研究者詳細 - 植木　尚子

論文 - 植木　尚子

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Differentiation of microbial community in coastal seawater samples before and during a Akashiwo sanguinea (Dinophyceae) bloom in urban area of Antofagasta city (northern Chile) 査読

Jingming Hua, Henry Camerónc, Joaquín I. Rillingd, Marco Camposd, Tay Ruiz-Gild, Mariela A. Gonzaleze, Gonzalo Gajardo, Karen Vergarad, Leonardo Guzmán, Oscar Espinoza-González, Gonzalo Fuenzalida, Carlos Riquelme, Shoko Ueki, Satoshi Nagai, Fumito Maruyama, So Fujiyoshi, Kyoko Yarimizum, Ishara Pereram, Andrés Ávilae, Jacquelinne J. Acuñad, Qian Zhanga, Milko A. Jorquera

Harmful Algae 2024年12月

The dataset of de novo assembly and inferred functional annotation of the transcriptome of Heterosigma akashiwo, a bloom-forming, cosmopolitan raphidophyte. 査読国際誌

Masanao Sato, Masahide Seki, Yutaka Suzuki, Shoko Ueki

Data in brief 48 109071 - 109071 2023年6月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Heterosigma akashiwo is a eukaryotic, cosmopolitan, and unicellular alga (class: Raphidophyceae), and produces fish-killing blooms. There is a substantial scientific and practical interest in its ecophysiological characteristics that determine bloom dynamics and its adaptation to broad climate zones. A well-annotated genomic/genetic sequence information enables researchers to characterize organisms using modern molecular technology. In the present study, we conducted H. akashiwo RNA sequencing, a de novo transcriptome assembly of 84,693,530 high-quality deduplicated short-read sequences. Obtained RNA reads were assembled by Trinity assembler and 144,777 contigs were identified with N50 values of 1085. Total 60,877 open reading frames with the length of 150 bp or greater were predicted. For further analyses, top Gene Ontology terms, pfam hits, and blast hits were annotated for all the predicted genes. The raw data were deposited in the NCBI SRA database (BioProject PRJDB6241 and PRJDB15108), and the assemblies are available in NCBI TSA database (ICRV01). The annotation information can be obtained in Dryad and can be accessed via doi: 10.5061/dryad.m0cfxpp56.

DOI： 10.1016/j.dib.2023.109071

The holobiome of marine harmful algal blooms (HABs): A novel ecosystem-based approach for implementing predictive capabilities and managing decisions 査読

Gonzalo Gajardo, Jesús Morón-López, Karen Vergara, Shoko Ueki, Leonardo Guzmán, Oscar Espinoza-González, Alondra Sandoval, Gonzalo Fuenzalida, Alejandro A. Murillo, Carlos Riquelme, Henry Camerón, Satoshi Nagai, Fumito Maruyama, So Fujiyoshi, Kyoko Yarimizu, Ishara Perera, Mikihiko Kawai, Andrés Ávila, Giovanni Larama, Mariela A. Gonzalez, Joaquín I. Rilling, Marco Campos, Tay Ruiz-Gil, Benjamin Durán-Vinet, Jacquelinne J. Acuña, Milko A. Jorquera

Environmental Science & Policy 143 44 - 54 2023年5月

Intraspecies variation of the mitochondrial genome: An evaluation for phylogenetic approaches based on the conventional choices of genes and segments on mitogenome. 査読国際誌

Jesús Morón-López, Karen Vergara, Masanao Sato, Gonzalo Gajardo, Shoko Ueki

PloS one 17 ( 8 ) e0273330 2022年

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Intraspecies nucleotide sequence variation is a key to understanding the evolutionary history of a species, such as the geographic distribution and population structure. To date, numerous phylogenetic and population genetics studies have been conducted based on the sequences of a gene or an intergenic region on the mitochondrial genome (mtDNA), such as cytochrome c oxidase subunits or the D-loop. To evaluate the credibility of the usage of such 'classic' markers, we compared the phylogenetic inferences based on the analyses of the partial and entire mtDNA sequences. Importantly, the phylogenetic reconstruction based on the short marker sequences did not necessarily reproduce the tree topologies based on the analyses of the entire mtDNA. In addition, analyses on the datasets of various organisms revealed that the analyses based on the classic markers yielded phylogenetic trees with poor confidence in all tested cases compared to the results based on full-length mtDNA. These results demonstrated that phylogenetic analyses based on complete mtDNA sequences yield more insightful results compared to those based on mitochondrial genes and segments. To ameliorate the shortcomings of the classic markers, we identified a segment of mtDNA that may be used as an 'approximate marker' to closely reproduce the phylogenetic inference obtained from the entire mtDNA in the case of mammalian species, which can be utilized to design amplicon-seq-based studies. Our study demonstrates the importance of the choice of mitochondrial markers for phylogenetic analyses and proposes a novel approach to choosing appropriate markers for mammalian mtDNA that reproduces the phylogenetic inferences obtained from full-length mtDNA.

DOI： 10.1371/journal.pone.0273330

Suitcase Lab: new, portable, and deployable equipment for rapid detection of specific harmful algae in Chilean coastal waters 査読

So Fujiyoshi, Kyoko Yarimizu, Yohei Miyashita, Joaquín Rilling, Jacquelinne J. Acuña, Shoko Ueki, Gonzalo Gajardo, Oscar Espinoza-González, Leonardo Guzmán, Milko A. Jorquera, Satoshi Nagai, Fumito Maruyama

Environmental Science and Pollution Research 2020年11月

掲載種別：研究論文（学術雑誌）出版者・発行元：Springer Science and Business Media LLC

<title>Abstract</title>Phytoplankton blooms, including harmful algal blooms (HABs), have serious impacts on ecosystems, public health, and productivity activities. Rapid detection and monitoring of marine microalgae are important in predicting and managing HABs. We developed a toolkit, the Suitcase Lab, to detect harmful algae species in the field. We demonstrated the Suitcase Lab’s capabilities for sampling, filtration, DNA extraction, and loop-mediated isothermal amplification (LAMP) detection in cultured <italic>Alexandrium catenella</italic> cells as well as Chilean coastal waters from four sites: Repollal, Isla García, Puerto Montt, and Metri. A LAMP assay using the Suitcase Lab in the field confirmed microscopic observations of <italic>A. catenella</italic> in samples from Repollal and Isla García. The Suitcase Lab allowed the rapid detection of <italic>A. catenella</italic>, within 2 h from the time of sampling, even at a single cell per milliliter concentrations, demonstrating its usefulness for quick and qualitative on-site diagnosis of target toxic algae species. This method is applicable not only to detecting harmful algae but also to other field studies that seek a rapid molecular diagnostic test.

DOI： 10.1007/s11356-020-11567-5

その他リンク： http://link.springer.com/article/10.1007/s11356-020-11567-5/fulltext.html

Protocols for Monitoring Harmful Algal Blooms for Sustainable Aquaculture and Coastal Fisheries in Chile 査読

Kyoko Yarimizu, So Fujiyoshi, Mikihiko Kawai, Luis Norambuena-Subiabre, Emma-Karin Cascales, Joaquin-Ignacio Rilling, Jonnathan Vilugrón, Henry Cameron, Karen Vergara, Jesus Morón-López, Jacquelinne J. Acuña, Gonzalo Gajardo, Oscar Espinoza-González, Leonardo Guzmán, Milko A. Jorquera, Satoshi Nagai, Gemita Pizarro, Carlos Riquelme, Shoko Ueki, Fumito Maruyama

International Journal of Environmental Research and Public Health 17 ( 20 ) 7642 - 7642 2020年10月

掲載種別：研究論文（学術雑誌）出版者・発行元：MDPI AG

Harmful algae blooms (HABs) cause acute effects on marine ecosystems due to their production of endogenous toxins or their enormous biomass, leading to significant impacts on local economies and public health. Although HAB monitoring has been intensively performed at spatiotemporal scales in coastal areas of the world over the last decades, procedures have not yet been standardized. HAB monitoring procedures are complicated and consist of many methodologies, including physical, chemical, and biological water sample measurements. Each monitoring program currently uses different combinations of methodologies depending on site specific purposes, and many prior programs refer to the procedures in quotations. HAB monitoring programs in Chile have adopted the traditional microscopic and toxin analyses but not molecular biology and bacterial assemblage approaches. Here we select and optimize the HAB monitoring methodologies suitable for Chilean geography, emphasizing on metabarcoding analyses accompanied by the classical tools with considerations including cost, materials and instrument availability, and easiness and efficiency of performance. We present results from a pilot study using the standardized stepwise protocols, demonstrating feasibility and plausibility for sampling and analysis for the HAB monitoring. Such specific instructions in the standardized protocol are critical obtaining quality data under various research environments involving multiple stations, different analysts, various time-points, and long HAB monitoring duration.

DOI： 10.3390/ijerph17207642

Phylogeographic characteristics of hypervariable regions in the mitochondrial genome of a cosmopolitan, bloom-forming raphidophyte, Heterosigma akashiwo 査読

Shoko Ueki

J Phycology 2019年

A unique, highly variable mitochondrial gene with coding capacity of Heterosigma akashiwo, class Raphidophyceae 査読

Aiko Higashi, Satoshi Nagai, Paulo S. Salomon, Shoko Ueki

JOURNAL OF APPLIED PHYCOLOGY 29 ( 6 ) 2961 - 2969 2017年12月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SPRINGER

Information related to geographical distribution and local strain composition is essential to an understanding of the dynamics of harmful algae in the environment. Previously, we identified a highly variable segment on the mitochondrial genome of Heterosigma akashiwo, a bloom-forming noxious unicellular algal species. Here, we assessed the utility of the mitochondrial hypervariable region for the phylogeographic study of the alga for different distance ranges. The sequences of H. akashiwo strains obtained from different geographic origins were successfully amplified and sequenced. We found differences among the sequences of the strains obtained from high-latitude regions of Northern Pacific/Atlantic; lower latitude regions of North America West Coast; and other regions including Brazil, Japan, Singapore, and North America East Coast. On the other hand, no strong geographic patterns for the sequences among Japanese strains were observed. Therefore, the hypervariable segment may be useful to distinguish H. akashiwo strains originated from distant regions (Atlantic/Pacific, high/low latitudes), rather than regions separated by shorter distances. The sequence contains an open reading frame coding for a protein with unknown function, and the transcription of the gene was confirmed by RNA-seq analysis. Despite the sequence variations observed among H. akashiwo strains originating in different parts of the world, three domains of the protein were highly conserved among all of the strains, suggesting that they may be important to the function of the protein.

DOI： 10.1007/s10811-017-1142-2

A hypervariable mitochondrial protein coding sequence associated with geographical origin in a cosmopolitan bloom-forming alga, Heterosigma akashiwo 査読

Aiko Higashi, Satoshi Nagai, Sergio Seone, Shoko Ueki

BIOLOGY LETTERS 13 ( 4 ) 2017年4月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ROYAL SOC

Geographical distributions of phytoplankton species can be defined by events on both evolutionary time and shorter scales, e.g. recent climate changes. Additionally, modern industrial activity, including the transport of live fish and spat for aquaculture and aquatic microorganisms in ship ballast water, may aid the spread of phytoplankton. Obtaining a reliable marker is key to gaining insight into the phylogeographic history of a species. Here, we report a hypervariable mitochondrial gene in the cosmopolitan bloom forming alga, Heterosigma akashiwo. We compared the entire mitochondrial genome sequences of seven H.. akashiwo strains from Japanese and North American coastal waters and identified a hypervariable segment. The region codes for a hypothetical protein with no defined function, and its variations between Japanese and North American isolates were prominent, while the sequences were more conserved among Japanese strains and North American isolates. Comparison of the sequence in isolates obtained from different geographical points in the Northern Hemisphere revealed that the sequence variations largely correlated with latitude and longitude (i.e. Pacific/Atlantic oceans). Our results demonstrate the usefulness of the sequence in determining the phylogeographic history of H. akashiwo.

DOI： 10.1098/rsbl.2016.0976

Chloroplast genome sequences of seven strains of the bloom-forming raphidophyte Heterosigma akashiwo 査読

Sergio Seoane, Kiwamu Hyodo, Shoko Ueki

Genome Announcements 5 ( 41 ) 2017年

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Society for Microbiology

We report here the complete chloroplast genome sequences of seven strains of the bloom-forming raphidophyte Heterosigma akashiwo. These ~160-kb sequences contain 124 protein-, 6 rRNA-, and 34 tRNA-coding sequences. Notable sequence variations were observed among these seven sequenced and two previously characterized strains.

DOI： 10.1128/genomeA.01030-17

Selective growth promotion of bloom-forming raphidophyte Heterosigma akashiwo by a marine bacterial strain 査読

Aiko Higashi, Yoshiko Fujitani, Natsuko Nakayama, Akio Tani, Shoko Ueki

HARMFUL ALGAE 60 150 - 156 2016年12月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE BV

Algal bloom is typically caused by aberrant propagation of a single species, resulting in its predomination in the local population. While environmental factors including temperature and eutrophication are linked to bloom, the precise mechanism of its formation process is still obscure. Here, we isolated a bacterial strain that promotes growth of Heterosigma akashiwo, a Raphidophyceae that causes harmful algal blooms. Based on 16S rRNA gene sequence, the strain was identified as Altererythrobacter ishigakiensis, a member of the class Alphaproteobacteria. When added to culture, this strain facilitated growth of H. akashiwo and increased its cell culture yield significantly. Importantly, this strain did not affect the growth of other raphidophytes, Chattonella ovate and C. antiqua, indicating that it promotes growth of H. akashiwo in a species-specific manner. We also found that, in co-culture, H. akashiwo suppressed the growth of C. ovate. When A. ishigakiensis was added to the mixed culture, H. akashiwo growth was facilitated while C ovate propagation was markedly suppressed, indicating that the presence of the bacterium enhances the dominance of H. akashiwo over C ovate. This is the first example of selective growth promotion of H. akashiwo by a marine bacterium, and may exemplify importance of symbiotic bacterium on algal bloom forming process in general. (C) 2016 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.hal.2016.11.009

Evolution and Phylogeny of Large DNA Viruses, Mimiviridae and Phycodnaviridae Including Newly Characterized Heterosigma akashiwo Virus 査読

Fumito Maruyama, Shoko Ueki

FRONTIERS IN MICROBIOLOGY 7 1942 2016年11月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：FRONTIERS MEDIA SA

Nucleocytoplasmic DNA viruses are a large group of viruses that harbor double stranded DNA genomes with sizes of several 100 kbp, challenging the traditional concept of viruses as small, simple 'organisms at the edge of life.' The most intriguing questions about them may be their origin and evolution, which have yielded the variety we see today. Specifically, the phyletic relationship between two giant dsDNA virus families that are presumed to be close, Mimiviridae, which infect Acantharnoeba, and Phycodnaviridae, which infect algae, is still obscure and needs to be clarified by indepth analysis. Here, we studied Mimiviridae Phycodnaviridae phylogeny including the newly identified Heterosigma akashiwo virus strain HaV53. Gene-to-gene comparison of HaV53 with other giant dsDNA viruses showed that only a small proportion of HaV53 genes show similarities with the others, revealing its uniqueness among Phycodnaviridae. Phylogenetic/genomic analysis of Phycodnaviridae including HaV53 revealed that the family can be classified into four distinctive subfamilies, namely, Megaviridae (Mimivirus-like), Chlorovirus-type, and Coccolitho/Phaeovirus-type groups, and HaV53 independent of the other three groups. Several orthologs found in specific subfamilies while absent from the others were identified, providing potential family marker genes. Finally, reconstruction of the evolutionary history of Phycodnaviridae and Mimiviridae revealed that these viruses are descended from a common ancestor with a small set of genes and reached their current diversity by differentially acquiring gene sets during the course of evolution. Our study illustrates the phylogeny and evolution of Mimiviridac Phycodnaviridae and proposes classifications that better represent phyletic relationships among the family members.

DOI： 10.3389/micb.2016.01942

Complete genome sequence of a phycodnavirus, Heterosigma akashiwo virus strain 53 査読

Yoshitoshi Ogura, Tetsuya Hayashi, Shoko Ueki

Genome Announcements 4 ( 6 ) 2016年

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Society for Microbiology

We report the complete genome sequence of Heterosigma akashiwo virus strain 53. The virus is a member of the Phycodnaviridae, one of the families regarded as giant double-stranded DNA viruses. The 274,793-bp genome contained 246 protein-coding and 3 tRNA-coding sequences.

DOI： 10.1128/genomeA.01279-16

Mitochondrial genome sequences of four strains of the bloom-forming raphidophyte Heterosigma akashiwo 査読

Yoshitoshi Ogura, Natsuko Nakayama, Tetsuya Hayashi, Shoko Ueki

Genome Announcements 4 ( 6 ) 2016年

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Society for Microbiology

We report here the complete mitochondrial genome sequences of four strains of bloom-forming raphidophytes from Heterosigma akashiwo. These 39-kb sequences contain 42 protein-, two rRNA-, and 26 tRNA-coding sequences. Notable sequence variations were observed among these four newly sequenced and three previously characterized strains, suggesting their potential usage as strain-specific markers.

DOI： 10.1128/genomeA.01288-16

Plasmodesmata-associated proteins: Can we see the whole elephant? 査読

Shoko Ueki, Vitaly Citovsky

Plant Signaling and Behavior 9 ( 2 ) e27899 2014年

担当区分：筆頭著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Landes Bioscience

Encased in rigid cell walls, plant cells have evolved unique channel structures, plasmodesma (Pd), to create a pathway for molecular exchange between adjacent cells. Pd are basically cytoplasmic channels through the cell wall, which are lined by plasma membrane, and contain a modified strand of ER that spans them. These structures provide cytoplasmic and membrane continuity between connected cells, and that continuity is utilized for short and long distance molecular trafficking. Pd sphincters, made from constricting the Pd openings by outer layers of callose, together with the ER strand that occludes the Pd lumen set the upper limit for the size of molecules that can freely diffuse through the cytoplasmic component of the Pd channel. This limit, called the size exclusion limit (SEL), is a major factor that restricts macromolecular transport through Pd. © 2014 Landes Bioscience.

DOI： 10.4161/psb.27899

Identification of a movement protein of Mirafiori lettuce big-vein ophiovirus 査読

Akihiro Hiraguri, Shoko Ueki, Hideki Kondo, Koji Nomiyama, Takumi Shimizu, Tamaki Ichiki-Uehara, Toshihiro Omura, Nobumitsu Sasaki, Hiroshi Nyunoya, Takahide Sasaya

Journal of General Virology 94 ( 5 ) 1145 - 1150 2013年5月

記述言語：英語掲載種別：研究論文（学術雑誌）

Mirafiori lettuce big-vein virus (MiLBVV) is a member of the genus Ophiovirus, which is a segmented negative-stranded RNA virus. In microprojectile bombardment experiments to identify a movement protein (MP) gene of ophioviruses that can trans-complement intercellular movement of an MP-deficient heterologous virus, a plasmid containing an infectious clone of a tomato mosaic virus (ToMV) derivative expressing the GFP was co-bombarded with plasmids containing one of three genes from MiLBVV RNAs 1, 2 and 4 onto Nicotiana benthamiana. Intercellular movement of the movement-defective ToMV was restored by co-expression of the 55 kDa protein gene, but not with the two other genes. Transient expression in epidermal cells of N. benthamiana and onion showed that the 55 kDa protein with GFP was localized on the plasmodesmata. The 55 kDa protein encoded in the MiLBVV RNA2 can function as an MP of the virus. This report is the first to describe an ophiovirus MP. © 2013 SGM.

DOI： 10.1099/vir.0.050005-0

Transient gene expression in epidermal cells of plant leaves by biolistic DNA delivery 査読

Shoko Ueki, Shimpei Magori, Benoît Lacroix, Vitaly Citovsky

Methods in Molecular Biology 940 17 - 26 2013年

担当区分：筆頭著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Transient gene expression is a useful approach for studying the functions of gene products. In the case of plants, Agrobacterium infiltration is a method of choice for transient introduction of genes for many species. However, this technique does not work efficiently in some species, such as Arabidopsis thaliana. Moreover, the infection of Agrobacterium is known to induce dynamic changes in gene expression patterns in the host plants, possibly affecting the function and localization of the proteins to be tested. These problems can be circumvented by biolistic delivery of the genes of interest. Here, we present an optimized protocol for biolistic delivery of plasmid DNA into epidermal cells of plant leaves, which can be easily performed using the Bio-Rad Helios gene gun system. This protocol allows efficient and reproducible transient expression of diverse genes in Arabidopsis, Nicotiana benthamiana and N. tabacum, and is suitable for studies of the biological function and subcellular localization of the gene products directly in planta. The protocol also can be easily adapted to other species by optimizing the delivery gas pressure. © 2013 Springer Science+Business Media, LLC.

DOI： 10.1007/978-1-62703-110-3_2

To Gate, or Not to Gate: Regulatory Mechanisms for Intercellular Protein Transport and Virus Movement in Plants 査読

Shoko Ueki, Vitaly Citovsky

MOLECULAR PLANT 4 ( 5 ) 782 - 793 2011年9月

担当区分：筆頭著者,　責任著者記述言語：英語出版者・発行元：OXFORD UNIV PRESS

Cell-to-cell signal transduction is vital for orchestrating the whole-body physiology of multi-cellular organisms, and many endogenous macromolecules, proteins, and nucleic acids function as such transported signals. In plants, many of these molecules are transported through plasmodesmata (Pd), the cell wall-spanning channel structures that interconnect plant cells. Furthermore, Pd also act as conduits for cell-to-cell movement of most plant viruses that have evolved to pirate these channels to spread the infection. Pd transport is presumed to be highly selective, and only a limited repertoire of molecules is transported through these channels. Recent studies have begun to unravel mechanisms that actively regulate the opening of the Pd channel to allow traffic. This macromolecular transport between cells comprises two consecutive steps: intracellular targeting to Pd and translocation through the channel to the adjacent cell. Here, we review the current knowledge of molecular species that are transported though Pd and the mechanisms that control this traffic. Generally, Pd traffic can occur by passive diffusion through the trans-Pd cytoplasm or through the membrane/lumen of the trans-Pd ER, or by active transport that includes protein-protein interactions. It is this latter mode of Pd transport that is involved in intercellular traffic of most signal molecules and is regulated by distinct and sometimes interdependent mechanisms, which represent the focus of this article.

DOI： 10.1093/mp/ssr060

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro 査読

Shoko Ueki, Benoit Lacroix, Vitaly Citovsky

JOVE-JOURNAL OF VISUALIZED EXPERIMENTS ( 54 ) 2011年8月

担当区分：筆頭著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：JOURNAL OF VISUALIZED EXPERIMENTS

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights.
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N-and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins' interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about proteinprotein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

DOI： 10.3791/2961

Biology of callose (β-1,3-glucan) turnover at plasmodesmata. 査読

Zavaliev R, Ueki S, Epel BL, Citovsky V

Protoplasma 248 ( 1 ) 117 - 130 2011年1月

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