the mutant, the phragmoplast MT array often fails to expand toward the cell periphery and MTs ultimately became disorganized [42 ]. Similarly when genes encoding augmin subunits are silenced in the moss P. patens, phragmoplast expansion is inhibited because of compromised MT formation in the array [43 ]. Thus, the MT-dependent MT nucleation mechanism, mediated by the augmin and g-tubulin complexes, is shared in both the spindle and phragmoplast for amplifying MTs.…”

Endogenous localizome identifies 43 mitotic kinesins in a plant cell

“…Another key advantage of the P. patens system is the ease of live cell microscopy in caulonemal stem cells that actively divide every 5-6 h (13,15). Caulonemal cells are found in protonemata, which are filamentous structures that appear after spore germination, and experience less autofluorescencerelated interference derived from chloroplasts; therefore, they are suitable for the live imaging of GFP-or red fluorescent protein (RFP)-tagged markers during cell division (14,19,20). Previous studies have characterized the process of mitotic spindle and phragmoplast formation in caulonemal cells by using GFP-tubulin, MAP65-GFP (anti-parallel MT-bundling protein), or EB1-GFP (MT plus-end-tracking protein) (14,19,22).…”

“…Caulonemal cells are found in protonemata, which are filamentous structures that appear after spore germination, and experience less autofluorescencerelated interference derived from chloroplasts; therefore, they are suitable for the live imaging of GFP-or red fluorescent protein (RFP)-tagged markers during cell division (14,19,20). Previous studies have characterized the process of mitotic spindle and phragmoplast formation in caulonemal cells by using GFP-tubulin, MAP65-GFP (anti-parallel MT-bundling protein), or EB1-GFP (MT plus-end-tracking protein) (14,19,22). Caulonemal cells, like many other plant cell types, do not have centrosomes or other discrete MT organizing centers; instead, they generate MTs predominantly through amplification mediated by a protein complex called augmin (14,23).…”

“…Previous studies have characterized the process of mitotic spindle and phragmoplast formation in caulonemal cells by using GFP-tubulin, MAP65-GFP (anti-parallel MT-bundling protein), or EB1-GFP (MT plus-end-tracking protein) (14,19,22). Caulonemal cells, like many other plant cell types, do not have centrosomes or other discrete MT organizing centers; instead, they generate MTs predominantly through amplification mediated by a protein complex called augmin (14,23). Moreover, unlike most other cell types in land plants, caulonemal cells do not form the preprophase band (PPB), a MT-based structure that transiently appears at the early stages of mitosis in the cell cortex and determines the future division plane (24)(25)(26).…”

“…The moss Physcomitrella patens is an emerging model land plant, for which a variety of molecular techniques can be used (12)(13)(14)(15)(16)(17)(18)(19)(20)(21). It has 27 chromosomes, and the genome sequence is available (12).…”