Screening and Identification of Host Factors Interacting with the Virulence Factor P0 Encoded by Sugarcane Yellow Leaf Virus by Yeast Two-Hybrid Assay
Abstract
:1. Introduction
2. Materials and Methods
2.1. Sequence Analysis
2.2. Plant Materials and Growth Conditions
2.3. Plasmid Construction
2.4. Agroinfiltration Assays
2.5. H2O2 Detection in Plants
2.6. Protein Extraction and Western Blot analysis
2.7. cDNA Expression Library Construction and Quality Assay
2.8. Assays of Auto-Activation and Toxicity of the Bait Plasmid
2.9. Yeast Two-Hybrid Screening with Co-Transformation
2.10. Validation of the Screened Interactions
2.11. Function Analysis of True Positive Prey
3. Results
3.1. Phylogenetic Analyses and Domain Architecture of Sugarcane-Yellow-Leaf-Virus-Encoded P0
3.2. P0 Is a Virulence Determinant and Elicits a Hypersensitive-Like Response (HLR) in N. benthamiana
3.3. Construction of cDNA Library and pGBKT7-P0 Bait Plasmid
3.4. Assay Results of Auto-Activation and Toxicity of the Bait Plasmid
3.5. Yeast Two-Hybrid Screening and Confirmation of the Interactions between pGBKT7-P0 and Screened Prey Plasmids
3.6. Sequencing and Analysis of Confirmed True Positive Prey Plasmids
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Liang, K.-L.; Liu, J.-Y.; Bao, Y.-Y.; Wang, Z.-Y.; Xu, X.-B. Screening and Identification of Host Factors Interacting with the Virulence Factor P0 Encoded by Sugarcane Yellow Leaf Virus by Yeast Two-Hybrid Assay. Genes 2023, 14, 1397. https://doi.org/10.3390/genes14071397
Liang K-L, Liu J-Y, Bao Y-Y, Wang Z-Y, Xu X-B. Screening and Identification of Host Factors Interacting with the Virulence Factor P0 Encoded by Sugarcane Yellow Leaf Virus by Yeast Two-Hybrid Assay. Genes. 2023; 14(7):1397. https://doi.org/10.3390/genes14071397
Chicago/Turabian StyleLiang, Kai-Li, Jing-Ying Liu, Ying-Ying Bao, Zhi-Yuan Wang, and Xiong-Biao Xu. 2023. "Screening and Identification of Host Factors Interacting with the Virulence Factor P0 Encoded by Sugarcane Yellow Leaf Virus by Yeast Two-Hybrid Assay" Genes 14, no. 7: 1397. https://doi.org/10.3390/genes14071397