Tzfira, T. et al. pSAT vectors: a modular series of plasmids for fluorescent protein tagging and expression of multiple genes in plants. Plant Mol. Biol. 57, 503-516
Hebrew University of Jerusalem, Yerushalayim, Jerusalem, Israel Plant Molecular Biology
(Impact Factor: 4.26).
04/2005; 57(4):503-16. DOI: 10.1007/s11103-005-0340-5
Autofluorescent protein tags represent one of the major and, perhaps, most powerful tools in modern cell biology for visualization of various cellular processes in vivo. In addition, advances in confocal microscopy and the development of autofluorescent proteins with different excitation and emission spectra allowed their simultaneous use for detection of multiple events in the same cell. Nevertheless, while autofluorescent tags are widely used in plant research, the need for a versatile and comprehensive set of vectors specifically designed for fluorescent tagging and transient and stable expression of multiple proteins in plant cells from a single plasmid has not been met by either the industrial or the academic communities. Here, we describe a new modular satellite (SAT) vector system that supports N- and C-terminal fusions to five different autofluorescent tags, EGFP, EYFP, Citrine-YFP, ECFP, and DsRed2. These vectors carry an expanded multiple cloning site that allows easy exchange of the target genes between different autofluorescence tags, and expression of the tagged proteins is controlled by constitutive promoters, which can be easily replaced with virtually any other promoter of interest. In addition, a series of SAT vectors has been adapted for high throughput Gateway recombination cloning. Furthermore, individual expression cassettes can be assembled into Agrobacterium binary plasmids, allowing efficient transient and stable expression of multiple autofluorescently tagged proteins from a single vector following its biolistic delivery or Agrobacterium-mediated genetic transformation.
Available from: Margherita Festa
- "Plant vacuoles from tpc1 knock-out plants appeared ideal to express animal TPC2; since plant vacuoles and animal lysosomes have several physiological functions in common, the working hypothesis was that the mechanisms of vacuolar/lysosomal targeting may be similar and conserved during evolution. Alex Costa, at that time in the group of Fiorella Lo Schiavo, cloned the human TPC2 fused with an EGFP to its C-terminus, into the plant expression vector pSAT6 . Transient transformation of isolated mesophyll protoplasts  showed, with great satisfaction, that hTPC2 at least partially localized to the vacuolar membrane . "
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ABSTRACT: Plant cells possess a large intracellular compartment that animal cells do not, the central vacuole, which has been investigated for a long time. The central vacuole can occupy up to 90% of the cellular volume and, differently from intracellular organelles from animal cells such as lysosomes or endosomes, it is easy to isolate. Because of its large dimension (up to 40μm diameter) it can be successfully studied using the classical patch-clamp technique. Following the idea that the vacuolar membrane could be used as a convenient model to characterize the functional properties of channel-forming peptides, we verified that the phytotoxic lipodepsipeptide Syringopeptin 25A from Pseudomonas syringae pv syringae was able to form ionic pores in sugar beet vacuoles and we performed a detailed biophysical analysis. Recently, we extended the use of plant vacuoles to the expression and functional characterisation of animal intracellular transporters, namely rat CLC-7, and channels, i.e. human TPC2. Since endo-lysosomal transporters and channels are still largely unexplored, principally because their intracellular localization renders them difficult to study, we believe that this novel approach will prove to be a powerful system for the investigation of the molecular mechanisms of exogenous transporters and channels.
- "was subcloned into the commercial pMOS vector (GE Healthcare) by PCR using the following pair of primers, both containing BamHI restriction sites (underlined): 59-GTAACTGGATCCACAGGAAGATGG-39 and 59-AGCCAAGGATCCAGTATCTATGAG-39. The complementary DNA sequence was PCR amplified and cloned into pGEMHE (Liman et al., 1992) for oocyte expression and into pSAT6-EGFP-N1 (Tzfira et al., 2005) for transientPlant Physiol. Vol. "
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ABSTRACT: Trans-plasma-membrane electron transfer is achieved by b-type cytochromes of different families, and plays a fundamental role in diverse cellular processes involving two interacting redox couples that are physically separated by a phospholipid bilayer, such as iron uptake and redox signalling. Despite their importance, no direct recordings of trans-plasma-membrane electron currents have been described in plants. In this work, we provide robust electrophysiological evidence of trans-plasma-membrane electron flow mediated by a soybean CYBDOM, a member of the cytochrome b561 family, which localizes to the plasma membrane in transgenic Arabidopsis plants and CYBDOM cRNA-injected Xenopus oocytes. In oocytes, two-electrode voltage-clamp experiments showed that CYBDOM-mediated currents were activated by extracellular electron acceptors in a concentration- and type-specific manner. Current amplitudes were voltage-dependent, strongly potentiated in oocytes pre-injected with ascorbate, the canonical electron donor for cytochromes b561, and abolished by mutating a highly conserved histidine residue (H292L) predicted to coordinate the cytoplasmic heme b group. We believe that this novel approach opens new perspectives in plant trans-membrane electron transport and beyond.
Copyright © 2015, Plant Physiology.
- "We employed a simple visual assay to assess the transfection efficiency using fluorescence microscopy. We transfected protoplasts with the pSAT plasmid, expressing the nuclear-targeted redshifted GFP (EGFP) driven by two cauliflower mosaic virus 35S promoters, oriented in tandem (Tzfira et al., 2005). Protoplasts were transfected using a poly(ethylene glycol) (PEG)-calcium-mediated transfection procedure as previously described (Yoo et al., 2007), except that we used 10 times more protoplasts than detailed in (Yoo et al., 2007). "
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ABSTRACT: The protoplast transient assay system has been widely used for rapid functional analyses of genes using cellular and biochemical approaches. This system has been increasingly employed for functional genetic studies using double-stranded (ds) RNA interference (RNAi). Here, we describe a modified procedure for the isolation of protoplasts from leaf mesophyll cells of 14-day-old Arabidopsis thaliana. This modification significantly simplifies and speeds up functional studies without compromising the yield and the viability of protoplasts. We also present the procedure for the isolation and transfection of protoplasts from mesophyll cells of an emerging model grass species, Brachypodium distachyon. Further, we detail procedures for RNAi-based functional studies of genes using transient expression of in vitro synthesized dsRNA in protoplasts.
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