A dual-site gateway cloning system for simultaneous cloning of two genes for plant transformation

Aboulela, Mostafa; Tanaka, Yuji; Nishimura, Kohji; Mano, Shoji; Kimura, Takeshi; Nakagawa, Tsuyoshi

doi:10.1016/j.plasmid.2017.05.001

Cited by 9 publications

(5 citation statements)

References 59 publications

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“…To further reduce the number of plasmids that were required for experiments, we incorporated a tRNA-gRNA-tRNA design 5 directly into the synthesized fragments (Figure 1B, D). Our initial attempts at building a dual-Gateway plasmid followed earlier examples and simply duplicated the standard ccdB negative selection gene in two separate attR-attR cassettes 8 . We found, however, that every plasmid recovered during cloning carried a mutation in one of the ccdB genes, suggesting that high ccdB dosage is deleterious to E. coli 4 .…”

Section: Resultsmentioning

confidence: 99%

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The Janelia Atalanta plasmids provide a simple and efficient CRISPR/Cas9-mediated homology directed repair platform forDrosophila

Stern,

Kim,

Behrman

2023

Preprint

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Homology-directed repair (HDR) is a powerful tool for modifying genomes in precise ways to address many biological questions. Use of Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)-Cas9 induced targeted DNA double-strand breakage has substantially simplified use of homology-directed repair to introduce specific perturbations in Drosophila, but existing platforms for CRISPR-Cas9-mediated HDR in Drosophila involve multiple cloning steps and have low efficiency. To simplify cloning of HDR plasmids, we designed a new plasmid platform, the Janelia Atalanta (pJAT) series, that exploits recent advances in dsDNA synthesis to facilitate Gateway cloning of gRNA sequences and homology arms in one step. Surprisingly, the pJAT plasmids yielded considerably higher HDR efficiency (approximately 25%) than we have observed with other approaches. pJAT plasmids work in multiple Drosophila species and exhibited such high efficiency that previously impossible experiments in Drosophila, such as driving targeted chromosomal inversions, were made possible. We provide pJAT plasmids for a range of commonly performed experiments including targeted insertional mutagenesis, insertion of phiC31-mediated attP landing sites, generation of strains carrying a germ-line source of Cas9, and induction of chromosomal rearrangements. We also provide "empty" pJAT plasmids with multiple cloning sites to simplify construction of plasmids with new functionality. The pJAT platform is generic and may facilitate improved efficiency CRISPR-Cas9 HDR in a wide range of model and non-model organisms.

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Section: Resultsmentioning

confidence: 99%

“…We initially attempted to build the double-Gateway system using multiple ccdB containing cassettes, as has been demonstrated by others 8 . However, we discovered during subsequent cloning that in every case one of the two ccdB genes had been inactivated.…”

Section: Discussionmentioning

confidence: 99%

The Janelia Atalanta plasmids provide a simple and efficient CRISPR/Cas9-mediated homology directed repair platform forDrosophila

Stern,

Kim,

Behrman

2023

Preprint

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“…According to TAIR, the genome of Arabidopsis thaliana contains 27,416 protein-coding genes ( Table S3 ). The main portion of PPIs was identified in several databases, while an additional 920 PPIs were identified through a PubMed search for studies of interactions in Arabidopsis published over the last twenty years [ 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , …”

Section: Resultsmentioning

confidence: 99%

Interactome of Arabidopsis Thaliana

Yılmaz

Paulic

Seidel

2022

Plants

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More than 95,000 protein–protein interactions of Arabidopsis thaliana have been published and deposited in databases. This dataset was supplemented by approximately 900 additional interactions, which were identified in the literature from the years 2002–2021. These protein–protein interactions were used as the basis for a Cytoscape network and were supplemented with data on subcellular localization, gene ontologies, biochemical properties and co-expression. The resulting network has been exemplarily applied in unraveling the PPI-network of the plant vacuolar proton-translocating ATPase (V-ATPase), which was selected due to its central importance for the plant cell. In particular, it is involved in cellular pH homeostasis, providing proton motive force necessary for transport processes, trafficking of proteins and, thereby, cell wall synthesis. The data points to regulation taking place on multiple levels: (a) a phosphorylation-dependent regulation by 14-3-3 proteins and by kinases such as WNK8 and NDPK1a, (b) an energy-dependent regulation via HXK1 and the glucose receptor RGS1 and (c) a Ca2+-dependent regulation by SOS2 and IDQ6. The known importance of V-ATPase for cell wall synthesis is supported by its interactions with several proteins involved in cell wall synthesis. The resulting network was further analyzed for (experimental) biases and was found to be enriched in nuclear, cytosolic and plasma membrane proteins but depleted in extracellular and mitochondrial proteins, in comparison to the entity of protein-coding genes. Among the processes and functions, proteins involved in transcription were highly abundant in the network. Subnetworks were extracted for organelles, processes and protein families. The degree of representation of organelles and processes reveals limitations and advantages in the current knowledge of protein–protein interactions, which have been mainly caused by a high number of database entries being contributed by only a few publications with highly specific motivations and methodologies that favor, for instance, interactions in the cytosol and the nucleus.

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“…Dual site Gateway cloning ( 24 ) was utilized to clone two gene construction into a single plant expression vector. For generation of double overexpression lines, the entry vector carrying a Ubiquitin 10 (pUBI) promoter of Arabidopsis was recombined into modified R4pDD607-mCitrine vector together with pDONR221:PTST2 , generating R4pDD607-pUbi:PTST2mCitrine .…”

Section: Methodsmentioning

confidence: 99%

MFP1 defines the subchloroplast location of starch granule initiation

Sharma,

Abt,

Eicke

et al. 2024

Proc. Natl. Acad. Sci. U.S.A.

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Starch is one of the major carbohydrate storage compounds in plants. The biogenesis of starch granules starts with the formation of initials, which subsequently expand into granules. Several coiled-coil domain-containing proteins have been previously implicated with the initiation process, but the mechanisms by which they act remain largely elusive. Here, we demonstrate that one of these proteins, the thylakoid-associated MAR-BINDING FILAMENT-LIKE PROTEIN 1 (MFP1), specifically determines the subchloroplast location of initial formation. The expression of MFP1 variants “mis”-targeted to specific locations within chloroplasts in Arabidopsis results in distinctive shifts in not only how many but also where starch granules are formed. Importantly, “re” localizing MFP1 to the stromal face of the chloroplast’s inner envelope is sufficient to generate starch granules in this aberrant position. These findings provide compelling evidence that a single protein MFP1 possesses the capacity to direct the initiation and biosynthesis machinery of starch granules.

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A dual-site gateway cloning system for simultaneous cloning of two genes for plant transformation

Cited by 9 publications

References 59 publications

The Janelia Atalanta plasmids provide a simple and efficient CRISPR/Cas9-mediated homology directed repair platform forDrosophila

The Janelia Atalanta plasmids provide a simple and efficient CRISPR/Cas9-mediated homology directed repair platform forDrosophila

Interactome of Arabidopsis Thaliana

MFP1 defines the subchloroplast location of starch granule initiation

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