Preface
Contents
Contributors
Chapter 1: Recent Advances in Plant Gene Silencing Methods
1 Introduction
2 Posttranscriptional and Transcriptional Gene Silencing
2.1 Components and Mechanism of Posttranscriptional Gene Silencing (PTGS)
2.1.1 siRNAs
Endogenous siRNA
siRNAs Induced by Foreign DNA
2.1.2 miRNA
2.2 Components and Mechanisms of Transcriptional Gene Silencing (TGS)
3 Methods of Gene Silencing
3.1 Posttranscriptional Gene Silencing (PTGS) Based Approaches
3.1.1 Hairpin RNAi
3.1.2 Virus-Induced Gene Silencing (VIGS)
3.1.3 MicroRNA-induced Gene Silencing (MIGS)
3.1.4 Artificial microRNAs
3.1.5 VIGS Using Artificial miRNAs (MIR-VIGS)
3.1.6 Host-induced Gene Silencing (HIGS)
3.1.7 Other Methods
3.2 Transcriptional Gene Silencing Methods
4 Limitations of the Gene Silencing Methods
4.1 Gene Editing Using CRISPR-Cas
5 Conclusions and Future Perspectives
References
Chapter 2: Strategies for Efficient RNAi-Based Gene Silencing of Viral Genes for Disease Resistance in Plants
1 Introduction
2 Antiviral Plant Defence Responses
3 Engineering Based on Pathogen-Derived Resistance
3.1 Engineering RNAi-Based Virus Resistance
4 Designing Effective Antiviral hp-RNAi Construct
4.1 Selection of Suitable Target Viral Sequence(s)
4.1.1 RNA Virus Genes
4.1.2 DNA Virus Genes
4.2 Size of Target Gene Sequences
4.3 Selection of Conserved Gene Sequences
4.4 Selection of Viral Genomic Regions Producing the Desired Level of siRNAs
4.5 Identification of Viral Target Region Producing Antiviral Effective siRNAs
5 Approaches for Reducing Off-Target Silencing
6 Conclusion
References
Chapter 3: Genome Editing and Designer Crops for the Future
1 Introduction
2 Genome Editing Approaches in Plants and Their Application in Crop Improvement
2.1 Meganucleases (MN)
2.2 Zinc-Finger Nuclease (ZFN)
2.3 Transcription Activator-Like Effector Nucleases (TALENs)
2.4 Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and CRISPR-Associated Protein 9 (Cas9)
2.5 CRISPR/Nuclease Dead Cas9 (dCas9)
3 Cis-Regulatory Elements Mediated Genome Editing: A Breakthrough
4 Comparing the Transgene-Based and Genome Editing Approaches
5 Applications and Prospects of Genome Editing and Bottlenecks
6 Conclusions
References
Chapter 4: In Silico Methods for the Identification of Viral-Derived Small Interfering RNAs (vsiRNAs) and Their Application in...
1 Introduction
1.1 What Are Virus-Derived Small Interfering RNAs?
1.2 Why Study vsiRNAs?
1.3 Biogenesis of vsiRNAs
1.4 vsiRNAs Influence on the Host Plant
2 Current Status of vsiRNAs Study in Plants and Current Databases
2.1 Role of Bioinformatics in the Detection of vsiRNAs
2.2 In Silico Methods for vsiRNAs Detection
2.2.1 Raw sRNA Read Generation
2.2.2 Read Preparation
2.2.3 Read Alignment
2.2.4 Database Searches Using Software Tools
2.2.5 Virus-Related Databases
2.2.6 Align sRNA Reads to Contigs and Uses to Detect Known and Novel Viruses
2.2.7 Putting It Together: Pipelines
3 Conclusion
3.1 Challenges
3.2 Limitations
3.3 Future Perspectives
References
Chapter 5: Barley Stripe Mosaic Virus (BSMV)-Based Virus-Induced Gene Silencing to Functionally Characterize Genes in Wheat an...
1 Introduction
2 Materials
2.1 Multiplication of BSMV-Based VIGS Plasmids
2.2 Construction of Gene-Specific VIGS Plasmid
2.3 Preparation of BSMV In Vitro Transcription Reactions
2.4 Plant Inoculation
3 Methods
3.1 Multiplication of BSMV Plasmids pΞ±, pΞ²ΞΞ²a, and pΞ³.MCS
3.2 Construction of Gene-of-Interest Plasmid (pΞ³GOI)
3.3 Growing Plants
3.4 Preparation of In Vitro Transcripts
3.5 Plant Inoculation with Viral Transcripts
4 Notes
References
Chapter 6: Virus-Induced Gene Silencing in Wheat and Related Monocot Species
1 Introduction
2 Materials
2.1 Plants and Plant Growth Materials
2.2 BSMV VIGS Construct Development
2.3 Transformation of BSMV VIGS Vectors into A. tumefaciens
2.4 Agrobacterium-Mediated Inoculation of N. benthamiana Plants
2.5 Mechanical (Rub)Inoculation of Monocot Plants
3 Methods
3.1 BSMV VIGS Vector Construction
3.2 Preparing A. tumefaciens Strains for Agroinfiltration
3.3 Agroinfiltration of N. benthamiana Leaves
3.4 Wheat Inoculation
4 Notes
References
Chapter 7: Virus-Induced Gene Silencing in Sorghum Using Brome Mosaic Virus
1 Introduction
2 Materials
2.1 Plant Growth Materials
2.2 Construct Preparation
2.3 Inoculation Preparation
3 Methods
3.1 Primer Design
3.2 Construct Preparation
3.3 Plant Germination and Growth
3.4 Virus Inoculum and Infection Confirmation
4 Notes
References
Chapter 8: RTBV-Based VIGS Vector for Functional Genomics in Rice: Methodology, Advances, Challenges, and Future Implications
1 Introduction
2 Materials
2.1 PCR Amplification of the Target Gene
2.2 Cloning the PCR-Amplified Target Gene into the pRTBV MVIGS Vector
2.3 Transformation of Recombinant VIGS Vector into A. tumefaciens
2.4 Seed Germination and Growth Conditions
2.5 Growth of A. tumefaciens Containing Recombinant VIGS Vector
2.6 Syringe Inoculation of A. tumefaciens Suspension in Rice Plants
2.7 Evaluation of VIGS-Mediated Silencing in Agroinoculated Plants
3 Methods
3.1 Cloning the Target Gene in the TA Vector
3.1.1 Sequence Analysis and Designing of Primers
3.1.2 RNA Isolation
3.1.3 cDNA Synthesis
3.1.4 A-Tailing of PCR-Amplified Product
3.1.5 Screening of Recombinant Clone
3.1.6 Cloning and Screening in the VIGS Vector
3.2 Transformation of Recombinant VIGS Vector into A. tumefaciens
3.2.1 Preparation of Chemical-Competent A. tumefaciens Cells
3.2.2 Transformation of Recombinant VIGS Vector to A. tumefaciens
3.3 Agrobacterium-Mediated Inoculation in Rice Plants
3.3.1 Rice Plant Growth
3.3.2 Preparation of A. tumefaciens Suspension for Inoculation
3.4 Preparation for Agroinoculation
3.4.1 Syringe Inoculation of A. tumefaciens Suspension in Rice
3.5 Observation and Validation of Silencing Phenotype Using Real-Time PCR
4 Notes
References
Chapter 9: Optimization of Tobacco Rattle Virus (TRV)-Based Virus-Induced Gene Silencing (VIGS) in Tomato
1 Introduction
2 Materials
2.1 Transformation of Recombinant VIGS Vector into Competent A. tumefaciens
2.1.1 Preparation of Competent Agrobacterium Cells (Chemical CaCl2 Method)
2.1.2 Transformation of Recombinant TRV Clones in A. tumefaciens
2.2 Agroinfiltration in Tomato Plants
2.2.1 Making Tomato Plants Ready for Infiltration
2.2.2 Preparation of Agroinoculum for Infiltration
2.2.3 Agroinfiltration in Tomato Plants
2.3 Confirmation of Gene Silencing
2.3.1 Collection of Leaf Samples
2.3.2 RNA Isolation
2.3.3 cDNA Synthesis
2.3.4 Semiquantitative RT-PCR (sqRT-PCR)
2.3.5 Measuring the Efficiency of Gene Silencing Via Quantitative Real Time RT-PCR (qRT-PCR)
3 Methods
3.1 Cloning of Target Gene in TRV-Based VIGS Vector
3.2 Transformation of Recombinant VIGS Vector into Competent A. tumefaciens Strain GV3110
3.2.1 Preparation of Competent A. tumefaciens Cells (Chemical CaCl2 Method)
3.2.2 Transformation of Recombinant TRV Clones in Agrobacterium
3.3 Agroinfiltration in Tomato Plants
3.3.1 Making Tomato Plants Ready for Infiltration
3.3.2 Preparation of Agroinoculum for Infiltration
3.3.3 Agroinfiltration of Tomato Plants
3.4 Confirmation of Gene Silencing
3.4.1 Phenotyping
3.4.2 Validation of Gene Silencing
Collection of Leaf Samples
RNA Isolation
cDNA Synthesis
sqRT-PCR
3.4.3 Measuring the Efficiency of Gene Silencing Via Quantitative Real Time RT-PCR (qRT-PCR)
4 Notes
References
Chapter 10: Virus-Induced Gene Silencing for Functional Genomics of Specialized Metabolism in Medicinal Plants
1 Introduction
2 Materials
2.1 PCR Amplification and Cloning
2.2 Transformation of Cloned Constructs
2.3 Growing of Plants
2.4 Agroinfiltration
2.5 Evaluation of VIGS
3 Methods
3.1 Cloning the Gene of Interest (GOI) into pTRV2 Vector
3.2 Transformation of Recombinant VIGS Vector into A. tumefaciens (GV3101) Competent Cells
3.2.1 Preparation of A. tumefaciens Competent Cells
3.2.2 Transformation of pTRV Plasmids and pTRV-Derived Constructs into A. tumefaciens
3.3 Virus-Induced Gene Silencing
3.3.1 Seed Germination and Preparation of Plant Material for VIGS
3.3.2 Preparation of A. tumefaciens Suspension for Inoculation
3.3.3 Agroinfiltration of C. roseus, R. serpentina, C. gigantea, and O. basilicum
3.3.4 Isolation of RNA from VIGS Leaves for Evaluation of Gene Silencing
3.3.5 cDNA Synthesis and Semi or Quantitative Reverse Transcriptase-PCR (qRT-PCR) Analysis
4 Notes
References
Chapter 11: VIGS-Based Gene Silencing for Assessing Mineral Nutrient Acquisition
1 Introduction
2 Materials
2.1 Plant Material and Growth Conditions
2.2 Vectors
2.3 PCR Amplification
2.4 Cloning of VIGS-Fragment
2.5 Mobilization of VIGS Vectors
2.6 Initiation of Agrobacterium Cultures Containing Recombinant VIGS Vectors
2.7 Agroinfiltration of pTRV2 VIGS Vectors and Plant Growth Conditions
2.8 Evaluation of VIGS-Mediated Silencing by PCR and Quantitative Real-Time PCR
2.9 Pi Estimation
3 Methods
3.1 Identification of Suitable Gene Fragment for VIGS
3.2 Cloning of the VIGS-Fragment in the pTRV2 VIGS Vector
3.3 Transformation of VIGS Vectors into Agrobacterium tumefaciens (GV3101) Competent Cells
3.4 Seed Germination
3.5 Preparation of Agrobacterium Cultures for Infiltration
3.6 Screening of VIGS Plants Either by Observation or by PCR Analysis Using TRV Coat Protein-Specific Primers
3.7 Validation of Downregulation of Target Gene Transcript by Quantitative Real-Time PCR
3.8 Determination of Total Soluble Pi Content in the Silenced Plants
4 Notes
References
Chapter 12: High-Throughput Analysis of Gene Function under Multiple Abiotic Stresses Using Leaf Disks from Silenced Plants
1 Introduction
2 Materials
2.1 Plants and Growth Conditions
2.2 Plasmids, Cloning, and Agrobacterium Strain
2.3 Infiltration Components
2.4 Stress Treatments
3 Methods
3.1 Preparation of Plant Material
3.2 Vector Construction
3.3 Preparation of Agrobacterium for Infiltration
3.4 Infiltration of N. benthamiana Seedlings with Agrobacterium
3.5 Stress Treatments and Stress Effect Quantification
3.5.1 Leaf Disk-Based Assays
3.5.2 Detached Leaf Assay for Drought Avoidance
4 Notes
References
Chapter 13: A Method for Developing RNAi-Derived Resistance in Cowpea Against Geminiviruses
1 Introduction
2 Materials
2.1 Plant Material
2.2 Agrobacterium tumefaciens Strain and Vector
2.3 Stock Solutions
2.3.1 MES Buffer for Agroinfiltration
2.3.2 Media Components
2.3.3 Phytohormone Stocks
2.3.4 Antibiotic Stocks
2.3.5 Other Solutions
2.4 Culture Media
2.4.1 For Agrobacterium tumefaciens
2.4.2 For Cowpea Transformation
3 Methods
3.1 Collection of Symptomatic Virus-Infected Plant Materials and Extraction of Genomic DNA
3.2 Rolling Circle Amplification
3.3 Cloning of Viral DNA Components
3.4 Identification of Cowpea Isolates of Begomoviruses
3.5 Construction of Agroinfectious Dimeric Clones
3.6 Mobilization of Dimeric Clones to Agrobacterium tumefaciens
3.7 Method of Inoculation of Infectious Dimeric Clones into Cowpea
3.7.1 Sap Inoculation
3.7.2 Agroinfiltration of Infectious Dimers
3.7.3 Insect Bioassay
3.8 RNAi in Plants
3.8.1 Construction of RNAi Vectors
3.9 Generation of hpRNA Cowpea Transgenic Lines
3.9.1 Seed Sterilization and Germination
3.9.2 Preparation of Agrobacterium Culture and Inoculation of Explants
3.9.3 Cocultivation, Selection, and Regeneration
3.9.4 Rooting and Hardening of Transformed Plants
3.10 Molecular Characterization of Putative RNAi Plants
3.10.1 PCR and Southern Blotting Analysis
3.10.2 siRNA Accumulation Analysis in Single-Copy Transgenic Cowpea Plants
3.10.3 RT-PCR Analysis of Transgenic Cowpea Plants
3.11 Transgenic Plant Inoculation with MYMIV and Symptom Analysis
3.12 Detection of Viral DNA in Transgenic Cowpea Plants
3.13 Rolling Circle Amplification (RCA) for Detection of Viral DNA Components
4 Notes
References
Chapter 14: In Vitro Method for Synthesis of Large-Scale dsRNA Molecule as a Novel Plant Protection Strategy
1 Introduction
2 Materials
2.1 In Vitro dsRNA Synthesis
2.1.1 PCR-Based Method
2.1.2 Traditional Cloning Method for the Development of Multi-gene Target dsRNA Constructs and Large Scale Synthesis of dsRNA ...
2.1.3 Bioassay of dsRNA and dsRNA Nano Clay Formulation Against Pest and Pathogens
3 Methods
3.1 In Vitro dsRNA Synthesis: One-Step PCR Method
3.2 Method II: Traditional Cloning Method of Multi-gene Target dsRNA Construct
3.2.1 In Vivo dsRNA Synthesis: Production of dsRNA Using T7 Express Cells
3.2.2 Use of Nano Clay Particle and Development of dsRNA Formulation
3.2.3 Bioassay of dsRNA Against Target Pest Pathogens
3.2.4 In Vitro Bioassay of dsRNA Against Pest and Pathogens: Case Study Potato Phytophthora infestans and Potato Cyst Nematode
In the Case of Fungal Assay
dsRNA Assay Using Detached Leaf Against Phytophthora infestans
In the Case of Nematode Assay
Exogenous Application of Nano Clay dsRNA Formulation Assay: Case Study Against Potato Phytophthora infestans and Potato Cyst N...
In the Case of Nematode, Soil Drenching of dsRNA Formulation
In the Case of Viral Disease
4 Notes
References
Chapter 15: Fine-Tuning Plant Gene Expression with Synthetic Trans-Acting Small Interfering RNAs
1 Introduction
2 Materials
2.1 Syn-tasiRNA Design
2.2 Syn-tasiRNA Cloning
3 Methods
3.1 Generation of Syn-tasiRNA Constructs
3.1.1 Syn-tasiRNA Design
3.1.2 Oligonucleotides for Syn-tasiRNA Cloning
3.1.3 Syn-tasiRNA Cloning
3.2 Generation of Syn-tasiRNA Constructs for Fine-Tuning Plant Gene Expression
3.2.1 Expression of a Single Syn-tasiRNA from Different Precursor Positions
3.2.2 Expression of Syn-tasiRNAs that Base-Pair with Target RNAs to Different Degrees
4 Notes
References
Chapter 16: Trans-Kingdom RNA Silencing in Plant-Fungal Disease Control
1 Introduction
2 Materials
2.1 Cotton Infection
2.2 Fungal Recovery
2.3 sRNA Gel Blotting
3 Methods
3.1 Cotton Infection
3.2 Fungal Recovery
3.3 sRNA Gel Blotting
4 Notes
References
Chapter 17: An Integrated Bioinformatics and Functional Approach for miRNA Validation
1 Introduction
2 Materials
2.1 Small RNA NGS Dataset Analysis and miRNA Identification Using miR-PREFeR Tool
2.2 Homology-Based In Silico Identification of miRNA169 Family Members in Tomato (a Case Study)
2.3 Validation of Predicted Tomato MIR169 Loci
2.3.1 Validating the Putative MIR Loci by Small RNA Data Support
2.3.2 Validation of Precursor by Cloning
Preparation of cDNA
Cloning and Sequencing
2.3.3 qRT-PCR Based Validation of miRNAs Precursors
2.4 Expression Analysis of miRNAs
2.4.1 Isolation of Small RNAs by Lithium Chloride Precipitation
2.4.2 Poly A-Tailing of the Small RNA
2.4.3 Preparation of cDNA from Small RNA
2.4.4 TaqMan qRT-PCR Based Expression Analysis of miRNAs
2.5 miRNA Target Identification Using Degradome Datasets Following CleaveLand Tool
2.6 Validation of miRNA-Mediated Target Cleavage:
2.6.1 5β² RLM-RACE:
2.6.2 Transient Assays for Target Cleavage
2.7 Functional Delineation of miRNA:Target Pair in Planta
2.7.1 Short Tandem Target Mimic (STTM):
2.7.2 Resistant Target
3 Methods
3.1 Small RNA Dataset Analysis for miRNA Identification Using miR-PREFeR Tool
3.1.1 Adapter Trimming
3.1.2 miR-PREFeR Analysis
Testing miR-PREfeR
Running miR-PREFeR on your Datasets
Pre-processing to Convert Uncollapsed Fasta Files into Collapsed Fasta Files
Alignment of RNA-Seq Fasta Files with Genome Using Bowtie
Preparation of Configuration File for the Pipeline
Running the Pipeline
3.2 Homology-Based In Silico Identification of miRNA169 Family Members in Plant Genomes (tomato MIR169 as a Case Study)
3.3 Validation of Predicted MIR169 Genes in Tomato
3.3.1 Validating the Putative MIR169 Loci by Small RNA Data Support
3.3.2 Validation of Precursor by Cloning and Sequencing
3.3.3 qRT-PCR Based Validation of miRNAs Precursors
3.4 Expression Analysis of miRNAs
3.4.1 Enrichment of Small RNAs by Lithium Chloride Precipitation
3.4.2 Poly A-Tailing of the Small RNA
3.4.3 Preparation of cDNA from Small RNA
3.4.4 TaqMan qRT-PCR Based Expression Analysis of miRNAs
3.5 miRNA Target Identification Using degradome Datasets Following CleaveLand Tool
3.6 Validation of miRNA-Mediated Cleavage
3.6.1 Mapping of miRNA Targets Cleavage Sites by 5β² RLM-RACE
3.6.2 Transient Assays for Validating Target Cleavage
Transient Assays for Target Cleavage
3.7 Functional Delineation of miRNA-Target Pair in Planta
3.7.1 Short Tandem Target Mimic (STTM) to Sponge miRNA: A Case Study of STTM169 in Tomato
3.7.2 Resistant Target: Target with Mutated miRNA Binding Site
4 Notes
References
Chapter 18: Development of a Ligation-Independent Cloning-Based Dual Vector System for RNA Interference in Plants
1 Introduction
2 Materials
2.1 Gene Cloning and Plasmid Construction
2.2 PCR Confirmation of RNAi Constructs
2.3 DNA Sequencing of RNAi Constructs and Agrobacterium Transformation
3 Methods
3.1 Construction of RNAi Vector
3.2 PCR Confirmation of RNAi Constructs
3.3 DNA Sequencing of RNAi Constructs and Agrobacterium Transformation
4 Notes
References
Chapter 19: Multigene Transformation Through Cre-lox Mediated Site-Specific Integration in Rice
1 Introduction
2 Material
2.1 DNA Vectors
2.2 Transformation by Gene Gun
2.3 Analysis of Transgenic Plants
3 Methods
3.1 Generation of Site-Specific Integration Lines
3.1.1 Preparation of Target Line Callus
3.1.2 Preparation of Gold Particles
3.1.3 Bombardment of Callus Plates
3.1.4 Tissue Culture
3.2 Molecular Characterization
4 Notes
References
Chapter 20: An Improvised Hairy Root Transformation Method for Efficient Gene Silencing in Roots and Nodules of Arachis hypoga...
1 Introduction
2 Materials
2.1 Plant and Bacteria
2.2 General
2.3 Media and Antibiotics
2.4 Plasmids and Primers
3 Methods
3.1 Preparation of Chemically Competent A. rhizogenes R1000 Cells
3.2 Transformation of Binary Plasmids into R1000 Competent Cells
3.3 A. rhizogenes Mediated Transformation and Hairy Root Induction
3.4 Infection of Composite Plants with Bradyrhizobia
3.5 Screening for Transformed Roots and Transformation Efficiency Calculation
4 Notes
References
Chapter 21: A Method to Reduce off-Targets in CRISPR/Cas9 System in Plants
1 Introduction
2 Materials
2.1 Functionalized Mesoporous Silica Nanoparticles (MSNs)
2.2 Plasmid Midipreparation
3 Methods
3.1 Synthesis of MSNs
3.2 Functionalization of the MSNs
3.3 Characterization of Functionalized MSNs
3.4 Minipreparation of Plasmid
3.5 Plasmid Binding to the Functionalized MSNs
3.6 Plant Transient Transformation Using MSNs Containing pDNA
3.7 Plant Stable Transformation Using MSNs Containing pDNA
4 Notes
References
Index