A New Set of Golden-Gate-Based Organelle Marker Plasmids for Colocalization Studies in Plants
In vivo localization of proteins using fluorescence-based approaches by fusion of the protein of interest (POI) to a fluorescent protein is a cost- and time-effective tool to gain insights into its physiological function in a plant cell. Determining the proper localization, however, requires the co-...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-10-01
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| Series: | Plants |
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| Online Access: | https://www.mdpi.com/2223-7747/11/19/2620 |
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| author | Hagen Stellmach Robert Hose Antonia Räde Sylvestre Marillonnet Bettina Hause |
| author_facet | Hagen Stellmach Robert Hose Antonia Räde Sylvestre Marillonnet Bettina Hause |
| author_sort | Hagen Stellmach |
| collection | DOAJ |
| description | In vivo localization of proteins using fluorescence-based approaches by fusion of the protein of interest (POI) to a fluorescent protein is a cost- and time-effective tool to gain insights into its physiological function in a plant cell. Determining the proper localization, however, requires the co-expression of defined organelle markers (OM). Several marker sets are available but, so far, the procedure requires successful co-transformation of POI and OM into the same cell and/or several cloning steps. We developed a set of vectors containing markers for basic cell organelles that enables the insertion of the gene of interest (GOI) by a single cloning step using the Golden Gate cloning approach and resulting in POI–GFP fusions. The set includes markers for plasma membrane, tonoplast, nucleus, endoplasmic reticulum, Golgi apparatus, peroxisomes, plastids, and mitochondria, all labelled with mCherry. Most of them were derived from well-established marker sets, but those localized in plasma membrane and tonoplast were improved by using different proteins. The final vectors are usable for localization studies in isolated protoplasts and for transient transformation of leaves of <i>Nicotiana benthamiana</i>. Their functionality is demonstrated using two enzymes involved in biosynthesis of jasmonic acid and located in either plastids or peroxisomes. |
| first_indexed | 2024-03-09T21:17:21Z |
| format | Article |
| id | doaj.art-db01a0dd8c92445daf84fc9bab15d9a2 |
| institution | Directory Open Access Journal |
| issn | 2223-7747 |
| language | English |
| last_indexed | 2024-03-09T21:17:21Z |
| publishDate | 2022-10-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Plants |
| spelling | doaj.art-db01a0dd8c92445daf84fc9bab15d9a22023-11-23T21:30:28ZengMDPI AGPlants2223-77472022-10-011119262010.3390/plants11192620A New Set of Golden-Gate-Based Organelle Marker Plasmids for Colocalization Studies in PlantsHagen Stellmach0Robert Hose1Antonia Räde2Sylvestre Marillonnet3Bettina Hause4Department of Cell and Metabolic Biology, Leibniz Institute of Plant Biochemistry, 06120 Halle (Saale), GermanyDepartment of Cell and Metabolic Biology, Leibniz Institute of Plant Biochemistry, 06120 Halle (Saale), GermanyDepartment of Cell and Metabolic Biology, Leibniz Institute of Plant Biochemistry, 06120 Halle (Saale), GermanyDepartment of Cell and Metabolic Biology, Leibniz Institute of Plant Biochemistry, 06120 Halle (Saale), GermanyDepartment of Cell and Metabolic Biology, Leibniz Institute of Plant Biochemistry, 06120 Halle (Saale), GermanyIn vivo localization of proteins using fluorescence-based approaches by fusion of the protein of interest (POI) to a fluorescent protein is a cost- and time-effective tool to gain insights into its physiological function in a plant cell. Determining the proper localization, however, requires the co-expression of defined organelle markers (OM). Several marker sets are available but, so far, the procedure requires successful co-transformation of POI and OM into the same cell and/or several cloning steps. We developed a set of vectors containing markers for basic cell organelles that enables the insertion of the gene of interest (GOI) by a single cloning step using the Golden Gate cloning approach and resulting in POI–GFP fusions. The set includes markers for plasma membrane, tonoplast, nucleus, endoplasmic reticulum, Golgi apparatus, peroxisomes, plastids, and mitochondria, all labelled with mCherry. Most of them were derived from well-established marker sets, but those localized in plasma membrane and tonoplast were improved by using different proteins. The final vectors are usable for localization studies in isolated protoplasts and for transient transformation of leaves of <i>Nicotiana benthamiana</i>. Their functionality is demonstrated using two enzymes involved in biosynthesis of jasmonic acid and located in either plastids or peroxisomes.https://www.mdpi.com/2223-7747/11/19/2620colocalizationGolden Gate cloninggreen fluorescent proteinmCherryplant organellesprotoplasts |
| spellingShingle | Hagen Stellmach Robert Hose Antonia Räde Sylvestre Marillonnet Bettina Hause A New Set of Golden-Gate-Based Organelle Marker Plasmids for Colocalization Studies in Plants Plants colocalization Golden Gate cloning green fluorescent protein mCherry plant organelles protoplasts |
| title | A New Set of Golden-Gate-Based Organelle Marker Plasmids for Colocalization Studies in Plants |
| title_full | A New Set of Golden-Gate-Based Organelle Marker Plasmids for Colocalization Studies in Plants |
| title_fullStr | A New Set of Golden-Gate-Based Organelle Marker Plasmids for Colocalization Studies in Plants |
| title_full_unstemmed | A New Set of Golden-Gate-Based Organelle Marker Plasmids for Colocalization Studies in Plants |
| title_short | A New Set of Golden-Gate-Based Organelle Marker Plasmids for Colocalization Studies in Plants |
| title_sort | new set of golden gate based organelle marker plasmids for colocalization studies in plants |
| topic | colocalization Golden Gate cloning green fluorescent protein mCherry plant organelles protoplasts |
| url | https://www.mdpi.com/2223-7747/11/19/2620 |
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