Experimental Plant Virology

Cover......Page 1
Advanced Topics in Science and Technology in China......Page 2
Experimental Plant\rVirology......Page 4
ISBN 9783642141188......Page 5
Preface......Page 6
Contents......Page 8
1.1 Introduction......Page 16
1.2 A Tomato Strain of Cucumber Mosaic Virus, a Natural Reassortant Between Subgroups IA and II......Page 20
1.3 The Araceae Strain of Cucumber Mosaic Virus Infecting Pinellia ternate Suggested to be a Novel Class Unit Under Subgroup I......Page 25
1.3.2 Phylogenetice and Sequence Divergence Analysis of 5′ UTR and 3′ UTR, 2a and 2b ORFs of RNA3......Page 30
1.4 The Potyvirus Infecting Pinellia ternata is a Recombinant Contributed by Soybean Mosaic Virus and Lettuce Mosaic Virus......Page 32
1.4.1 DAS-ELISA Analysis of Field Samples for Detecting the Potyvirus......Page 33
1.4.2 Sequencing and Nucleotide Sequence Analysis of the Potyvirus Infecting Pinellia......Page 35
1.4.3 Amino Acid Sequence Analysis for CP of the Potyvirus Infecting Pinellia......Page 37
1.4.4 Nucleotide Sequence Analysis for CP N-terminal of the Potyvirus Infecting Pinellia......Page 39
1.4.5 Amino Acid Sequences for N-terminal and for the Conserved Region of the Potyvirus Infecting Pinellia......Page 40
1.4.7 The General Character and Possible Origin of the Potyvirus Infecting Pinellia......Page 42
1.5 The 5′ Terminal and a Single Nucleotide Determine the Accumulation of Cucumber Mosaic Virus Satellite RNA......Page 46
1.5.1 GUUU- in 5′ Terminal is Necessary to Initiate Replicationof 2msatRNA......Page 47
1.5.2 Typical Structure at the 5′ Terminal is Necessary for Long-distance Movement or High Accumulation of 2msatRNA......Page 49
1.5.3 Low Accumulation of 2mF5sat Mutants is Related to Single Nucleotide Mutation......Page 51
1.5.4 Secondary Structure of 2mF5sat Impaired its Replication Capacity......Page 53
1.6.1 Purification of CMV Virions from Plant Tissue......Page 55
1.6.2 RT-PCR and cDNA Cloning for Full-length Genomic RNAs of Cucumber Mosaic Virus......Page 56
1.6.3 RT-PCR and Gene Cloning for 3\'-end of Viral Genome of Soybean Mosaic Virus......Page 57
1.6.5 Pseudo-recombination of Satellite RNA of Cucumber Mosaic Virus and the Helper Virus......Page 58
References......Page 59
2.1 Introduction......Page 62
2.2 Multiplex RT-PCR System for Simultaneous Detection of Five Potato Viruses......Page 66
2.2.1 Comparison of 18S rRNA and nad2 mRNA as Internal Controls......Page 67
2.2.2 The Optimized System for Simultaneous Detection of Potato Viruses with Multiplex RT-PCR......Page 69
2.2.3 Sensitivities of Multiplex RT-PCR and DAS-ELISA in Detecting Potato Viruses......Page 70
2.3 Detection of Cucumber Mosaic Virus Subgroups and Tobamoviruses Infecting Tomato......Page 72
2.3.1 Multiplex RT-PCR for Simultaneous Detection of Strains of CMV and ToMV in Tomato......Page 73
2.3.3 Identification of CMV Subgroups by Restriction Enzymes......Page 77
2.4 A Novel Glass Slide Hybridization for Detecting Plant RNA Viruses and Viroids......Page 80
2.4.1 Preparation of Highly Sensitive Fluorescent-labeled Probes......Page 81
2.4.2 Effect of Spotting Solutions on Spot Quality......Page 82
2.4.3 Effect of Glass Surface Chemistries on Efficiencies of RNA Binding......Page 83
2.4.4 Detection Limits of Glass Slide Hybridization and Nylon Membrane Hybridization......Page 84
2.4.5 Specificity of Glass Slide Hybridization......Page 86
2.4.6 Detection of PVY and PSTVd from Field Potato Samples......Page 87
2.5 Quantitative Determination of CMV Genome RNAs in Virions by Real-time RT-PCR......Page 89
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......Page 91
2.6 Accurate and Efficient Data Processing for Quantitative Real-time PCR......Page 96
2.6.1 Quantification of CMV RNAs in Virions with Standard Curves......Page 97
2.6.2 Quantification of CMV RNAs in Virions by SCF......Page 98
2.6.3 Quantification of CMV RNAs in Virions by LinReg PCR and DART Programs......Page 100
2.6.4 Determination of the Suppression Effect of Satellite RNA on CMV Accumulation in Plant Tissues Using N 0 Values......Page 102
2.7.1 Primers Design and Specificity Tests in RT-PCR......Page 103
2.7.2 Comparison of 18S rRNA and nad2 mRNA as Internal Controls......Page 104
2.7.3 Optimization of Multiplex RT-PCR......Page 105
2.7.5 Glass Slide Hybridization......Page 106
References......Page 109
3.1 Introduction......Page 112
3.2 Cucumber Mosaic Virus-mediated Regulation of Disease Development Against Tomato Mosaic Virus in the Tomato......Page 113
3.2.1 ToMV-N5 Initiated Necrosis on Tomato Can be Protected by Previous Inoculation with Wild-type CMV......Page 115
3.2.2 ToMV-N5 Initiated Necrosis on Tomato Cannot be Protected by Previous Inoculation with CMVΔ2b......Page 117
3.2.3 ToMV-N5-initiated Necrosis on Tomato Cannot be Protected by Previous Inoculation with Potato Virus X......Page 118
3.2.4 CMV-initiated Protection against ToMV-N5 is Related to the Replication and Accumulation of Challenging Virus......Page 119
3.3.1 Wildtype and Pseudo-recombinants and with or without satRNA Induce Different Symptoms on N. glutinosa......Page 120
3.3.2 Wildtype and Pseudo-recombinants with or without satRNA Induce Different Symptoms on N. benthamiana......Page 122
3.3.3 Wildtype and Pseudo-recombinants with or without satRNA Induce Different Symptoms on Tomato Varieties......Page 123
3.3.4 The Pathogenicity of Wildtype and Pseudorecombinants with or without satRNA-Tsh are Related to Viral Accumulation......Page 125
3.4 Synergy via Cucumber Mosaic Virus and Zucchini Yellow Mosaic Virus on Cucurbitaceae Hosts......Page 126
3.4.1 Assessment of Symptom and Synergic Interaction by Cucumber Mosaic Virus and Zucchini Yellow Mosaic Virus......Page 127
3.4.2 Accumulation Kinetics for CMV ORFs in Single or Complex Infection......Page 128
3.4.3 Accumulation Kinetics of ZYMV CP ORF in Single or Complex Infection......Page 131
3.5 Methodology......Page 132
3.5.1 The Interaction Study on CMV and ToMV Interaction......Page 133
3.5.2 Pseudo-recombination of CMV Subgroups......Page 134
3.5.3 Synergy between CMV and ZYMV on Cucurbitaceae......Page 136
References......Page 137
4.1 Introduction......Page 140
4.2 The 2b Protein of Cucumber Mosaic Virus is a Determinant of Pathogenicity and Controls Symptom Expression......Page 142
4.2.1 Infectivity and Stability of Fny-CMV Derived Mutants......Page 143
4.2.2 Replacement of the 2b ORF Affected Capsidation of Viral RNA 2......Page 145
4.2.4 Divertive Virulence is Mediated by the 2b Protein Rather than by the C-terminal Overlapping Parts of the 2a Protein......Page 147
4.2.5 Virulence is Associated with the Accumulation of Viral Progeny RNAs Affected by 2b Protein......Page 149
4.3 Function of CMV 2b Protein and the C-terminus of 2a Protein in Determining Viral RNA Accumulation and Symptom Development......Page 152
4.3.1 The Systemic Necrosis-inducing Domain is Related to a 125-nucleotide Region of RNA 2......Page 153
4.3.2 Effect of 2b Protein Amino Acid 55 on Viral Accumulation and Symptom Development......Page 155
4.3.3 Sequence Analyses of the 2b Proteins and the C-top of the 2a Proteins......Page 156
4.3.4 Effect of the C-terminus of 2a Protein on Symptom Expression and Virus Accumulation......Page 158
4.4.1 Symptom Expression on N. Tabacum Inoculated with CMV-Fsat......Page 161
4.4.3 Symptom Expression on the Host Plants Inoculated with CMV-FnyΔ2b......Page 163
4.4.4 Accumulation of CMV-FnyΔ2b Genomic RNAs and the Effect of satRNAs......Page 164
4.4.5 Accumulation of CMV-Fny Genomic RNAs in the Inoculated Leaves and the Effect of satRNAs......Page 166
4.4.6 The Effect of satRNAs on Long-distance Movement of CMV-Fny Genomic RNAs......Page 167
4.5.1 Plants, Viruses and Plasmid Constructs......Page 168
4.5.2 Plant Inoculation and Viral Progeny RNA Analysis......Page 173
References......Page 174
5.1 Introduction......Page 178
5.2.1 Computational Prediction of miRNAs and Their Target Genes for Plant Species with Known Genome Sequences......Page 180
5.2.2 Use Plant miRNA Microwares to Identify Conservative miRNAs from New Host Plants......Page 182
5.2.3 Use Plant miRNA Microarrays to Identify Conservative miRNAs Response to Virus Infection......Page 184
5.2.4 Quantitative Determination of miRNAs by Stem-loop Real-time RT-PCR......Page 185
5.2.5 Design of Plant miRNA-array and Data Analysis......Page 188
5.3 Tomato miRNAs Predicted from Known Genomic Sequences and Discovered by miRNA Microarray......Page 189
5.3.1 Potential Tomato miRNAs Predicted Computationally According to Known Genomic Sequences......Page 190
5.3.2 Potential Targets of Newly Predicted miRNAs and Their Function......Page 193
5.3.3 Confirmation of Tomato miRNAs Expression and Survey by Microarray......Page 195
5.4 Mechanisms Involved in Plant miRNA Expression with Regard to Infection of ssRNA Viruses......Page 200
5.4.1 Phenotype in Tomato Under Infection with CMV/satRNA Combinations and ToMV......Page 201
5.4.2 Response of Tomato miRNA Expression to Virus Infection......Page 202
5.4.3 MiRNA Expression Profiles between CMV-Fny and CMV-FnyΔy2b Infections......Page 208
5.4.4 MiRNAs Expression Profiles Altered with Addition of satRNAs......Page 209
5.4.5 A Comparison of miRNAs Expression Profiles between CMV and ToMV Infections......Page 210
5.5 Tomato miRNA Response to Virus Infection Quantified by Real-time RT-PCR......Page 212
5.5.1 Identification of Tomato ARF8- and AGO1-like Genes......Page 214
5.5.2 Analytical Validation of Real-time RT-PCR for Amplification of miRNAs......Page 215
5.5.3 Quantification of Tomato miRNAs Expression by Stem-loop Real-time RT-PCR......Page 217
5.5.4 Quantification of miRNAs Targets in Tomato under Cucumovirus Infection......Page 219
References......Page 221
6.1 Introduction......Page 226
6.2 Novel dsRNA Viruses Infecting Raphanus Sativus......Page 227
6.2.1 Yellow Edge Symptoms and dsRNA Patterns in the Radish......Page 228
6.2.2 Genome Characterization of Raphanus Sativus Cryptic Virus 1......Page 232
6.2.3 Genome Characterization of Raphanus Sativus Cryptic Virus 2......Page 237
6.2.4 Correlation of Raphanus Sativus Cryptic Virus 2 with Raphanus Sativus Cryptic Virus 1......Page 239
6.2.5 Genome Characterization of Suggested Raphanus Sativus Cryptic Virus 3......Page 241
6.3 Double Stranded Viruses in Vicia Faba......Page 244
6.3.1 Two dsRNA Viruses Infecting V. faba......Page 245
6.3.2 A Partitiviruss Infecting Aspergilus sp. Associated with Leaf Tissue of Vicia faba......Page 252
6.4 A Novel dsRNA Virus Infecting Primula Malacoides Franch......Page 258
6.5 Derivation and Evolutionary Relationship of dsRNA Viruses Infecting plants......Page 264
6.6 Conclusion......Page 272
6.7.1 Plant Material and dsRNA Extraction......Page 273
6.7.3 Amplification of Unknown dsRNA Sequence by Modified Single-primer Amplification Technique (SPAT)......Page 275
6.7.4 Sequence Analysis......Page 276
References......Page 277
Index......Page 282