Biology subjects

Wakazaki, M.

Publications and source records attributed to Wakazaki, M..

5 recordsLinked to original sources

bioRxiv2024

Coatomer complex I is required for the transport of SARS-CoV-2 progeny virions from the endoplasmic reticulum-Golgi intermediate compartment

SARS-CoV-2 undergoes budding within the lumen of the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) and delivers progeny virions to the cell surface by employing vesicular transport. However, the molecular mechanisms remain poorly understood. Using three-dimensional electron microscopic analysis, such as array tomography and electron tomography, we found that virion-transporting vesicles possessed a coated protein on their membrane and demonstrated that the coated protein was coatomer complex I (COPI). During the later stages of SARS-CoV-2 infection, we observed a notable alteration in the distribution of COPI and ERGIC throughout the cytoplasm. Depletion of COPB2, a key component of COPI, led to the confinement of SARS-CoV-2 structural proteins in the perinuclear region, where progeny virions were accumulated within the ERGIC. While the expression levels of viral proteins within cells were comparable, this depletion significantly reduced the efficiency of virion release, leading to the significant inhibition of viral replication. Hence, our findings suggest COPI as a critical player in facilitating the transport of SARS-CoV-2 progeny virions from the ERGIC. Thus, COPI could be a promising target for the development of antivirals against SARS-CoV-2.

microbiology↗

bioRxiv2020

Three-dimensional reconstructions of the internal structures of haustoria in parasitic Orobanchaceae

Parasitic plants infect other plants by forming haustoria, specialized multicellular organs consisting of several cell types each of which has unique morphological features and physiological roles associated with parasitism. Understanding the spatial organization of cell types is, therefore, of great importance in elucidating the functions of haustoria. Here, we report a three-dimensional (3-D) reconstruction of haustoria from two Orobanchaceae species, the obligate parasite Striga hermonthica infecting rice and the facultative parasite Phtheirospermum japonicum infecting Arabidopsis. Our images reveal the spatial arrangements of multiple cell types inside haustoria and their interaction with host roots. The 3-D internal structures of haustoria highlight differences between the two parasites, particularly at the xylem connection site with the host. Our study provides structural insights into how organs interact between hosts and parasitic plants. One-sentence summaryThree-dimensional image reconstruction was used to visualize the spatial organization of cell types in the haustoria of parasitic plants with special reference to their interaction with host roots.

plant biology↗

bioRxiv2020

GSK3 activity is a cell fate switch that balances the ratio of vascular cell type

The phloem transports photosynthetic assimilates and signalling molecules. It mainly consists of sieve elements (SEs), which act as "highways" for transport, and companion cells (CCs), which serve as "gates" to load/unload cargos. Though SEs and CCs function together, it remains unknown what determines the ratio of SE/CC in the phloem. In this study, we develop a novel culture system for CC differentiation named VISUAL-CC, which reconstitutes the SE-CC complex formation. Comparative expression analysis in VISUAL-CC reveals that SE and CC differentiation tends to show negative correlation, while total phloem differentiation is unchanged. This varying SE/CC ratio is largely dependent on GSK3 kinase activity. Indeed, gsk3 hextuple mutants possess much more SEs and less CCs in planta. Conversely, gsk3 gain-of-function mutants induced by phloem-specific promoter partially increased the CC ratio. Taken together, GSK3 activity appears to function as a cell fate switch in the phloem, thereby balancing the SE/CC ratio.

plant biology↗

bioRxiv2019

Excessive assimilation of ammonium by plastidic glutamine synthetase is a major cause of ammonium toxicity in Arabidopsis thaliana

Plants use nitrate and ammonium in the soil as their main nitrogen sources. Recently, ammonium has attracted attention due to evidence suggesting that, in C3 species, an elevated CO2 environment inhibits nitrate assimilation. However, high concentrations of ammonium as the sole nitrogen source for plants causes impaired growth, i.e. ammonium toxicity. Although ammonium toxicity has been studied for a long time, the primary cause remains to be elucidated. Here, we show that ammonium assimilation in plastids rather than ammonium accumulation is a primary cause for toxicity. Our genetic screen of ammonium-tolerant Arabidopsis lines with enhanced shoot growth identified plastidic GLUTAMINE SYNTHETASE 2 (GLN2) as the causal gene. Our reciprocal grafting of wild-type and GLN2 or GLN1;2-deficient lines suggested that shoot GLN2 activity results in ammonium toxicity, whilst root GLN1;2 activity prevents it. With exposure to toxic levels of ammonium, the shoot GLN2 reaction produced an abundance of protons within cells, thereby elevating shoot acidity and stimulating expression of acidic stress-responsive genes. Application of an alkaline ammonia solution to the toxic ammonium medium efficiently alleviated the ammonium toxicity with a concomitant reduction in shoot acidity. Consequently, we conclude that a primary cause of ammonium toxicity is acidic stress in the shoot. This fundamental insight provides a framework for enhanced understanding of ammonium toxicity in plants.

plant biology↗

bioRxiv2018

The RopGEF KARAPPO is Essential for the Initiation of Vegetative Reproduction in Marchantia

Many plants can reproduce vegetatively, producing clonal progeny from vegetative cells; however, little is known about the molecular mechanisms underlying this process. Liverwort (Marchantia polymorpha), a basal land plant, propagates asexually via gemmae, which are clonal plantlets formed in gemma cups on the dorsal side of the vegetative thallus [1]. The initial stage of gemma development involves elongation and asymmetric divisions of a specific type of epidermal cell, called a gemma initial, which forms on the floor of the gemma cup [2, 3]. To investigate the regulatory mechanism underlying gemma development, we focused on two allelic mutants in which no gemma initial formed; these mutants were named karappo, meaning \"empty\". We used whole-genome sequencing of both mutants, and molecular genetic analyses to identify the causal gene, KARAPPO (KAR), which encodes a Rop guanine nucleotide exchange factor (RopGEF) carrying a PRONE catalytic domain. In vitro GEF assays showed that the full-length KAR protein and the PRONE domain have significant GEF activity toward MpRop, the only Rop GTPase in M. polymorpha. Moreover, genetic complementation experiments showed a significant role for the N- and C-terminal variable regions in gemma development. Our investigation demonstrated an essential role for KAR/RopGEF in the initiation of plantlet development from a differentiated cell, which may involve cell polarity formation and subsequent asymmetric cell division via activation of Rop signaling, implying a similar developmental mechanism in vegetative reproduction of various land plants.

plant biology↗