1 Gene Cloning of Cucumber Mosaic Virus and Some Related Viral Agents 1
1.1 Introduction 1
1.2 A Tomato Strain of Cucumber Mosaic Virus,a Natural Reassortant Between Subgroups IA and Ⅱ 5
1.3 The Araceae Strain of Cucumber Mosaic Virus Infecting Pinellia ternate Suggested to be a Novel Class Unit Under Subgroup Ⅰ 10
1.3.1 Phylogenetice and Sequence Divergence Analysis of 3a and CP ORFs 15
1.3.2 Phylogenetice and Sequence Divergence Analysis of 5′ UTR and 3′UTR,2a and 2b ORFs of RNA3 15
1.4 The Potyvirus Infecting Pinellia ternata is a Recombinant Contributed by Soybean Mosaic Virus and Lettuce Mosaic Virus 17
1.4.1 DAS-ELISA Analysis of Field Samples for Detecting the Potyvirus 18
1.4.2 Sequencing and Nucleotide Sequence Analysis of the Potyvirus Infecting Pinellia 20
1.4.3 Amino Acid Sequence Analysis for CP of the Potyvirus Infecting Pinellia 22
1.4.4 Nucleotide Sequence Analysis for CP N-terminal of the Potyvirus Infecting Pinellia 24
1.4.5 Amino Acid Sequences for N-terminal and for the Conserved Region of the Potyvirus Infecting Pinellia 25
1.4.6 Nucleotide Sequences for 3′UTR of the Potyvirus Infecting Pinellia 27
1.4.7 The General Character and Possible Origin of the Potyvirus Infecting Pinellia 27
1.5 The 5′Terminal and a Single Nucleotide Determine the Accumulation of Cucumber Mosaic Virus Satellite RNA 31
1.5.1 GUUU-in 5′Terminal is Necessary to Initiate Replication of 2msatRNA 32
1.5.2 Typical Structure at the 5′Terminal is Necessary for Long-distance Movement or High Accumulation of 2msatRNA 34
1.5.3 Low Accumulation of 2mF5sat Mutants is Related to Single Nucleotide Mutation 36
1.5.4 Secondary Structure of 2mF5sat Impaired its Replication Capacity 38
1.6 Methodology 40
1.6.1 Purification of CMV Virions from Plant Tissue 40
1.6.2 RT-PCR and cDNA Cloning for Full-length Genomic RNAs of Cucumber Mosaic Virus 41
1.6.3 RT-PCR and Gene Cloning for 3′-end of Viral Genome of Soybean Mosaic Virus 42
1.6.4 Sequence Analysis and Phylogenetice Analysis 43
1.6.5 Pseudo-recombination of Satellite RNA of Cucumber Mosaic Virus and the Helper Virus 43
References 44
2 Molecular Detection of Cucumber Mosaic Virus and Other RNA Viruses Based on New Techniques 47
2.1 Introduction 47
2.2 Multiplex RT-PCR System for Simultaneous Detection of Five Potato Viruses 51
2.2.1 Comparison of 18S rRNA and nad2 mRNA as Internal Controls 52
2.2.2 The Optimized System for Simultaneous Detection of Potato Viruses with Multiplex RT-PCR 54
2.2.3 Sensitivities of Multiplex RT-PCR and DAS-ELISA in Detecting Potato Viruses 55
2.3 Detection of Cucumber Mosaic Virus Subgroups and Tobamoviruses Infecting Tomato 57
2.3.1 Multiplex RT-PCR for Simultaneous Detection of Strains of CMV and ToMV in Tomato 58
2.3.2 Field Detection of Tomato Viruses by Multiplex RT-PCR 62
2.3.3 Identification of CMV Subgroups by Restriction Enzymes 62
2.4 A Novel Glass Slide Hybridization for Detecting Plant RNA Viruses and Viroids 65
2.4.1 Preparation of Highly Sensitive Fluorescent-labeled Probes 66
2.4.2 Effect of Spotting Solutions on Spot Quality 67
2.4.3 Effect of Glass Surface Chemistries on Efficiencies of RNA Binding 68
2.4.4 Detection Limits of Glass Slide Hybridization and Nylon Membrane Hybridization 69
2.4.5 Specificity of Glass Slide Hybridization 71
2.4.6 Detection of PVY and PSTVd from Field Potato Samples 72
2.5 Quantitative Determination of CMV Genome RNAs in Virions by Real-time RT-PCR 74
2.5.1 Optimization of Real-time RT-PCR and the Specificity 76
2.5.2 Quantification of CMV Genomic RNAs by RT-PCR and Comparison of the Quantification with Lab-on-a-Chip and Northern Blot Hybridization Assays 76
2.6 Accurate and Efficient Data Processing for Quantitative Real-time PCR 81
2.6.1 Quantification of CMV RNAs in Virions with Standard Curves 82
2.6.2 Quantification of CMV RNAs in Virions by SCF 83
2.6.3 Quantification of CMV RNAs in Virions by LinReg PCR and DART Programs 85
2.6.4 Determination of the Suppression Effect of Satellite RNA on CMV Accumulation in Plant Tissues Using N0 Values 87
2.7 Methodology 88
2.7.1 Primers Design and Specificity Tests in RT-PCR 88
2.7.2 Comparison of 18S rRNA and nad2 mRNA as Internal Controls 89
2.7.3 Optimization of Multiplex RT-PCR 90
2.7.4 Comparison of Sensitivities for Multiplex RT-PCR and DAS-ELISA 91
2.7.5 Glass Slide Hybridization 91
References 94
3 Infectious Clones and Chimerical Recombination of Cucumber Mosaic Virus and its Satellite RNAs 97
3.1 Introduction 97
3.2 Cucumber Mosaic Virus-mediated Regulation of Disease Development Against Tomato Mosaic Virus in the Tomato 98
3.2.1 ToMV-N5 Initiated Necrosis on Tomato Can be Protected by Previous Inoculation with Wild-type CMV 100
3.2.2 ToMV-N5 Initiated Necrosis on Tomato Cannot be Protected by Previous Inoculation with CMV△2b 102
3.2.3 ToMV-N5-initiated Necrosis on Tomato Cannot be Protected by Previous Inoculation with Potato Virus X 103
3.2.4 CMV-initiated Protection against ToMV-N5 is Related to the Replication and Accumulation of Challenging Virus 104
3.3 Pseudo-recombination between Subgroups of Cucumber Mosaic Virus Demonstrates Different Pathotypes and Satellite RNA Support Characters 105
3.3.1 Wildtype and Pseudo-recombinants and with or without satRNA Induce Different Symptoms on N.glutinosa 105
3.3.2 Wildtype and Pseudo-recombinants with or without satRNA Induce Different Symptoms on N.benthamiana 107
3.3.3 Wildtype and Pseudo-recombinants with or without satRNA Induce Different Symptoms on Tomato Varieties 108
3.3.4 The Pathogenicity of Wildtype and Pseudorecombinants with or without satRNA-Tsh are Related to Viral Accumulation 110
3.4 Synergy via Cucumber Mosaic Virus and Zucchini Yellow Mosaic Virus on Cucurbitaceae Hosts 111
3.4.1 Assessment of Symptom and Synergic Interaction by Cucumber Mosaic Virus and Zucchini Yellow Mosaic Virus 112
3.4.2 Accumulation Kinetics for CMV ORFs in Single or Complex Infection 113
3.4.3 Accumulation Kinetics of ZYMV CP ORF in Single or Complex Infection 116
3.5 Methodology 117
3.5.1 The Interaction Study on CMV and ToMV Interaction 118
3.5.2 Pseudo-recombination of CMV Subgroups 119
3.5.3 Synergy between CMV and ZYMV on Cucurbitaceae 121
References 122
4 Gene Function of Cucumber Mosaic Virus and its Satellite RNA Regarding Viral-host Interactions 125
4.1 Introduction 125
4.2 The 2b Protein of Cucumber Mosaic Virus is a Determinant of Pathogenicity and Controls Symptom Expression 127
4.2.1 Infectivity and Stability of Fny-CMV Derived Mutants 128
4.2.2 Replacement of the 2b ORF Affected Capsidation of Viral RNA 2 130
4.2.3 Intraspecies Hybrid Viruses by Changing 2b Gene Induce Different Virulence 132
4.2.4 Divertive Virulence is Mediated by the 2b Protein Rather than by the C-terminal Overlapping Parts of the 2a Protein 132
4.2.5 Virulence is Associated with the Accumulation of Viral Progeny RNAs Affected by 2b Protein 134
4.3 Function of CMV 2b Protein and the C-terminus of 2a Protein in Determining Viral RNA Accumulation and Symptom Development 137
4.3.1 The Systemic Necrosis-inducing Domain is Related to a 125-nucleotide Region of RNA 2 138
4.3.2 Effect of 2b Protein Amino Acid 55 on Viral Accumulation and Symptom Development 140
4.3.3 Sequence Analyses of the 2b Proteins and the C-top of the 2a Proteins 141
4.3.4 Effect of the C-terminus of 2a Protein on Symptom Expression and Virus Accumulation 143
4.4 Satellite RNA-mediated Reduction in Accumulation of CMV Genomic RNAs in Tobacco Related to 2b Gene of the Helper Virus 146
4.4.1 Symptom Expression on N.Tabacum Inoculated with CMV-Fsat 146
4.4.2 Effect of satRs on the Accumulation of CMV-Fny Genomic RNAs 148
4.4.3 Symptom Expression on the Host Plants Inoculated with CMV-Fny△2b 148
4.4.4 Accumulation of CMV-Fny△2b Genomic RNAs and the Effect of satRNAs 149
4.4.5 Accumulation of CMV-Fny Genomic RNAs in the Inoculated Leaves and the Effect of satRNAs 151
4.4.6 The Effect of satRNAs on Long-distance Movement of CMV-Fny Genomic RNAs 152
4.5 Methodology 153
4.5.1 Plants,Viruses and Plasmid Constructs 153
4.5.2 Plant Inoculation and Viral Progeny RNA Analysis 158
4.5.3 Quantifying the Accumulation of Viral RNAs in Leaf Tissue 159
References 159
5 Plant MicroRNAs and Their Response to Infection of Plant Viruses 163
5.1 Introduction 163
5.2 Methodology 165
5.2.1 Computational Prediction of miRNAs and Their Target Genes for Plant Species with Known Genome Sequences 165
5.2.2 Use Plant miRNA Microwarrays to Identify Conservative miRNAs from New Host Plants 167
5.2.3 Use Plant miRNA Microarrays to Identify Conservative miRNAs Response to Virus Infection 169
5.2.4 Quantitative Determination of miRNAs by Stem-loop Real-time RT-PCR 170
5.2.5 Design of Plant miRNA-array and Data Analysis 173
5.2.6 Confirmation of miRNAs by Northern Blotting and Target mRNA by 3′-RACE 174
5.3 Tomato miRNAs Predicted from Known Genomic Sequences and Discovered by miRNA Microarray 174
5.3.1 Potential Tomato miRNAs Predicted Computationally According to Known Genomic Sequences 175
5.3.2 Potential Targets of Newly Predicted miRNAs and Their Function 178
5.3.3 Confirmation of Tomato miRNAs Expression and Survey by Microarray 180
5.4 Mechanisms Involved in Plant miRNA Expression with Regard to Infection of ssRNA Viruses 185
5.4.1 Phenotype in Tomato Under Infection with CMV/satRNA Combinations and ToMV 186
5.4.2 Response of Tomato miRNA Expression to Virus Infection 187
5.4.3 MiRNA Expression Profiles between CMV-Fny and CMV-Fny△2b Infections 193
5.4.4 MiRNAs Expression Profiles Altered with Addition of satRNAs 194
5.4.5 A Comparison of miRNAs Expression Profiles between CMV and ToMV Infections 195
5.5 Tomato miRNA Response to Virus Infection Quantified by Real-time RT-PCR 197
5.5.1 Identification of Tomato ARF8-and AGO1-like Genes 199
5.5.2 Analytical Validation of Real-time RT-PCR for Amplification of miRNAs 200
5.5.3 Quantification of Tomato miRNAs Expression by Stem-loop Real-time RT-PCR 202
5.5.4 Quantification of miRNAs Targets in Tomato under Cucumovirus Infection 204
References 206
6 Genomic Characterization of New Viruses with Double Stranded RNA Genomes 211
6.1 Introduction 211
6.2 Novel dsRNA Viruses Infecting Raphanus sativus 212
6.2.1 Yellow Edge Symptoms and dsRNA Patterns in the Radish 213
6.2.2 Genome Characterization of Raphanus sativus Cryptic Virus 1 217
6.2.3 Genome Characterization of Raphanus sativus Cryptic Virus 2 222
6.2.4 Correlation of Raphanus sativus Cryptic Virus 2 with Raphanus sativus Cryptic Virus 1 224
6.2.5 Genome Characterization of Suggested Raphanus sativus Cryptic Virus 3 226
6.2.6 The Possible Existence of More dsRNA Viruses in Radish 229
6.3 Double Stranded Virnses in Vicia faba 229
6.3.1 Two dsRNA Viruses Infecting V.faba 230
6.3.2 A Partitiviruss Infecting Aspergilus sp.Associated with Leaf Tissue of Vicia faba 237
6.4 A Novel dsRNA Virus Infecting Primula malacoides Franch 243
6.5 Derivation and Evolutionary Relationship of dsRNA Viruses Infecting plants 249
6.6 Conclusion 257
6.7 Methodology 258
6.7.1 Plant Material and dsRNA Extraction 258
6.7.2 Purification of Virus Particles 260
6.7.3 Amplification of Unknown dsRNA Sequence by Modified Single-primer Amplification Technique(SPAT) 260
6.7.4 Sequence Analysis 261
6.7.5 Dot-Blot Hybridization 262
References 262
Index 267