Vectors for multi-color bimolecular fluorescence complementation to investigate protein-protein interactions in living plant cells

Lee, Lan‐Ying; Fang, Mei‐Jane; Kuang, Lin‐Yun; Gelvin, Stanton B.

doi:10.1186/1746-4811-4-24

Cited by 203 publications

(193 citation statements)

References 22 publications

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“…As RHC1 also functions specifically in the CO 2 response pathway, we examined a possible relationship among RHC1, CAs and HT1. We first performed a bimolecular fluorescence complementation (BiFC) assay in Arabidopsis protoplasts 46,47 . The combination of RHC1-nVenus and CA4-cCFP or RHC1-nVenus and HT1-cCFP produced a green fluorescent signal in the plasma membrane (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The resulting cDNAs were inserted into pCAMBIA 1302 to produce the 35S::RHC1 (or SLAC1)-GFP constructs. For BiFC in Arabidopsis mesophyll protoplasts, the full-length RHC1, OST1, HT1 and CA4 were subcloned into pSAT1-nVenus-N (or pSAT1-cCFP-N) 46 to produce 35S::RHC1-nVenus, 35S::OST1-cCFP, 35S::HT1-cCFP, 35S::CA4-cCFP and 35S::OST1-nVenus with gene-specific primers. For two-electrode voltage-clamp analysis in Xenopus oocytes, RHC1, HT1, HT1(K133W), SLAC1, CA4, OST1 and CPK3 cDNAs encoding full-length proteins were inserted into pGEMHE with gene-specific primers.…”

Section: Methodsmentioning

confidence: 99%

“…The indicated pairs of expression constructs 46 were co-transformed into Arabidopsis mesophyll protoplasts by PEG-mediated transfection 54 . Protoplast images were taken by confocal microscopy (Zeiss 5-LIVE) operated with LSM Image Browser software.…”

Section: Methodsmentioning

confidence: 99%

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A molecular pathway for CO2 response in Arabidopsis guard cells

et al. 2015

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Increasing carbon dioxide (CO 2 ) levels in the atmosphere have caused global metabolic changes in diverse plant species. CO 2 is not only a carbon donor for photosynthesis but also an environmental signal that regulates stomatal movements and thereby controls plant-water relationships and carbon metabolism. However, the mechanism underlying CO 2 sensing in stomatal guard cells remains unclear. Here we report characterization of Arabidopsis RESISTANT TO HIGH CO 2 (RHC1), a MATE-type transporter that links elevated CO 2 concentration to repression of HT1, a protein kinase that negatively regulates CO 2 -induced stomatal closing. We also show that HT1 phosphorylates and inactivates OST1, a kinase which is essential for the activation of the SLAC1 anion channel and stomatal closing. Combining genetic, biochemical and electrophysiological evidence, we reconstituted the molecular relay from CO 2 to SLAC1 activation, thus establishing a core pathway for CO 2 signalling in plant guard cells.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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A molecular pathway for CO2 response in Arabidopsis guard cells

et al. 2015

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“…BiFC can be performed with various derivatives of both GFP and red FPs (31,(34)(35)(36)(37)(38)(39). This allows selection of an FP that will facilitate co-localising the RNA of interest with other fluorescent markers that may already be available.…”

Section: Choice Of Fpmentioning

confidence: 99%

Pumilio-Based RNA In Vivo Imaging

Tilsner

2014

Methods in Molecular Biology

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SummarySubcellular, sequence-specific detection of RNA in vivo is a powerful tool to study the macromolecular transport that occurs through plasmodesmata. The RNA binding domain of Pumilio proteins can be engineered to bind RNA sequences of choice and fused to fluorescent proteins for RNA imaging. This chapter describes the construction of a Pumilio-based imaging system to track the RNA of Tobacco mosaic virus in vivo, and practical aspects of RNA live-cell imaging.

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“…For more than two decades, considerable efforts have been made to create improved binary vectors that facilitate the generation of transgenic plants. These efforts have typically included minimizing the plasmid backbone size for ease of genetic manipulation, improving the properties of the T-DNA region by including more unique restriction endonuclease sites facilitating gene of interest insertion, incorporating a variety of plant selectable marker genes and/or integrating novel reporters or sequence tags to generate proteins for tissue/cellular localization or protein-protein interaction studies (Lee et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

Transformation of <i>Lesquerella Fendleri</i> with the New Binary Vector pGPro4-35S

M.¹

2011

OnLine Journal of Biological Sciences

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Problem statement:Crop genetic engineering requires the use of various promoters to control the expression of introduced transgenes. Some of the binary vectors currently available for promoter characterization in dicotyledonous plants have pitfalls due to their construction, such as containing a selectable marker cassette with enhancer sequences that can potentially interfere with the expression specificity of nearby promoters. Also, many binary vectors are quite large in size and contain few useful restriction sites making their in vitro manipulation technically challenging. Approach: A small (7698 bp) and flexible binary vector named pGPro4 was constructed to possess unique features favorable for promoter analysis in dicot plants. A nopaline synthase (nos) promoter was used to control the expression of the selectable marker of pGPro4 to prevent the problem of interference with the neighboring promoter-reporter fusion. In pGPro4, the nos promoter and hygromycin phosphotransferase II (hptII) sequences are flanked by loxP sites, which allow for Cre recombinase-mediated removal when hygromycin resistance is no longer desired. pGPro4 also contains a bifunctional ß-glucuronidase-enhanced Green Fluorescent Protein (gusA-eGFP) reporter gene that provides visual detection of reporter gene expression using either fluorescence in live cells or histochemical detection of β-glucuronidase activity. Results and Conclusion: To demonstrate the usefulness of the pGPro4 vector, a CaMV35S promoter was fused to gusA-eGFP and the resulting plasmid, pGPro4-35S, was used to transform Lesquerella fendleri. Primary shoots were generated from explants at an expected frequency of 10-27.5%, indicating that the nos promoter drove sufficient hptII expression to generate hygromycin resistant plants. Six independent transgenic L. fendleri lines were grown to maturity and generated T 1 seeds. The bifunctionality of the gusA-eGFP reporter gene was verified by detecting both green fluorescence and β-glucuronidase activity in multiple T 1 L. fendleri seedlings from 5 of the 6 the independent transgenic lines.

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Vectors for multi-color bimolecular fluorescence complementation to investigate protein-protein interactions in living plant cells

Cited by 203 publications

References 22 publications

A molecular pathway for CO2 response in Arabidopsis guard cells

A molecular pathway for CO2 response in Arabidopsis guard cells

Pumilio-Based RNA In Vivo Imaging

Transformation of <i>Lesquerella Fendleri</i> with the New Binary Vector pGPro4-35S

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