Fusarium wilt: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Pure Culture and DNA Sequence-Based Identification of Fusarium from Symptomatic Plants and Diverse Substrates
1 Introduction
2 Materials
2.1 Isolation and Pure Culture of Strains from Symptomatic Hosts
2.2 Culturing Strains for Isolation of Total Genomic DNA
2.3 PCR Amplification, DNA Sequencing, and Strain Identification
3 Methods
3.1 Isolation and Pure Culture of Strains from Symptomatic Hosts
3.2 Culturing Strains for Isolation of Total Genomic DNA
3.3 PCR Amplification, DNA Sequencing, and Strain Identification via BLASTn Queries of Publicly Accessible DNA Sequence Databa...
4 Notes
References
Chapter 2: Isolation of High-Molecular-Weight (HMW) DNA from Fusarium oxysporum for Long-Read Sequencing
1 Introduction
2 Materials and Equipment
2.1 Materials
2.2 Equipment
3 Methods
3.1 DNA Preparation
3.1.1 Prepare the DNA for PCR
3.1.2 Prepare the DNA for MinION Sequencing
3.2 Check the DNA Quality by CHEF Gel
3.3 MinION Sequencing
3.3.1 Construction of MinION DNA Library
3.3.2 SRE Purification
3.3.3 MinION Library Construction (See Note 7) and Sequencing
4 Notes
References
Chapter 3: Karyotyping of Fusarium oxysporum by Pulsed-Field Gel Electrophoresis and the Germ Tube Burst Method
1 Introduction
2 Materials
2.1 Protoplasting
2.2 Preparation of Agarose Plugs
2.3 Pulsed-Field Gel Electrophoresis (PFGE)
2.4 Southern Hybridization
2.5 Germ Tube Burst Method
3 Methods
3.1 PFGE
3.1.1 Preparation of Protoplasts-Containing Agarose Plugs
3.1.2 Separation of Chromosomal DNAs by PFGE
3.1.3 Southern Hybridization
3.2 Cytological Karyotyping
3.2.1 Preparation of Mitotic Chromosome Spreads by the Germ Tube Burst Method (GTBM)
3.2.2 Fluorescence Staining and Fluorescence Microscopy of Mitotic Chromosomes
DAPI Staining
DAPI/PI Double Staining
3.2.3 Karyotyping
4 Notes
References
Chapter 4: RNA-seq Data Analysis for Differential Expression
1 Introduction
2 Datasets and Directory Setup
3 Methods
3.1 RNA-seq Data Analysis
3.2 Differential Gene Expression
4 Notes
References
Chapter 5: Quantification and Isolation of Spontaneous Colony Growth Variants
1 Introduction
2 Materials
3 Methods
3.1 Quantification of Colony Growth Sectors
3.2 Isolation and Phenotypic Characterization of Colony Sectors
4 Notes
References
Chapter 6: Agrobacterium tumefaciens-Mediated Transformation Method for Fusarium oxysporum
1 Introduction
2 Materials
2.1 Biological Organisms
2.2 Agrobacterium-Mediated Transformation
2.3 Screening and Validation of Transformants
3 Methods
3.1 Assessment of Isolate Sensitivity to Hygromycin B
3.2 A. tumefaciens-Mediated Transformation of F. oxysporum
3.3 Confirmation of Generation of Transformants
3.3.1 Observation of Green Fluorescence
3.3.2 DNA Extraction and PCR Analyses
3.4 Mitotic Stability Test
4 Notes
References
Chapter 7: Targeted Gene Disruption Via CRISPR/Cas9 Ribonucleoprotein Complexes in Fusarium oxysporum
1 Introduction
2 Materials
2.1 Cas9 Protein Extraction and Purification
2.2 sgRNA Design and Synthesis
2.3 In Vitro Cleavage Assay
2.4 Preparation of Donor DNA
2.4.1 ~1 kb Overlapping Flanking Region for Homologous Recombination Strategy
2.4.2 50 bp Overlapping Region Strategy
2.5 Growth of Fusarium oxysporum
2.6 Making Protoplasts
2.7 Fusarium Transformation
3 Methods
3.1 Cas9 Purification
3.2 sgRNA Design and Synthesis
3.3 In Vitro Cleavage Assay
3.4 Preparation of Donor DNA
3.4.1 1000 bp Overlapping Region Strategy
3.4.2 50-70 bp Overlapping Region Strategy
3.5 Protoplasting
3.6 Transformation
4 Notes
References
Chapter 8: CRISPR/Cas9 RNP-Mediated Gene Fusion to Assess Protein Quantification and Subcellular Localization in Fusarium oxys...
1 Introduction
2 Materials
2.1 Fungal Isolates and Bacterial Strains
2.2 Fungal and Bacterial Culture Media
2.3 Donor DNA Construction
2.4 Transformation Reagents
2.5 Microscopy
3 Methods
3.1 Construction of Donor DNA
3.2 In Vitro Cas9 RNP Cleavage Assay
3.3 Generating Fungal Protoplasts
3.4 PEG-Mediated Transformation
3.5 Evaluation of Protein Expression and Subcellular Localization by Microscopy
4 Notes
References
Chapter 9: In Vivo Monitoring of Cytosolic pH Using the Ratiometric pH Sensor pHluorin
1 Introduction
2 Materials
2.1 Fungal Growth and Inoculation
2.2 Measurement of Cytosolic pH in a Fungal Cell Population
2.3 Single-Cell Analysis of Cytosolic pH
3 Methods
3.1 Fungal Growth and Inoculation
3.2 Measurement of Cytosolic pH in a Fungal Population
3.3 Single-Cell Analysis of Cytosolic pH
3.4 Measurement of Cytosolic pH
3.4.1 Calibration Curve
3.4.2 Experimental Samples
4 Notes
References
Chapter 10: Multi-Pronged Investigation of Volatile Compound-Mediated Interactions of Fusarium oxysporum with Plants, Fungi, a...
1 Introduction
2 Materials
2.1 Biological Materials
2.2 Culture Media and Solutions
2.3 Equipment and Supplies
2.4 Homemade System for Assaying Bioactivities of Fungal VCs (Fig. 1; See Note 3)
3 Methods
3.1 I Plate-Based Assay for Investigating How F. oxysporum VCs Affect the Growth and Development of A. thaliana
3.2 Determination of How F. oxysporum VCs Affect A. thaliana Grown in Soils
3.3 Assessment of Various Changes in A. thaliana After Exposure to F. oxysporum VCs
3.4 Sandwiched Plate Assay Used to Study VC-Mediated Interactions Between F. oxysporum and T. virens
3.4.1 Assessment of the Inhibitory Activity of T. virens VCs Against F. oxysporum
3.4.2 Assessment of the Inhibitory Activity of F. oxysporum VCs Against T. virens
3.4.3 Assessment of VC-Mediated Recognition of F. oxysporum by T. virens (Fig. 6)
3.4.4 Assessment of VC-Mediated Recognition of T. virens by F. oxysporum
3.5 Sandwiched Plate Assay to Study the VC-Mediated Interaction Between F. oxysporum and E. coli
3.5.1 Assessment of the Inhibitory Activity of E. coli VCs Against F. oxysporum
3.5.2 Assessment of the Inhibitory Activity of F. oxysporum VCs Against E. coli
3.5.3 Assessment to Determine if Bacterial VCs Alter the Biosynthesis of Antibacterial Molecules Secreted by F. oxysporum
3.6 Extraction of VCs Produced by F. oxysporum for GC-MS Analysis
4 Notes
References
Chapter 11: Isolation of Mitochondria from Fusarium Species
1 Introduction
2 Materials
3 Methods
3.1 Conidial Stocks
3.2 Vegetative Growth
3.3 Preparation of Fusarium Mitochondria
4 Notes
References
Chapter 12: In Planta Gene Expression Analysis and Colonization of Fusarium oxysporum
1 Introduction
2 Materials
2.1 Plant Inoculation with Fusarium oxysporum
2.1.1 Growth of Plants
2.1.2 Preparation of Conidia to Be Used as Inoculum
2.1.3 Inoculation of Phaseolus vulgaris
2.2 RT-qPCR Quantification and Analysis
2.2.1 Isolation of RNA
2.2.2 Determination of RNA Concentration and Quality
2.2.3 cDNA Synthesis
2.2.4 RT-qPCR
2.3 Confocal Laser Microscopy
3 Methods
3.1 Plant Inoculation with F. oxysporum
3.1.1 Growth of Plants
3.1.2 Preparation of the Inoculum
3.1.3 Inoculation of Phaseolus vulgaris
3.2 RT-qPCR Quantification and Analysis
3.2.1 Collection of Samples
3.2.2 Isolation of RNA
3.2.3 Determination of RNA Concentration and Quality
3.2.4 Reverse Transcription from RNA to cDNA
3.2.5 Quantitative Real-Time PCR (RT-qPCR)
3.2.6 Analysis of Gene Expression
3.3 Confocal Laser Microscopy
3.3.1 GFP-Expressing Fungal Strains
3.3.2 Non-GFP-Expressing Fungal Strains
3.3.3 Confocal Laser Microscopy
4 Notes
References
Chapter 13: Time-Lapse Imaging of Root Pathogenesis and Fungal Proliferation Without Physically Disrupting Roots
1 Introduction
2 Materials
2.1 Biological Materials
2.2 Culture Media and Solutions
2.3 Equipment and Supplies
2.4 Microscope Hardware and Software
3 Methods
3.1 Transformation of F. oxysporum
3.2 Transformation of A. thaliana
3.3 Setup, Inoculation, and Maintenance of Plants in Optical Growth Chambers
3.4 Confocal Microscopy of Root Pathogenesis
3.5 Imaging Ca2+ Signature in Planta
4 Notes
References
Chapter 14: High-Throughput Screening Assays to Identify Plant Natural Products with Antifungal Properties Against Fusarium ox...
1 Introduction
2 Materials
3 Methods
3.1 Cultivation and Preparation of Fungi
3.2 Developing High-Throughput Screening Assays
3.2.1 Resazurin Cell Viability Assay
3.2.2 Optical Density Assay
3.3 Screening Plant Cell Lines for Compounds with Antifungal Properties
3.3.1 Abiotic Elicitation Using Methyl Jasmonate
3.3.2 Biotic Elicitation Using F. oxysporum in a Bi-chamber Transwell Plate
4 Notes
References
Chapter 15: Screening and Assessment of Pisatin Demethylase Activity (PDA)
1 Introduction
2 Materials
2.1 Biological Samples
2.2 Screening of Pisatin Demethylase Activity (Scintillation Vial Assay)
2.3 Assessment of Pisatin Demethylase Activity over Time
3 Methods
3.1 Screening of Pisatin Demethylase Activity (Scintillation Vial Assay)
3.2 Assessment of Pisatin Demethylase Activity over Time
4 Notes
References
Chapter 16: Detection and Genotyping of Fov4 (Race 4, VCG0114), the Fusarium Wilt Pathogen of Cotton
1 Introduction
2 Materials
2.1 Culture Media and Isolation
2.2 DNA Extraction
2.3 PCR Detection of Fov4-Specific Polymorphisms
2.4 Gel Electrophoresis
3 Methods
3.1 Sample Collection
3.2 Selective Plating
3.3 DNA Extraction
3.3.1 DNA Extraction from Fungal Cultures
3.3.2 DNA Extraction from Cotton
3.4 Detection and Genotype Determination of Fov4
3.4.1 Detection and Genotype Determination of Fov4 by Multiplex PCR
3.4.2 Detection of Fov4 by Singleplex PCR
3.4.3 Detection and Genotype Determination of Fov4 by Singleplex PCR
4 Notes
References
Index