Identification and Molecular Mechanisms of Key Nucleotides Causing Attenuation in Pathogenicity of Dahlia Isolate of Potato Spindle Tuber Viroid
Abstract
:1. Introduction
2. Results
2.1. Infectivity and Genetic Stability of PSTVd Mutants
2.2. Mutation at Positions 42 and 64 Attenuated Symptoms
2.3. Impact of Mutation on PSTVd Secondary Structure
2.4. Identification of Three Tomato Genes Whose Expression Levels Fluctuate to Virulence of PSTVd Mutants
3. Discussion
4. Materials and Methods
4.1. Construction of PSTVd Dimeric cDNA Clones and Transcription of RNA Transcripts
4.2. Infection Assay to Analyze the Infectivity and Genetic Stability of Ten Types of PSTVd Mutants and Total RNA Extraction
4.3. Infection Assay to Compare the Pathogenicity of Selected PSTVd Mutants
4.4. Sequence Analysis of PSTVd Progeny
4.5. Secondary Structure Prediction of Mutants and Target Search for PSTVd-sRNA
4.6. Northern Blot Hybridization
4.7. Statistical Analysis
4.8. RT-Quantitative PCR (RT-qPCR)
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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PSTVd Construct | Nucleotide Mutated | Northern-Blot Assay | Genetic Stability (4 wpi) | Systemic Accumulation | Pathogenicity | |||||
---|---|---|---|---|---|---|---|---|---|---|
42 | 43 | 64 | 310 | 312 | 1st Passage | 2nd Passage | ||||
PSTVd-I | C | U | - | A | - | + | N.T. *1 | stable (13/13) | fast | severe |
PSTVd-D | U | C | U | C | UU | + | N.T. *1 | stable (10/10) | slow | mild |
I-C42U | U | U | - | A | - | + | stable (11/11) | stable (11/11) | fast | mild |
I-U43C | C | C | - | A | - | + | revert (2/13) | revert (10/10) | (fast) *2 | (severe) *2 |
I-64U | C | U | U | A | - | + | stable (14/14) | stable (15/15) | fast | mild |
I-A310C | C | U | - | C | - | + | stable (13/13) | stable (11/11) | fast | severe |
I-312UU | C | U | - | A | UU | + | unstable (UU > U; 10/12) | unstable (UU > U; 12/12) | (fast) *2 | (severe) *2 |
D-U42C | C | C | U | C | UU | + | covariation (C43U; 10/11) | covariation (C43U; 12/12) | (slow) *2 | (mild) *2 |
D-C43U | U | U | U | C | UU | + | stable (12/12) | stable (11/11) | slow | mild |
D-64Δ | U | C | - | C | UU | + | covariation (310CΔ; 5/12) | covariation (310CΔ; 10/10) | (slow) *2 | (mild) *2 |
D-C310A | U | C | U | A | UU | + | covariation (312UΔ; 5/10) | covariation (312UΔ; 10/10) | (slow) *2 | (mild) *2 |
D-312UUΔ | U | C | U | C | - | + | stable (12/12) | stable (10/10) | slow | mild |
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Kitabayashi, S.; Tsushima, D.; Adkar-Purushothama, C.R.; Sano, T. Identification and Molecular Mechanisms of Key Nucleotides Causing Attenuation in Pathogenicity of Dahlia Isolate of Potato Spindle Tuber Viroid. Int. J. Mol. Sci. 2020, 21, 7352. https://doi.org/10.3390/ijms21197352
Kitabayashi S, Tsushima D, Adkar-Purushothama CR, Sano T. Identification and Molecular Mechanisms of Key Nucleotides Causing Attenuation in Pathogenicity of Dahlia Isolate of Potato Spindle Tuber Viroid. International Journal of Molecular Sciences. 2020; 21(19):7352. https://doi.org/10.3390/ijms21197352
Chicago/Turabian StyleKitabayashi, Shoya, Daiki Tsushima, Charith Raj Adkar-Purushothama, and Teruo Sano. 2020. "Identification and Molecular Mechanisms of Key Nucleotides Causing Attenuation in Pathogenicity of Dahlia Isolate of Potato Spindle Tuber Viroid" International Journal of Molecular Sciences 21, no. 19: 7352. https://doi.org/10.3390/ijms21197352