Viroids: Methods and Protocols

Preface
Contents
Contributors
Part I: Biological Techniques
Chapter 1: Study and Detection of Citrus Viroids in Woody Hosts
1 Introduction
2 Materials
2.1 Greenhouse Indexing Facilities
2.2 Production of Indicator Plants
3 Methods
3.1 Experimental Design
3.2 Collection and Storage of Inoculum
3.3 Inoculation
3.4 Post-inoculation Care and Time for Symptom Expression
3.5 Symptom Expression
3.5.1 Symptom Expression of Viroids in `A-861-S-1´
3.5.2 Symptom Expression of HSVd Citrus Variants CVd-II in `Parson´s Special # 9´
3.5.3 Record Keeping
3.5.4 Disposal of Plant Materials
4 Notes
References
Chapter 2: Detection and Characterization of Viroids via Biological Assays on Herbaceous Hosts
1 Introduction
2 Materials
2.1 Materials for In Vitro Transcription and Inoculation
2.2 Agrobacterium Strain for Agroinoculation
2.3 Materials Required for Rub Inoculation
2.4 Materials Required for Grafting
2.5 Transformation of Agrobacteria and Agroinoculation
3 Methods
3.1 Plasmid DNA or Transcript Generation and Inoculation
3.2 Agroinfiltration of Viroid Gene Constructs
3.3 Mechanical Transmission from Sap
3.4 Grafting
3.5 Agroinfiltration of Viroid Gene Constructs
4 Notes
References
Chapter 3: Analysis on RNA Motif-Based RNA Trafficking in Plants
1 Introduction
2 Materials
2.1 Site-Directed Mutagenesis
2.2 Materials for DIG (Digoxigenin) Northern Blot
3 Methods
3.1 Generating Mutational Variants and Infection
3.2 Northern Blot Detection of Viroid Trafficking
4 Notes
References
Chapter 4: Isolation and Transfection of Citrus Protoplasts with Citrus Exocortis Viroid
1 Introduction
2 Materials
2.1 Special-Purpose Equipment
2.2 General-Purpose Equipment
2.3 Consumables
2.4 Source of Viroid RNA and Citrus Protoplast Material
2.5 Enzymes, Reagents, and Consumables
2.5.1 Suspension Cell Culture Media (H + H)
2.5.2 Stock Solution
2.5.3 Enzyme Solution
2.5.4 Stocks of CPW Salts (100x)
2.5.5 1 M MES, 1 M CaCl2, and 10 N KOH
2.5.6 25% SucroseM
2.5.7 13% MannitolM
2.5.8 0.6MMCPW
2.5.9 0.6MMCPWC
2.5.10 0.75MMCPWC
2.5.11 30% PEG-8000 Solution
2.5.12 Citrus Protoplast Culture Media (0.6MBH3)
3 Methods
3.1 In Vitro Transcription of CEVd RNAs
3.2 Suspension Cell Culture
3.3 Isolation of Citrus Protoplasts
3.3.1 Isolation of Citrus Protoplasts from Suspension Cell Culture
3.3.2 Isolation of Citrus Protoplasts from Callus Culture
3.3.3 Isolation of Citrus Protoplasts from Leaves
3.4 Cell Viability Count
3.5 Transfection of Citrus Protoplasts with In Vitro Transcripts of CEVd
3.6 Analysis of Replication and Population Profile of CEVd
4 Notes
References
Part II: Electrophoresis and Hybridization Techniques
Chapter 5: High-Throughput RNA Extraction from Citrus Tissues for the Detection of Viroids
1 Introduction
2 Materials
2.1 Tissue Preparation
2.2 Tissue Pulverization
2.3 RNA Extraction
3 Methods
3.1 Tissue Preparation
3.2 Tissue Pulverization
3.3 RNA Extraction and Purification
4 Notes
References
Chapter 6: Extraction and Purification of Viroids from Herbaceous Hosts
1 Introduction
2 Materials
2.1 RNA Isolation
2.2 Return Polyacrylamide Gel Electrophoresis (R-PAGE)
3 Methods
3.1 TRIzol or TRI Reagent Method
3.2 Enrichment for LiCl-Soluble Nucleic Acids (DNA, 4S RNA, dsRNAs, and Viroid RNAs) if Needed
3.3 Purification by Return Polyacrylamide Gel Electrophoresis (R-PAGE)
4 Notes
References
Additional Reading
Chapter 7: Detection of Viroids by sPAGE Gel Electrophoresis
1 Introduction
2 Materials
3 Methods
3.1 Cast the First (Non-denaturing) Gel
3.2 Load Samples and Begin Electrophoresis
3.3 Cast the Second (Denaturing/Urea-Containing) Gel
3.4 Apply Strip of the First Gel on the Second Gel and Begin Electrophoresis
3.5 Visualize Small Circular RNAs
4 Notes
References
Chapter 8: Two-Dimensional Polyacrylamide Gel Electrophoresis Analysis of Viroid RNAs
1 Introduction
2 Materials
2.1 Non-denaturing PAGE
2.2 Gel Staining
2.3 Denaturing PAGE Under High Ionic Strength
2.4 Denaturing PAGE Under Low Ionic Strength
2.5 RNA Blotting
2.6 Northern Hybridization
3 Methods
3.1 First Dimension PAGE in Non-denaturing Conditions
3.2 RNA Staining and Cutting Gel Lanes
3.3 First Dimension PAGE in Denaturing (High Ionic Strength) Conditions
3.4 Second Dimension PAGE in Denaturing (Low Ionic Strength) Conditions
3.5 Blotting the 2D-Separated RNA to a Membrane
3.6 Viroid RNA Hybridization
4 Notes
References
Chapter 9: A Simplified Dot-Blot Hybridization Protocol for Potato spindle tuber viroid Detection in Solanaceae
1 Introduction
2 Materials
3 Methods
3.1 Sample Preparation and Loading onto the Membrane, Nucleic Acid Fixing
3.2 Hybridization
3.3 Chemiluminescent Detection of Alkaline Phosphatase
3.4 Film Exposure and Development
4 Notes
References
Chapter 10: Gel Blot Hybridization for Viroids
1 Introduction
2 Materials
2.1 8 M Urea 5% Polyacrylamide 89 mM TBE Gel
2.2 Electroblotting
2.3 Hybridization
2.4 Membrane Treatment
3 Methods
3.1 Gel Electrophoresis
3.2 Electroblotting
3.3 Hybridization
3.4 Membrane Treatment
4 Notes
References
Chapter 11: Tissue Print Hybridization for Detection and Characterization of Viroids
1 Introduction
2 Materials
2.1 Protocol 1: Modification from
2.1.1 DIG-DNA Probe Synthesis and Purification
2.1.2 Printing Samples on Membrane
2.1.3 Prehybridization and Hybridization
2.1.4 Washes and Detection
2.2 Protocol 2: Modification from
2.2.1 DIG-RNA Probe Synthesis and Purification
2.2.2 Printing Samples on Membrane
2.2.3 Prehybridization and Hybridization
2.2.4 Washes and Detection
3 Methods
3.1 Protocol 1: Modification from
3.1.1 DIG-Labeled DNA Probe Synthesis and Purification
3.1.2 Printing Samples on Membrane
3.1.3 Prehybridization and Hybridization
3.1.4 Washes and Detection
3.2 Protocol 2: Modification from
3.2.1 DIG-Labeled RNA Probe Synthesis and Purification
3.2.2 Printing Samples on Membrane
3.2.3 Prehybridization and Hybridization
3.2.4 Washes and Detection
4 Notes
References
Chapter 12: Fluorescein-Based Electrophoretic Mobility Shift Assay
1 Introduction
2 Materials
2.1 Reagents for TFIIIA Purification
2.2 Reagents for EMSA
3 Methods
3.1 TFIIIA Purification
3.2 RNA Substrate Preparation
3.3 EMSA
4 Notes
References
Part III: PCR Techniques
Chapter 13: Detection of Viroids by RT-PCR
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 14: Detection of Viroids Using RT-qPCR
1 Introduction
2 Materials
2.1 Plant Material
2.2 RNA Isolation
2.2.1 TRIzol Method (Invitrogen Kit)
2.2.2 Commercial Kit (Spectrum Plant RNA Isolation Kit by Sigma-Aldrich)
2.3 Reverse Transcriptase-Quantitative Polymerase Chain Reaction (RT-qPCR)
3 Methods
3.1 Infectivity Assay
3.2 RNA Isolation (SeeNotes 1-4)
3.2.1 TRIzol Method (Invitrogen Kit Manual)
3.2.2 Commercial Kit (Spectrum Plant RNA Isolation Kit by Sigma-Aldrich)
3.3 Reverse Transcriptase-Quantitative Polymerase Chain Reaction (RT-qPCR)
3.3.1 First-Strand cDNA Synthesis
3.3.2 SYBR Green-Based qPCR
3.3.3 Analysis of RT-qPCR Data
4 Notes
References
Chapter 15: Multiplex RT-PCR
1 Introduction
2 Materials
2.1 Reverse Transcription Reaction
2.2 Polymerase Chain Reaction
2.3 One-Step-One-Tube Kit
2.4 Detection
2.5 Laboratory Equipment
3 Methods
3.1 Primer Design: Parameter for Multiplex RT-PCR
3.2 Sample Preparation (Tissue Selection, RNA Extraction, DNase)
3.3 Mixture Setup and Development of the Method
3.3.1 Mixture Setup
3.3.2 Cycling Condition
3.4 Preparation of the Assay
3.4.1 Two Steps-Two Tubes
3.4.2 One Step-One Tube
3.5 Discrimination Methods
3.6 Evaluation of the Performance
4 Notes
References
Chapter 16: Real-Time Detection of Viroids Using Singleplex and Multiplex Quantitative Polymerase Chain Reaction
1 Introduction
2 Materials
3 Methods
3.1 RT-qPCR Assay Design: Primers and qPCR Probes
3.2 Resuspension of Primers and qPCR Probes for RT-qPCR Assay Preparation
3.3 Singleplex RT-qPCR Procedure
3.4 Multiplex RT-qPCR Procedure
3.5 Validation of Singleplex and Multiplex RT-qPCR Assays
3.5.1 Specificity and Cross-Reactivity
3.5.2 Efficiency
3.5.3 Sensitivity and Viroid Load
3.5.4 Inter- and Intra-Assay Variation
3.6 Evaluation of Singleplex and Multiplex RT-qPCR Assays
3.6.1 Use with a Large Number of Diverse Host and Viroid Titer Samples
3.6.2 Robustness
3.6.3 Applicability, Practicability, and Transferability
3.7 RT-qPCR Swab Test and Decontamination Practices
4 Notes
References
Chapter 17: Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) at the Service of Viroid Detection
1 Introduction
2 Materials
2.1 RT-LAMP Reaction Components
2.1.1 2x LAMP Reaction Buffer
2.1.2 RT-LAMP Primers (Table 1)
2.1.3 Template
2.1.4 Enzymes (See Note 7)
2.1.5 Other Reagents
2.2 Detection
2.2.1 Endpoint Detection of RT-LAMP Products by Gel Electrophoresis
2.2.2 Endpoint Detection of RT-LAMP Products Using Fluorescent Metal Indicators (See Note 8)
2.2.3 Real-Time Detection
2.3 Specificity Test of RT-LAMP Products
2.4 Use of RT-LAMP in the Field (Fig. 2)
3 Methods
3.1 Primer Design
3.2 RT-LAMP
3.3 Detection
3.3.1 Endpoint Detection of RT-LAMP Products by Gel Electrophoresis
3.3.2 Endpoint Detection of RT-LAMP Products Using Fluorescent Metal Indicators
3.3.3 Real-Time Detection
3.4 Specificity Test of RT-LAMP Products
3.5 RT-LAMP in the Field
4 Notes
References
Chapter 18: SYBR Green RT-qPCR for the Universal Detection of Citrus Viroids
1 Introduction
2 Materials
3 Methods
3.1 RT-qPCR Preparation
3.2 Interpreting RT-qPCR Results
4 Notes
References
Chapter 19: Detection of Avocado Sunblotch and Other Viroids Using RNA Filter Paper Capture and RT-PCR
1 Introduction
2 Materials
3 Methods
3.1 Tissue Collection
3.1.1 Nursery Trees
3.1.2 Young Field Trees
3.1.3 Mature Field Trees
3.1.4 Fruit
3.2 Tissue Preparation
3.3 RNA Extraction
3.4 RT-PCR
3.5 Gel Electrophoresis
4 Notes
References
Part IV: Cloning and Sequencing Techniques
Chapter 20: Cloning and Sequencing of Viroids
1 Introduction
2 Materials
2.1 cDNA Cloning of Viroid RNAs Using Random Primers
2.2 cDNA Cloning of Viroid RNAs Using Specific Primers and PCR Amplification
3 Methods
3.1 cDNA Cloning of Viroid RNAs Using Random Primers
3.2 cDNA Cloning of Viroid RNAs Using Specific Primers and PCR Amplification
3.3 Cloning of cDNA Products Produced in Subheadings 3.1 and 3.2 for Sequence Analysis
4 Notes
References
Chapter 21: QuantiGene Plex Assay: A Method for High-Throughput Multiplex Citrus Viroid Detection and Identification
1 Introduction
2 Materials
3 Methods
3.1 Target Detection with Luminex MAGPIX Instrument
4 Notes
References
Chapter 22: Culture-Independent Discovery of Viroids by Deep Sequencing and Computational Algorithms
1 Introduction
2 Materials
2.1 Sample, Reagents, and Equipment for RNA Extraction and Purification
2.2 Hardware
2.3 Software
2.4 Data Source
3 Methods
3.1 RNA Extraction and Purification
3.1.1 Grapevine
3.1.2 Potato
3.2 Enrichment of Viroid and Viroid-Specific Small RNAs
3.3 DNase 1 Treatment
3.4 Two-Dimensional PAGE (2D-PAGE)
3.5 Northern Blot Hybridization
3.6 RNA Cleanup
3.6.1 RNeasy MinElute Cleanup Kit
3.6.2 Removal of Ribosomal RNA by Ribo-Zero rRNA Removal Kit
Magnetic Bead Preparation
RNA Sample Preparation/Hybridization of Probes
Concentration of rRNA-Free RNA Using Ethanol Precipitation
3.7 Circular RNA Enrichment
3.8 Small RNA and RNA-seq Library Construction
3.8.1 First-Strand cDNA Synthesis
3.8.2 Second-Strand cDNA Synthesis
3.8.3 Fragment Selection
3.8.4 PCR Amplification
3.8.5 Library Purification and Sequencing
3.9 Quality Control and Adaptor Trimming and Mapping
3.10 Working of PFOR
3.11 Evaluation of PFOR2
3.12 Homology Search for PFOR/PFOR2 Assembled Contigs
3.13 Validation of PFOR/PFOR2 Assembled Contigs
4 Notes
References
Chapter 23: An In Silico Detection of a Citrus Viroid from Raw High-Throughput Sequencing Data
1 Introduction
2 Materials
3 Methods
3.1 Tissue Preparation and RNA Extraction
3.2 Library Preparation (from Oxford Nanopore)
3.3 Priming and Loading cDNA on the MinION Flow Cell R9.4.1
3.4 Data Processing and Analysis with MiFi
4 Notes
References
Part V: RNA Silencing Techniques
Chapter 24: Detection of Viroid RNA and vd-siRNA in N. benthamiana Plants: Northern Blot Analyses for Viroid and vd-siRNAs
1 Introduction
2 Materials
2.1 Agroinfiltration
2.1.1 Material-Equipment
2.1.2 Solutions
2.2 RNA Extraction
2.2.1 Material-Equipment
2.2.2 Solutions
2.3 Formaldehyde Denaturing Agarose Gel
2.3.1 Material-Equipment
2.3.2 Solutions
2.4 DIG Labeling
2.4.1 Material-Equipment
2.4.2 Solutions
2.5 Polyacrylamide Gel
2.5.1 Material-Equipment
2.5.2 Buffers
2.6 Solutions for Radioactive Work
2.6.1 Material-Equipment
2.6.2 Solutions
3 Methods
3.1 Agroinfiltration
3.1.1 Agrobacteria Growth
3.1.2 Preparation of Agrobacteria for Inoculation
3.1.3 Infiltration
3.2 RNA Extraction
3.2.1 General Recommendation When Working with RNA
3.2.2 Tissue Collection
3.2.3 Tissue Homogenization
3.2.4 Phase Separation
3.2.5 RNA Precipitation
3.2.6 Total (Large) RNA Precipitation
3.2.7 Total RNA Precipitation with Small RNA Enrichment
3.2.8 RNA Washing
3.2.9 RNA Drying and Resuspension
3.2.10 Spectrometric Analysis of RNA
3.3 Northern Blot for PSTVd
3.3.1 Denaturating Formaldehyde Agarose/Gel (1.4%)
3.3.2 Prepare Samples
3.3.3 Capillary Transfer of RNAs
3.3.4 Prepare the Gel
3.3.5 Preparation of the Bridge
3.3.6 Transfer Mounting
3.3.7 Membrane Staining
3.3.8 Prepare Viroid Probe for DIG labeling
3.3.9 DIG Protocol for Viroids
3.3.10 Anticipated Results
3.4 Northern Blot for vd-siRNAs of PSTVd
3.4.1 17% Polyacrylamide Gel
3.4.2 Prepare Samples
3.4.3 Semidry Transfer of RNAs
3.4.4 Preparation of the Gel
3.4.5 Mounting for Semidry Transfer
3.4.6 Membrane Staining
3.4.7 Prepare Viroid Template for Radioactive Probe
3.4.8 Radioactivity to Detect vd-siRNAs
3.4.9 Anticipated Results
3.4.10 Stripping of the Membrane
4 Notes
References
Part VI: Splicing Techniques
Chapter 25: Production of Recombinant RNA in Escherichia coli Using Eggplant Latent Viroid as a Scaffold
1 Introduction
2 Materials
2.1 Bacterial Strains
2.2 Culture Media
2.3 Plasmids
2.4 Oligonucleotides
2.5 Enzymes
2.6 Recombinant RNA Extraction Purification
3 Methods
3.1 Plasmid Construction
3.2 Transformation of the E. coli Expression Strain
3.3 Analysis of Recombinant RNA Production
3.4 Large-Scale Production of Recombinant RNA
3.5 Extraction of Recombinant RNA
3.6 Chromatographic Purification of Recombinant RNA
3.7 2D-PAGE Separation of the Circular Form of the Recombinant RNA
3.8 Elution of the Circular Form of the Recombinant RNA from the Polyacrylamide Gel
4 Notes
References
Part VII: Structural Analysis
Chapter 26: Predicting the Structure of a Viroid
1 Introduction
2 Materials
2.1 Installation and Web Usage
3 Methods
3.1 The Structure of Minimum Free Energy Is One of the Myriad of Structures from the Structure Distribution
3.1.1 mfe Structure
3.1.2 Partition Folding
3.2 The Consensus Structure
3.2.1 Sequence Alignment
3.2.2 RNAalifold
3.2.3 ConStruct
3.3 Drawing
3.3.1 Sequence and Structure Logos
3.3.2 Sequence Logo
3.3.3 Structure Logo
3.3.4 RNAplot
3.3.5 VARNA
3.3.6 R2R
References
Index