Plant Virology: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: In Vivo Detection of Double-Stranded RNA by dRBFC Assay
1 Introduction
2 Materials
2.1 Plant and Planting Materials
2.2 Plasmids, Enzymes, and Bacteria
2.3 Reagents
2.4 Equipment
3 Methods
3.1 Grow N. benthamiana Seedlings
3.2 Plasmid Propagation
3.3 A. tumefaciens Transformation
3.4 Virus Propagation
3.5 dsRNA Labeling
3.6 Confocal Microscopy
4 Notes
References
Chapter 2: Developing Rice Mutants Using CRISPR/Cas9-Based Genome Editing Technology
1 Introduction
2 Materials
2.1 Material for Constructing Vectors
2.2 Materials for Constructing Transgenic Rice
2.3 Instrumentation
2.4 Primers
3 Methods
3.1 Construction of CRISPR/Cas9 Vector
3.2 Construction of CRISPR Transgenic Rice
4 Notes
References
Chapter 3: Microscale Thermophoresis Assay: A Powerful Method to Quantify Protein-Nucleic Acid and Protein-Protein Interactions
1 Introduction
2 Materials
3 Methods
3.1 Labeling the Tested Protein Using Monolith Series Protein Labeling Kit RED-NHS Second Generation
3.2 Assay Setup Pretests
3.3 Assay Setup
3.4 Data Interpretation
4 Notes
References
Chapter 4: Transient Expression-Mediated Gene Silencing in Plants and Suppression of Gene Silencing with Viral Suppressors
1 Introduction
2 Materials
2.1 Plant Growth and Data Analysis
2.2 Construction of Transient Expression Vectors
3 Methods
3.1 Preparation of Plant Materials
3.2 Construction of the Transient Gene Expression Vector (Fig. 1a)
3.3 Agroinfiltration for Single-Stranded GFP (ssGFP) Induced Silencing
3.4 Agroinfiltration for Double-Stranded GFP (dsGFP) Induced Silencing
3.5 Observations and Photographs under UV Light
3.6 RNA Extraction, Northern Blot and siRNA Blot Analysis
4 Notes
References
Chapter 5: Isolation and Transfection of Plant Mesophyll Protoplasts for Virology Research
1 Introduction
2 Materials
2.1 Plant Material and Plasmid DNA
2.2 Equipment
2.3 Enzymes and Plasmid Kit
2.4 Reagent Set-up
3 Methods
3.1 Preparation of Plasmid DNA (TuMV cDNA Infectious Clone)
3.2 Protoplast Isolation
3.3 PEG-Mediated Transfection
3.4 Protoplast Harvest
4 Notes
References
Chapter 6: Split-Luciferase Complementation for Analysis of Virus-Host Protein Interactions
1 Introduction
2 Materials
2.1 Construction of Expression Vectors
2.2 Agrobacterium Culture and Infiltration
2.3 Luminescence Imaging
3 Methods
3.1 Construction of Binary Protein Expression Vector for Agroinfiltration
3.2 Agrobacterium-Mediated Transient expression in N. benthamiana Leaves
3.3 Measuring Luminescence with CCD Imaging System
4 Notes
References
Chapter 7: Monitoring Virus Intercellular Movement from Primary Infected Cells to Neighboring Cells in Plants
1 Introduction
2 Materials
2.1 Plasmids
2.2 Plant Material and Bacterial Strains
2.3 Equipment
2.4 Reagents
3 Methods
3.1 Construction of the Dual Fluorescent TuMV Vector pCBTuMV-GFP//mCherry
3.2 Mutagenesis for Generating TuMV Mutants
3.3 Introduction of Viral Constructs into Plant Epidermal Cells Through Agroinfiltration
3.4 Confocal Microscopy Analysis of Cell-to-Cell Movement of Wild-Type and Mutant TuMV
3.5 Analysis of Systemic Infection of Wild-Type and Mutant TuMV
4 Notes
References
Chapter 8: Simultaneous Determination and Subcellular Localization of Protein-Protein Interactions in Plant Cells Using Bimole...
1 Introduction
2 Materials
2.1 Plant Materials and Bacterial Strains
2.2 Instrumentations
2.3 Reagents
3 Methods
3.1 BiFC Vector Construction
3.2 Transient Coexpression of BiFC Vectors in N. benthamiana Leaves
3.3 Confocal Microscopy of BiFC Results
4 Notes
References
Chapter 9: A Powerful Method for Studying Protein-Protein Interactions in Plants: Coimmunoprecipitation (Co-IP) Assay
1 Introduction
2 Materials
2.1 Plant Material
2.2 Reagents
3 Methods
3.1 Vector Construction and Preparation of Plant Materials
3.2 Protein Extraction and Coimmunoprecipitation
4 Notes
References
Chapter 10: Membrane and Nuclear Yeast Two-Hybrid Systems
1 Introduction
2 Materials
2.1 Materials for Nuclear Yeast Two-Hybrid
2.2 Materials for Membrane Yeast Two-Hybrid
3 Methods
3.1 Methods for Nuclear Yeast Two-Hybrid
3.1.1 Construction of a Bait Clone Using One-Step PCR Cloning
3.1.2 Preparation of Yeast Competent Cells
3.1.3 Transformation of the CP Bait Construct into AH109 and Test for Self-Activation
3.1.4 cDNA Library Screening and the Selection of Interactors
3.1.5 Confirmation of Interactors
3.1.6 Identification of the Bait-Prey Interaction
3.2 Methods for Membrane Yeast Two-Hybrid
3.2.1 Bait Construction
3.2.2 Transform Yeast Competent Cells with Bait and Test for Self-Activation
3.2.3 cDNA Library Screening and Selection of Interactors
3.2.4 Confirmation of Interactors
3.2.5 Identification of Bait-Prey Interaction
4 Notes
References
Chapter 11: Tag-Based Pull-Down Assay
1 Introduction
2 Materials
2.1 Plant Material and Competent Cells
2.2 Instruments
2.3 Plasmids and Reagents
3 Methods
3.1 Plant Preparation
3.2 Expression Vector Construction and Expression Assessment
3.3 Transient Express Proteins in N. benthamiana Leaves
3.4 Protein Extraction
3.5 Pull-Down of the Target Protein Complex
3.6 Elution of the Protein Complex
3.7 Analysis of Eluted Proteins
4 Notes
References
Chapter 12: Purification of Plasmodesmata-Enriched Fraction for Proteomic Analyses
1 Introduction
2 Materials
2.1 Plant Material, Agrobacterial Strains, and Plasmids
2.2 Instrumentation
2.3 Reagents and Consumable Supplies
3 Methods
3.1 Cell Wall Purification
3.2 PD Preparation
3.3 Protein Quantification Assay
3.4 Purity Assessment
3.5 Subcellular Validation of Potential PD Candidates
4 Notes
References
Chapter 13: Purification and Proteomics Analysis of Phloem Tissues from Virus-Infected Plants
1 Introduction
2 Materials
2.1 Phloem Tissue/Protein Isolation
2.2 Peptide Precipitation and Lysis
2.3 Peptide Desalting
2.4 Mass Spectrometry Analysis
2.5 Data Analysis
3 Methods
3.1 Phloem Tissue Isolation
3.2 Phloem Protein Exudation (See Note 7)
3.3 Phloem Protein Extraction
3.4 Protein Precipitation and Digestion
3.5 Removal of Salts from Digested Samples
3.6 Mass Spectrometry of Digested Samples (See Note 16)
3.7 Data Conversion (See Note 18)
3.8 Data Analysis with MaxQuant (See Note 19)
3.9 Imputation of MaxQuant Results with Perseus (See Note 23)
4 Notes
References
Chapter 14: Rapid and Specific Purification of Argonaute-Small RNA Complexes from Rice for Slicer Activity
1 Introduction
2 Materials
2.1 Plant Material and Reagents
2.2 Equipment
3 Methods
3.1 Immunoprecipitation of Argonaute-small RNA Complex (see Note 1)
3.2 Argonaute Slicer Activity Test (Fig. 2, see Notes 2 and 3)
3.3 Small RNA Sequencing and Bioinformatic Analysis (Fig. 2)
3.4 Perl Script Used for Statistical Analysis of miRNA Expression
4 Notes
References
Chapter 15: Silencing Specific Genes in Plants Using Virus-Induced Gene Silencing (VIGS) Vectors
1 Introduction
2 Materials
2.1 Plants and Growth Conditions
2.2 Vector and Constructs
2.3 Cloning cDNA Fragment into pCMV201-2bN81
2.4 Agroinoculation of ZMBJ-CMV VIGS Vectors on N. benthamiana
2.5 Vascular Puncture Inoculation (VPI) of Maize Seeds
2.6 Quantitative RT-PCR
3 Methods
3.1 Select a Specific Fragment Using Software Tools
3.2 Cloning the Selected Fragment into VIGS Vector by Using Standard Molecular Cloning Procedure
3.3 Transformation of VIGS Constructs into Agrobacterium Tumefaciens
3.4 Agroinoculation of N. benthamiana Plants
3.5 Preparation of Crude Sap Containing Virions from Agroinoculated N. benthamiana Leaves
3.6 Inoculate Maize Seeds Through Vascular Puncture Inoculation (VPI) (See Note 16)
3.7 Observation Silencing Phenotypes and Validation Gene Silencing Using Real-Time Quantitative Reverse Transcription-PCR (qRT...
4 Notes
References
Chapter 16: A Versatile Expression Platform in Insects and Cereals Based on a Cytorhabdovirus
1 Introduction
2 Materials
2.1 Biological Materials
2.2 Insertion of Target Gene
2.3 Agroinfiltration
2.4 Crude Extraction
2.5 Insect-Injection and Transmission Assays
3 Methods
3.1 Insertion of Target Gene into BYSMV cDNA Infectious Clones
3.2 Coinfiltration into N. benthamiana Leaves
3.3 Crude Extraction
3.4 Insect Injection and Transmission Assays
4 Notes
References
Chapter 17: Production of Virus-Free Chrysanthemum (Chrysanthemum morifolium Ramat) by Tissue Culture Techniques
1 Introduction
2 Materials
2.1 Plant Material
2.2 Virus Detection Equipment and Materials
2.3 Tissue Culture Equipment and Materials
3 Methods
3.1 Virus Detection by Negative Staining TEM
3.2 Virus Detection by Thin Sectioning and Imaging TEM
3.3 Virus Detection by DAS-ELISA
3.4 RT-PCR Detection of Virus
3.5 Virus-Free Plantlet Production
3.5.1 Collection and Surface Sterilization of Explants
3.5.2 Culturing Aseptic Plantlets
3.5.3 Production of Virus-Free Plants
3.5.4 Propagation of Healthy Virus-Free Plants
3.5.5 Rooting
3.5.6 Acclimation
4 Notes
References
Chapter 18: Long-Term Preservation of Plant Viruses in Cryopreserved Shoot Tips
1 Introduction
2 Materials
2.1 General Equipment
2.2 Reagents
2.3 Media and Solutions
3 Methods
4 Notes
References
Chapter 19: Establishment of White-Backed Planthopper Cell Lines
1 Introduction
2 Materials
2.1 Equipment and Insect Eggs
2.2 Reagents
2.3 Heat-Treatment of the FBS Before Use
3 Methods
3.1 Establishment of Primary WBPH Cell Culture
3.2 Subculture of Primary Cell Cultures
3.3 Maintenance of Insect Cell Lines
3.4 Storage and Recovery of Insect Cell Lines
4 Notes
References
Chapter 20: Biolistic Inoculation of Fruit Trees with Full-Length Infectious cDNA Clones of RNA Viruses
1 Introduction
2 Materials
2.1 Plant Material
2.2 Construction of Full-Length PPV Infectious cDNA Clone
2.3 Biolistic Inoculation
3 Methods
3.1 Preparation of Peach Seedlings
3.2 Construction of a PPV Full-Length cDNA Infectious Clone
3.3 Biolistic Inoculation of Peach Seedlings
3.3.1 Coating Microcarriers with DNA
3.3.2 Cartridge Preparation with Tubing Prep Station
3.3.3 Plant Preparation Prior to Bombardment
3.3.4 Biolistic Bombardment
3.3.5 Plant Recovery Post Bombardment (Fig. 1)
3.4 Evaluate the Infectivity of Biolistic Bombardment
4 Notes
References
Chapter 21: Purification of Total RNAs and Small RNAs from Fruit Tree Leaf Tissues
1 Introduction
2 Materials
2.1 Materials for Total RNA Extraction
2.2 Materials for Small RNA Purification
3 Methods
3.1 Total RNAs Extraction Using a Modified TRIzol Method
3.2 Small RNA Purification Using mirVana miRNA Isolation Kit (Ambion/Life Technologies)
4 Notes
References
Chapter 22: Diagnosis of Viral Diseases Using Deep Sequencing and Metagenomics Analyses
1 Introduction
2 Materials
2.1 Plant Samples
2.2 RNA Extraction and Purification Suppliers
2.3 Ribosomal RNA Depletion Suppliers
2.4 Ethanol Precipitation Suppliers
2.5 RNA Cleanup Suppliers
2.6 Electrophoresis for RNA Integrity Evaluation Suppliers
2.7 Software for Bioinformatics Analysis
3 Methods
3.1 Total RNA Extraction
3.2 Small RNA (sRNA) Extraction by mirVana Kit
3.3 Ribosomal RNA Depletion
3.3.1 Hybridization Step
3.3.2 Preparing Beads
3.3.3 Removing rRNA
3.4 Concentration of rRNA-Free RNA Using Ethanol Precipitation
3.5 RNA Integrity Check and RNA Purification
3.6 Library Preparation and High-Throughput Sequencing
3.7 Data Quality Control by FastQC
3.8 Removal of Adapter Sequences
3.9 Merging Forward and Reverse Reads (for Total RNA Dataset Only)
3.10 De Novo Transcriptome Assembly from Total RNA-Seq
3.11 De Novo Transcriptome Assembly from sRNA-Seq
3.12 Viral Genome Identification and Taxonomic Assignment
3.13 Read Mapping
4 Notes
References
Chapter 23: Immunocapture-Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay for Detection of Plant RNA Viruses
1 Introduction
2 Materials
2.1 Plant Material and CTV Isolates
2.2 Immunocapture of Virus Particles (Virions)
2.3 IC-RT-LAMP Assay
3 Methods
3.1 Immunocapture of Viral Particles (Virions)
3.2 IC-RT-LAMP Assay
4 Notes
References
Chapter 24: Detection of Tomato Spotted Wilt Virus (TSWV) Infection in Plants Using DAS-ELISA and Dot-ELISA
1 Introduction
2 Materials
2.1 Plant Materials
2.2 DAS-ELISA Reagents and Equipment
2.3 Dot-ELISA Reagents and Equipment
3 Methods
3.1 Preparation of AP-MAb or AP-PAb Conjugate for DAS-ELISA
3.2 DAS-ELISA Procedure (Fig. 1)
3.3 Dot-ELISA Procedure (Fig. 2)
4 Notes
References
Chapter 25: Tube-Capture (TC) RT-PCR and Multiplex RT-PCR for Diagnosis and Characterization of Viruses in Fruit Trees
1 Introduction
2 Materials
2.1 Plant Material Preparation
2.2 Total RNA Extraction Using CTAB Method
2.3 RNA Extraction Using RNArose Reagent RNA/DNA Kit
2.4 Tube Capture
2.5 Reverse Transcription
2.6 Polymerase Chain Reaction (PCR)
2.7 One-Step Multiplex Reverse Transcription/PCR Amplification of Viral RNA
2.8 Electrophoretic Analysis
2.9 General Laboratory Equipment
3 Methods
3.1 Extraction of Total Nucleic Acids by CTAB
3.2 Extraction of Total Nucleic Acids by RNArose Reagent RNA/DNA Kit (See Note 8)
3.3 Reverse Transcription (RT)-PCR
3.3.1 Tube Capture
3.3.2 Total RNA as Template
3.3.3 First-Strand DNA (cDNA) Synthesis Using M-MLV
3.3.4 PCR Using Taq DNA Polymerase
3.4 One-Step RT-PCR Using SuperScript Ill Kit (See Note 18)
3.5 One-Step Multiplex RT-PCR (See Note 19)
3.6 Electrophoresis Analyses
3.6.1 Agarose Gel Electrophoresis Analysis
3.6.2 PAGE Electrophoresis Analysis (See Note 21)
4 Notes
References
Chapter 26: Detection of Cucumber green mottle mosaic virus (CGMMV) in Cucurbitaceous Crop Seeds by RT-PCR
1 Introduction
2 Materials and Equipment
2.1 Plant Materials
2.2 Materials and Equipment Needed for Total RNA Isolation from Seeds
2.3 Materials and Equipment Needed for One-Step RT-PCR
2.4 Materials and Equipment Needed for Two-Step RT-PCR
3 Methods
3.1 Isolation of Total RNA
3.2 Detection of CGMMV by One-Step RT-PCR and Electrophoresis Detection
3.3 Detection of CGMMV by Two-Step RT-PCR and Electrophoresis Detection
4 Notes
References
Chapter 27: RNA In Situ Hybridization of Detecting Cucumber Mosaic Virus in Shoots of Nicotiana benthamiana Plants
1 Introduction
2 Materials
2.1 Plant and Virus Materials
2.2 Synthesis and Hybridization Efficiency Detection of DIG-Labeled Probe
2.3 Fixation, Paraffin Embedding, and Sectioning of Plant Tissue
2.4 Section Pretreatment and Hybridization
2.5 Posthybridization and Detection
3 Methods
3.1 Plant Materials and Virus Inoculation
3.2 Transcriptional Labeling of RNA Probe
3.3 Checking the Hybridization Efficiency of DIG-Labeled Probe
3.4 Fixation, Embedding, and Sectioning of Plant Tissue
3.5 Prehybridization and Hybridization
3.6 Posthybridization and Detection of Samples
4 Notes
References
Chapter 28: Transmission Electron Microscopic Methods for Plant Virology
1 Introduction
2 Materials
2.1 Equipment
2.2 Supplies
2.3 Support Films
2.4 Reagents and Solutions
2.4.1 Sodium Phosphate Buffer (Sörensen)
2.4.2 Fixatives
2.4.3 Dehydration Solvents
2.4.4 Embedding Resin
2.4.5 Solution for Staining Ultrathin Sections
2.4.6 Solution for Staining Semithin Sections
2.4.7 Negative Stain Solution
3 Methods
3.1 Single Droplet Negative Staining TEM
3.1.1 Sample Preparation
3.1.2 Negative Staining Method I
3.1.3 Negative Staining Method II
3.2 Immuno-negative Staining TEM
3.2.1 Immunosorbent
3.2.2 Immunodecoration
3.2.3 Immunosorbent-Decoration
3.2.4 Analysis of Negative Stained Samples by TEM
3.3 Ultrathin Sectioning TEM
3.3.1 Sampling (Fig. 4)
3.3.2 Fixation
3.3.3 Dehydration
3.3.4 Infiltration and Embedding
3.3.5 Ultrathin Sectioning
3.3.6 Staining
3.3.7 Observation of Ultrathin Section
4 Notes
References
Index