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
Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, NY 11794-5215, USA.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.
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- "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). "
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.Methods in molecular biology (Clifton, N.J.) 03/2015; 1284:433-52. DOI:10.1007/978-1-4939-2444-8_22 · 1.29 Impact Factor
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- "Plasmid construction and transformation with ZmPIP2;4 The ZmPIP2;4 was cloned into the vector pSAT4(A)-MCS (Tzfira et al. 2005) using the enzyme XhoI. All expression cassettes contained the constitutive CaMV 35Sx2 promoter . "
ABSTRACT: Main conclusion: Enhancing the membrane content of PtdInsP 2 , the already-recognized protein-regulating lipid, increased the osmotic water permeability of tobacco protoplasts, apparently by increasing the abundance of active aquaporins in their membranes. While phosphoinositides are implicated in cell volume changes and are known to regulate some ion channels, their modulation of aquaporins activity has not yet been reported for any organism. To examine this, we compared the osmotic water permeability (P f) of protoplasts isolated from tobacco (Nicotiana tabacum) cultured cells (NT1) with different (genetically lowered or elevated relative to controls) levels of inositol trisphosphate (InsP3) and phosphatidyl inositol [4,5] bisphosphate (PtdInsP2). To achieve this, the cells were transformed with, respectively, the human InsP3 5-phosphatase ('Ptase cells') or human phosphatidylinositol (4) phosphate 5-kinase ('PIPK cells'). The mean P f of the PIPK cells was several-fold higher relative to that of controls and Ptase cells. Three results favor aquaporins over the membrane matrix as underlying this excessive P f: (1) transient expression of the maize aquaporin ZmPIP2;4 in the PIPK cells increased P f by 12-30 μm s(-1), while in the controls only by 3-4 μm s(-1). (2) Cytosol acidification-known to inhibit aquaporins-lowered the P f in the PIPK cells down to control levels. (3) The transcript of at least one aquaporin was elevated in the PIPK cells. Together, the three results demonstrate the differences between the PIPK cells and their controls, and suggest a hitherto unobserved regulation of aquaporins by phosphoinositides, which could occur through direct interaction or indirect phosphoinositides-dependent cellular effects.Planta 12/2014; 241(3). DOI:10.1007/s00425-014-2216-x · 3.26 Impact Factor
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- "We constitutively expressed the fusion protein OsNAR2.1 and GFP using the pSAT6-EGFP-C1 and pSAT6A-EGFP-N1 expression vector (Tzfira et al., 2005) to investigate the subcellular localization of OsNAR2.1. We transfected OsNAR2.1-GFP and GFP-OsNAR2.1 fusions into rice blade protoplasts under control of the cauliflower mosaic virus 35S promoter, and GFP expression was determined using confocal microscopy. "
ABSTRACT: A partner protein, NAR2, is essential for high-affinity nitrate transport of the NRT2 protein in plants. However, the NAR2 motifs that interact with NRT2s for their plasma membrane (PM) localization and nitrate transporter activity have not been functionally characterized. In this study, OsNAR2.1 mutations with different carbon (C)-terminal deletions and nine different point mutations in the conserved regions of NAR2 homologs in plants were generated to explore the essential motifs involved in the interaction with OsNRT2.3a. Screening using the membrane yeast two-hybrid system and Xenopus oocytes for nitrogen-15 (15N) uptake demonstrated that either R100G or D109N point mutations impaired the OsNAR2.1 interaction with OsNRT2.3a. Western blotting and visualization using green fluorescent protein fused to either the N- or C-terminus of OsNAR2.1 indicated that OsNAR2.1 is expressed in both the PM and cytoplasm. The split-yellow fluorescent protein (YFP)/BiFC analyses indicated that OsNRT2.3a was targeted to the PM in the presence of OsNAR2.1, while either R100G or D109N mutation resulted in the loss of OsNRT2.3a-YFP signal in the PM. Based on these results, arginine 100 and aspartic acid 109 of the OsNAR2.1 protein are key amino acids in the interaction with OsNRT2.3a, and their interaction occurs in the PM but not cytoplasm.New Phytologist 08/2014; 204(1). DOI:10.1111/nph.12986 · 7.67 Impact Factor
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