Preface
Contents
Contributors
Part I: Screening for Novel Small Molecules
Chapter 1: Chemical Genetics to Uncover Mechanisms Underlying Lipid-Mediated Signaling Events in Plants
1 Introduction
2 Materials
2.1 Preparing Arabidopsis Seeds for Screening
2.2 Preparing Media for Screening
2.3 Preparing for Chemical Screening
2.4 Imaging
3 Method
3.1 Primary Chemical Screen
3.1.1 Preparation of Seeds and Growth Media
3.1.2 Dilution of Chemicals in the Library
3.1.3 Treating Seedlings with the Chemical Compounds
3.1.4 Scoring Treatment Plates for Putative ``Hits´´
3.2 Secondary Chemical Screen
3.3 Third Chemical Screen
4 Notes
References
Chapter 2: Method for Phenotypic Chemical Screening to Identify Cryptochrome Inhibitors
1 Introduction
2 Materials
2.1 Plant Material
2.2 Culture Media and Growth Conditions
2.3 Chemical Library
2.4 Image Acquisition
2.5 Cell-Free Protein Synthesis
2.6 In Vitro Beads Binding Assay
3 Methods
3.1 Prepare Plant Material
3.2 Chemical Screening
3.2.1 First Screening for Blue-Light-Signaling Inhibitors
3.2.2 Phenotype Quantification by Image Processing
3.2.3 Second Screening of Compounds That Specifically Inhibit Seedling Growth Only Under Blue-Light Irradiation
3.3 Target Identification
3.3.1 Prediction of the Action Pathway or Target of a Compound Using Light-Signaling Mutant Lines
3.3.2 Recombinant Protein Synthesis Using Wheat Germ Extract
3.3.3 In Vitro Binding Assay and Immunoblot Analysis
4 Notes
References
Chapter 3: Whole-Seedling-Based Chemical Genetic Screens in Arabidopsis
1 Introduction
2 Materials
3 Methods
3.1 Search for an Optimum Screening Condition
3.1.1 Choice of Mutant Lines
3.1.2 Types of Screening Plate
3.1.3 Temperature
3.2 Preparation of Seeds and Culture Media
3.3 Chemical Genetic Screen
4 Notes
References
Chapter 4: Identification of Type III Secretion Inhibitors for Plant Disease Management
1 Introduction
2 Materials
2.1 Plant Growth
2.2 Bacterial Strains and Growth
2.3 T3SS Inhibition Test In Vitro
2.4 Effect of the Tested Compound on Bacterial T3E Secretion
2.5 In Planta Experiments
3 Methods
3.1 Plant and Bacterial Growth
3.1.1 N. benthamiana/N. tabacum
3.1.2 Solanum lycopersicum cv. Marmande
3.1.3 Ralstonia solanacearum
3.2 In Vitro T3SS Inhibitor Screening in Ralstonia solanacearum
3.3 Effect of the Tested Compound on Bacterial T3E Secretion
3.4 In Vivo T3E Translocation Test Using Hypersensitive Response Assays
3.5 Compound Effect on Bacterial Fitness In Planta
3.6 Effect of the T3 Secretion Inhibitor on Bacterial Virulence to Plants
4 Notes
References
Chapter 5: Investigation of Drug Efficacy by Screening Bioactive Chemical Effects on Plant Cell Subcellular Architecture
1 Introduction
2 Materials
2.1 Chemicals
2.2 Transgenic Plants
2.3 Microscopy
3 Methods
3.1 Plantlet Treatment and Observation
4 Notes
References
Part II: New Approaches to Variation: Peptide Screen for Novel Effectors
Chapter 6: In Vivo Chemical Genomics with Random Cyclized Peptides
1 Introduction
2 Materials
2.1 Library Construction
2.2 Gateway Cloning
2.3 Floral Dip Transformation of Arabidopsis thaliana
2.4 Transgenic Seed Selection
3 Methods
3.1 Library Construction
3.2 Gateway Cloning
3.3 Floral Dip Transformation of Arabidopsis thaliana
3.4 Transgenic Seedling Selection
3.4.1 Surface Sterilization of Seeds
3.4.2 Plating
3.4.3 Germination
3.4.4 Selection
4 Phenotyping and Genotyping
4.1 Phase I Screening
4.2 Phase II Screening
4.3 Additional Activities
5 Notes
References
Chapter 7: Interfering Peptides Targeting Protein-Protein Interactions in the Ethylene Plant Hormone Signaling Pathway as Tool...
1 Introduction
2 Materials
2.1 Plant Material
2.2 Instrumentation
2.3 Software Stack for Data Analysis
3 Methods
3.1 Preparation of Peptide Stock for Senescence Studies
3.2 Setup of Senescence Studies
3.3 Continuous Monitoring of Senescence Studies
3.4 Processing of Image Files Acquired in Senescence Studies
3.5 Analysis of Processed Image Data
3.6 Troubleshooting
4 Notes
References
Part III: Chemical Genomics in Hormone Signaling
Chapter 8: The Screening for Novel Inhibitors of Auxin-Induced Ca2+ Signaling
1 Introduction
2 Materials
3 Methods
3.1 BY-2 Transformation
3.2 Growing the BY-2 Cells
3.3 Preparation of BY-2 Cells for Ca2+ Measurements
3.4 Primary Screen Setup
3.5 Confirmation Screen Setup
4 Notes
References
Chapter 9: Identification of ABA Receptor Agonists Using a Multiplexed High-Throughput Chemical Screening
1 Introduction
2 Materials
2.1 Protein Purification
2.2 PP2C Phosphatase Assay
3 Methods
3.1 Preparation of Stock Chemical Library Plates
3.2 Protein Purification
3.3 Chemical Screening Using a PP2C Phosphatase Assay in 96-Well Plate Format
3.4 Chemical Screening Using a PP2C Phosphatase Multiplexed Assay in 384-Well Plate Format
3.4.1 Adaptation of the PP2C Phosphatase Assay to 384-Well Plate Format
3.4.2 Assay Multiplexing
3.4.3 A Screening Example Using the Multiplexed 384-Well Plate Assay
4 Notes
References
Chapter 10: A Luciferase Reporter Assay to Identify Chemical Activators of ABA Signaling
1 Introduction
2 Materials
2.1 Generation of Transgenic Lines
2.2 Chemical Screening
3 Methods
3.1 Generate pMAPKKK18::LUC+ Reporter Line
3.2 Grow Transgenic Plants
3.3 Prepare Chemical Plates
3.4 Chemical Screening
3.5 Process the Images and Identify Candidate Compounds
4 Notes
References
Chapter 11: Identification of Novel Molecular Regulators Modulating Ethylene Biosynthesis Using EMS-Based Genetic Screening
1 Introduction
2 Materials
2.1 Seed Sterilization
2.2 Suppressor Screening, Ethylene Measurement, and Other Physiological Assays
3 Methods
3.1 Screening for eto3 Suppressors in Response to Cytokinin
3.1.1 Seed Sterilization
3.1.2 Suppressor Screening
3.2 Observation of Triple Response and Measurement of Ethylene Production in the Selected Suppressor Mutants from the Root Ass...
3.3 Identification of Suppressors that Show ACC-Mediated Rescued Triple Responses and Root Phenotype
4 Notes
References
Chapter 12: Investigation of Auxin Biosynthesis and Action Using Auxin Biosynthesis Inhibitors
1 Introduction
2 Materials
2.1 E. coli Strains
2.2 Plant Material and Growth Medium
2.3 Hardware and Software
2.4 Standard and Internal Standard
3 Methods
3.1 Prepare Recombinant Protein
3.2 Enzyme Inhibition Assay of TAA1
3.3 Enzyme Inhibition Assay of YUCCA
3.4 Inhibitor Treatment for Arabidopsis Seedlings
3.5 Inhibitor Treatment for Rice Seedlings
3.6 Analyzing IAA-Related Compounds except for IPyA and IAAld
3.7 Analyses for IPyA and IAAld
4 Notes
References
Part IV: Target Identification and Confirmation Approaches
Chapter 13: Target Profiling of an Anticancer Drug Curcumin by an In Situ Chemical Proteomics Approach
1 Introduction
2 Materials
2.1 Cell Culture and Extraction of Cellular Proteins
2.2 Click Chemistry
2.3 SDS-PAGE and Fluorescence Scanning
2.4 Protein Affinity Enrichment Using Streptavidin Beads
2.5 iTRAQ Labeling of the Tryptic Peptides
2.6 Strong Cation Exchange (SCX) Chromatography to Clean Up Samples
2.7 Desalting of Labeled Samples by C18 Column
2.8 Nano-LC Electrospray Ionization MS
2.9 Protein Identification and iTRAQ Quantification
3 Method
3.1 Profiling of Curcumin-Specific Target Proteins with Fluorescence Labeling
3.1.1 Cell Culture and Extraction of Cellular Proteins
3.1.2 Click Chemistry Tagging with Rhodamine B-Azide
3.1.3 SDS-PAGE and Fluorescence Scanning
3.2 Identifying the Targets of Curcumin with iTRAQ-Based Quantitative Chemical Proteomics Approach
3.2.1 Cell Culture and Extraction of Cell Protein
3.2.2 Click Chemistry Tagging with Biotin-Azide Tag
3.2.3 Affinity Enrichment Using Streptavidin Beads
3.2.4 iTRAQ Labeling of the Tryptic Peptides
3.3 LC-MS/MS Procedures
3.3.1 Strong Cation Exchange (SCX) Chromatography to Clean Up Samples
3.3.2 Desalting of Labeled Samples by C18 Column
3.3.3 Nano-LC Electrospray Ionization MS
3.4 Protein Identification and iTRAQ Quantification
4 Notes
References
Chapter 14: Label-Free Target Identification and Confirmation Using Thermal Stability Shift Assays
1 Introduction
2 Materials
2.1 Protein Extraction
2.2 Thermal Shift
2.3 TPP
2.3.1 Protein Digestion and TMT Labeling
2.4 CETSA and ITDRF
2.5 Western Blot
3 Methods
3.1 Protein Extraction
3.2 General Thermal Shift Protocol
3.3 TPP
3.4 CETSA
3.5 ITDRF
4 Notes
References
Chapter 15: Drug Affinity Responsive Target Stability (DARTS) Assay to Detect Interaction Between a Purified Protein and a Sma...
1 Introduction
2 Materials
3 Method
3.1 Purification of AtEXO70A1 Protein
3.2 Protein Proteolysis by Pronase
3.3 Protein Detection by Silver Staining
4 Notes
References
Chapter 16: Using Differential Scanning Fluorimetry (DSF) to Detect Ligand Binding with Purified Protein
1 Introduction
2 Materials
2.1 Reagents
2.2 Instrument
3 Methods
3.1 Prepare Reaction Mix
3.2 Setting Up the Thermal Shift Program
3.3 Data Analysis
4 Notes
References
Chapter 17: Microscale Thermophoresis (MST) to Detect the Interaction Between Purified Protein and Small Molecule
1 Introduction
2 Materials
3 Methods
3.1 Purification of AtEXO70A1 Protein
3.2 Pretests for Protein Quality, Fluorescence Intensity, and Possible Ligand-Induced Effects
3.3 Binding Affinity Test
3.4 Data Analysis
4 Notes
References
Index