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Particle bombardment and subcellular protein localization analysis in the aquatic plant Egeria densa

Particle bombardment is a powerful and relatively easy method for transient expression of genes of interest in plant cells, especially those that are recalcitrant to other transformation methods. This method has facilitated numerous analyses of subcellular localization of fluorescent fusion protein...

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Autores principales: Osaki, Yasuhide, Kodama, Yutaka
Formato: Online Artículo Texto
Lenguaje:English
Publicado: PeerJ Inc. 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5592081/
https://www.ncbi.nlm.nih.gov/pubmed/28894649
http://dx.doi.org/10.7717/peerj.3779
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author Osaki, Yasuhide
Kodama, Yutaka
author_facet Osaki, Yasuhide
Kodama, Yutaka
author_sort Osaki, Yasuhide
collection PubMed
description Particle bombardment is a powerful and relatively easy method for transient expression of genes of interest in plant cells, especially those that are recalcitrant to other transformation methods. This method has facilitated numerous analyses of subcellular localization of fluorescent fusion protein constructs. Particle bombardment delivers genes to the first layer of plant tissue. In leaves of higher plants, epidermal cells are the first cell layer. Many studies have used the epidermal cell layer of onion bulb (Allium cepa) as the experimental tissue, because these cells are relatively large. However, onion epidermal cells lack developed plastids (i.e., chloroplasts), thereby precluding subcellular localization analysis of chloroplastic proteins. In this study, we developed a protocol for particle bombardment of the aquatic plant Egeria densa, and showed that it is a useful system for subcellular localization analysis of higher plant proteins. E. densa leaflets contain only two cell layers, and cells in the adaxial layer are sufficiently large for observation. The cells in both layers contain well-developed chloroplasts. We fused fluorescent proteins to conventional plant localization signals for the nucleus, cytosol, mitochondria, peroxisome, and chloroplast, and used particle bombardment to transiently express these fusion constructs in E. densa leaves. The plant subcellular localization signals functioned normally and displayed the expected distributions in transiently transformed E. densa cells, and even chloroplastic structures could be clearly visualized.
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spelling pubmed-55920812017-09-11 Particle bombardment and subcellular protein localization analysis in the aquatic plant Egeria densa Osaki, Yasuhide Kodama, Yutaka PeerJ Cell Biology Particle bombardment is a powerful and relatively easy method for transient expression of genes of interest in plant cells, especially those that are recalcitrant to other transformation methods. This method has facilitated numerous analyses of subcellular localization of fluorescent fusion protein constructs. Particle bombardment delivers genes to the first layer of plant tissue. In leaves of higher plants, epidermal cells are the first cell layer. Many studies have used the epidermal cell layer of onion bulb (Allium cepa) as the experimental tissue, because these cells are relatively large. However, onion epidermal cells lack developed plastids (i.e., chloroplasts), thereby precluding subcellular localization analysis of chloroplastic proteins. In this study, we developed a protocol for particle bombardment of the aquatic plant Egeria densa, and showed that it is a useful system for subcellular localization analysis of higher plant proteins. E. densa leaflets contain only two cell layers, and cells in the adaxial layer are sufficiently large for observation. The cells in both layers contain well-developed chloroplasts. We fused fluorescent proteins to conventional plant localization signals for the nucleus, cytosol, mitochondria, peroxisome, and chloroplast, and used particle bombardment to transiently express these fusion constructs in E. densa leaves. The plant subcellular localization signals functioned normally and displayed the expected distributions in transiently transformed E. densa cells, and even chloroplastic structures could be clearly visualized. PeerJ Inc. 2017-09-07 /pmc/articles/PMC5592081/ /pubmed/28894649 http://dx.doi.org/10.7717/peerj.3779 Text en ©2017 Osaki and Kodama http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited.
spellingShingle Cell Biology
Osaki, Yasuhide
Kodama, Yutaka
Particle bombardment and subcellular protein localization analysis in the aquatic plant Egeria densa
title Particle bombardment and subcellular protein localization analysis in the aquatic plant Egeria densa
title_full Particle bombardment and subcellular protein localization analysis in the aquatic plant Egeria densa
title_fullStr Particle bombardment and subcellular protein localization analysis in the aquatic plant Egeria densa
title_full_unstemmed Particle bombardment and subcellular protein localization analysis in the aquatic plant Egeria densa
title_short Particle bombardment and subcellular protein localization analysis in the aquatic plant Egeria densa
title_sort particle bombardment and subcellular protein localization analysis in the aquatic plant egeria densa
topic Cell Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5592081/
https://www.ncbi.nlm.nih.gov/pubmed/28894649
http://dx.doi.org/10.7717/peerj.3779
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