Host-Pathogen Interactions: Methods and Protocols

Dedication
Preface
Contents
Contributors
Part I: Introduction
	Chapter 1: Host-Pathogen Interaction: Biology and Public Health
		1 Introduction
		2 Mechanisms of the Evolution of Pathogen-Host Interaction
		3 Stages of Pathogen-Host Interaction
			3.1 Host Invasion
			3.2 Evasion of the Immune System
			3.3 Pathogen Replication in the Host
			3.4 Pathogen Control by the Immune System
		4 Therapeutic Approaches
			4.1 Tuberculosis
			4.2 HIV
			4.3 Malaria
			4.4 SARS-CoV-2
		5 Challenges in Public Health
			5.1 Tuberculosis
			5.2 COVID-19
			5.3 HIV
			5.4 Malaria
		6 Conclusion
		References
	Chapter 2: Genetic Association Studies in Host-Pathogen Interaction Analysis
		1 Introduction
		2 Case and Control Selection and Power Estimation
		3 Gene Variant Selection
		4 Data Analysis Strategies
		5 Limitations of Genetic Association Studies
		6 Illustrative Examples
		7 Conclusions
		References
Part II: Viral-Host Interactions
	Chapter 3: Live-Cell Fluorescence Imaging for Virus-Host Interactions
		1 Introduction
		2 Materials
			2.1 Cell Culture
			2.2 Lentivirus Packaging and Concentration
			2.3 Lentivirus Transduction
			2.4 Virus Activation and Infection
			2.5 Live-Cell Imaging
		3 Methods
			3.1 Preparation of Imaging Slides/Plates
			3.2 GCaMP6s Lentivirus Packaging and Concentration
			3.3 Generation of GCaMP6s-Expressing Cell Lines
			3.4 Validation of GCaMP-Expressing Cell Lines
			3.5 Virus Infection
			3.6 Epifluorescence Microscope Setup
		4 Notes
		References
	Chapter 4: Construction of a Mycoviral Infectious Clone for Reverse Genetics in Botrytis cinerea
		1 Introduction
		2 Materials
			2.1 Cultivation of B. cinerea
			2.2 RNA Extraction and Detection by Conventional RT-PCR
			2.3 RNA Genome Amplification by PCR and Cloning in pUC19 Vector
			2.4 Sequencing of the pUC19-BVF Infectious Clone
			2.5 Transfection of B. cinerea Using the BVF Infectious Clone
			2.6 General Equipment
		3 Methods
			3.1 Detection of BVF in Field Isolates
				3.1.1 Cultivation of B. cinerea
				3.1.2 RNA Total Extraction
				3.1.3 Viral Detection by Complementary DNA (cDNA) Synthesis Followed by Conventional PCR (See Notes 20 and 21)
			3.2 Construction of the BVF Infectious Clone
				3.2.1 Obtention of Full-Length cDNA Derived from BVF Genomic RNA
				3.2.2 Cloning of Amplified Fragments into pUC19 Vector (See Note 27)
				3.2.3 Sequencing of the pUC19-BVF Infectious Clone
			3.3 Transfection of the BVF Infectious Clone
				3.3.1 Preparation of BVF Template
				3.3.2 Preparation of BVF Transcripts
				3.3.3 Transfection of B. cinerea Protoplasts
				3.3.4 Generation of Fungal Single-Spore Infected Isolates
		4 Notes
		References
Part III: Plant-Pathogens Interactions
	Chapter 5: Exopolysaccharide Production and Precipitation Method as a Tool to Study Virulence Factors
		1 Introduction
		2 Materials
			2.1 Aaa Strains
			2.2 Bacterial Media
			2.3 Chemicals and Laboratory Equipment
		3 Methods
			3.1 Bacterial Growth
			3.2 EPS Precipitation
			3.3 Weight and Calculations
		4 Notes
		References
	Chapter 6: Virulence-Related Assays for Investigation of the Acidovorax citrulli-Cucurbitaceae Pathosystem
		1 Introduction
		2 Materials
			2.1 Bacterial Strains, Vectors, and Media
			2.2 Enzymes, Compounds, and Kits for Molecular Biology
			2.3 Plant Inoculation
			2.4 Assessment of Virulence Determinants
		3 Methods
			3.1 Marker-Free Gene Deletion Using the sacB-Containing Plasmid pK18mobsacB
			3.2 Plant Inoculation Assays
				3.2.1 Preparation of Bacterial Inoculum
				3.2.2 Seed-to-Seedling Transmission Assay
				3.2.3 Stem Inoculation Assay
				3.2.4 Foliage Inoculation Assay
			3.3 Assessment of Twitching Motility
			3.4 Biofilm Formation Assay
		4 Notes
		References
	Chapter 7: Dual-Fluorescence Chromosome-Located Labeling System for Accurate In Vivo Single-Cell Gene Expression Analysis in P...
		1 Introduction
		2 Materials
			2.1 Bacterial Growth
			2.2 Tetraparental Mating
			2.3 P. syringae Transformation
			2.4 Southern Blot Analysis
			2.5 Single-Cell Analysis
		3 Methods
			3.1 Generating a Labeled Strain Constitutively Expressing Fluorescent Proteins by Tetraparental Mating
				3.1.1 Bacterial Cultures
				3.1.2 Tetraparental Mating Preparation
				3.1.3 Recovering and Selection of Strains
			3.2 Generating Transcriptional Fusions to Target Genes by Introducing a Promoterless Fluorescent Reporter Gene into Selected S...
				3.2.1 P. syringae Electrocompetent Cell Preparation
				3.2.2 P. syringae Transformation by Electroporation
				3.2.3 Selection of Clones Carrying Transcriptional Fusions Without Chromosomal Plasmid Integration
			3.3 Validating Chromosomal Integrations by Southern Blot Analysis
			3.4 Single-Cell Analysis of T3SS Gene Expression in Pseudomonas syringae
				3.4.1 P. syringae Cultures in  HIM
				3.4.2 Flow Cytometry Analysis of the Strains
				3.4.3 Visualization by Confocal Microscopy of the Strains
			3.5 Analysis of the Strains
		4 Notes
		References
	Chapter 8: A Robust Method to Perform In Vitro and In Planta Interbacterial Competition Assays: Killing Plant Pathogens by a P...
		1 Introduction
		2 Materials
			2.1 General Reagents and Culture Media
			2.2 Vectors
			2.3 Bacterial Strains
			2.4 Plants
			2.5 Molecular Biology Reagents
		3 Methods
			3.1 Preparation of Antibiotic Resistance Strains
			3.2 Preparation of Samples for Interbacterial Competition
			3.3 Interbacterial Competition
				3.3.1 In Vitro Competition Assay
				3.3.2 In Planta Competition Assay
			3.4 Quantification and Analysis of Data
			3.5 Competitive Index Calculation
		4 Notes
		References
Part IV: Molecular Methods for Plant-Interacting Bacteria
	Chapter 9: Quantification of Mixed-Linkage β-Glucan (MLG) in Bacteria
		1 Introduction
		2 Materials
			2.1 Culture Media and Bacterial Strains
			2.2 Plate Format and Equipment
			2.3 Buffers, Solutions, Reagents, and Enzymes
		3 Methods
			3.1 Bacterial Growth Conditions
			3.2 Lichenase Digestion
			3.3 MLG Quantification
		4 Notes
		References
	Chapter 10: Surface Plasmon Resonance as a Tool to Elucidate the Molecular Determinants of Key Transcriptional Regulators Cont...
		1 Introduction
		2 Materials
			2.1 General Equipment and Materials
			2.2 Protein Expression
			2.3 Protein Purification
			2.4 Protein Quality and Quantity Determination
			2.5 Generation of Biotinylated Double-Stranded Promoter Regions
			2.6 Surface Plasmon Resonance with Biacore Technology (SPR)
		3 Methods
			3.1 Expression of Intein-Chitin-Tagged Proteins
				3.1.1 Transformation of Expression Plasmids into E. coli ER2566
				3.1.2 Optimization of Conditions for Maximum Expression and Solubility
			3.2 Purification of Intein-Chitin-Tagged Proteins
				3.2.1 Cells Production for Protein Overexpression
				3.2.2 Protein Purification
			3.3 Generation of Biotinylated Double-Stranded DNA Probes
			3.4 Biotinylated Promoter Immobilization
			3.5 DNA-Protein Binding Assay
			3.6 Data Analyses
		4 Notes
		References
	Chapter 11: Microscope Subcellular Localization of Plant-Interacting Bacterial Effectors in Animal Cell Cultures
		1 Introduction
		2 Materials
			2.1 Bacteria, Cell Handling, and Infection Conditions
			2.2 Protein Immunodetection
			2.3 Cell Transfection
			2.4 Cell Cycle Analysis
		3 Methods
			3.1 Detection of Effectors Overproduced and Released from Salmonella in HeLa Cells
				3.1.1 Infection of HeLa Cells and Effector Protein Induction
				3.1.2 Controlled Autolysis of Intracellular Bacteria
				3.1.3 Detection of Released Proteins by Immunofluorescence
			3.2 Detection of Effectors in Transfected HeLa Cells
				3.2.1 Transfection of HeLa Cells and Effector Protein Induction
				3.2.2 Cell Cycle Analysis in Flow Cytometer
		4 Notes
		References
	Chapter 12: A Workflow for the Functional Characterization of Noncoding RNAs in Legume Symbiotic Bacteria
		1 Introduction
		2 Materials
			2.1 Culture, Harvest of Bacteria, and Cell Lysis
			2.2 Total RNA Extraction
			2.3 Northern Blot Hybridization
			2.4 Rapid Amplification of the 5′/3′-cDNA Ends (RACE)
			2.5 Evaluation of Promoter Activity
			2.6 Identification of Proteins Regulating sRNA Transcription
			2.7 SDS-Page
			2.8 Construction of sRNA Mutants and Validation of mRNA Targets
			2.9 Bioinformatic Tools
		3 Methods
			3.1 Detection and Molecular Characterization of sRNAs
				3.1.1 Cell Growth and Isolation of Total  RNA
				3.1.2 Isolation of Total RNA from Nodules
				3.1.3 Northern Blot Analysis of sRNA Expression
				3.1.4 5′/3′-RACE
			3.2 Transcriptional Regulation of sRNAs
				3.2.1 Determination of Promoter Activity
				3.2.2 Bacteroid Isolation to Track sRNA Transcription in Planta
				3.2.3 Promoter Pull-Down Assays
			3.3 sRNA Function and Target Regulation
				3.3.1 Construction of S. meliloti sRNA Mutants
				3.3.2 Validation of Candidate Target mRNAs
		4 Notes
		References
	Chapter 13: Methods for Studying Swimming and Surface Motilities in Rhizobia
		1 Introduction
		2 Materials
			2.1 Preparation of Media
			2.2 Motility Assays
			2.3 Image Acquisition
			2.4 Sample Preparation for TEM
		3 Methods
			3.1 Swimming Motility Assays
			3.2 Surface Motility Assays
			3.3 Sample Preparation for Visualization of Bacterial Flagella by TEM
		4 Notes
		References
	Chapter 14: Isolation, Quantification, and Visualization of Extracellular Membrane Vesicles in Rhizobia Under Free-Living Cond...
		1 Introduction
		2 Materials
			2.1 Bacterial Strains and Culture Medium
			2.2 Centrifugation Process
			2.3 Filtration Process
			2.4 Ultrafiltration Process
			2.5 Ultracentrifugation Process
			2.6 Integrity Evaluation and Quantification Assays
		3 Methods
			3.1 EMVs Isolation
			3.2 Microscopy for EMVs Integrity Evaluation
			3.3 Lipid Dye for EMVs Quantification
			3.4 Nanoparticle Tracking Analysis for EMVs Quantification
		4 Notes
		References
	Chapter 15: Isolation of Rhizobial Extracellular Membrane Vesicles from Bacteroids
		1 Introduction
		2 Materials
			2.1 Inoculum Preparation
			2.2 Plant Inoculation Assay
			2.3 Nodule EMV Fraction Collection
			2.4 EMV Isolation and Concentration by Ultracentrifugation
		3 Methods
			3.1 Culture Preparation
			3.2 Plant Inoculation Assay
				3.2.1 Seed Germination
				3.2.2 Seed Inoculation and Nodule Harvesting
			3.3 Symbiosome Space Collection
			3.4 Isolation and Concentration of EMVs by Ultracentrifugation
			3.5 Integrity and Quantification of EMVs
		4 Notes
		References
	Chapter 16: Nod Factor Lipopolysaccharide Purification to Study Nitrogen-Fixing Bacteria Symbiosis with Legumes
		1 Introduction
		2 Materials
			2.1 Rhizobial Growth for NF Production
			2.2 Radiolabeling and Thin-Layer Chromatography
			2.3 NF Purification
		3 Methods
			3.1 Rhizobial Growth for NF Production
			3.2 Radiolabeling and Thin-Layer Chromatography
				3.2.1 Radiolabeling of NF
				3.2.2 NF Extraction
				3.2.3 Thin-Layer Chromatography
			3.3 NF Purification
				3.3.1 Generating a Large Volume of NF-Producing Rhizobia
				3.3.2 NF Extraction and Purification
		4 Notes
		References
	Chapter 17: A Novel System to Selective Tagging of Sinorhizobium fredii Symbiotic Plasmids
		1 Introduction
		2 Materials
			2.1 Bacterial Strains, Vectors, Media, and Antibiotics
			2.2 PCR Analysis
			2.3 Bi- or Triparental Mating
			2.4 Agarose Gel Components
			2.5 Symbiotic Plasmids Electrophoresis
		3 Methods
			3.1 Sinorhizobium Strain pSym´s Tagging by Biparental Mating
			3.2 Validation of pSym´s Tagging by PCR Analysis
			3.3 pSyms Transference from Tagged Strains to S. fredii Cured Strains
			3.4 Validation of pSyms Transference by PCR Analysis
			3.5 Preparation of Agarose Gel for in-well Cell Lysis
			3.6 Culture Preparation for pSyms Electrophoresis
		4 Notes
		References
	Chapter 18: New Inoculation Strategy for Legume Based on Rhizobium-Metabolite Co-encapsulation
		1 Introduction
		2 Materials
			2.1 Bacterial Strain
			2.2 Encapsulation
			2.3 Conservation
		3 Methods
			3.1 Culture Conditions
			3.2 Encapsulation Technique
			3.3 Cell Count in Alginate Beads
		4 Notes
		References
Index