Development of a New Tomato Torrado Virus-Based Vector Tagged with GFP for Monitoring Virus Movement in Plants
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plasmid Construction
2.2. Plant Material, Agroinfiltration, and Sap Inoculation
2.3. Virus Detection by RT-PCR
2.4. Fluorescence Monitoring in Plants
2.5. Immunodetection (IP) of the Recombined GFP Protein
3. Results
3.1. The New Generation of Infectious Clones of ToTV Retains Their Biological Activity
3.2. GFP-Tagged ToTV Infects N. Benthamiana, Spreads Efficiently in the Host and Can Be Mechanically Passaged
3.3. GFP-Derived Fluorescence is Detected in Plants Infected with ToTV-GFP
3.4. GFP is Produced in N. Benthamiana Infected by ToTV-GFP
4. Discussion
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Primer Name | Sequence 5′→3′ | Purpose |
---|---|---|
pJL89ToT1_R | TATATTCTCAAAATAACTCTTTTAACCTCTCCAAATGAAATGAACTTCC | Amplification of plasmid backbone for cloning of cDNA copy of the ToTV-Kra RNA1 |
pJL89ToT1i2_F | TTTAAAAAAAAAAAAAAAAAAAAAAAAAGGGTCGGCATGGCATCTC | |
pJL89ToT2_R | TATTGTATAAAATTATTCTTTTAAACCTCTCCAAATGAAATGAACTTCC | Amplification of plasmid backbone for cloning of cDNA copy of the ToTV-Kra RNA2 |
pJL89ToT1i2_F | TTTAAAAAAAAAAAAAAAAAAAAAAAAAGGGTCGGCATGGCATCTC | |
asTo1A_pJL_FW | TTTCATTTGGAGAGGTTAAAAGAGTTATTTTGAGAATATAAC | Amplification of cDNA copy of ToTV RNA1 |
asTo2C_pJL_RV | ATGCCATGCCGACCCTTTTTTTTTTTTTTTTTTTTTTTTTAAAAT | |
asTo2A_pJL_FW | TTTCATTTGGAGAGGTTTAAAAGAATAATTTTATACAATATTTATGT | Amplification of cDNA copy of ToTV RNA2 |
asTo2C_pJL_RV | ATGCCATGCCGACCCTTTTTTTTTTTTTTTTTTTTTTTTTAAAAT | |
pJLRNA2_CASF4 | AGCAAGACAGCGCTTCTATAAAGAAGCAGCGAAAGCGCAAGTGAAAAACAAGGTGGCCCAAAC | Preparation ToTV-Kra copy of RNA2 variant with duplicated protease recognition site |
pJLRNA_CASR4 | GGAATCTCCTCGACGGAGGTCTGCGCTACTTTATTCTTAACCTGAGCCTTGGCCGCCTC | |
EGFP_CASF3 | ACCTCCGTCGAGGAGATTCCGTCAACGAGCTTTGCGACCATGGTGAGCAAGGGCGAG | Amplification of sGFP coding sequence |
EGFP_CASR3 | TATAGAAGCGCTGTCTTGCTTGCTCTTTCGCTACGCGTTCCTTGTACAGCTCGTCCAT | |
2TT5 | GATGAGAAAGGAAAGAAGCAG | Reverse transcription-polymerase chain reaction (RT-PCR) for detection of ToTV variants in plants |
2TT6 | CATATCACCCAAATGCTTCTC | |
3A/Vp35seqF | CCCTTTGATTGTTATGATGGCTT | |
3A/Vp35seqR | TGGGCCTTACAGCTTCATTG | |
GFP_F | ATGGTGAGCAAGGGCGAG | |
GFP_R | CTTGTACAGCTCGTCC |
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Wieczorek, P.; Budziszewska, M.; Frąckowiak, P.; Obrępalska-Stęplowska, A. Development of a New Tomato Torrado Virus-Based Vector Tagged with GFP for Monitoring Virus Movement in Plants. Viruses 2020, 12, 1195. https://doi.org/10.3390/v12101195
Wieczorek P, Budziszewska M, Frąckowiak P, Obrępalska-Stęplowska A. Development of a New Tomato Torrado Virus-Based Vector Tagged with GFP for Monitoring Virus Movement in Plants. Viruses. 2020; 12(10):1195. https://doi.org/10.3390/v12101195
Chicago/Turabian StyleWieczorek, Przemysław, Marta Budziszewska, Patryk Frąckowiak, and Aleksandra Obrępalska-Stęplowska. 2020. "Development of a New Tomato Torrado Virus-Based Vector Tagged with GFP for Monitoring Virus Movement in Plants" Viruses 12, no. 10: 1195. https://doi.org/10.3390/v12101195