Effector-Triggered Immunity: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: A Brief Introduction to Effector-Triggered Immunity
	1 Introduction
	2 ETI in Plants
	3 ETI in Mammals
	4 Conclusion
	References
Chapter 2: Lambda Red Recombineering in Shigella flexneri
	1 Introduction
	2 Materials
		2.1 Oligo Design, Amplicon Construction, and Purification
		2.2 Counterselection of Plasmids and Excision of Antibiotic Cassettes
		2.3 Recharging of Electroporation Cuvettes
	3 Methods
		3.1 PCR
		3.2 Mating
		3.3 Electroporation
		3.4 Counterselection and Diagnostic PCR
		3.5 Removal of Cassette
		3.6 Recharging of Electroporation Cuvettes (See Note 6)
	4 Notes
	References
Chapter 3: Using Genomic Deletion Mutants to Investigate Effector-Triggered Immunity During Legionella pneumophila Infection
	1 Introduction
	2 Materials
		2.1 Bioinformatics to Design the Genomic Mutants
		2.2 Cloning the Flanking Sequence Into Suicide Vector
		2.3 Legionella Mutagenesis
		2.4 Measuring the Growth of Legionella mutants In Vitro
		2.5 Infection of A-Strain  Mice
		2.6 Enumeration of CFU and Cytokine Bead Array (CBA)
	3 Methods
		3.1 Creation of L. pneumophila Genomic Deletion Mutants
			3.1.1 Design of Genomic Regions to Be Deleted
			3.1.2 Clone the Flanking Sequence of Designed Genomic Regions into Suicide Vector pSR47s
				Amplification and Ligation of the Flanking Regions
				Restriction Digest and Ligation
				Transformation into Chemically Competent pir-Positive Escherichia coli
			3.1.3 Legionella mutagenesis
				Legionella Transformation
				Sucrose Selection
		3.2 Validate the Growth of Legionella mutants In Vitro
		3.3 Infection of A-Strain Mice and Harvesting Lungs
		3.4 Measure the Bacterial Numbers After In Vivo Infection
		3.5 Measure the Levels of Cytokines in Lung Homogenate
		3.6 Identification of Dot/Icm Effectors that Trigger the Mammalian Immune Response
	4 Notes
	References
Chapter 4: Methods of Bacterial Membrane Vesicle Production, Purification, Quantification, and Examination of Their Immunogeni...
	1 Introduction
	2 Materials
		2.1 Production of BMVs from Planktonic Cultures
		2.2 Concentration of Bacterial-Free Supernatant Using a Tangential Flow  Unit
		2.3 Isolation of BMVs by Ultracentrifugation
		2.4 Purification of BMVs by Density Gradient Ultracentrifugation
		2.5 Verification of BMV-Containing Fractions and Examination of Their Purity by Transmission Electron Microscopy
		2.6 Quantification of BMVs
			2.6.1 Nanoparticle Counting
			2.6.2 Protein Assay
		2.7 Quantification of BMV Protein, DNA, and RNA Contents Using Qubit Assays
		2.8 Fluorescent Labeling of BMVs for Confocal Microscopy
		2.9 Stimulation of Eukaryotic Cells with BMVs
	3 Methods
		3.1 Production of BMVs from Planktonic Cultures
		3.2 Concentration of Bacterial-Free Supernatant Using a Tangential Flow  Unit
		3.3 Isolation of BMVs by Ultracentrifugation
		3.4 Purification of BMVs by Density Gradient Ultracentrifugation
			3.4.1 Washing BMVs to Remove OptiPrep Medium Using Ultracentrifugation
			3.4.2 Washing BMVs Using Centrifugal Filtration Units
		3.5 Verification of BMV-Containing Fractions and Examination of Their Purity by Transmission Electron Microscopy
		3.6 Quantification of BMVs
			3.6.1 Nanoparticle Counting
			3.6.2 Protein Assay
		3.7 Quantification of BMV Protein, DNA, and RNA Contents Using Qubit Fluorometric Quantitation
		3.8 Fluorescent Labeling of BMVs for Use in Biological Assays
		3.9 Stimulation of Cells with BMVs
	4 Notes
	References
Chapter 5: Gene Gun-Mediated Transient Gene Expression for Functional Studies in Plant Immunity
	1 Introduction
	2 Materials
		2.1 General
		2.2 Bombardment of Barley Leaves
			2.2.1 Consumables for the PDS-1000/He Particle Delivery System
		2.3 Bgh Infection Assay
		2.4 Evaluation
	3 Methods
		3.1 Bombardment of Barley Leaves
			3.1.1 Preparation of Microcarriers
			3.1.2 Coating Microcarriers with DNA
			3.1.3 Bombardment
		3.2 Infection of Bombarded Barley Leaves
		3.3 Evaluation
			3.3.1 GUS-Staining of Barley Leaves
			3.3.2 Microscopic Evaluation
	4 Notes
	References
Chapter 6: Host-Induced Gene Silencing Using BPMV on Soybean to Study Genes in the Soybean Rust Fungus Phakopsora pachyrhizi
	1 Introduction
	2 Materials
		2.1 Cloning Silencing Constructs
		2.2 Biolistic Transformation of BPMV into Soybean and Production of Virus Inoculum
		2.3 Rub Inoculation with BPMV Silencing Constructs, Superinoculation with P. pachyrhizi
		2.4 RT-qPCR to Quantify Reductions in Fungal Growth and Gene Silencing
	3 Methods
		3.1 Cloning Silencing Constructs
			3.1.1 Cloning Silencing Constructs for a Single Target  Gene
			3.1.2 Cloning Silencing Constructs for Two or Multiple Target Genes (See Note 4)
		3.2 Biolistic Transformation of BPMV into Soybean and Production of Virus Inoculums
			3.2.1 Growing Plants for Biolistic Transformation
			3.2.2 Biolistic Inoculation and Production of Virus Inoculum
		3.3 Rub Inoculation with BPMV Silencing Constructs, Superinoculation with P. pachyrhizi
			3.3.1 Growing Plants for Rub Inoculation
			3.3.2 Rub Inoculation
			3.3.3 Superinoculation with P. pachyrhizi
		3.4 RT-qPCR to Quantify Reductions in Fungal Growth and Gene Silencing
	4 Notes
	References
Chapter 7: Virus-Mediated Protein Overexpression (VOX) in Monocots to Identify and Functionally Characterize Fungal Effectors
	1 Introduction
	2 Materials
		2.1 Plants and Plant Growth Materials
		2.2 General Materials and Apparatus
		2.3 Materials for the Development of BSMV VOX Constructs
		2.4 Materials for the Development of FoMV VOX Constructs
		2.5 Materials for Transformation of VOX Vectors into Agrobacterium tumefaciens
		2.6 Materials for Agrobacterium-Mediated Inoculation of N. benthamiana Plants
		2.7 Virus (Rub)Inoculation of Monocot Plant Leaves
	3 Methods
		3.1 BSMV VOX Constructs Development
		3.2 FoMV VOX Constructs Development
		3.3 Preparing Agrobacterium tumefaciens Strains for Agroinfiltration
		3.4 Agroinfiltration of N. benthamiana Leaves
		3.5 Wheat Inoculation
	4 Notes
	References
Chapter 8: Time-Resolved Fluorescence Microscopy Screens on Host Protein Subversion During Bacterial Cell Invasion
	1 Introduction
	2 Materials, Solutions, Cell Lines, and Bacterial Strains
		2.1 Bacterial Strains
		2.2 Bacterial Culture
		2.3 Cell Culture
		2.4 Plasmid Library Maintenance, Purification, and Transfection
		2.5 Infection and Microscopy
		2.6 Data Processing and Analysis
	3 Methods
		3.1 Plasmid Library Preparation and Handling
		3.2 Cell Culture and Transfection
		3.3 Bacterial Inoculum Preparation
		3.4 Bacterial Infection
		3.5 Image Acquisition
		3.6 Data Analysis of Bacterial Infection Foci and Bacteria-Triggered Compartments
	4 Notes
	References
Chapter 9: Assaying RIPK2 Activation by Complex Formation
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Bacterial Strains and Reagents for Culture
		2.3 Polyacrylamide Gel Electrophoresis (PAGE)
		2.4 Western Blot (WB)
		2.5 Immunostaining and Chemiluminescent Detection of Proteins
		2.6 Fluorescence Microscopy and Live Cell Imaging
	3 Methods
		3.1 Cell Culture
		3.2 Induction of RIPosomes by Bacterial Infection
		3.3 Induction of RIPosomes by Inhibitor Treatment
		3.4 Electromobility Change of RIPK2 via Western Blot Analysis
		3.5 Microscopic Analysis of RIPosomes
		3.6 Microscopic Analysis of RIPosomes by Live Cell Imaging
	4 Notes
	References
Chapter 10: Identification of Novel Endogenous NOD Ligands: Quantitative Analysis of Binding Affinities of NOD1 or NOD2 with S...
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Protein Purification
		2.3 SDS-PAGE and Western Blotting
		2.4 Protein Quantitation
		2.5 Microscale Thermophoresis Assay
	3 Methods
		3.1 Induction of NOD1-GFP and NOD2-GFP Expression
		3.2 Purification of NOD1-GFP and NOD2-GFP
		3.3 Quantification of Purified NOD1-GFP and NOD2-GFP
		3.4 Quantification of Binding Affinity of NOD1-GFP or NOD2-GFP with S1P by MST
	4 Notes
	References
Chapter 11: A Surface Plasmon Resonance-Based Strategy to Characterize Interactions of NLR Proteins with Associated Factors
	1 Introduction
	2 Materials
		2.1 Cloning of Dual Expression Vector and Preparation of Bacmid
		2.2 Transfection of Insect Cells, Protein Expression, and Purification
		2.3 SPR Measurement
	3 Methods
		3.1 Cloning of pFastBac Dual with BirA and Avi-MBP-tev Expression Cassettes
		3.2 Transfection of Insect Cells and Expression of Protein
		3.3 Purification of Biotinylated Protein
		3.4 Surface Plasmon Resonance Measurement
	4 Notes
	References
Chapter 12: Functional Assessment of Disease-Associated Pyrin Variants
	1 Introduction
	2 Materials
		2.1 Pyrin-Expressing U937 Cell Lines
		2.2 Primary Monocytes
		2.3 Pyrin Phosphorylation Level
		2.4 ASC Speck Immunofluorescence
		2.5 Inflammasome Stimuli
		2.6 Real-Time Cell Death Assay
		2.7 ELISA
	3 Methods
		3.1 Determination of Pyrin Phosphorylation Level by Immunoprecipitation Followed by Western  Blot
		3.2 ASC Speck Visualization by Immunofluorescence (IF)
		3.3 Real-Time Cell Death Analysis by Propidium Iodide (PI) Incorporation
		3.4 Quantification of IL-1β and IL-18 Release by ELISA
	4 Notes
	References
Chapter 13: An Assay for the Seeding of Homotypic Pyrin Domain Filament Transitions
	1 Introduction
	2 Materials
		2.1 Cloning, Expression, and Purification of Monomeric NLRP3PYD
		2.2 Cloning, Expression, and Purification of Monomeric ASC-mCherry and Filament Polymerization
		2.3 Negative Stain EM
	3 Methods
		3.1 Cloning, Expression, and Purification of Monomeric NLRP3PYD
		3.2 Cloning, Expression, and Purification of Monomeric ASC-mCherry
		3.3 NLRP3PYD Seeded ASC-mCherry Filament Polymerization
		3.4 Negative Stain EM
	4 Notes
	References
Chapter 14: Detection of Gasdermin Activation and Lytic Cell Death During Pyroptosis and Apoptosis
	1 Introduction
		1.1 Inflammasomes and Pyroptosis
		1.2 Apoptosis and Inflammation
		1.3 Cross Talk Between Apoptosis and Pyroptosis
		1.4 Objectives of This Chapter
			1.4.1 LDH Release as a Measure of Lytic Cell Death, But Not GSDMD Pore Formation
			1.4.2 Diverse GSDMD Fragments Detected by Immunoblotting After Processing by Inflammatory Caspase-1 or Apoptotic Caspases-3/-7...
			1.4.3 GSDME Processing Does Not Translate Into Lytic Cell Death Suring Extrinsic Apoptosis in Primary Murine Macrophages
	2 Materials
		2.1 Pyroptosis and Apoptosis Induction
		2.2 Quantification of LDH Release
		2.3 Quantification of IL-1β Release by ELISA
		2.4 SDS-PAGE
		2.5 Gel Transfer
		2.6 Immunoblotting
	3 Methods
		3.1 Pyroptosis (LPS/Nigericin) and Apoptosis (TNF/TAK1i) Induction in Bone Marrow-Derived Macrophages
		3.2 Quantification of LDH Release in the Supernatant
		3.3 Quantification of IL-1β Release in the Supernatant by ELISA
		3.4 Protein Preparation for SDS Gel Electrophoresis (Cell Lysate and Supernatant)
		3.5 SDS Polyacrylamide Gel Preparation
		3.6 SDS Polyacrylamide Gel Electrophoresis
		3.7 Gel Transfer
		3.8 Immunoblotting
	4 Notes
	References
Chapter 15: Visualizing Effector Triggered Immunity in Response to Pore-Forming Toxins by Live-Cell Imaging
	1 Introduction
	2 Materials
		2.1 Macrophages Preparation
		2.2 Live-Cell Imaging
		2.3 Monitoring Apoptosis Using Live-Cell Imaging
	3 Methods
		3.1 Macrophages Culture Preparation
		3.2 Imaging Macrophage Cell Death and Mitochondrial Membrane Potential
		3.3 Imaging the Activity of Apoptotic Caspases
	4 Notes
	References
Chapter 16: Non-apoptotic Cell Death Control of Neutrophil Extracellular Trap Formation
	1 Introduction
	2 Materials
		2.1 Solutions
		2.2 Percoll Stock Solutions
		2.3 Antibodies and Fluorescent Stains for NET Detection
		2.4 Reagents for EM Embedding
	3 Methods
		3.1 Mouse Neutrophil Purification
		3.2 Human Neutrophil Purification
		3.3 Flow Cytometry
			3.3.1 Gating Strategy
		3.4 Electron Microscopy
			3.4.1 Transmission Electron Microscopy
			3.4.2 Immunogold Electron Microscopy (Tokuyasu Method)
	4 Notes
	References
17: Analysis of Bacteria-Triggered Inflammasome: Activation in Neutrophils by Immunoblot
	1 Introduction
	2 Materials
		2.1 Generation of Murine Bone Marrow Neutrophils (BMNs)
		2.2 Purification of Murine Bone Marrow Neutrophils (BMNs)
		2.3 Seeding Murine Bone Marrow Neutrophils (BMNs)
		2.4 Infection/Stimulation of Murine Bone Marrow Neutrophils (BMNs)
		2.5 Processing BMN Samples for Immunoblotting
		2.6 Western Blot of Murine Bone Marrow Neutrophil (BMN) Samples
	3 Methods
		3.1 Isolation of Bone Marrow Neutrophils (BMNs)
		3.2 Seeding Murine Bone Marrow Neutrophils (BMNs)
		3.3 Infection of Murine Bone Marrow Neutrophils (BMNs)
		3.4 Processing BMN Samples for Immunoblotting
		3.5 Precipitation of Cell Supernatant
		3.6 Immunoblotting
	4 Notes
	References
Chapter 18: Quantifying Regulated Mitochondrial Fission in Macrophages
	1 Introduction
	2 Materials
		2.1 Macrophage Culture
		2.2 Quantification of Mitochondrial Dynamics and mitoROS Production
		2.3 Quantification of DRP1 Phosphorylation by Western Blotting
	3 Methods
		3.1 Differentiating Bone Marrow Progenitor Cells into  BMMs
		3.2 Measuring Mitochondrial Fission by Microscopy After LPS Stimulation
		3.3 Staining with MitoTracker Red  FM
		3.4 Immunostaining for Mitochondrial Proteins (Drp1/Tom20)
		3.5 Assessing Fission by Immunofluorescence: Image Acquisition
		3.6 Assessing Fission by Microscopy: Image Quantification
		3.7 Assessing Fission by Electron Microscopy (EM): Image Acquisition and Analysis
	4 Assessing Fission Response by Quantifying Drp1 Post-translational Modifications (PTM)
	5 Measuring mitoROS Production After LPS Stimulation
	6 Controls to Assess Mitochondrial Fission Versus Mitochondrial Biogenesis and/or Mitophagy
	7 Notes
	References
Chapter 19: Live Cell Imaging of T Cell Pyroptosis
	1 Introduction
	2 Materials
	3 Methods
	4 Notes
	References
Index