DNA Methylation Suppression by Bhendi Yellow Vein Mosaic Virus
Abstract
:1. Introduction
2. Results
2.1. BYVMV Interferes with DNA Methylation
2.2. BYVMV Gene(s) Involved in Methylation Suppression
2.3. BYVMV C4 and C2 Hinders Transcriptional Gene Silencing
2.4. C4 Mutation Impairs Viral Movement
3. Discussion
4. Materials and Methods
4.1. Cloning
4.2. Agroinfiltration
4.3. Viral DNA Isolation
4.4. Chop-PCR
4.5. Southern Hybridization
4.6. Quantitative PCR (qPCR)
4.7. Protoplast Isolation and Transfection
4.8. Bisulfite Conversion
4.9. RCA
4.10. DNA Processing for Illumina Sequencing and Library Preparation
4.11. Mapping Cytosine Methylation Using MethGo
Supplementary Materials
Author Contributions
Acknowledgments
Conflicts of Interest
References
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S. No | Primers | Sequences | Target DNA & Nucleotide Coordinate | Details of Experimental Use |
---|---|---|---|---|
1 | BYVMV DNA A IR Forward | 5′ GTTGACTATTTTTGAGACCCGATT 3′ | BYVMV DNA (1–24) | Chop-PCR |
2 | BYVMV DNA A IR Reverse | 5′ GGATCCCACATTTTTTGAATTG 3′ | BYVMV DNA A (285–264) | Chop-PCR |
3 | BglII BYVMV AV2 Forward | 5′ AGATCTATGTGGGATCCACTATTAAACG 3′ | BYVMV DNA A (119–131) | Chop-PCR |
4 | HindIII BYVMV AV2 Reverse | 5′ AAGCTTTCACATCCCCTTGGAACATCC 3′ | BYVMV DNA A (485–465) | Chop-PCR |
5 | HindIII BYVMV C2 Forward | 5′ AAGCTTATGCAGCATTCGTCTTTC 3′ | BYVMV DNA A (1584–1601) | Chop-PCR |
6 | BglII BYVMV C2 Reverse | 5′ AGATCTTTAGAGATATTTGAGGAC 3′ | BYVMV DNA A (1170–1190) | Chop-PCR |
7 | BamHI BYVMV AC4 Forward | 5′ GGATCCTTATCTTTAAGAACTCTAAGAGC 3′ | BYVMV DNA A (2130–2154) | Chop-PCR |
8 | HindIII BYVMV AC4 Reverse | 5′ AGCTTATGAAAATGGGGAACCTCATCTTCACG 3′ | BYVMV DNA A (2438–2412) | Chop-PCR |
9 | BglII BYVMV βC1 Forward | 5′ AGATCTAATTATTATCTTATTATCAATAGTAC 3′ | BYVMV DNA β (180–207) | Chop-PCR |
10 | HindIII BYVMV βC1 Reverse | 5′ AAGCTTATGAAAATATCTATACATTTCATC 3′ | BYVMV DNA β (602–578) | Chop-PCR |
11 | βC1 Promoter Forward | 5′ ATAATGTGTGGATGAAATTTATAGA 3′ | BYVMV DNA β 603–627 | Chop-PCR |
12 | βC1 Promoter Reverse | 5′ CTCATTTACCTATCGGTGTCTG 3′ | BYVMV DNA β 1080–1059 | Chop-PCR |
13 | BYVMV V2 RT Forward | 5′ GCAACTTTTGTCGCAGGATT 3′ | BYVMV DNA A (194–213) | Quantitative PCR |
14 | BYVMV V2 RT Reverse | 5′ ATAGGCCTGTTTGTCCATGC 3′ | BYVMV DNA A (406–425) | Quantitative PCR |
15 | BYVMV β RT Forward | 5′ CGCGCGGTAAATGGTAAATA 3′ | BYVMV DNA β (1017–1036) | Quantitative PCR |
16 | BYVMV β RT Reverse | 5′ CTACGACGCGCGATATAACT 3′ | BYVMV DNA β (1158–1177) | Quantitative PCR |
17 | C4 stop 1 Forward | 5′ ATT TTCCTTCGAACTGGATTAGCACGTGAAGATGAGGTT 3′ | BYVMV DNA A (2386–2426) | Site-specific mutagenesis |
18 | C4 stop 1 Reverse | 5′ AACCTCATCTTCACGTGCTAATCCAGTTCGAAGGAAAAT 3′ | BYVMV DNA A (2387–2426) | Site-specific mutagenesis |
19 | C2 stop 1 Forward | 5′ CCGAGTACAACGGTTGGGTTAGAAAGACGAATGCTGCAT 3′ | BYVMV DNA A 1563–1600 | Site-specific mutagenesis |
20 | C2 stop 1 Reverse | 5′ ATGCAGCATTCGTCTTTCTAACCCAACCGTTGTACTCGG 3′ | BYVMV DNA A 1600–1563 | Site-specific mutagenesis |
21 | C2 stop 2 Forward | 5′CGCTTCCTTGTGTTGAACTTAGATTGGTACCCGAGTACA 3′ | BYVMV DNA A 1533–1571 | Site-specific mutagenesis |
22 | C2 stop 2 Reverse | 5′ TGTACTCGGGTACCAATCTAAGTTCAACACAAGGAAGCG 3′ | BYVMV DNA A 1571–1533 | Site-specific mutagenesis |
S.No | Type of Infiltration | Total No. of Plants Infiltrated | Severe Symptomatic Plants | Mild Symptomatic Plants | Asymptomatic |
---|---|---|---|---|---|
1 | APTR alone | 22 | 0 | 13 | 9 |
2 | APTR + βPTR | 27 | 13 | 4 | 10 |
3 | C4MAPTR alone | 10 | 0 | 0 | 10 |
4 | C4MAPTR + βPTR | 10 | 0 | 0 | 10 |
5 | C2MAPTR alone | 10 | 0 | 0 | 10 |
6 | C2MAPTR + βPTR | 10 | 0 | 0 | 10 |
7 | βPTR alone | 14 | 0 | 0 | 14 |
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Babu, K.S.D.; Guria, A.; Karanthamalai, J.; Srikakulam, N.; Kumari, K.; Sharma, P.; Chandran, S.A.; Barnabas, A.D.; Tennyson, J.; Pandi, G. DNA Methylation Suppression by Bhendi Yellow Vein Mosaic Virus. Epigenomes 2018, 2, 7. https://doi.org/10.3390/epigenomes2020007
Babu KSD, Guria A, Karanthamalai J, Srikakulam N, Kumari K, Sharma P, Chandran SA, Barnabas AD, Tennyson J, Pandi G. DNA Methylation Suppression by Bhendi Yellow Vein Mosaic Virus. Epigenomes. 2018; 2(2):7. https://doi.org/10.3390/epigenomes2020007
Chicago/Turabian StyleBabu, Kandhalu Sagadevan Dinesh, Ashirbad Guria, Jeyalakshmi Karanthamalai, Nagesh Srikakulam, Kamlesh Kumari, Priyanka Sharma, Sam Aldrin Chandran, Anburaj Daniel Barnabas, Jebasingh Tennyson, and Gopal Pandi. 2018. "DNA Methylation Suppression by Bhendi Yellow Vein Mosaic Virus" Epigenomes 2, no. 2: 7. https://doi.org/10.3390/epigenomes2020007