Plant-Pathogen Interactions (Methods in Molecular Biology, 2659)

✍ Scribed by Nora A. Foroud (editor), Jonathan A. D. Neilson (editor)

• Publisher: Humana
• Year: 2023
• Tongue: English
• Leaves: 242
• Category: Library

✦ Synopsis

This detailed collection focuses on current methodologies for studying various crop diseases. The book explores phytopathogen detection, host-pathogen interactions at the earliest stages of colonization, RNA-seq analyses, as well as host responses downstream of, or coincidental to, gene expression and a newly established protocol for gene editing of Medicago sativa. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical, Plant-Pathogen Interactions serves as an ideal guide for researchers working in the vital area of plant diseases.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Specific Detection and Quantification of Major Fusarium spp. Associated with Cereal and Pulse Crops
1 Introduction
2 Materials
2.1 Media
2.2 DNA Extraction
2.3 PCR Amplification
2.4 PCR-RFLP
2.5 Gel Electrophoresis
2.6 qPCR Reagents
2.7 Software and Databases
3 Methods
3.1 Recovering Fusarium spp. from Different Plant Tissues
3.2 DNA Extraction
3.2.1 Extracting DNA from Pure Fusarium Cultures
3.2.2 Extracting DNA from Infected Plant Tissues
3.3 Detection Methods
3.3.1 PCR Amplification
3.3.2 Multiplex PCR
3.3.3 Gel Electrophoresis
3.3.4 PCR-RFLP
3.3.5 qPCR Quantification
3.4 Quantify Fusarium spp. in Cereal and/or Pulse Host Tissues
3.4.1 Master Mix Preparation
3.4.2 Cycling Conditions and Obtaining Results
4 Notes
References
Chapter 2: Distinguishing Puccinia striiformis f. sp. tritici Isolates Using Genomic Sequencing: A Case Study
1 Introduction
2 Materials
2.1 Plant Rearing and Rust Isolate Inoculation and Collection
2.2 DNA Extraction from Stripe Rust Spores and Sequencing
2.3 Software
3 Methods
3.1 Identification of Stripe Rust Isolates
3.2 Single Spore Isolation of Stripe Rust
3.3 Protocol for Spore Amplification
3.4 Determination of Virulence/Avirulence Pattern of Stripe Rust Isolates
3.5 DNA Isolation from Stripe Rust Spores and DNA Sequencing
3.6 Case Study
4 Notes
References
Chapter 3: DNA-Barcoding Identification of Plant Pathogens for Disease Diagnostics
1 Introduction
2 Materials
2.1 Sample Preparation and DNA Extraction
2.2 DNA Concentration and Quality Control
2.3 PCR Amplification
2.4 Agarose Gel Electrophoresis
3 Methods
3.1 Sample Preparation and DNA Extraction
3.2 Measurement of DNA Concentration and Quality Control
3.3 PCR Amplification of the ITS Barcode Region
3.4 Assessment of PCR Product Size and Quality by Agarose Gel Electrophoresis
3.5 Sequence Editing and Taxon Assignment to the DNA Barcode
4 Notes
References
Chapter 4: Real-Time Portable LAMP Assay for a Rapid Detection of Xylella fastidiosa In-Field
1 Introduction
2 Materials
2.1 Sample Collection
2.2 DNA Extraction from Plant
2.3 Real-Time Portable LAMP Amplification
3 Methods
3.1 Sample Collection
3.2 DNA Extraction
3.3 Real-Time Portable LAMP Amplification
4 Notes
References
Chapter 5: Selective Quantification of Chemotropic Responses of Fusarium graminearum
1 Introduction
2 Materials
2.1 Fungal Culturing and Macroconidia Preparation
2.2 Plant Growth Material
2.3 Chemotropism Plate Assay
2.4 Preparation of Chemical Stimuli
2.5 Counting Macroconidia and Measuring Hyphal Angles and Lengths Under Microscope
2.6 Installation of Software
3 Methods
3.1 Fungal Culture Conditions and Macroconidia Harvest
3.2 Wheat Growth Conditions
3.3 Quantitative Chemotropism Plate Assay
3.4 Preparation of ``Ligand´´ Containing Samples to Use as Test Compounds
3.5 Hyphal Angle Measurements
3.6 Hyphal Length Measurements
4 Notes
References
Chapter 6: Live-Cell Visualization of Early Stages of Root Colonization by the Vascular Wilt Pathogen Fusarium oxysporum
1 Introduction
2 Materials
2.1 Generation of a Fluorescent Protein-Encoding Construct for Fungal Transformation
2.2 Fungal Cultures
2.3 Screening of Fungal Transformants and Live-Cell Imaging
2.4 Software
3 Methods
3.1 Generation of Fo-mClover3 Expressing Transformants of F. oxysporum
3.2 Screening of the Obtained Fungal Transformants
3.3 Live-Cell Microscopy of Early Root Colonization Stages of F. oxysporum
4 Notes
References
Chapter 7: Transfection of Barley Leaf Protoplasts with a Fluorescently Tagged Fungal Effector for In Planta Localization Stud...
1 Introduction
2 Materials
2.1 Plant Growth
2.2 Protoplast Isolation
2.3 Protoplast Transfection and Microscopy
3 Methods
3.1 Preparing Plants for Protoplast Isolation
3.2 Isolation of Mesophyll Protoplasts from Barley Leaves
3.3 Transfection of Barley Protoplasts with DNA
3.4 Localization of Fluorescently Tagged Effectors
4 Notes
References
&spi1;Chapter 8: A Bioinformatic Guide to Identify Protein Effectors from Phytopathogens
1 Introduction
2 Identification of Putative Effector Proteins
3 Notes
References
Chapter 9: Unraveling Plant-Pathogen Interactions in Cereals Using RNA-seq
1 Introduction
2 Recent Advances in RNA Sequencing and Best Practices
2.1 Technologies for Gathering Transcriptome Data, Past and Present
2.2 RNA-seq Mapping and Assembly
2.2.1 RNA Isolation, Sequencing, and Data Filtering
2.2.2 Read Mapping, Gene Annotation, and Variant Detection
2.3 Analysis of Differentially Expressed Genes
2.4 Advanced Applications of RNA-seq in Co-expression Networks and Systems Biology
3 A Novel Example of Using GCNs to Connect Genes to Biological Pathways in F. graminearum
4 Conclusions and Future Directions
References
Chapter 10: RNA-Seq Data Processing in Plant-Pathogen Interaction System: A Case Study
1 Introduction
2 Materials
2.1 Datasets
2.2 Genome References and Corresponding Annotation Data
2.3 Computer Hardware
2.4 Software
3 Method
3.1 Mapping Strategies on Simulated Datasets
3.1.1 Generate Simulation Data
3.1.2 Strategy A: Perform Dual-Genome Alignment Using Simulated Datasets
3.1.3 Strategy B: Perform Sequential Alignments Using Simulated Datasets
3.1.4 Strategy C: Perform Sequential Alignment in Reverse Order of Strategy B Using Simulated Datasets
3.2 Mapping Strategies for the F. graminearum-Wheat Interaction Datasets
3.2.1 Preprocess RNA-Seq Reads
3.2.2 Obtain and Combine Wheat and F. graminearum Reference Genome and Annotation Files
3.3 Technical Insights
4 Notes
References
Chapter 11: Differential Expression Feature Extraction (DEFE): A Case Study in Wheat FHB RNA-Seq Data Analysis
1 Introduction
2 Materials
2.1 RNA-Seq Dataset
2.2 Computational Hardware
2.3 Computational Software
3 Methods
3.1 Obtaining the Raw Read Count Data
3.2 Perform Gene Differential Expression Analysis Using DESeg2
3.3 Perform DEFE Analyses
3.4 Perform Functional Group Analyses
3.4.1 To Identify Groups of Genes Associated with FHB Resistance
3.4.2 To Identify Groups of Genes Associated with FHB Susceptibility
3.5 Technical Insights
4 Notes
References
Chapter 12: Proteomic Profiling of Host Response in the Cereal Crop Triticum aestivum to the Mycotoxin, 15-Acetyldeoxynivaleno...
1 Introduction
2 Materials
2.1 Mock and Deoxynivalenol Inoculation Solutions
2.2 Plant Material
2.3 Proteome Extraction
2.4 Stop-and-Go Extraction (STAGE) Tip Desalting
2.5 TMT Labeling
2.6 TMT Desalting
2.7 LC-MS/MS Analysis
3 Methods
3.1 Inoculation and Sample Preparation of T. aestivum with Deoxynivalenol
3.2 Proteome Extraction and Digestion
3.3 Preparing C18 STAGE Tips and Desalting Samples
3.4 TMT Labeling
3.5 LC-MS/MS Analysis
3.6 Proteome Data Analysis
4 Notes
References
Chapter 13: Quantitative Phosphoproteome Analysis of the Interaction Between Fusarium graminearum and Triticum aestivum
1 Introduction
2 Materials
2.1 Media for Inoculation
2.2 Plant Materials
2.3 Inoculation of T. aestivum
2.4 Harvesting Materials
2.5 Proteome Extraction
2.6 Phosphopeptide Enrichment by Immobilized Affinity Chromatography (IMAC)
2.7 Stop-and-Go Extraction Tips (STAGE-Tip) Desalting
2.8 Mass Spectrometry
3 Methods
3.1 Culturing F. graminearum and Inoculating T. aestivum
3.2 Protein Extraction and Solubilization
3.3 Protein Quantification and Digestion
3.4 Phosphopeptide Enrichment by IMAC
3.5 Preparing the C18 Stop-and-Go Extraction Tips (STAGE-Tip)
3.6 Desalting Samples with Prepared STAGE-Tip
3.7 LC-MS/MS Analysis
3.8 Phosphoproteome (and Total Proteome) Data Analysis
4 Notes
References
Chapter 14: Fatty Acid Profiling of Grapevine Extracellular Compartment
1 Introduction
2 Materials
2.1 Plant Material and Apoplastic Fluid Isolation Buffer
2.2 Fatty Acid Profiling Reagents
2.3 Equipment and Consumables
3 Methods
3.1 Apoplastic Fluid (APF) Extraction
3.2 Fatty Acid Analysis Through Direct Methylation of APF and Leaf Tissue and Gas Chromatography
3.2.1 Preparation of Fatty Acid Methyl Esters (FAMEs) from Grapevine APF and Leaf Samples
3.2.2 Analysis of FAMEs by Gas Chromatography
3.3 Fatty Acid Analysis Through Direct Methylation and Gas Chromatography
3.4 Apoplast Fluid Purity Assessment Through Fatty Acid Profiling
4 Notes
References
Chapter 15: Identifying Fungal Secondary Metabolites and Their Role in Plant Pathogenesis
1 Introduction
2 Materials
2.1 Preparation of Knockout Constructs
2.1.1 Genomic DNA Extraction
2.1.2 Minipreparation of Plasmid DNA
2.1.3 Knockout Construct Preparation
2.2 Polyethylene Glycol (PEG)-Mediated Protoplast Transformation
2.3 Confirmation of Deletion Mutants
2.3.1 PCR Confirmation
2.3.2 Southern Blot Confirmation
2.4 Host Colonization Assays
2.4.1 Preparation of Fresh Spore Suspension
2.4.2 Colonization of Plant Species or Fruit and Vegetable Commodities
2.5 RNA Extraction and Sequencing Experiments
2.6 Comparative Metabolome Analyses
3 Methods
3.1 Knockout of Fungal Global Transcriptional Regulators
3.1.1 Design of Knockout Constructs
3.1.2 Genomic DNA Extraction
3.1.3 Minipreparation of Plasmid DNA
3.1.4 PCR-Mediated Generation of Gene Knockout Construct
3.1.5 PEG-Mediated Protoplast Transformation
3.1.6 Identification of Positive Transformants by PCR
3.1.7 Southern Blot Confirmation Screening
3.2 In Vivo Colonization Assays
3.2.1 Preparation of Fresh Spore Suspension
3.2.2 Colonization of Plant Species
3.2.3 Colonization of Fruit or Vegetable Commodities
3.3 Comparative Transcriptome Analysis: RNA Extraction from Infected Plant or Vegetable Materials
3.4 Identification of Biosynthetic Gene Targets for Knockout Assays
3.5 Comparative Metabolome Analyses
3.6 Isolation and Identification of Fungal Secondary Metabolites
3.7 In Vivo Colonization Assays
4 Notes
References
Chapter 16: Eliciting Targeted Mutations in Medicago sativa Using CRISPR/Cas9-Mediated Genome Editing: A Potential Tool for th...
1 Introduction
2 Materials
2.1 Construct Assembly
2.2 Agrobacterium-Mediated Transformation of Alfalfa Leaf and Petiole Explants
2.3 PCR Validation of Transgenic Genotypes
2.4 Identification of Edited Genotypes
2.4.1 T7E1 Assays
2.4.2 Droplet Digital PCR
2.4.3 Sanger Sequencing
3 Methods
3.1 Selection of Target Sequence and Guide RNA Design
3.2 Construct Assembly
3.3 Transformation of Agrobacterium tumefaciens
3.4 Agrobacterium-Mediated Transformation of Alfalfa Leaf/Petiole Explants
3.5 PCR Validation of Transgenic Genotypes
3.6 Identification of Edited Genotypes
3.6.1 T7E1 Assays
3.6.2 Droplet Digital PCR
3.6.3 Sanger Sequencing
4 Notes
References
Index