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Rubisco Extraction and Purification from Diatoms

Featured protocol,  Authors: Jodi N. Young
Jodi N. YoungAffiliation: Department of Oceanography, University of Washington, Seattle, USA
For correspondence: youngjn@uw.edu
Bio-protocol author page: a4026
Ana M. C. Heureux
Ana M. C. HeureuxAffiliation: Department of Earth Sciences, University of Oxford, Oxford, UK
Bio-protocol author page: a4027
Rosalind E. M. Rickaby
Rosalind E. M. RickabyAffiliation: Department of Earth Sciences, University of Oxford, Oxford, UK
Bio-protocol author page: a4028
François M. M. Morel
François M. M. MorelAffiliation: Department of Geosciences, Princeton University, Princeton, USA
Bio-protocol author page: a4029
Spencer M. Whitney
Spencer M. WhitneyAffiliation: Plant Science Division, Research School of Biology, the Australian National University, Canberra, Australia
Bio-protocol author page: a4030
 and Robert E. Sharwood
Robert E. SharwoodAffiliation: Plant Science Division, Research School of Biology, the Australian National University, Canberra, Australia
Bio-protocol author page: a4031
date: 3/20/2017, 134 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2191.

Brief version appeared in J Exp Bot, May 2016
This protocol describes a method to extract ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) from diatoms (Bacillariophyta) to determine catalytic performance. This protocol has been adapted from use in cyanobacteria and higher plants (Andrews, 1988; Whitney and Sharwood, 2007). First part (steps A1-A3) of the extraction provides a crude extract of Rubisco that is sufficient for carboxylation assays to measure the Michaelis constant for CO2 (KC) and the catalytic turnover rate (kcatc). However, the further purification steps outlined (steps B1-B4) are needed for measurements of Rubisco CO2/O2 Specificity (SC/O, [Kane et al., 1994]).

Extraction and Analysis of Carotenoids from Escherichia coli in Color Complementation Assays

Featured protocol,  Authors: Andreas Blatt
Andreas BlattAffiliation: Institut für Molekulare Physiologie - Pflanzenbiochemie, Johannes Gutenberg-Universität, Mainz, Germany
Bio-protocol author page: a4223
 and Martin Lohr
Martin LohrAffiliation: Institut für Molekulare Physiologie - Pflanzenbiochemie, Johannes Gutenberg-Universität, Mainz, Germany
For correspondence: lohr@uni-mainz.de
Bio-protocol author page: a4224
date: 3/20/2017, 109 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2179.

Brief version appeared in Plant J, May 2015
A common method to investigate the function of genes putatively involved in carotenoid biosynthesis is the so called color complementation assay in Escherichia coli (see, e.g., Cunningham and Gantt, 2007). In this assay, the gene under investigation is expressed in E. coli strains genetically engineered to synthesize potential carotenoid substrates, followed by analysis of the pigment changes in the carotenogenic bacteria via high-performance liquid chromatography (HPLC). Two crucial steps in this method are (i) the quantitative extraction of the carotenoids out of E. coli and (ii) the reproducible and complete separation of the pigments by HPLC.

Ribosomal RNA N-glycosylase Activity Assay of Ribosome-inactivating Proteins

Featured protocol,  Authors: Rosario Iglesias
Rosario IglesiasAffiliation: Department of Biochemistry and Molecular Biology and Physiology, Faculty of Sciences, University of Valladolid, Valladolid, Spain
Bio-protocol author page: a4233
Lucía Citores
Lucía CitoresAffiliation: Department of Biochemistry and Molecular Biology and Physiology, Faculty of Sciences, University of Valladolid, Valladolid, Spain
Bio-protocol author page: a4234
 and José M. Ferreras
José M. FerrerasAffiliation: Department of Biochemistry and Molecular Biology and Physiology, Faculty of Sciences, University of Valladolid, Valladolid, Spain
For correspondence: rosario@bio.uva.es
Bio-protocol author page: a4232
date: 3/20/2017, 106 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2180.

Brief version appeared in Mol Plant Pathol, Feb 2016
Ribosome-inactivating proteins (RIPs) are enzymes that irreversibly inactivate ribosomes as a consequence of their N-glycosylase (EC 3.2.2.22) activity. The enzyme cleaves the N-glycosidic bond between the adenine No. 4324 from the 28S rRNA and its ribose in rat ribosomes (or the equivalent adenine in sensitive ribosomes from other organisms). This adenine is located in the α-sarcin-ricin loop (SRL) that is crucial for anchoring the elongation factor (EF) G and EF2 on the ribosome during mRNA-tRNA translocation in prokaryotes and eukaryotes, respectively. RIPs have been isolated mainly from plants and examples of these proteins are ricin or Pokeweed Antiviral Protein (PAP). These proteins, either alone or as a part of immunotoxins, are useful tools for cancer therapy. The following protocol describes a method to detect the RNA fragment released when the RIP-treated apurinic RNA from rabbit reticulocyte lysate is incubated in the presence of acid aniline by electrophoresis on polyacrylamide gels. The fragment released (Endo’s fragment) is diagnostic of the action of RIPs.

Extraction, Purification and Quantification of Diffusible Signal Factor Family Quorum-sensing Signal Molecules in Xanthomonas oryzae pv. oryzae

Featured protocol,  Authors: Lian Zhou
Lian ZhouAffiliation 1: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
Affiliation 2: Zhiyuan Innovative Research Center, Shanghai Jiao Tong University, Shanghai, China
Bio-protocol author page: a4225
Xing-Yu Wang
Xing-Yu WangAffiliation: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
Bio-protocol author page: a4226
Wei Zhang
Wei ZhangAffiliation: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
Bio-protocol author page: a4227
Shuang Sun
Shuang SunAffiliation: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
Bio-protocol author page: a4228
 and Ya-Wen He
Ya-Wen HeAffiliation: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
For correspondence: yawenhe@sjtu.edu.cn
Bio-protocol author page: a4229
date: 3/20/2017, 108 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2190.

Brief version appeared in Mol Plant Microbe Interact, Mar 2016
Bacteria use quorum-sensing (QS) systems to monitor and regulate their population density. Bacterial QS involves small molecules that act as signals for bacterial communication. Many Gram-negative bacterial pathogens use a class of widely conserved molecules, called diffusible signal factor (DSF) family QS signals. The measurement of DSF family signal molecules is essential for understanding DSF metabolic pathways, signaling networks, as well as regulatory roles. Here, we describe a method for the extraction of DSF family signal molecules from Xanthomonas oryzae pv. oryzae (Xoo) cell pellets and Xoo culture supernatant. We determined the levels of DSF family signals using ultra-performance liquid chromatographic system (UPLC) coupled with accurate mass time-of-flight mass spectrometer (TOF-MS). With the aid of UPLC/MS system, the detection limit of DSF was as low as 1 µM, which greatly improves the ability to detect DSF DSF family signal molecules in bacterial cultures and reaction mixtures.

Wheat Root-dip Inoculation with Fusarium graminearum and Assessment of Root Rot Disease Severity

Featured protocol,  Authors: Qing Wang
Qing Wang Affiliation: Department of Plant Breeding, IFZ Research Centre for Biosystems, Land Use and Nutrition, Justus Liebig University, Giessen, Germany
Bio-protocol author page: a4241
 and Sven Gottwald
Sven GottwaldAffiliation: Department of Plant Breeding, IFZ Research Centre for Biosystems, Land Use and Nutrition, Justus Liebig University, Giessen, Germany
For correspondence: sv.gottwald@t-online.de
Bio-protocol author page: a4240
date: 3/20/2017, 127 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2189.

Brief version appeared in Mol Plant Microbe Interact, Dec 2015
Fusarium graminearum is one of the most common and potent fungal pathogens of wheat (Triticum aestivum) and other cereals, known for causing devastating yield losses and mycotoxin contaminations of food and feed. The pathogen is mainly considered as a paradigm for the floral disease Fusarium head blight, while its ability to colonize wheat plants via root infection has been examined recently. F. graminearum has a unique infection strategy which comprises complex, specialized structures and processes. Root colonisation negatively affects plant development and leads to systemic plant invasion by tissue-adapted fungal strategies. The pathosystem wheat root - F. graminearum makes available an array of research areas, such as (i) the relatively unknown root interactions with a necrotrophic pathogen; (ii) genes and pathways contributing to (overall) Fusarium resistance; (iii) induced systemic (whole-plant) resistance; (iv) pathogenic strategies in a variety of host tissues; and (v) age-related changes in the single-genotype responses to seedling and adult plant (root/spike) infection. The presented Fusarium root rot bioassay allows for efficient infection of wheat roots, evaluation of disease severity and progress as well as statistical analysis of disease dynamics.

Xylem Sap Extraction Method from Hop Plants

Featured protocol,  Authors: Marko Flajšman*
Marko FlajšmanAffiliation: Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
Bio-protocol author page: a4167
Stanislav Mandelc*
Stanislav MandelcAffiliation: Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
Bio-protocol author page: a4168
Sebastjan Radišek*
Sebastjan RadišekAffiliation: Slovenian Institute of Hop Research and Brewing, Zalec, Slovenia
Bio-protocol author page: a4169
 and Branka Javornik
Branka JavornikAffiliation: Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
For correspondence: branka.javornik@bf.uni-lj.si
Bio-protocol author page: a4170
 (*contributed equally to this work) date: 3/20/2017, 121 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2172.

Brief version appeared in Mol Plant Microbe Interact, May 2016
Verticillium wilt is one of the most important diseases on hop that significantly influence continuation of production on affected areas. It is caused by the soil borne vascular pathogen Verticillium nonalfalfae, which infects plants through the roots and then advances through the vascular (xylem) system. During infection, V. nonalfalfae secretes many different virulence factors. Xylem sap of infected plants is therefore a rich source for investigating the molecules that are involved in molecular interactions of Verticillium – hop plants. This protocol provides instructions on how to infect hop plants with V. nonalfalfae artificially and how to obtain xylem sap from hop plants.

Pathogenicity Assay of Verticillium nonalfalfae on Hop Plants

Featured protocol,  Authors: Marko Flajšman*
Marko FlajšmanAffiliation: Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
Bio-protocol author page: a4167
Sebastjan Radišek*
Sebastjan RadišekAffiliation: Slovenian Institute of Hop Research and Brewing, Zalec, Slovenia
Bio-protocol author page: a4169
 and Branka Javornik
Branka JavornikAffiliation: Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
For correspondence: branka.javornik@bf.uni-lj.si
Bio-protocol author page: a4170
 (*contributed equally to this work) date: 3/20/2017, 106 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2171.

Brief version appeared in Mol Plant Microbe Interact, May 2016
Verticillium nonalfalfae is a soil-borne plant pathogen that infects its hosts through roots. It spreads in the plant’s xylem and causes wilt disease symptoms by secreting different virulence factors. Hop (Humulus lupulus) is a primary host of V. nonalfalfae, so it is used as a model plant for studying this phytopathogenic fungus. Artificial infections of hop plants and disease scoring are prerequisites for studying the pathogen’s virulence/pathogenicity and its interaction with hop plants. In this protocol we describe the root dipping inoculation method for conducting pathogenicity assay of V. nonalfalfae on hop plants.

Knock-in Blunt Ligation Utilizing CRISPR/Cas9

Featured protocol,  Authors: Jonathan M. Geisinger
Jonathan M. GeisingerAffiliation: Department of Biology, Stanford University, Stanford, USA
For correspondence: jonmg54@stanford.edu
Bio-protocol author page: a4146
 and Michele P. Calos
Michele P. CalosAffiliation: Department of Genetics, Stanford University, Stanford, USA
Bio-protocol author page: a4147
date: 3/5/2017, 197 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2163.

Brief version appeared in Nucleic Acids Res, May 2016
The incorporation of the CRISPR/Cas9 bacterial immune system into the genetic engineering toolbox has led to the development of several new methods for genome manipulation (Auer et al., 2014; Byrne et al., 2015). We took advantage of the ability of Cas9 to generate blunt-ended double-strand breaks (Jinek et al., 2012) to introduce exogenous DNA in a highly precise manner through the exploitation of non-homologous end-joining DNA repair machinery (Geisinger et al., 2016). This protocol has been successfully applied to traditional immortalized cell lines and human induced pluripotent stem cells. Here we present a generalized protocol for knock-in blunt ligation, using HEK293 cells as an example.

In Gel Kinase Assay

Featured protocol,  Authors: Gaston A. Pizzio
Gaston A. PizzioAffiliation: Department of Molecular Genetics, Centre for Research in Agricultural Genomics (CRAG; consortium CSIC-IRTA-UAB-UB), Barcelona, Spain
For correspondence: gapizzio@gmail.com
Bio-protocol author page: a2769
 and Pedro L. Rodriguez
Pedro L. RodriguezAffiliation: Instituto de Biologia Molecular y Celular de Plantas, Consejo Superior de Investigaciones Cientificas-Universidad Politecnica de Valencia, Valencia, Spain
Bio-protocol author page: a4211
date: 3/5/2017, 233 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2170.

Brief version appeared in Plant Cell, Jun 2012
Proper spatiotemporal regulation of protein phosphorylation in cells and tissues is required for normal development and homeostasis. We present the protocol ‘In Gel Kinase Assay’, which is useful for protein kinase activity measurements from crude protein extracts. We have successfully used ‘In Gel Kinase Assay’ protocol to show that the Arabidopsis thaliana sextuple mutant in the PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYR/PYL/RCAR-ABA receptors; line pyr/pyl112458) is impaired in ABA-mediated activation of SnRK2.2, SnRK2.3 and OST1/SnRK2.6, as much as the triple mutant snrk2.2/2.3/2.6 (Gonzalez-Guzman et al., 2012).

Laser Scanning Confocal Microcopy for Arabidopsis Epidermal, Mesophyll, and Vascular Parenchyma Cells

Featured protocol,  Authors: Christian Elowsky*
Christian ElowskyAffiliation: Department of Agronomy and Horticulture, University of Nebraska, Lincoln, USA
Bio-protocol author page: a4164
Yashitola Wamboldt*
Yashitola WamboldtAffiliation: Department of Agronomy and Horticulture, University of Nebraska, Lincoln, USA
Bio-protocol author page: a4165
 and Sally Mackenzie
Sally MackenzieAffiliation: Department of Agronomy and Horticulture, University of Nebraska, Lincoln, USA
For correspondence: smackenzie2@unl.edu
Bio-protocol author page: a4166
 (*contributed equally to this work) date: 3/5/2017, 288 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2150.

Brief version appeared in Mol Plant, Feb 2016
Investigation of protein targeting to plastids in plants by confocal laser scanning microscopy (CLSM) can be complicated by numerous sources of artifact, ranging from misinterpretations from in vivo protein over-expression, false fluorescence in cells under stress, and organellar mis-identification. Our studies have focused on the plant-specific gene MSH1, which encodes a dual targeting protein that is regulated in its expression and resides within the nucleoid of a specialized plastid type (Virdi et al., 2016). Therefore, our methods have been optimized to study protein dual targeting to mitochondria and plastids, spatial and temporal regulation of protein expression, and sub-organellar localization, producing a protocol and set of experimental standards that others may find useful for such studies.

Direct Visualization and Quantification of the Actin Nucleation and Elongation Events in vitro by TIRF Microscopy

Featured protocol,  Authors: Yuxiang Jiang
Yuxiang JiangAffiliation 1: Center for Plant Biology, School of Life Sciences, Tsinghua University, Beijing, China
Affiliation 2: Institute of Botany, Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a4178
 and Shanjin Huang
Shanjin HuangAffiliation 1: Center for Plant Biology, School of Life Sciences, Tsinghua University, Beijing, China
Affiliation 2: Institute of Botany, Chinese Academy of Sciences, Beijing, China
For correspondence: sjhuang@tsinghua.edu.cn
Bio-protocol author page: a966
date: 3/5/2017, 187 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2146.

Brief version appeared in Mol Plant, Dec 2015
Total internal reflection fluorescence (TIRF) microscopy is a powerful tool for visualizing the dynamics of actin filaments at single-filament resolution in vitro. Thanks to the development of various fluorescent probes, we can easily monitor all kinds of events associated with actin dynamics, including nucleation, elongation, bundling, fragmentation and monomer dissociation. Here we present a detailed protocol regarding the visualization and quantification of actin nucleation and filament elongation events by TIRF microscopy in vitro, which is based on the methods previously reported (Liu et al., 2015; Yang et al., 2011) .

Acetyl Bromide Soluble Lignin (ABSL) Assay for Total Lignin Quantification from Plant Biomass

Featured protocol,  Authors: William J. Barnes
William J. BarnesAffiliation 1: Department of Biology, The Pennsylvania State University, University Park, PA, USA
Affiliation 2: Center for Lignocellulose Structure and Formation, The Pennsylvania State University, University Park, PA, USA
Bio-protocol author page: a4185
 and Charles T. Anderson
Charles T. AndersonAffiliation 1: Department of Biology, The Pennsylvania State University, University Park, PA, USA
Affiliation 2: Center for Lignocellulose Structure and Formation, The Pennsylvania State University, University Park, PA, USA
For correspondence: cta3@psu.edu
Bio-protocol author page: a4143
date: 3/5/2017, 216 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2149.

Brief version appeared in J Exp Bot, Jul 2015
Lignin is the second most abundant biopolymer on Earth, providing plants with mechanical support in secondary cell walls and defense against abiotic and biotic stresses. However, lignin also acts as a barrier to biomass saccharification for biofuel generation (Carroll and Somerville, 2009; Zhao and Dixon, 2011; Wang et al., 2013). For these reasons, studying the properties of lignin is of great interest to researchers in agriculture and bioenergy fields. This protocol describes the acetyl bromide method of total lignin extraction and quantification, which is favored among other methods for its high recovery, consistency, and insensitivity to different tissue types (Johnson et al., 1961; Chang et al., 2008; Moreira-Vilar et al., 2014; Kapp et al., 2015). In brief, acetyl bromide digestion causes the formation of acetyl derivatives on free hydroxyl groups and bromide substitution of α-carbon hydroxyl groups on the lignin backbone to cause a complete solubilization of lignin, which can be quantified using known extinction coefficients and absorbance at 280 nm (Moreira-Vilar et al., 2014).

Surface Inoculation and Quantification of Pseudomonas syringae Population in the Arabidopsis Leaf Apoplast

Featured protocol,  Authors: Cristián Jacob*
Cristián JacobAffiliation: Department of Plant Sciences, University of California, Davis, USA
For correspondence: cjjacob@ucdavis.edu
Bio-protocol author page: a4204
Shweta Panchal*
Shweta PanchalAffiliation: Centre for Genome Research, Faculty of Science, the Maharaja Sayajirao University of Baroda, Baroda, India
For correspondence: shwetapanchal84@gmail.com
Bio-protocol author page: a4158
 and Maeli Melotto
Maeli MelottoAffiliation: Department of Plant Sciences, University of California, Davis, USA
For correspondence: melotto@ucdavis.edu
Bio-protocol author page: a4160
 (*contributed equally to this work) date: 3/5/2017, 239 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2167.

Brief version appeared in Front Plant Sci, Jun 2016
Bacterial pathogens must enter the plant tissue in order to cause a successful infection. Foliar bacterial pathogens that are not able to directly penetrate the plant epidermis rely on wounds or natural openings to internalize leaves. This protocol describes a procedure to estimate the population size of Pseudomonas syringae in the leaf apoplast after surface inoculation of Arabidopsis rosettes.

An HPLC-based Method to Quantify Coronatine Production by Bacteria

Featured protocol,  Authors: Shweta Panchal
Shweta PanchalAffiliation: Centre for Genome Research, Department of Microbiology and Biotechnology Centre, the Maharaja Sayajirao University of Baroda, Baroda, India
For correspondence: shwetapanchal84@gmail.com
Bio-protocol author page: a4158
Zachary S. Breitbach
Zachary S. BreitbachAffiliation: Department of Chemistry, University of Texas, Arlington, USA
For correspondence: zachbreitbach@yahoo.com
Bio-protocol author page: a4159
 and Maeli Melotto
Maeli MelottoAffiliation: Department of Plant Sciences, University of California, Davis, USA
For correspondence: melotto@ucdavis.edu
Bio-protocol author page: a4160
date: 3/5/2017, 232 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2147.

Brief version appeared in Front Plant Sci, Jun 2016
Coronatine is a polyketide phytotoxin produced by several pathovars of the plant pathogenic bacterium Pseudomonas syringae. It is one of the most important virulence factors determining the success of bacterial pathogenesis in the plant at both epiphytic and endophytic stages of the disease cycle. This protocol describes an optimized procedure to culture bacterial cells for coronatine production and to quantify the amount of coronatine secreted in the culture medium using an HPLC-based method.

Multiplexed GuideRNA-expression to Efficiently Mutagenize Multiple Loci in Arabidopsis by CRISPR-Cas9

Featured protocol,  Authors: Julia Schumacher
Julia SchumacherAffiliation 1: Institute for Biochemistry and Biology, University of Potsdam, Potsdam, Germany
Affiliation 2: Department for Plant Cell and Molecular Biology, Institute for Biology, Humboldt-Universität zu Berlin, Berlin, Germany
Bio-protocol author page: a4201
Kerstin Kaufmann
Kerstin KaufmannAffiliation: Department for Plant Cell and Molecular Biology, Institute for Biology, Humboldt-Universität zu Berlin, Berlin, Germany
Bio-protocol author page: a4202
 and Wenhao Yan
Wenhao YanAffiliation 1: Institute for Biochemistry and Biology, University of Potsdam, Potsdam, Germany
Affiliation 2: Department for Plant Cell and Molecular Biology, Institute for Biology, Humboldt-Universität zu Berlin, Berlin, Germany
For correspondence: yanwen@uni-potsdam.de
Bio-protocol author page: a4203
date: 3/5/2017, 230 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2166.

Brief version appeared in Plant Methods, Apr 2016
Since the discovery of the CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein (Cas) as an efficient tool for genome editing in plants (Li et al., 2013; Shan et al., 2013; Nekrasov et al., 2013), a large variety of applications, such as gene knock-out, knock-in or transcriptional regulation, has been published. So far, the generation of multiple mutants in plants involved tedious crossing or mutagenesis followed by time-consuming screening of huge populations and the use of the Cas9-system appeared a promising method to overcome these issues. We designed a binary vector that combines both the coding sequence of the codon optimized Streptococcus pyogenes Cas9 nuclease under the control of the Arabidopsis thaliana UBIQUITIN10 (UBQ10)-promoter and guideRNA (gRNA) expression cassettes driven by the A. thaliana U6-promoter for efficient multiplex editing in Arabidopsis (Yan et al., 2016). Here, we describe a step-by-step protocol to cost-efficiently generate the binary vector containing multiple gRNAs and the Cas9 nuclease based on classic cloning procedure.

Rubisco Extraction and Purification from Diatoms

Authors: Jodi N. Young
Jodi N. YoungAffiliation: Department of Oceanography, University of Washington, Seattle, USA
For correspondence: youngjn@uw.edu
Bio-protocol author page: a4026
Ana M. C. Heureux
Ana M. C. HeureuxAffiliation: Department of Earth Sciences, University of Oxford, Oxford, UK
Bio-protocol author page: a4027
Rosalind E. M. Rickaby
Rosalind E. M. RickabyAffiliation: Department of Earth Sciences, University of Oxford, Oxford, UK
Bio-protocol author page: a4028
François M. M. Morel
François M. M. MorelAffiliation: Department of Geosciences, Princeton University, Princeton, USA
Bio-protocol author page: a4029
Spencer M. Whitney
Spencer M. WhitneyAffiliation: Plant Science Division, Research School of Biology, the Australian National University, Canberra, Australia
Bio-protocol author page: a4030
 and Robert E. Sharwood
Robert E. SharwoodAffiliation: Plant Science Division, Research School of Biology, the Australian National University, Canberra, Australia
Bio-protocol author page: a4031
date: 3/20/2017, 134 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2191.

[Abstract] This protocol describes a method to extract ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) from diatoms (Bacillariophyta) to determine catalytic performance. This protocol has been adapted from use in cyanobacteria and higher plants (Andrews, 1988; Whitney and Sharwood, 2007). First part (steps A1-A3) of the extraction provides a crude extract ...

Extraction and Analysis of Carotenoids from Escherichia coli in Color Complementation Assays

Authors: Andreas Blatt
Andreas BlattAffiliation: Institut für Molekulare Physiologie - Pflanzenbiochemie, Johannes Gutenberg-Universität, Mainz, Germany
Bio-protocol author page: a4223
 and Martin Lohr
Martin LohrAffiliation: Institut für Molekulare Physiologie - Pflanzenbiochemie, Johannes Gutenberg-Universität, Mainz, Germany
For correspondence: lohr@uni-mainz.de
Bio-protocol author page: a4224
date: 3/20/2017, 109 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2179.

[Abstract] A common method to investigate the function of genes putatively involved in carotenoid biosynthesis is the so called color complementation assay in Escherichia coli (see, e.g., Cunningham and Gantt, 2007). In this assay, the gene under investigation is expressed in E. coli strains genetically engineered to synthesize potential carotenoid substrates, ...

Ribosomal RNA N-glycosylase Activity Assay of Ribosome-inactivating Proteins

Authors: Rosario Iglesias
Rosario IglesiasAffiliation: Department of Biochemistry and Molecular Biology and Physiology, Faculty of Sciences, University of Valladolid, Valladolid, Spain
Bio-protocol author page: a4233
Lucía Citores
Lucía CitoresAffiliation: Department of Biochemistry and Molecular Biology and Physiology, Faculty of Sciences, University of Valladolid, Valladolid, Spain
Bio-protocol author page: a4234
 and José M. Ferreras
José M. FerrerasAffiliation: Department of Biochemistry and Molecular Biology and Physiology, Faculty of Sciences, University of Valladolid, Valladolid, Spain
For correspondence: rosario@bio.uva.es
Bio-protocol author page: a4232
date: 3/20/2017, 106 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2180.

[Abstract] Ribosome-inactivating proteins (RIPs) are enzymes that irreversibly inactivate ribosomes as a consequence of their N-glycosylase (EC 3.2.2.22) activity. The enzyme cleaves the N-glycosidic bond between the adenine No. 4324 from the 28S rRNA and its ribose in rat ribosomes (or the equivalent adenine in sensitive ribosomes from other organisms). This ...

Extraction, Purification and Quantification of Diffusible Signal Factor Family Quorum-sensing Signal Molecules in Xanthomonas oryzae pv. oryzae

Authors: Lian Zhou
Lian ZhouAffiliation 1: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
Affiliation 2: Zhiyuan Innovative Research Center, Shanghai Jiao Tong University, Shanghai, China
Bio-protocol author page: a4225
Xing-Yu Wang
Xing-Yu WangAffiliation: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
Bio-protocol author page: a4226
Wei Zhang
Wei ZhangAffiliation: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
Bio-protocol author page: a4227
Shuang Sun
Shuang SunAffiliation: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
Bio-protocol author page: a4228
 and Ya-Wen He
Ya-Wen HeAffiliation: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
For correspondence: yawenhe@sjtu.edu.cn
Bio-protocol author page: a4229
date: 3/20/2017, 108 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2190.

[Abstract] Bacteria use quorum-sensing (QS) systems to monitor and regulate their population density. Bacterial QS involves small molecules that act as signals for bacterial communication. Many Gram-negative bacterial pathogens use a class of widely conserved molecules, called diffusible signal factor (DSF) family QS signals. The measurement of DSF family signal ...

Wheat Root-dip Inoculation with Fusarium graminearum and Assessment of Root Rot Disease Severity

Authors: Qing Wang
Qing Wang Affiliation: Department of Plant Breeding, IFZ Research Centre for Biosystems, Land Use and Nutrition, Justus Liebig University, Giessen, Germany
Bio-protocol author page: a4241
 and Sven Gottwald
Sven GottwaldAffiliation: Department of Plant Breeding, IFZ Research Centre for Biosystems, Land Use and Nutrition, Justus Liebig University, Giessen, Germany
For correspondence: sv.gottwald@t-online.de
Bio-protocol author page: a4240
date: 3/20/2017, 127 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2189.

[Abstract] Fusarium graminearum is one of the most common and potent fungal pathogens of wheat (Triticum aestivum) and other cereals, known for causing devastating yield losses and mycotoxin contaminations of food and feed. The pathogen is mainly considered as a paradigm for the floral disease Fusarium head blight, while its ability to colonize wheat plants via ...

Xylem Sap Extraction Method from Hop Plants

Authors: Marko Flajšman*
Marko FlajšmanAffiliation: Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
Bio-protocol author page: a4167
Stanislav Mandelc*
Stanislav MandelcAffiliation: Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
Bio-protocol author page: a4168
Sebastjan Radišek*
Sebastjan RadišekAffiliation: Slovenian Institute of Hop Research and Brewing, Zalec, Slovenia
Bio-protocol author page: a4169
 and Branka Javornik
Branka JavornikAffiliation: Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
For correspondence: branka.javornik@bf.uni-lj.si
Bio-protocol author page: a4170
 (*contributed equally to this work) date: 3/20/2017, 121 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2172.

[Abstract] Verticillium wilt is one of the most important diseases on hop that significantly influence continuation of production on affected areas. It is caused by the soil borne vascular pathogen Verticillium nonalfalfae, which infects plants through the roots and then advances through the vascular (xylem) system. During infection, V. nonalfalfae secretes many ...

Pathogenicity Assay of Verticillium nonalfalfae on Hop Plants

Authors: Marko Flajšman*
Marko FlajšmanAffiliation: Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
Bio-protocol author page: a4167
Sebastjan Radišek*
Sebastjan RadišekAffiliation: Slovenian Institute of Hop Research and Brewing, Zalec, Slovenia
Bio-protocol author page: a4169
 and Branka Javornik
Branka JavornikAffiliation: Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
For correspondence: branka.javornik@bf.uni-lj.si
Bio-protocol author page: a4170
 (*contributed equally to this work) date: 3/20/2017, 106 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2171.

[Abstract] Verticillium nonalfalfae is a soil-borne plant pathogen that infects its hosts through roots. It spreads in the plant’s xylem and causes wilt disease symptoms by secreting different virulence factors. Hop (Humulus lupulus) is a primary host of V. nonalfalfae, so it is used as a model plant for studying this phytopathogenic fungus. Artificial infections ...

Knock-in Blunt Ligation Utilizing CRISPR/Cas9

Authors: Jonathan M. Geisinger
Jonathan M. GeisingerAffiliation: Department of Biology, Stanford University, Stanford, USA
For correspondence: jonmg54@stanford.edu
Bio-protocol author page: a4146
 and Michele P. Calos
Michele P. CalosAffiliation: Department of Genetics, Stanford University, Stanford, USA
Bio-protocol author page: a4147
date: 3/5/2017, 197 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2163.

[Abstract] The incorporation of the CRISPR/Cas9 bacterial immune system into the genetic engineering toolbox has led to the development of several new methods for genome manipulation (Auer et al., 2014; Byrne et al., 2015). We took advantage of the ability of Cas9 to generate blunt-ended double-strand breaks (Jinek et al., 2012) to introduce exogenous DNA in ...

In Gel Kinase Assay

Authors: Gaston A. Pizzio
Gaston A. PizzioAffiliation: Department of Molecular Genetics, Centre for Research in Agricultural Genomics (CRAG; consortium CSIC-IRTA-UAB-UB), Barcelona, Spain
For correspondence: gapizzio@gmail.com
Bio-protocol author page: a2769
 and Pedro L. Rodriguez
Pedro L. RodriguezAffiliation: Instituto de Biologia Molecular y Celular de Plantas, Consejo Superior de Investigaciones Cientificas-Universidad Politecnica de Valencia, Valencia, Spain
Bio-protocol author page: a4211
date: 3/5/2017, 233 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2170.

[Abstract] Proper spatiotemporal regulation of protein phosphorylation in cells and tissues is required for normal development and homeostasis. We present the protocol ‘In Gel Kinase Assay’, which is useful for protein kinase activity measurements from crude protein extracts. We have successfully used ‘In Gel Kinase Assay’ protocol to show that the Arabidopsis ...

Laser Scanning Confocal Microcopy for Arabidopsis Epidermal, Mesophyll, and Vascular Parenchyma Cells

Authors: Christian Elowsky*
Christian ElowskyAffiliation: Department of Agronomy and Horticulture, University of Nebraska, Lincoln, USA
Bio-protocol author page: a4164
Yashitola Wamboldt*
Yashitola WamboldtAffiliation: Department of Agronomy and Horticulture, University of Nebraska, Lincoln, USA
Bio-protocol author page: a4165
 and Sally Mackenzie
Sally MackenzieAffiliation: Department of Agronomy and Horticulture, University of Nebraska, Lincoln, USA
For correspondence: smackenzie2@unl.edu
Bio-protocol author page: a4166
 (*contributed equally to this work) date: 3/5/2017, 288 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2150.

[Abstract] Investigation of protein targeting to plastids in plants by confocal laser scanning microscopy (CLSM) can be complicated by numerous sources of artifact, ranging from misinterpretations from in vivo protein over-expression, false fluorescence in cells under stress, and organellar mis-identification. Our studies have focused on the plant-specific gene ...
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Detection of Hydrogen Peroxide by DAB Staining in Arabidopsis Leaves Updates
The author made some updates (highlighted in blue) to the protocol on 09/19/2016.

Authors: Arsalan Daudi
Arsalan DaudiAffiliation 1: Department of Biological Sciences, Royal Holloway University of London, Egham, UK
Affiliation 2: Department of Plant Pathology, University of California, Davis, CA, USA
For correspondence: aadaudi@ucdavis.edu
Bio-protocol author page: a107
 and Jose A. O’Brien
Jose A. O’BrienAffiliation: Department of Biological Sciences, Royal Holloway University of London, Egham, UK
Bio-protocol author page: a108
date: 9/20/2012, 31945 views, 16 Q&A
DOI: https://doi.org/10.21769/BioProtoc.263.

[Abstract] In this protocol, the in situ detection of hydrogen peroxide (one of several reactive oxygen species) is described in mature Arabidopsis rosette leaves by staining with 3,3'-diaminobenzidine (DAB) using an adaptation of previous methods (Thordal-Christensen et al., 1997; Bindschedler et al., 2006; Daudi ...

[Bio101] Infiltration of Nicotiana benthamiana Protocol for Transient Expression via Agrobacterium

Author: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
date: 7/20/2011, 30895 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.95.

[Abstract] Transient expression in tobacco plant (Nicotiana benthamiana) is used to determine the subcellular location of a protein of interest when tagged with a reporter such as green fluorescent protein (GFP), or to mass produce proteins without making transgenic plants. The root tumor bacteria, Agrobacteria, ...

[Bio101] Pollen Fertility/viability Assay Using FDA Staining

Author: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
date: 5/20/2011, 16263 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.75.

[Abstract] Pollen grains can be fertile or sterile by nature. This method stains pollen grains for an enzyme as the vital indicator of membrane integrity. Only fertile grains fluoresce under microscopic examination....

[Bio101] Histostaining for Tissue Expression Pattern of Promoter-driven GUS Activity in Arabidopsis

Author: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
date: 7/5/2011, 15476 views, 4 Q&A
DOI: https://doi.org/10.21769/BioProtoc.93.

[Abstract] Promoter-driven GUS (beta-glucuronidase) activity is the most commonly used technique for tissue-specific expression patterns in Arabidopsis. In this procedure, GUS enzyme converts 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc) to a blue product. The staining is very sensitive. Processed samples can ...

[Bio101] Arabidopsis Pollen Tube Aniline Blue Staining

Author: Yongxian Lu
Yongxian LuAffiliation: Carnegie Institution for Scienc, Stanford University, Stanford, USA
For correspondence: yxlu@stanford.edu
Bio-protocol author page: a28
date: 6/20/2011, 15126 views, 4 Q&A
DOI: https://doi.org/10.21769/BioProtoc.88.

[Abstract] The aim of this experiment is to track pollen tube growth in vivo in the female tissues after pollination. This can be used to phenotype pollen germination, tube growth and guidance, and reception....

[Bio101] Extract Genomic DNA from Arabidopsis Leaves (Can be Used for Other Tissues as Well)

Author: Yongxian Lu
Yongxian LuAffiliation: Carnegie Institution for Science, Stanford University, Stanford, USA
For correspondence: yxlu@stanford.edu
Bio-protocol author page: a28
date: 7/5/2011, 14500 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.90.

[Abstract] This is a simple protocol for isolating genomic DNA from fresh plant tissues. DNA from this experiment can be used for all kinds of genetics studies, including genotyping and mapping. This protocol uses Edward’s extraction buffer to isolate DNA....

In vitro Protein Ubiquitination Assays

Authors: Qingzhen Zhao
Qingzhen ZhaoAffiliation: College of Life Science, Liaocheng University, Liaocheng, China
Bio-protocol author page: a887
 and Qi Xie
Qi XieAffiliation: State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology Chinese Academy of Sciences, Beijing, China
For correspondence: qxie@genetics.ac.cn
Bio-protocol author page: a888
date: 10/5/2013, 14311 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.928.

[Abstract] Ubiquitin can be added to substrate protein as a protein tag by the concerted actions of ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2) and ubiquitin protein ligase (E3). At the present of E1 and ubiquitin, E2 activity can be determined by the thio-ester formation. The E3 activity ...

[Bio101] Arabidopsis Growing Protocol – A General Guide

Author: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
date: 9/5/2011, 13192 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.126.

[Abstract] Arabidopsis as the model organism for higher plants is widely studied among plant biology labs around the world. However, taking care of this tiny plant may not be trivial. Here is a general guide used for the Heven Sze lab at the University of Maryland, College Park. A lot of efforts have been taken ...

Seed Germination and Viability Test in Tetrazolium (TZ) Assay

Authors: Pooja Verma
Pooja VermaAffiliation: NIPGR, National Institute of Plant Genome Research, New Delhi, India
Bio-protocol author page: a799
 and Manoj Majee
Manoj MajeeAffiliation: NIPGR, National Institute of Plant Genome Research, New Delhi, India
For correspondence: manojmajee@nipgr.ac.in
Bio-protocol author page: a800
date: 9/5/2013, 12795 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.884.

[Abstract] Tetrazolium (TZ) assay is the fast evaluation for seed viability and alternative quick method for seed’s germinability (Porter et al., 1947; Wharton, 1955). All respiring tissues are capable of converting a colourless compound, TZ (2,3,5 triphenyl tetrazolium chloride) to a carmine red coloured water-insoluble ...

[Bio101] A Transient Expression Assay Using Arabidopsis Mesophyll Protoplasts

Author: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
date: 5/20/2011, 11145 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.70.

[Abstract] This method can be used to free and separate the mesophyll cells from Arabidopsis leaves. The protoplasts that are generated in this way can be used for transient expression for protein activity and subcellular localization assays....
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