A highly efficient transient protoplast system for analyzing defence gene expression and protein–protein interactions in rice
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SUMMARY The transient assay system based on mesophyll or cultured cell-derived protoplasts has been exploited in several plant species and has become a powerful tool for rapid gene functional analysis and biochemical manipulations. However, the system has not been widely used in rice owing to the difficulties in large-scale isolation of viable rice protoplasts from leaves or suspension-cultured cells. Here, we describe a significantly improved method to isolate a large number of protoplasts from stem and sheath tissues of both young and mature plants. High-level coexpression of multiple constructs and efficient suppression of exogenous and endogenous genes were observed in the stem- and sheath-derived protoplasts. A transient green fluorescent protein and luciferase-based reporter system for defence-related genes expression analysis has been established, which is useful for screening and characterizing genes involved in rice defence signalling pathways. Furthermore, a protoplast-based bimolecular fluorescence complementation (BiFC) system for the detection of protein-protein interactions in living rice cells was developed. The YFP complementation of two split-YFP halves mediated by homodimerization of the GUS and SPIN1, a cell-death related protein, was observed in transfected protoplasts. In combination with genetic, genomic and proteomic approaches, the established versatile protoplast transient assay system will facilitate large-scale functional analysis of defence-related genes in rice.Keywords:
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Bimolecular fluorescence complementation
Protein-fragment complementation assay
Plant cell
Bimolecular fluorescence complementation
Protein-fragment complementation assay
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Bimolecular fluorescence complementation
Protein-fragment complementation assay
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Yellow fluorescent protein
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Bimolecular fluorescence complementation
Protein-fragment complementation assay
Fluorescent protein
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Bimolecular fluorescence complementation
Protein-fragment complementation assay
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Yellow fluorescent protein
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Journal Article The Plant Protoplast: A Useful Tool for Plant Research and Student Instruction Get access George J. Wagner, George J. Wagner Search for other works by this author on: Oxford Academic Google Scholar Henry C. Butcher, IV, Henry C. Butcher, IV Search for other works by this author on: Oxford Academic Google Scholar Harold W. Siegelman Harold W. Siegelman Search for other works by this author on: Oxford Academic Google Scholar BioScience, Volume 28, Issue 2, February 1978, Pages 95–101, https://doi.org/10.2307/1307422 Published: 01 February 1978 Article history Accepted: 28 September 1977 Published: 01 February 1978
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Protein-protein interactions play a central role in many cellular processes. Their characterisation is necessary in order to analyse these processes and for the functional identification of unknown proteins. Existing detection methods such as the yeast two-hybrid (Y2H) and tandem affinity purification (TAP) method provide a means to answer rapidly questions regarding protein-protein interactions, but have limitations which restrict their use to certain interaction networks; furthermore they provide little information regarding interaction localisation at the subcellular level. The development of protein-fragment complementation assays (PCA) employing a fluorescent reporter such as a member of the green fluorescent protein (GFP) family has led to a new method of interaction detection termed Bimolecular Fluorescent Complementation (BiFC). These assays have become important tools for understanding protein interactions and the development of whole genome interaction maps. BiFC assays have the advantages of very low background signal coupled with rapid detection of protein-protein interactions in vivo while also providing information regarding interaction compartmentalisation. Modified forms of the assay such as the use of combinations of spectral variants of GFP have allowed simultaneous visualisation of multiple competing interactions in vivo. Advantages and disadvantages of the method are discussed in the context of other fluorescence-based interaction monitoring techniques. Keywords: Protein-protein interactions, green fluorescent protein, protein complementation assay, bimolecular fluorescent complementation, EGFP, two hybrid screen
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Techniques are now available for isolating plant protoplasts in any quantity from leaves or from cultured cells by enzymatic removal of the cell walls. The culture conditions for regenerating dividing cells and complete plants has been established for a few species. Intact viruses enter plant protoplasts and multiply. Spontaneous fusion occurs in protoplasts of the same species. Mitosis frequently takes place synchronously in the nuclei of the multinucleated protoplasts. Induced fusion of widely different species such as wheat and soybean has been demonstrated. Hybrid plants of two tobacco species have been produced by cell fusion. Transformation has been achieved by feeding isolated DNA to plants. Isolated DNA supplied to protoplasts is absorbed. The progress in plant cell and protoplast research demonstrates the feasibility of transferring genetic information and makes hybrid plant production by somatic cell hybridization a realistic objective.
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Techniques to detect and verify interactions between proteins in vivo have become invaluable tools in functional genomic research. While many of the initially developed interaction assays (e.g., yeast two-hybrid system and split-ubiquitin assay) usually are conducted in heterologous systems, assays relying on bimolecular fluorescence complementation (BiFC; also referred to as split-YFP assays) are applicable to the analysis of protein-protein interactions in most native systems, including plant cells. Like all protein-protein interaction assays, BiFC can produce false positive and false negative results. The purpose of this commentary is to (1) highlight shortcomings of and potential pitfalls in BiFC assays, (2) provide guidelines for avoiding artifactual interactions, and (3) suggest suitable approaches to scrutinize potential interactions and validate them by independent methods.
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Abstract The bimolecular fluorescence complementation (BiFC) assay has been widely accepted for studying in vivo detection of protein–protein interactions in several organisms. To facilitate the application of the BiFC assay to yeast research, we have created a series of plasmids that allow single‐step, PCR‐based C‐ or N‐terminal tagging of yeast proteins with yellow fluorescent protein fragments for BiFC assay. By examination of several interacting proteins (Sis1–Sis1, Net1–Sir2, Cet1–Cet1 and Pho2–Pho4), we demonstrate that the BiFC assay can be used to reliably analyse the occurrence and subcellular localization of protein–protein interactions in living yeast cells. The sequences for the described plasmids were submitted to the GenBank under Accession Nos: EF210802, pFA6a‐VN‐His3MX6; EF210803, pFA6a‐VC‐His3MX6; EF210804, pFA6a‐VN‐TRP1; EF210807, pFA6a‐VC‐TRP1; EF210808, pFA6a‐VN‐kanMX6; EF210809, pFA6a‐VC‐kanMX6; EF210810, pFA6a‐His3MX6‐P GAL1 ‐VN; EF210805, pFA6a‐His3MX6‐P GAL1 ‐VC; EF210806, pFA6a‐TRP1‐P GAL1 ‐VN; EF210811, pFA6a‐TRP1‐P GAL1 ‐VC; EF210812, pFA6a‐kanMX6‐P GAL1 ‐VN; EF210813, pFA6a‐kanMX6‐P GAL1 ‐VC; EF521883, pFA6a‐His3MX6‐P CET1 ‐VN; EF521884, pFA6a‐His3MX6‐P CET1 ‐VC; EF521885, pFA6a‐TRP1‐P CET1 ‐VN; EF521886, pFA6a‐TRP1‐P CET1 ‐VC; EF521887, pFA6a‐kanMX6‐P CET1 ‐VN; EF521888, pFA6a‐kanMX6‐P CET1 ‐VC. Copyright © 2007 John Wiley & Sons, Ltd.
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