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Dot Blot Analysis of N6-methyladenosine RNA Modification Levels

Featured protocol,  Authors: Lisha Shen
Lisha ShenAffiliation: Temasek Life Sciences Laboratory and Department of Biological Sciences, National University of Singapore, Singapore
Bio-protocol author page: a4000
Zhe Liang
Zhe Liang Affiliation: Temasek Life Sciences Laboratory and Department of Biological Sciences, National University of Singapore, Singapore
Bio-protocol author page: a4001
 and Hao Yu
Hao YuAffiliation: Temasek Life Sciences Laboratory and Department of Biological Sciences, National University of Singapore, Singapore
For correspondence: dbsyuhao@nus.edu.sg
Bio-protocol author page: a4002
date: 1/5/2017, 85 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2095.

Brief version appeared in Dev Cell, Jul 2016
N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic messenger RNA (mRNA). The total amount of m6A can be detected by several methods, such as dot blot analysis using specific m6A antibodies and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Fu et al., 2014; Shen et al., 2016). Here we describe the method for fast detection of total m6A levels in mRNA by dot blot analysis using a specific m6A antibody.

Cation (Ca2+ and Mn2+) Partitioning Assays with Intact Arabidopsis Chloroplasts

Featured protocol,  Authors: Anna Harms
Anna HarmsAffiliation: Biozentrum der LMU München, Department Biologie I, Munich, Germany
Bio-protocol author page: a3963
Iris Steinberger
Iris SteinbergerAffiliation: Biozentrum der LMU München, Department Biologie I, Munich, Germany
Bio-protocol author page: a2369
 and Anja Schneider
Anja SchneiderAffiliation: Biozentrum der LMU München, Department Biologie I, Munich, Germany
For correspondence: anja.schneider@lrz.uni-muenchen.de
Bio-protocol author page: a2371
date: 1/5/2017, 90 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2094.

Brief version appeared in Plant J, Apr 2014
Determination of the relative distribution of Ca2+ and Mn2+ is an important tool for analyzing mutants showing altered levels of calcium and/or manganese transporters in the chloroplast envelope or thylakoid membrane. The method described in this protocol allows quantitative analyses of the relative distribution of calcium and manganese ions between chloroplast stroma and thylakoids using the isotopes [45Ca] and [54Mn] as radioactive tracers. To avoid contaminations with non chloroplastidic membrane systems, the method is designed for isolating pure and intact chloroplasts of Arabidopsis thaliana. Intact chloroplasts are isolated via Percoll gradient centrifugation. Chloroplasts are then allowed to take up [45Ca] or [54Mn] during a light incubation step. After incubation, chloroplasts are either kept intact or osmotically/mechanically treated to release thylakoids. The amount of incorporated [45Ca] or [54Mn] can be determined by liquid scintillation counting and the relative distribution calculated.

In vitro Ubiquitin Dimer Formation Assay

Featured protocol,  Authors: Sheng Wang
Sheng WangAffiliation: Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada
Bio-protocol author page: a1483
Ling Cao
Ling CaoAffiliation 1: Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada
Affiliation 2: National Key laboratory of Crop Genetic Improvement, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, China
Bio-protocol author page: a1482
 and Hong Wang
Hong WangAffiliation: Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada
For correspondence: hong.wang@usask.ca
Bio-protocol author page: a1485
date: 1/5/2017, 108 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2082.

Brief version appeared in J Exp Bot, May 2016
The process of protein ubiquitination typically consists of three sequential steps to add an ubiquitin (Ub) or Ub chain to a substrate protein, requiring three different enzymes, ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin protein ligase (E3). Most E2s possess the classical E2 activity in forming E2-Ub complex through a thioester linkage, in presence of an E1 and Ub. Additionally, some E2s have the ability of catalyzing the formation of free Ub dimer. Such activity indicates an important role of these E2s in ubiquitination pathway. Thus, we developed an in vitro Ub dimer formation assay to determine the activity of certain E2s. Moreover, by using Ub mutants, in which different lysine residues are mutated, the specific linkage of dimer can also be determined.

Inoculation of Rice with Different Pathogens: Sheath Blight (Rhizoctonia solani), Damping off Disease (Pythium graminicola) and Barley Powdery Mildew (Blumeria graminis f. sp. hordei)

Featured protocol,  Authors: Rhoda Delventhal
Rhoda DelventhalAffiliation: Department of Plant Physiology, RWTH Aachen University, Aachen, Germany
Bio-protocol author page: a3900
Marco Loehrer
Marco LoehrerAffiliation: Department of Plant Physiology, RWTH Aachen University, Aachen, Germany
Bio-protocol author page: a3899
Denise Weidenbach
Denise WeidenbachAffiliation: Department of Plant Physiology, RWTH Aachen University, Aachen, Germany
Bio-protocol author page: a3901
 and Ulrich Schaffrath
Ulrich SchaffrathAffiliation: Department of Plant Physiology, RWTH Aachen University, Aachen, Germany
For correspondence: schaffrath@bio3.rwth-aachen.de
Bio-protocol author page: a3902
date: 12/20/2016, 147 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2070.

Brief version appeared in Mol Plant, Apr 2016
To prevent yield losses in plant cultivation due to plant pathogens, it is an important task to find new disease resistance mechanisms. Recently, Weidenbach et al. (2016) reported about the capacity of the rice gene OsJAC1 to enhance resistance in rice and barley against a broad spectrum of different pathogens. Here, we describe the respective protocols used by Weidenbach and colleagues for inoculation of rice with the basidiomycete Rhizoctonia solani, the oomycete Pythium graminicola and the ascomycete Blumeria graminis f. sp. hordei (Bgh).

Bacterial Growth Inhibition Assay for Xanthomonoas oryzae pv. oryzae or Escherichia coli K12 Grown together with Plant Leaf Extracts

Featured protocol,  Authors: Marco Loehrer
Marco LoehrerAffiliation: Department of Plant Physiology, RWTH Aachen University, Aachen, Germany
Bio-protocol author page: a3899
Rhoda Delventhal
Rhoda DelventhalAffiliation: Department of Plant Physiology, RWTH Aachen University, Aachen, Germany
Bio-protocol author page: a3900
Denise Weidenbach
Denise WeidenbachAffiliation: Department of Plant Physiology, RWTH Aachen University, Aachen, Germany
Bio-protocol author page: a3901
 and Ulrich Schaffrath
Ulrich SchaffrathAffiliation: Department of Plant Physiology, RWTH Aachen University, Aachen, Germany
For correspondence: schaffrath@bio3.rwth-aachen.de
Bio-protocol author page: a3902
date: 12/20/2016, 171 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2069.

Brief version appeared in Mol Plant, Apr 2016
We performed a growth inhibition assay to test antibacterial compounds in leaf extracts from transgenic rice plants. The assay is based on over-night co-incubation of a defined concentration of colony forming units (cfu) of the respective bacteria together with aqueous extracts of ground leaf tissue. 

Plant Tissue Trypan Blue Staining During Phytopathogen Infection

Featured protocol,  Authors: Nuria Fernández-Bautista
Nuria Fernández-BautistaAffiliation: Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) – Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Parque Científico y Tecnológico, UPM, Campus de Montegancedo, Ctra M-40, km 38, 28223, Pozuelo de Alarcón (Madrid), Spain
Bio-protocol author page: a3925
José Alfonso Domínguez-Núñez
José Alfonso Domínguez-NúñezAffiliation: Departamento de Sistemas y Recursos Naturales, Escuela Técnica Superior de Ingeniería de Montes, Forestal y del Medio Natural, Ciudad Universitaria s/n, 28040, Madrid, Spain
Bio-protocol author page: a3926
M. Mar Castellano Moreno
M. Mar Castellano MorenoAffiliation: Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) – Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Parque Científico y Tecnológico, UPM, Campus de Montegancedo, Ctra M-40, km 38, 28223, Pozuelo de Alarcón (Madrid), Spain
Bio-protocol author page: a3927
 and Marta Berrocal-Lobo
Marta Berrocal-LoboAffiliation 1: Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) – Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Parque Científico y Tecnológico, UPM, Campus de Montegancedo, Ctra M-40, km 38, 28223, Pozuelo de Alarcón (Madrid), Spain
Affiliation 2: Departamento de Sistemas y Recursos Naturales, Escuela Técnica Superior de Ingeniería de Montes, Forestal y del Medio Natural, Ciudad Universitaria s/n, 28040, Madrid, Spain
For correspondence: m.berrocal@upm.es
Bio-protocol author page: a3928
date: 12/20/2016, 214 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2078.

Brief version appeared in Plant J, Jan 2002
In this protocol plant tissue is stained with trypan blue dye allowing the researcher to visualize cell death. Specifically this method avoids the use of the carcinogen compound chloral hydrate, making this classical method of staining safer and faster than never. The protocol is applied specifically to detect cell death on Arabidopsis leaves during the course of infection with necrotrophic fungus Botrytis cinerea.

Electro-fusion of Gametes and Subsequent Culture of Zygotes in Rice

Featured protocol,  Authors: Erika Toda
Erika TodaAffiliation 1: Department of Biological Sciences, Tokyo Metropolitan University, Hachioji, Tokyo, Japan
Affiliation 2: Plant Breeding Innovation Laboratory, RIKEN Innovation Center, Tsurumi-ku, Yokohama, Japan
Bio-protocol author page: a3915
Yukinosuke Ohnishi
Yukinosuke OhnishiAffiliation: Department of Biological Sciences, Tokyo Metropolitan University, Hachioji, Tokyo, Japan
Bio-protocol author page: a2105
 and Takashi Okamoto
Takashi OkamotoAffiliation: Department of Biological Sciences, Tokyo Metropolitan University, Hachioji, Tokyo, Japan
For correspondence: okamoto-takashi@tmu.ac.jp
Bio-protocol author page: a2106
date: 12/20/2016, 135 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2074.

Brief version appeared in Plant Physiol, May 2016
Electro-fusion system with isolated gametes has been utilized to dissect fertilization-induced events in angiosperms, such as egg activation, zygote development and early embryogenesis, since the female gametophytes of plants are deeply embedded within ovaries. In this protocol, procedures for isolation of rice gametes, electro-fusion of gametes, and culture of the produced zygotes are described.

Assessment of Wheat Resistance to Fusarium graminearum by Automated Image Analysis of Detached Leaves Assay

Featured protocol,  Authors: Alexandre Perochon
Alexandre PerochonAffiliation: University College Dublin Earth Institute and School of Biology and Environmental Science, College of Science, University College Dublin, Belfield, Dublin, Ireland
For correspondence: alexandre.perochon@ucd.ie
Bio-protocol author page: a3890
 and Fiona M. Doohan
Fiona M. DoohanAffiliation: University College Dublin Earth Institute and School of Biology and Environmental Science, College of Science, University College Dublin, Belfield, Dublin, Ireland
Bio-protocol author page: a2308
date: 12/20/2016, 340 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2065.

Brief version appeared in Plant Physiol, Dec 2015
Fusarium head blight (FHB) caused by Fusarium pathogens is a globally important cereal disease. To study Fusarium pathogenicity and host disease resistance, robust methods for disease assessment and quantification are needed. Here we describe the procedure of a detached leaves assay emphasizing the image analysis. The protocol provides the different steps of a rapid, automatic and quantitative image analysis to evaluate leaf area infected by Fusarium graminearum.

Quantitative Determination of Ascorbate from the Green Alga Chlamydomonas reinhardtii by HPLC

Featured protocol,  Authors: László Kovács
László KovácsAffiliation: Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary
Bio-protocol author page: a3895
André Vidal-Meireles
André Vidal-MeirelesAffiliation: Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary
Bio-protocol author page: a3896
Valéria Nagy
Valéria NagyAffiliation: Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary
Bio-protocol author page: a3897
 and Szilvia Z. Tóth
Szilvia Z. TóthAffiliation: Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary
For correspondence: toth.szilviazita@brc.mta.hu
Bio-protocol author page: a3898
date: 12/20/2016, 161 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2067.

Brief version appeared in Plant Cell Environ, Jul 2016
Ascorbate (Asc, also called vitamin C) is of vital importance to the cellular functions of both animals and plants. During evolution, Asc has become one of the most abundant metabolites in seed plants; however, Asc contents in cyanobacteria, green algae and bryophytes are very low. Here we describe a sensitive and reliable HPLC method for the quantitative determination of cellular Asc content in the green alga Chlamydomonas reinhardtii.

Expression, Purification and Crystallization of Recombinant Arabidopsis Monogalactosyldiacylglycerol Synthase (MGD1)

Featured protocol,  Authors: Joana Rocha
Joana RochaAffiliation: CERMAV-CNRS, University of Grenoble, Grenoble, France
Bio-protocol author page: a3887
Valerie Chazalet
Valerie ChazaletAffiliation: CERMAV-CNRS, University of Grenoble, Grenoble, France
Bio-protocol author page: a3888
 and Christelle Breton
Christelle BretonAffiliation: CERMAV-CNRS, University of Grenoble, Grenoble, France
For correspondence: breton@cermav.cnrs.fr
Bio-protocol author page: a3889
date: 12/20/2016, 148 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2064.

Brief version appeared in Plant J, Mar 2016
In plant cells, galactolipids are predominant, representing up to 50% of the lipid content in photosynthetic tissues. Galactolipid synthesis is initiated by MGDG synthases (MGDs), which use UDP-galactose as a donor sugar and diacylglycerol (DAG) as acceptor, to form monogalactosyldiacylglycerol (MGDG). This protocol is used to produce a recombinant form of Arabidopsis thaliana (A. thaliana) monogalactosyldiacylglycerol synthase 1 (MGD1) protein, in Escherichia coli (E. coli), using a two-step chromatographic purification procedure. The protein is easily expressed and purified to milligram quantities, suitable for biochemical and structural studies. The crystallization of MGD1 is also described.

Infection of Nicotiana benthamiana Plants with Potato Virus X (PVX)

Featured protocol,  Authors: Emmanuel Aguilar
Emmanuel AguilarAffiliation: Department of Environmental Biology, Centro de Investigaciones Biológicas (CIB-CSIC), Madrid, Spain
Bio-protocol author page: a3882
Francisco J. del Toro
Francisco J. del ToroAffiliation: Department of Environmental Biology, Centro de Investigaciones Biológicas (CIB-CSIC), Madrid, Spain
Bio-protocol author page: a3883
Bong-Nam Chung
Bong-Nam ChungAffiliation: National Institute of Horticultural & Herbal Science, Agricultural Research Center for Climate Change, Jeju Island, South Korea
Bio-protocol author page: a3884
Tomás Canto
Tomás CantoAffiliation: Department of Environmental Biology, Centro de Investigaciones Biológicas (CIB-CSIC), Madrid, Spain
Bio-protocol author page: a3885
 and Francisco Tenllado
Francisco TenlladoAffiliation: Department of Environmental Biology, Centro de Investigaciones Biológicas (CIB-CSIC), Madrid, Spain
For correspondence: tenllado@cib.csic.es
Bio-protocol author page: a3886
date: 12/20/2016, 151 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2063.

Brief version appeared in MPMI, Dec 2015
Potato Virus X (PVX) is the type member of Potexvirus genus, a group of plant viruses with a positive-strand RNA genome (~6.4 kb). PVX is able to establish compatible infections in Nicotiana benthamiana, a commonly used host in plant virology, leading to mild symptoms, such as chlorotic mosaic and mottling. PVX has been widely used as a viral vector for more than two decades (Chapman et al., 1992; Baulcombe et al., 1995; Aguilar et al., 2015). It provides a feasible means for the systemic expression in plants of heterologous proteins, such as avirulence factors, proteins with pharmacological properties, etc., (Hammond-Kosack et al., 1995; Gleba et al., 2014), and also as a tool to help decipher the function of genes in plants by virus-induced gene silencing (VIGS) (Lacomme and Chapman, 2008). Two different protocols, i.e., rubbing (A) and agroinfiltration (B), that allow efficient multiplication and propagation of PVX in N. benthamiana are described here in detail. The rubbing method requires previous infected sap, and infection is achieved by inducing mechanical damages to leaf tissue, allowing viral particles to penetrate the plant surface. Agroinfiltration needs previously modified agrobacterium to carry and deliver T-DNA with PVX sequences into the plant cell. Agrobacterium is grown until saturation and infection is established by infiltrating it into plant tissue with a syringe. Any of these two methods can be successfully applied, and the choice should be based mainly on the availability of material and time, but it is recommended to use agroinfiltration when chimeric viruses are being used.

Detection of Reactive Oxygen Species in Oryza sativa L. (Rice)

Featured protocol,  Authors: Navdeep Kaur
Navdeep KaurAffiliation: Department of Biotechnology, Guru Nanak Dev University, Amritsar, Punjab, India
Bio-protocol author page: a3877
Isha Sharma
Isha SharmaAffiliation: Department of Biotechnology, Guru Nanak Dev University, Amritsar, Punjab, India
Bio-protocol author page: a3878
Kamal Kirat
Kamal KiratAffiliation: Department of Biotechnology, Guru Nanak Dev University, Amritsar, Punjab, India
Bio-protocol author page: a3879
 and Pratap Kumar Pati
Pratap Kumar PatiAffiliation: Department of Biotechnology, Guru Nanak Dev University, Amritsar, Punjab, India
For correspondence: pkpati@yahoo.com
Bio-protocol author page: a3876
date: 12/20/2016, 199 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2061.

Brief version appeared in BMC Plant Biol, Jun 2016
Superoxide ions (O2-) and hydrogen peroxide (H2O2) are the reactive oxygen species (ROS) that play a significant role in regulation of many plant processes. The level of O2- ions is determined qualitatively using nitrobluetetrazolium (NBT) assay while the H2O2 is qualitatively estimated using 3,3-diaminobenzidine (DAB) and 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA) assay. Further the aqueous content of H2O2 is estimated quantitatively using ferrous oxidation-xylenol orange (FOX) assay.

Protocol for Increasing Carotenoid Levels in the Roots of Citrus Plants

Featured protocol,  Authors: Matías Manzi
Matías ManziAffiliation 1: Ecofisiologia i Biotecnologia, Dept. Ciencies Agraries i del Medi Natural, Universitat Jaume I, Castello de la Plana, Spain
Affiliation 2: Fertilidad de Suelos, Dept. Suelos y Agua, Facultad de Agronomía, Universidad de la República, Salto, Uruguay
Bio-protocol author page: a3922
Marta Pitarch-Bielsa
Marta Pitarch-BielsaAffiliation: Ecofisiologia i Biotecnologia, Dept. Ciencies Agraries i del Medi Natural, Universitat Jaume I, Castello de la Plana, Spain
Bio-protocol author page: a3921
Vicent Arbona
Vicent ArbonaAffiliation: Ecofisiologia i Biotecnologia, Dept. Ciencies Agraries i del Medi Natural, Universitat Jaume I, Castello de la Plana, Spain
Bio-protocol author page: a3923
 and Aurelio Gómez-Cadenas
Aurelio Gómez-CadenasAffiliation: Ecofisiologia i Biotecnologia, Dept. Ciencies Agraries i del Medi Natural, Universitat Jaume I, Castello de la Plana, Spain
For correspondence: aurelio.gomez@uji.es
Bio-protocol author page: a3924
date: 12/20/2016, 153 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2077.

Brief version appeared in Plant Sci, Nov 2016
Carotenoids in plants play several key functions such as acting as light-harvesters, antioxidants (Lado et al., 2016) or being precursors of strigolactones, abscisic acid, volatiles and other signaling compounds (Arbona et al., 2013). Although those functions are well-known in light-exposed tissues, information in belowground organs is limited because of reduced abundance of these pigments. In order to better understand the role of carotenoids in roots, we developed a methodology to increase the abundance of these pigments in underground tissues. We took advantage of the fact that citrus roots exposed to light develop pigmentation in order to increase the carotenoid content. Therefore, here we describe a simple method to increase carotenoids in citrus roots.

Dot Blot Analysis of N6-methyladenosine RNA Modification Levels

Authors: Lisha Shen
Lisha ShenAffiliation: Temasek Life Sciences Laboratory and Department of Biological Sciences, National University of Singapore, Singapore
Bio-protocol author page: a4000
Zhe Liang
Zhe Liang Affiliation: Temasek Life Sciences Laboratory and Department of Biological Sciences, National University of Singapore, Singapore
Bio-protocol author page: a4001
 and Hao Yu
Hao YuAffiliation: Temasek Life Sciences Laboratory and Department of Biological Sciences, National University of Singapore, Singapore
For correspondence: dbsyuhao@nus.edu.sg
Bio-protocol author page: a4002
date: 1/5/2017, 85 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2095.

[Abstract] N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic messenger RNA (mRNA). The total amount of m6A can be detected by several methods, such as dot blot analysis using specific m6A antibodies and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Fu ...

Cation (Ca2+ and Mn2+) Partitioning Assays with Intact Arabidopsis Chloroplasts

Authors: Anna Harms
Anna HarmsAffiliation: Biozentrum der LMU München, Department Biologie I, Munich, Germany
Bio-protocol author page: a3963
Iris Steinberger
Iris SteinbergerAffiliation: Biozentrum der LMU München, Department Biologie I, Munich, Germany
Bio-protocol author page: a2369
 and Anja Schneider
Anja SchneiderAffiliation: Biozentrum der LMU München, Department Biologie I, Munich, Germany
For correspondence: anja.schneider@lrz.uni-muenchen.de
Bio-protocol author page: a2371
date: 1/5/2017, 90 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2094.

[Abstract] Determination of the relative distribution of Ca2+ and Mn2+ is an important tool for analyzing mutants showing altered levels of calcium and/or manganese transporters in the chloroplast envelope or thylakoid membrane. The method described in this protocol allows quantitative analyses of the relative ...

In vitro Ubiquitin Dimer Formation Assay

Authors: Sheng Wang
Sheng WangAffiliation: Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada
Bio-protocol author page: a1483
Ling Cao
Ling CaoAffiliation 1: Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada
Affiliation 2: National Key laboratory of Crop Genetic Improvement, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, China
Bio-protocol author page: a1482
 and Hong Wang
Hong WangAffiliation: Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada
For correspondence: hong.wang@usask.ca
Bio-protocol author page: a1485
date: 1/5/2017, 108 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2082.

[Abstract] The process of protein ubiquitination typically consists of three sequential steps to add an ubiquitin (Ub) or Ub chain to a substrate protein, requiring three different enzymes, ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin protein ligase (E3). Most E2s possess ...

Inoculation of Rice with Different Pathogens: Sheath Blight (Rhizoctonia solani), Damping off Disease (Pythium graminicola) and Barley Powdery Mildew (Blumeria graminis f. sp. hordei)

Authors: Rhoda Delventhal
Rhoda DelventhalAffiliation: Department of Plant Physiology, RWTH Aachen University, Aachen, Germany
Bio-protocol author page: a3900
Marco Loehrer
Marco LoehrerAffiliation: Department of Plant Physiology, RWTH Aachen University, Aachen, Germany
Bio-protocol author page: a3899
Denise Weidenbach
Denise WeidenbachAffiliation: Department of Plant Physiology, RWTH Aachen University, Aachen, Germany
Bio-protocol author page: a3901
 and Ulrich Schaffrath
Ulrich SchaffrathAffiliation: Department of Plant Physiology, RWTH Aachen University, Aachen, Germany
For correspondence: schaffrath@bio3.rwth-aachen.de
Bio-protocol author page: a3902
date: 12/20/2016, 147 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2070.

[Abstract] To prevent yield losses in plant cultivation due to plant pathogens, it is an important task to find new disease resistance mechanisms. Recently, Weidenbach et al. (2016) reported about the capacity of the rice gene OsJAC1 to enhance resistance in rice and barley against a broad spectrum of different ...

Bacterial Growth Inhibition Assay for Xanthomonoas oryzae pv. oryzae or Escherichia coli K12 Grown together with Plant Leaf Extracts

Authors: Marco Loehrer
Marco LoehrerAffiliation: Department of Plant Physiology, RWTH Aachen University, Aachen, Germany
Bio-protocol author page: a3899
Rhoda Delventhal
Rhoda DelventhalAffiliation: Department of Plant Physiology, RWTH Aachen University, Aachen, Germany
Bio-protocol author page: a3900
Denise Weidenbach
Denise WeidenbachAffiliation: Department of Plant Physiology, RWTH Aachen University, Aachen, Germany
Bio-protocol author page: a3901
 and Ulrich Schaffrath
Ulrich SchaffrathAffiliation: Department of Plant Physiology, RWTH Aachen University, Aachen, Germany
For correspondence: schaffrath@bio3.rwth-aachen.de
Bio-protocol author page: a3902
date: 12/20/2016, 171 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2069.

[Abstract] We performed a growth inhibition assay to test antibacterial compounds in leaf extracts from transgenic rice plants. The assay is based on over-night co-incubation of a defined concentration of colony forming units (cfu) of the respective bacteria together with aqueous extracts of ground leaf tissue. ...

Plant Tissue Trypan Blue Staining During Phytopathogen Infection

Authors: Nuria Fernández-Bautista
Nuria Fernández-BautistaAffiliation: Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) – Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Parque Científico y Tecnológico, UPM, Campus de Montegancedo, Ctra M-40, km 38, 28223, Pozuelo de Alarcón (Madrid), Spain
Bio-protocol author page: a3925
José Alfonso Domínguez-Núñez
José Alfonso Domínguez-NúñezAffiliation: Departamento de Sistemas y Recursos Naturales, Escuela Técnica Superior de Ingeniería de Montes, Forestal y del Medio Natural, Ciudad Universitaria s/n, 28040, Madrid, Spain
Bio-protocol author page: a3926
M. Mar Castellano Moreno
M. Mar Castellano MorenoAffiliation: Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) – Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Parque Científico y Tecnológico, UPM, Campus de Montegancedo, Ctra M-40, km 38, 28223, Pozuelo de Alarcón (Madrid), Spain
Bio-protocol author page: a3927
 and Marta Berrocal-Lobo
Marta Berrocal-LoboAffiliation 1: Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) – Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Parque Científico y Tecnológico, UPM, Campus de Montegancedo, Ctra M-40, km 38, 28223, Pozuelo de Alarcón (Madrid), Spain
Affiliation 2: Departamento de Sistemas y Recursos Naturales, Escuela Técnica Superior de Ingeniería de Montes, Forestal y del Medio Natural, Ciudad Universitaria s/n, 28040, Madrid, Spain
For correspondence: m.berrocal@upm.es
Bio-protocol author page: a3928
date: 12/20/2016, 214 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2078.

[Abstract] In this protocol plant tissue is stained with trypan blue dye allowing the researcher to visualize cell death. Specifically this method avoids the use of the carcinogen compound chloral hydrate, making this classical method of staining safer and faster than never. The protocol is applied specifically ...

Electro-fusion of Gametes and Subsequent Culture of Zygotes in Rice

Authors: Erika Toda
Erika TodaAffiliation 1: Department of Biological Sciences, Tokyo Metropolitan University, Hachioji, Tokyo, Japan
Affiliation 2: Plant Breeding Innovation Laboratory, RIKEN Innovation Center, Tsurumi-ku, Yokohama, Japan
Bio-protocol author page: a3915
Yukinosuke Ohnishi
Yukinosuke OhnishiAffiliation: Department of Biological Sciences, Tokyo Metropolitan University, Hachioji, Tokyo, Japan
Bio-protocol author page: a2105
 and Takashi Okamoto
Takashi OkamotoAffiliation: Department of Biological Sciences, Tokyo Metropolitan University, Hachioji, Tokyo, Japan
For correspondence: okamoto-takashi@tmu.ac.jp
Bio-protocol author page: a2106
date: 12/20/2016, 135 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2074.

[Abstract] Electro-fusion system with isolated gametes has been utilized to dissect fertilization-induced events in angiosperms, such as egg activation, zygote development and early embryogenesis, since the female gametophytes of plants are deeply embedded within ovaries. In this protocol, procedures for isolation ...

Assessment of Wheat Resistance to Fusarium graminearum by Automated Image Analysis of Detached Leaves Assay

Authors: Alexandre Perochon
Alexandre PerochonAffiliation: University College Dublin Earth Institute and School of Biology and Environmental Science, College of Science, University College Dublin, Belfield, Dublin, Ireland
For correspondence: alexandre.perochon@ucd.ie
Bio-protocol author page: a3890
 and Fiona M. Doohan
Fiona M. DoohanAffiliation: University College Dublin Earth Institute and School of Biology and Environmental Science, College of Science, University College Dublin, Belfield, Dublin, Ireland
Bio-protocol author page: a2308
date: 12/20/2016, 340 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2065.

[Abstract] Fusarium head blight (FHB) caused by Fusarium pathogens is a globally important cereal disease. To study Fusarium pathogenicity and host disease resistance, robust methods for disease assessment and quantification are needed. Here we describe the procedure of a detached leaves assay emphasizing the ...

Quantitative Determination of Ascorbate from the Green Alga Chlamydomonas reinhardtii by HPLC

Authors: László Kovács
László KovácsAffiliation: Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary
Bio-protocol author page: a3895
André Vidal-Meireles
André Vidal-MeirelesAffiliation: Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary
Bio-protocol author page: a3896
Valéria Nagy
Valéria NagyAffiliation: Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary
Bio-protocol author page: a3897
 and Szilvia Z. Tóth
Szilvia Z. TóthAffiliation: Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary
For correspondence: toth.szilviazita@brc.mta.hu
Bio-protocol author page: a3898
date: 12/20/2016, 161 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2067.

[Abstract] Ascorbate (Asc, also called vitamin C) is of vital importance to the cellular functions of both animals and plants. During evolution, Asc has become one of the most abundant metabolites in seed plants; however, Asc contents in cyanobacteria, green algae and bryophytes are very low. Here we describe ...

Expression, Purification and Crystallization of Recombinant Arabidopsis Monogalactosyldiacylglycerol Synthase (MGD1)

Authors: Joana Rocha
Joana RochaAffiliation: CERMAV-CNRS, University of Grenoble, Grenoble, France
Bio-protocol author page: a3887
Valerie Chazalet
Valerie ChazaletAffiliation: CERMAV-CNRS, University of Grenoble, Grenoble, France
Bio-protocol author page: a3888
 and Christelle Breton
Christelle BretonAffiliation: CERMAV-CNRS, University of Grenoble, Grenoble, France
For correspondence: breton@cermav.cnrs.fr
Bio-protocol author page: a3889
date: 12/20/2016, 148 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2064.

[Abstract] In plant cells, galactolipids are predominant, representing up to 50% of the lipid content in photosynthetic tissues. Galactolipid synthesis is initiated by MGDG synthases (MGDs), which use UDP-galactose as a donor sugar and diacylglycerol (DAG) as acceptor, to form monogalactosyldiacylglycerol (MGDG). ...
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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, 30523 views, 15 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, 29587 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, 15562 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, 14922 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, 14667 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, 13820 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, 13276 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, 12486 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, 11146 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, 10754 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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