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Penetration Assays, Fungal Recovery and Pathogenicity Assays for Verticillium dahliae

Featured protocol,  Authors: Yun-Long Zhao
Yun-Long ZhaoAffiliation: State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
For correspondence: zyl714@126.com
Bio-protocol author page: a4092
Tao Zhang
Tao Zhang Affiliation: State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a4093
 and Hui-Shan Guo
Hui-Shan GuoAffiliation: State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a2721
date: 2/20/2017, 46 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2133.

Brief version appeared in PLoS Pathog, Jul 2016
Verticillium dahliae is a soil-borne phytopathogenic fungus that infects host roots and proliferates in vascular tissues. The great loss of economically important crop caused by V. dahliae has raised worldwide concern, however, little is known about the mechanism of its pathogenicity (Klosterman et al., 2011; Yadeta and Thomma, 2013). Our recent work has shown that V. dahliae develops hyphopodium as an infection structure to breach plant root cell wall (Zhao et al., 2016). Here, we provide a detailed protocol to analyze the penetration ability and the pathogenicity of V. dahliae as well as recover fungal hyphae from infected cotton stems developed from our previous studies (Zhang et al., 2016a and 2016b; Zhao et al., 2016). Cellophane membrane has been used in inducing appressorium development of foliar pathogens but not root pathogens (Bourett and Howard, 1990). We adopted the method of using the cellophane membrane to induce and assess the development of hyphopodium. Hopefully, it will greatly promote the research of molecular events involved in recognition of the host that regulate infectious development. This protocol is also helpful to identify the key component controlling the pathogenicity of V. dahliae and widen our understanding of the mechanism of plant-microbe interaction.

Penetration Assays, Fungal Recovery and Pathogenicity Assays for Verticillium dahliae

Authors: Yun-Long Zhao
Yun-Long ZhaoAffiliation: State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
For correspondence: zyl714@126.com
Bio-protocol author page: a4092
Tao Zhang
Tao Zhang Affiliation: State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a4093
 and Hui-Shan Guo
Hui-Shan GuoAffiliation: State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a2721
date: 2/20/2017, 46 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2133.

[Abstract] Verticillium dahliae is a soil-borne phytopathogenic fungus that infects host roots and proliferates in vascular tissues. The great loss of economically important crop caused by V. dahliae has raised worldwide concern, however, little is known about the mechanism of its pathogenicity (Klosterman et ...

In vitro Histone H3 Cleavage Assay for Yeast and Chicken Liver H3 Protease

Authors: Sakshi Chauhan
Sakshi ChauhanAffiliation: Laboratory of Chromatin Biology, Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, India
Bio-protocol author page: a3946
Gajendra Kumar Azad
Gajendra Kumar AzadAffiliation 1: Laboratory of Chromatin Biology, Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, India
Affiliation 2: Department of Genetics, Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem, Israel
Bio-protocol author page: a3947
 and Raghuvir Singh Tomar
Raghuvir Singh TomarAffiliation: Laboratory of Chromatin Biology, Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, India
For correspondence: rst@iiserb.ac.in
Bio-protocol author page: a3948
date: 1/5/2017, 353 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2085.

[Abstract] Histone proteins are subjected to a wide array of reversible and irreversible post-translational modifications (PTMs) (Bannister and Kouzarides, 2011; Azad and Tomar, 2014). The PTMs on histones are known to regulate chromatin structure and function. Histones are irreversibly modified by proteolytic ...

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, 609 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 ...

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, 548 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 ...

Highly Accurate Real-time Measurement of Rapid Hydrogen-peroxide Dynamics in Fungi

Authors: Michael Mentges
Michael MentgesAffiliation: Biocenter Klein Flottbek, Department of Molecular Phytopathology, University of Hamburg, Hamburg, Germany
Bio-protocol author page: a3933
 and Jörg Bormann
Jörg BormannAffiliation: Biocenter Klein Flottbek, Department of Molecular Phytopathology, University of Hamburg, Hamburg, Germany
For correspondence: joerg.bormann@uni-hamburg.de
Bio-protocol author page: a3387
date: 12/20/2016, 346 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2080.

[Abstract] Reactive oxygen species (ROS) are unavoidable by-products of aerobic metabolism. Despite beneficial aspects as a signaling molecule, ROS are principally recognized as harmful agents that act on nucleic acids, proteins and lipids. Reactive oxygen species, and, in particular, hydrogen peroxide (H2O2), ...

Single-step Marker Switching in Schizosaccharomyces pombe Using a Lithium Acetate Transformation Protocol

Authors: Simon David Brown
Simon David BrownAffiliation: Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK
Bio-protocol author page: a3916
 and Alexander Lorenz
Alexander LorenzAffiliation: Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK
For correspondence: a.lorenz@abdn.ac.uk
Bio-protocol author page: a3917
date: 12/20/2016, 506 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2075.

[Abstract] The ability to utilize different selectable markers for tagging or mutating multiple genes in Schizosaccharomyces pombe is hampered by the historical use of only two selectable markers, ura4+ and kanMX6; the latter conferring resistance to the antibiotic G418 (geneticin). More markers have been described ...

Fusarium graminearum Maize Stalk Infection Assay and Associated Microscopic Observation Protocol

Authors: Juan He
Juan HeAffiliation: National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
Bio-protocol author page: a3791
Tinglu Yuan
Tinglu Yuan Affiliation: National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
Bio-protocol author page: a3792
 and Wei-Hua Tang
Wei-Hua TangAffiliation: National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
For correspondence: whtang@sibs.ac.cn
Bio-protocol author page: a2037
date: 12/5/2016, 367 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2034.

[Abstract] The ascomycete fungus Fusarium graminearum (previously also called Gibberella zeae) causes Gibberella stalk rot in maize (Zea mays) and results in lodging and serious yield reduction. To develop methods to assess the fungal growth and symptom development in maize stalks, we present here a protocol of ...

Uptake Assay for Radiolabeled Peptides in Yeast

Authors: Melinda Hauser
Melinda HauserAffiliation: Department of Microbiology, University of Tennessee, Knoxville, USA
Bio-protocol author page: a3756
Houjian Cai
Houjian CaiAffiliation: Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, USA
Bio-protocol author page: a3755
Fred Naider
Fred NaiderAffiliation 1: Department of Chemistry and Macromolecular Assembly Institute, College of Staten Island of the City University of New York, Staten Island, New York, USA
Affiliation 2: Ph.D. Programs in Biochemistry and Chemistry, The Graduate Center of the City University of New York, New York, USA
Bio-protocol author page: a3757
 and Jeffrey M. Becker
Jeffrey M. BeckerAffiliation: Department of Microbiology, University of Tennessee, Knoxville, USA
For correspondence: jbecker@utk.edu
Bio-protocol author page: a3758
date: 11/20/2016, 419 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2026.

[Abstract] We describe an assay for measuring the uptake of radioactive peptides into the yeast Saccharomyces cerevisiae. The methods presented here can be adapted to measure a variety of substrates transported into any bacterial or fungal cell via specific carrier-mediated systems....

Halo Assay for Toxic Peptides and Other Compounds in Microorganisms

Authors: Houjian Cai
Houjian CaiAffiliation: Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, USA
Bio-protocol author page: a3755
Melinda Hauser
Melinda HauserAffiliation: Department of Microbiology, University of Tennessee, Knoxville, USA
Bio-protocol author page: a3756
Fred Naider
Fred NaiderAffiliation 1: Department of Chemistry and Macromolecular Assembly Institute, College of Staten Island of the City University of New York, Staten Island, USA
Affiliation 2: Ph.D. Programs in Biochemistry and Chemistry, The Graduate Center of the City University of New York, New York, USA
Bio-protocol author page: a3757
 and Jeffrey M. Becker
Jeffrey M. BeckerAffiliation: Department of Microbiology, University of Tennessee, Knoxville, USA
For correspondence: jbecker@utk.edu
Bio-protocol author page: a3758
date: 11/20/2016, 460 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2025.

[Abstract] We describe an assay for determination of toxicity in S. cerevisiae involving spotting of a toxic peptide on a lawn of yeast cells. This assay may be generalized to determine toxicity of a variety of compounds by substituting a putative toxic compound in place of the peptide. The general protocol may ...

An Assay to Study Botrytis cinerea-infected Grapevine Leaves Primed with Pseudomonas fluorescens

Authors: Charlotte Gruau
Charlotte GruauAffiliation: URVVC EA4707, UFR Sciences, University of Reims, Reims, France
Bio-protocol author page: a3539
Patricia Trotel-Aziz
Patricia Trotel-AzizAffiliation: URVVC EA4707, UFR Sciences, University of Reims, Reims, France
Bio-protocol author page: a3540
Bas Verhagen
Bas VerhagenAffiliation: URVVC EA4707, UFR Sciences, University of Reims, Reims, France
Bio-protocol author page: a3541
Sandra Villaume
Sandra VillaumeAffiliation: URVVC EA4707, UFR Sciences, University of Reims, Reims, France
Bio-protocol author page: a3542
Fanja Rabenoelina
Fanja RabenoelinaAffiliation: URVVC EA4707, UFR Sciences, University of Reims, Reims, France
Bio-protocol author page: a3543
Barbara Courteaux
Barbara CourteauxAffiliation: URVVC EA4707, UFR Sciences, University of Reims, Reims, France
Bio-protocol author page: a3544
Christophe Clément
Christophe ClémentAffiliation: URVVC EA4707, UFR Sciences, University of Reims, Reims, France
Bio-protocol author page: a3545
Fabienne Baillieul
Fabienne BaillieulAffiliation: URVVC EA4707, UFR Sciences, University of Reims, Reims, France
Bio-protocol author page: a3546
 and Aziz Aziz
Aziz AzizAffiliation: URVVC EA4707, UFR Sciences, University of Reims, Reims, France
For correspondence: aziz.aziz@univ-reims.fr
Bio-protocol author page: a3547
date: 10/5/2016, 634 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1943.

[Abstract] Grapevine (Vitis vinifera L.) is susceptible to an array of diseases among them the grey mold caused by the necrotrophic fungus Botrytis cinerea that decreases grape productivity and quality. To ensure a satisfactory yield and harvest quality numerous chemical fungicides are required, but they have ...
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In vitro Protein Kinase Assay

Author: Yuehua Wei
Yuehua WeiAffiliation: Department of Pharmacology, Cancer Institute of New Jersey, UMDNJ Robert Wood Johnson Medical School, Piscataway, USA
For correspondence: weiyh.sjtu.edu@gmail.com
Bio-protocol author page: a49
date: 6/5/2012, 21038 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.212.

[Abstract] This protocol will describe experimental procedures for an in vitro kinase assay of the yeast protein kinase Sch9. This protocol can be tailored to detect kinase activity of other yeast protein kinase....

[Bio101] Making Yeast Competent Cells and Yeast Cell Transformation

Author: Yongxian Lu
Yongxian LuAffiliation: Carnegie Institution for Science, Stanford University, Stanford, USA
For correspondence: yxlu@stanford.edu
Bio-protocol author page: a28
date: 7/20/2011, 20369 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.96.

[Abstract] This is a quite simple but reliable protocol to make very high transformation efficiency yeast competent cells. By express your gene of interest, protein function can be studied in yeast cells....

[Bio101] Protocol for Whole Cell Lysis of Yeast

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
date: 1/5/2011, 17969 views, 4 Q&A
DOI: https://doi.org/10.21769/BioProtoc.14.

[Abstract] This protocol describes how to perform lysis on whole yeast cell samples using NaOH. The lysed cells can then be used for downstream applications such as the extraction of total proteins. ...

Spot Assay for Yeast

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
date: 1/5/2012, 17549 views, 3 Q&A
DOI: https://doi.org/10.21769/BioProtoc.16.

[Abstract] This protocol can be used to compare the cell growth rate of yeast under different growth conditions. It involves the serial dilution and spotting of yeast colonies....

[Bio101] Yeast Vacuole Staining with FM4-64

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
date: 1/5/2011, 14022 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.18.

[Abstract] The lipophilic probe, FM 4-64 does not fluoresce much in water but fluoresces strongly after binding to the outer plasma membrane, providing clear and distinguishable plasma membrane staining. The binding is rapid and reversible. In this protocol vacuoles in yeast cells are stained with the FM4-64 dye, ...

[Bio101] How to Use an Avestin Emulsiflex C3 Homogenizer to Disrupt Cells

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
date: 1/5/2011, 11138 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.11.

[Abstract] The EmulsiFlex-C3 homogenizer is powered by an electric motor. The pump does not require a compressor for it to run. This equipment can be used to disrupt cells at a large scale. The EmulsiFlex-C3 has a fixed flow-through capacity of 3 L/h. It has the ability to process samples as small as 10 ml. The ...

[Bio101] Large Scale Native Affinity Purifications of Solubilized Membrane Proteins from Yeast

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicin, Baltimore , USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
date: 1/5/2011, 10769 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.12.

[Abstract] This protocol can be used to purify membrane proteins from yeast samples under native conditions at a large scale. This protocol has been developed primarily for FLAG-tagged proteins. This protocol can however be slightly modified and applied to other tags, such as GST or HA....

[Bio101] Small Scale Native Affinity Purifications of Solubilized Membrane Proteins from Yeast

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
date: 1/5/2011, 10229 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.15.

[Abstract] In this protocol, we show how to purify membrane proteins from yeast using affinity purification under native conditions at a small scale. ...

Illumina Sequencing Library Construction from ChIP DNA

Author: Wei Zheng
Wei ZhengAffiliation: Keck Biotech Services, Yale University, New Haven, USA
For correspondence: wei.zheng.madison@gmail.com
Bio-protocol author page: a10
date: 2/20/2012, 9619 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.91.

[Abstract] The Illumina sequencing platform is very popular among next-generation sequencing platforms. However, the DNA sequencing library construction kit provided by Illumina is considerably expensive. The protocol described here can be used to construct high-quality sequencing libraries from chromatin immunoprecipitated ...

Co-immunoprecipitation in Yeast

Author: Olesya O. Panasenko
Olesya O. PanasenkoAffiliation: Department of Microbiology and Molecular Medicine, University of Geneva, Faculty of Medicine, CMU, Geneva, Switzerland
For correspondence: olesya.panasenko@unige.ch
Bio-protocol author page: a88
date: 8/20/2012, 9082 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.250.

[Abstract] This protocol describes investigation of protein-protein interactions in baker yeast by co-immunoprecipitation (CoIP). CoIP is a technique to identify physiologically relevant protein-protein interactions in the cell. The interesting protein can be isolated out of solution using antibody that specifically ...
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