Supporting Information - PNAS
6
Supporting Information Butterbach et al. 10.1073/pnas.1400894111 SI Materials and Methods Plant Material and Disease Assay. Tomato cultivars with Ty-1 and Ty-3 are derived from introgressions from Solanum chilense ac- cession numbers LA1969 and LA2779 (Tomato Genetics Re- source Center, Davis, CA) as described in Verlaan et al. (1, 2), respectively, whereas the cultivar with Ty-2 contains an in- trogression derived from S. habrochaites accession B6013 as described in Kallo and Banerjee 1990 (3) (additional in- formation about B6013 such as the collection site, date of col- lection, and other passport data are unknown or not available). Tomato plants of Moneymaker (MM), Ty-1, Ty-2, and Ty-3 were grown in the greenhouse at 22 °C. Agrobacterium-mediated inoculation of the infectious tomato yellow leaf curl virus (TYLCV)-IL clone (pTYCz40a) was performed as described (2). Three-week-old seedlings were infiltrated by pressure inoculation in the leaves with a 30-gauge needle-less syringe. Top leaves were sampled after 37 and 41 d postinoculation (dpi), frozen immedi- ately in liquid nitrogen, and stored at -80 °C until use. For GFP silencing assays, plants of Nicotiana benthamiana line 16C stably expressing a functional copy of GFP (kindly provided by David Baulcombe, Cambridge, United Kingdom) were grown and infiltrated with Agrobacterium tumefaciens containing con- struct pBinGFP (4). Fourteen days postagroinfiltration (dpi), leaf tissue was harvested from top leaves exhibiting strong si- lencing of GFP, as visually monitored by UV light exposure. Three-week-old seedlings were agroinoculated with binary clones of the DNA A and B components of tomato severe rugose bego- movirus (ToSRV) isolate 1164 (kindly provided by Alice Inoue- Nagata, Empresa Brasileira de Pesquisa Agropecuária, Brasilia, Brazil). Top leaves were sampled after 40 dpi, frozen immediately in liquid nitrogen, and stored at -80 °C until use. Mixed infection with cucumber mosaic virus (CMV) isolate PRI_TOM5-4 (Plant Research International, Wageningen Uni- versity and Research Centre) was performed on 3-wk-old seed- lings by agroinfiltration with TYLCV immediately followed by mechanical inoculation of the same leaves using fresh inoculum of CMV. Top leaves were sampled after 14 dpi, frozen imme- diately in liquid nitrogen, and stored at -80 °C until use. Nucleic Acid Isolation. Total DNA and RNA were isolated from frozen leaf tissue by phenol/chloroform extraction and purified by ethanol precipitation. Nucleic acid content was checked by gel electrophoresis and quantified using a NanoDrop (Thermo Fisher Scientific). Subsequently, low molecular weight (LMW) RNA molecules were separated from total DNA/RNA prepa- rations by PEG precipitation. The DNA/RNA fraction pellet was resolved in 50 μL Tris-EDTA and stored at -20 °C for further use. For the purification of siRNAs, total LMW RNA was re- solved on a 15% denaturing polyacrylamide gel containing 8 M urea. After ethidium bromide staining, the small RNA fraction comigrating with a purified siRNA marker reference was excised from the gel, ground to small pieces, and incubated overnight at 4 °C in 3 M NaCl for diffusion extraction. After centrifugation at 500 × g for 1 min, the supernatant was collected and the siRNAs were precipitated by ethanol. ToSRV DNA samples were purified from high molecular weight plant DNA using purification columns of GF/F borosilicate glass fiber paper (GE Healthcare) as described by Borodina et al. in the protocol for purification of DNA fragments (5). For TaqMan PCR assays, DNA was extracted from young leaves using cetyltrimethyl ammonium bromide extraction (6). DNA concentration was estimated by a Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen). TaqMan PCR Assay. The TaqMan PCR assay was performed as described (7). The primers forward (F): TYLCV-SAR 1669F: 5′- GTCGAGATATTCTTTAAATGATGATTGTG-3′, reverse (R): TYLCV-cons 1756R: 5′-GGCAAGCCCATTCAAATTAAAGG-3′ were used in combination with the probe TYLCV-cons 1701T: HEX-5′-CCTGGATTGCAGAGGAAGATAGTGGGAATTC- 3′-Black Hole Quencher 1 to detect TYLCV. A TaqMan assay on COX (cytochrome oxidase 1) was used as a control as described (8). For this, primers COX F: 5′-CGTCGCATTCCAGATTA- TCCA-3′ and COX R: 5′-CAACTACGGATATATAAGRRC- CRRAACTG-3′ were used together with the probe COX-Sol 1511T: Yakima-Yellow-5′-AGGGCATTCCATCCAGCGTAA- GCA-3′-Black Hole Quencher 1. TaqMan PCR was performed on the ABI PRISM 7700 sequence detector (Applied Biosystems) with TaqMan Universal Master Mix (Applied Biosystems) using the following PCR conditions: 2 min at 50 °C followed by 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. ToSRV was detected by PCR using the primers 5′-AAGGC- GACGTCTTTGGAAGG-3′ and 5′-CTCAGCGGCCTTGTT- ATATT-3′ amplifying a fragment of 863 bp starting at position 2016 and ending at position 284 in the DNA A component within the ORFs encoding the replicase and the coat protein, respectively. The following PCR conditions were used: 2 min at 95 °C, followed by 35 cycles of 15 s at 95 °C, 30 s at 60 °C, and 1 min at 72 °C, with a final cycle of 7 min at 72 °C. Southern Blotting. siRNAs were dephosphorylated with calf intes- tinal alkaline phosphatase (Promega) and subsequently radio- labeled with [γ- 32 P]ATP by T4 polynucleotide kinase according to the manufacturer’s instructions (Invitrogen). Six sequence fragments representing the entire genome of TYLCV were amplified from the TYLCV amplicon as template using the primers 5′-TAATATTACCGGATGG-3′ and 5′-TCA- GGGCTTCGATACATTC-3′ (V2), 5′-TTACGCCTTATTGG-3′ and 5′-CCATGGAGACCTAATAG-3′ (C2), 5′-TCTCGTGG- AGTTCTCTGC-3′ and 5′-TACGGATGGCCGCTTTAATG-3′ (C1), 5′-TGTTCCCCGTGGATGTG-3′ and 5′-TTAATTTGA- TATTGAATC-3′ (V1), 5′ -TAAAATTTATATTTTATATCATG-3′ and 5′-GCGTGTAGACCTAGAC-3′ (C3), and 5′-CCGCGC- AGCGGAAGA-3′ and 5′-ATGGGGAACCACATCTC-3′ (C4). Primer sets C2 and C1 amplified the replicase sequence from the start codon of C2 to the start codon of C3, and from the stop codon of C4 to the start codon of C2, respectively. DNA con- centration of products was determined using a NanoDrop (Thermo Fisher Scientific). Equimolar amounts of purified PCR products were calculated and next resolved on a 1% agarose gel and subsequently blotted to Hybond-N membrane (Amersham Biosciences) by top-down blotting. Filters were subsequently hybridized overnight to γ- 32 P–labeled siRNAs in Church buffer at 48 °C as described by Sambrook et al. (9). Filters were washed in 2× SSC with 0.1% SDS at room temperature and subsequently exposed to a phosphor screen (Bio-Rad). Radioactive hybrid- ization signals were visualized by phosphoimaging (Molecular Dynamics Typhoon PhosphorImager; Amersham Biosciences). Bisulfite Reaction, PCR, and Cloning. Primer sets encompassing specified TYLCV genome regions were designed using the BiSearch program (10) and resulted in two primer sets: Butterbach et al. www.pnas.org/cgi/content/short/1400894111 1 of 6
Transcript of Supporting Information - PNAS
pnasSI201400894 1..6Supporting Information Butterbach et al.
10.1073/pnas.1400894111 SI Materials and Methods Plant Material and
Disease Assay. Tomato cultivars with Ty-1 and Ty-3 are derived from
introgressions from Solanum chilense ac- cession numbers LA1969 and
LA2779 (Tomato Genetics Re- source Center, Davis, CA) as described
in Verlaan et al. (1, 2), respectively, whereas the cultivar with
Ty-2 contains an in- trogression derived from S. habrochaites
accession B6013 as described in Kallo and Banerjee 1990 (3)
(additional in- formation about B6013 such as the collection site,
date of col- lection, and other passport data are unknown or not
available). Tomato plants of Moneymaker (MM), Ty-1, Ty-2, and Ty-3
were grown in the greenhouse at 22 °C. Agrobacterium-mediated
inoculation of the infectious tomato yellow leaf curl virus
(TYLCV)-IL clone (pTYCz40a) was performed as described (2).
Three-week-old seedlings were infiltrated by pressure inoculation
in the leaves with a 30-gauge needle-less syringe. Top leaves were
sampled after 37 and 41 d postinoculation (dpi), frozen immedi-
ately in liquid nitrogen, and stored at −80 °C until use. For GFP
silencing assays, plants of Nicotiana benthamiana line
16C stably expressing a functional copy of GFP (kindly provided by David Baulcombe, Cambridge, United Kingdom) were grown and infiltrated with Agrobacterium tumefaciens containing con- struct pBinGFP (4). Fourteen days postagroinfiltration (dpi), leaf tissue was harvested from top leaves exhibiting strong si- lencing of GFP, as visually monitored by UV light exposure. Three-week-old seedlings were agroinoculated with binary clones
of the DNA A and B components of tomato severe rugose bego- movirus (ToSRV) isolate 1164 (kindly provided by Alice Inoue- Nagata, Empresa Brasileira de Pesquisa Agropecuária, Brasilia, Brazil). Top leaves were sampled after 40 dpi, frozen immediately in liquid nitrogen, and stored at −80 °C until use. Mixed infection with cucumber mosaic virus (CMV) isolate
PRI_TOM5-4 (Plant Research International, Wageningen Uni- versity and Research Centre) was performed on 3-wk-old seed- lings by agroinfiltration with TYLCV immediately followed by mechanical inoculation of the same leaves using fresh inoculum of CMV. Top leaves were sampled after 14 dpi, frozen imme- diately in liquid nitrogen, and stored at −80 °C until use.
Nucleic Acid Isolation. Total DNA and RNA were isolated from frozen leaf tissue by phenol/chloroform extraction and purified by ethanol precipitation. Nucleic acid content was checked by gel electrophoresis and quantified using a NanoDrop (Thermo Fisher Scientific). Subsequently, low molecular weight (LMW) RNA molecules were separated from total DNA/RNA prepa- rations by PEG precipitation. The DNA/RNA fraction pellet was resolved in 50 μL Tris-EDTA and stored at −20 °C for further use. For the purification of siRNAs, total LMW RNA was re- solved on a 15% denaturing polyacrylamide gel containing 8 M urea. After ethidium bromide staining, the small RNA fraction comigrating with a purified siRNA marker reference was excised from the gel, ground to small pieces, and incubated overnight at 4 °C in 3 M NaCl for diffusion extraction. After centrifugation at 500 × g for 1 min, the supernatant was collected and the siRNAs were precipitated by ethanol. ToSRV DNA samples were purified from high molecular weight
plant DNA using purification columns of GF/F borosilicate glass fiber paper (GE Healthcare) as described by Borodina et al. in the protocol for purification of DNA fragments (5). For TaqMan PCR assays, DNA was extracted from young
leaves using cetyltrimethyl ammonium bromide extraction (6).
DNA concentration was estimated by a Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen).
TaqMan PCR Assay. The TaqMan PCR assay was performed as described (7). The primers forward (F): TYLCV-SAR 1669F: 5′- GTCGAGATATTCTTTAAATGATGATTGTG-3′, reverse (R): TYLCV-cons 1756R: 5′-GGCAAGCCCATTCAAATTAAAGG-3′ were used in combination with the probe TYLCV-cons 1701T: HEX-5′-CCTGGATTGCAGAGGAAGATAGTGGGAATTC- 3′-Black Hole Quencher 1 to detect TYLCV. A TaqMan assay on COX (cytochrome oxidase 1) was used as a control as described (8). For this, primers COX F: 5′-CGTCGCATTCCAGATTA- TCCA-3′ and COX R: 5′-CAACTACGGATATATAAGRRC- CRRAACTG-3′ were used together with the probe COX-Sol 1511T: Yakima-Yellow-5′-AGGGCATTCCATCCAGCGTAA- GCA-3′-Black Hole Quencher 1. TaqMan PCR was performed on the ABI PRISM 7700 sequence detector (Applied Biosystems) with TaqMan Universal Master Mix (Applied Biosystems) using the following PCR conditions: 2 min at 50 °C followed by 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. ToSRV was detected by PCR using the primers 5′-AAGGC-
GACGTCTTTGGAAGG-3′ and 5′-CTCAGCGGCCTTGTT- ATATT-3′ amplifying a fragment of 863 bp starting at position 2016 and ending at position 284 in the DNA A component within the ORFs encoding the replicase and the coat protein, respectively. The following PCR conditions were used: 2 min at 95 °C, followed by 35 cycles of 15 s at 95 °C, 30 s at 60 °C, and 1 min at 72 °C, with a final cycle of 7 min at 72 °C.
Southern Blotting. siRNAs were dephosphorylated with calf intes- tinal alkaline phosphatase (Promega) and subsequently radio- labeled with [γ-32P]ATP by T4 polynucleotide kinase according to the manufacturer’s instructions (Invitrogen). Six sequence fragments representing the entire genome of
TYLCV were amplified from the TYLCV amplicon as template using the primers 5′-TAATATTACCGGATGG-3′ and 5′-TCA- GGGCTTCGATACATTC-3′ (V2), 5′-TTACGCCTTATTGG-3′ and 5′-CCATGGAGACCTAATAG-3′ (C2), 5′-TCTCGTGG- AGTTCTCTGC-3′ and 5′-TACGGATGGCCGCTTTAATG-3′ (C1), 5′-TGTTCCCCGTGGATGTG-3′ and 5′-TTAATTTGA- TATTGAATC-3′ (V1), 5′-TAAAATTTATATTTTATATCATG-3′ and 5′-GCGTGTAGACCTAGAC-3′ (C3), and 5′-CCGCGC- AGCGGAAGA-3′ and 5′-ATGGGGAACCACATCTC-3′ (C4). Primer sets C2 and C1 amplified the replicase sequence from the start codon of C2 to the start codon of C3, and from the stop codon of C4 to the start codon of C2, respectively. DNA con- centration of products was determined using a NanoDrop (Thermo Fisher Scientific). Equimolar amounts of purified PCR products were calculated and next resolved on a 1% agarose gel and subsequently blotted to Hybond-N membrane (Amersham Biosciences) by top-down blotting. Filters were subsequently hybridized overnight to γ-32P–labeled siRNAs in Church buffer at 48 °C as described by Sambrook et al. (9). Filters were washed in 2× SSC with 0.1% SDS at room temperature and subsequently exposed to a phosphor screen (Bio-Rad). Radioactive hybrid- ization signals were visualized by phosphoimaging (Molecular Dynamics Typhoon PhosphorImager; Amersham Biosciences).
Bisulfite Reaction, PCR, and Cloning. Primer sets encompassing specified TYLCV genome regions were designed using the BiSearch program (10) and resulted in two primer sets:
Butterbach et al. www.pnas.org/cgi/content/short/1400894111 1 of 6
5′-TGGTCCCCAAGTATTTTGTC-3′ and 5′-TCGCTTGTTT- GTGCCTTGGA-3′ (V1) and 5′-CGGTGTATCGGTGTCT- TATT-3′ and 5′-CTCGTAAGTTTCCTCAACGG-3′ (V2). For ToSRV, primer set 5′-CAATTAAAGTAAAGTGATTGT-3′ and 5′-CTCAGCGGCCTTGTTATATT-3′ was used. Bisulfite modification was carried out on DNA isolated using
the Qiagen Bisulfite Kit according to the manufacturer’s proce- dures. PCR amplification was carried out using GoTaq (Promega),
and PCR products were purified and cloned into the pGEM-T Easy Vector System (Promega) for further analysis.
Sequence Analysis. Sequence analysis was done according to Sanger- dideoxy sequencing (Eurofins Genomics; MWG Operon). The re- trieved sequences were aligned to the TYLCV(Alm) (GenBank accession no. AJ489258) or ToSRV1164 reference sequences using BioEdit software (11) for the identification of transformed cytosines.
1. Verlaan MG, et al. (2011) Chromosomal rearrangements between tomato and Solanum chilense hamper mapping and breeding of the TYLCV resistance gene Ty-1. Plant J 68(6):1093–1103.
2. Verlaan MG, et al. (2013) The tomato yellow leaf curl virus resistance genes Ty-1 and Ty-3 are allelic and code for DFDGD-class RNA-dependent RNA polymerases. PLoS Genet 9(3):e1003399.
3. Kalloo G, Banerjee MK (1990) Transfer of tomato leaf curl virus resistance from Lycopersicon hirsutum f. glabratum to L. esculentum. Plant Breeding 105:156–159.
4. Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544. 5. Borodina TA, Lehrach H, Soldatov AV (2003) DNA purification on homemade silica
spin-columns. Anal Biochem 321(1):135–137. 6. Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh
leaf tissue. Phytochem Bull 19(1):11–15.
7. Powell ME, et al. (2012) First record of the Q biotype of the sweetpotato whitefly, Bemisia tabaci, intercepted in the UK. Eur J Plant Pathol 133(4):1–5.
8. van Gent-Pelzer MP, Krijger M, Bonants PJ (2010) Improved real-time PCR assay for detection of the quarantine potato pathogen, Synchytrium endobioticum, in zonal centrifuge extracts from soil and in plants. Eur J Plant Pathol 126(1):129–133.
9. Sambrook J, Frisch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab Press, Cold Spring Harbor, NY).
10. Tusnády GE, Simon I, Váradi A, Arányi T (2005) BiSearch: Primer-design and search tool for PCR on bisulfite treated genomes. Nucleic Acids Research 33:e9.
11. Hall TA (1999) BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98.
Fig. S1. Systemic leaves of plants that were mock inoculated or infected with TYLCV after local agroinfiltration with an infectious TYLCV clone, 21 dpi, as described in Table S1.
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Line, no. Virus Ct-TYLCV Ct-COX S*
MM 1 TYLCV 18.35 21.07 3 MM 2 TYLCV 18.76 21.88 3 MM 3 TYLCV 19.16 21.41 3 MM 4 TYLCV 19.43 21.08 3 MM 5 TYLCV 19.71 21.49 3 Ty-1 1 TYLCV 37.60 21.82 1 Ty-1 2 TYLCV No product 21.98 1 Ty-1 3 TYLCV 22.50 22.44 1 Ty-1 4 TYLCV 23.30 22.20 1 Ty-1 5 TYLCV 36.03 22.24 1 Ty-3 1 TYLCV 21.59 21.55 2 Ty-3 2 TYLCV 21.92 21.93 2 Ty-3 3 TYLCV 38.11 22.36 1 Ty-3 4 TYLCV No product 21.95 1 Ty-3 5 TYLCV 21.85 21.72 2 MM 1 Mock No product 21.85 —
MM 2 Mock No product 21.85 —
Ty-1 1 Mock 39.17 22.36 —
Ty-3 1 Mock 39.52 22.19 —
Results of TaqMan PCR for TYLCV and COX as cycle thresholds (Ct). Symp- toms of tomato plants are at 21 dpi. *Symptoms: 1, no symptoms; 2, yellowing on lower leaves, size as mock infected plants; 3, plants are clearly stunted and yellowing.
Table S2. Control experiment for bisulfite conversion efficiency for TYLCV and ToSRV using bacterial derived and therefore unmethylated virus DNA for bisulfite conversion and subsequent PCR, cloning, and sequencing of clones
Virus amplicon used
TYLCV 13 13 ToSRV 5 5
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Line, no. Virus Ct-TYLCV Ct-COX
MM 1 CMV 29.02 21.25 MM 2 CMV 29.45 20.84 MM 3 CMV 30.41 20.80 MM 4 CMV 30.01 21.36 MM 5 CMV 29.65 22.53 MM 1 TYLCV 11.95 20.73 MM 2 TYLCV 11.66 20.21 MM 3 TYLCV 11.69 20.29 MM 4 TYLCV 14.67 20.47 MM 1 CMV+TYLCV 11.90 20.04 MM 2 CMV+TYLCV 11.66 20.03 MM 3 CMV+TYLCV 11.90 20.93 MM 4 CMV+TYLCV 18.08 19.94 Ty-1 1 CMV 28.93 21.61 Ty-1 2 CMV 29.95 22.06 Ty-1 3 CMV 27.92 21.87 Ty-1 4 CMV 30.08 21.55 Ty-1 5 CMV 28.70 20.88 Ty-1 1 TYLCV 27.36 19.91 Ty-1 2 TYLCV 28.91 19.76 Ty-1 3 TYLCV 29.52 19.74 Ty-1 4 TYLCV 18.97 20.68 Ty-1 5 TYLCV 28.87 22.00 Ty-1 1 CMV+TYLCV 18.20 20.25 Ty-1 2 CMV+TYLCV 25.79 19.43 Ty-1 3 CMV+TYLCV 19.71 20.40 Ty-1 4 CMV+TYLCV 21.07 19.83 Ty-3 1 CMV 27.83 20.92 Ty-3 2 CMV 25.23 20.76 Ty-3 3 CMV 29.41 21.92 Ty-3 4 CMV 26.62 21.99 Ty-3 1 TYLCV 20.00 20.49 Ty-3 2 TYLCV 27.74 21.50 Ty-3 3 TYLCV 27.79 No product Ty-3 4 TYLCV 14.38 21.91 Ty-3 5 TYLCV 18.83 20.29 Ty-3 1 CMV+TYLCV 19.21 21.46 Ty-3 2 CMV+TYLCV 17.36 21.67 Ty-3 3 CMV+TYLCV 27.27 20.45 Ty-3 4 CMV+TYLCV 14.80 20.17 MM 1 Mock 35.17 21.83 Ty-1 1 Mock 37.01 22.75 Ty-3 1 Mock 31.91 22.39
Results of TaqMan PCR for TYLCV and COX on tomato plants at 14 d postinoculation.
Butterbach et al. www.pnas.org/cgi/content/short/1400894111 6 of 6
16C stably expressing a functional copy of GFP (kindly provided by David Baulcombe, Cambridge, United Kingdom) were grown and infiltrated with Agrobacterium tumefaciens containing con- struct pBinGFP (4). Fourteen days postagroinfiltration (dpi), leaf tissue was harvested from top leaves exhibiting strong si- lencing of GFP, as visually monitored by UV light exposure. Three-week-old seedlings were agroinoculated with binary clones
of the DNA A and B components of tomato severe rugose bego- movirus (ToSRV) isolate 1164 (kindly provided by Alice Inoue- Nagata, Empresa Brasileira de Pesquisa Agropecuária, Brasilia, Brazil). Top leaves were sampled after 40 dpi, frozen immediately in liquid nitrogen, and stored at −80 °C until use. Mixed infection with cucumber mosaic virus (CMV) isolate
PRI_TOM5-4 (Plant Research International, Wageningen Uni- versity and Research Centre) was performed on 3-wk-old seed- lings by agroinfiltration with TYLCV immediately followed by mechanical inoculation of the same leaves using fresh inoculum of CMV. Top leaves were sampled after 14 dpi, frozen imme- diately in liquid nitrogen, and stored at −80 °C until use.
Nucleic Acid Isolation. Total DNA and RNA were isolated from frozen leaf tissue by phenol/chloroform extraction and purified by ethanol precipitation. Nucleic acid content was checked by gel electrophoresis and quantified using a NanoDrop (Thermo Fisher Scientific). Subsequently, low molecular weight (LMW) RNA molecules were separated from total DNA/RNA prepa- rations by PEG precipitation. The DNA/RNA fraction pellet was resolved in 50 μL Tris-EDTA and stored at −20 °C for further use. For the purification of siRNAs, total LMW RNA was re- solved on a 15% denaturing polyacrylamide gel containing 8 M urea. After ethidium bromide staining, the small RNA fraction comigrating with a purified siRNA marker reference was excised from the gel, ground to small pieces, and incubated overnight at 4 °C in 3 M NaCl for diffusion extraction. After centrifugation at 500 × g for 1 min, the supernatant was collected and the siRNAs were precipitated by ethanol. ToSRV DNA samples were purified from high molecular weight
plant DNA using purification columns of GF/F borosilicate glass fiber paper (GE Healthcare) as described by Borodina et al. in the protocol for purification of DNA fragments (5). For TaqMan PCR assays, DNA was extracted from young
leaves using cetyltrimethyl ammonium bromide extraction (6).
DNA concentration was estimated by a Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen).
TaqMan PCR Assay. The TaqMan PCR assay was performed as described (7). The primers forward (F): TYLCV-SAR 1669F: 5′- GTCGAGATATTCTTTAAATGATGATTGTG-3′, reverse (R): TYLCV-cons 1756R: 5′-GGCAAGCCCATTCAAATTAAAGG-3′ were used in combination with the probe TYLCV-cons 1701T: HEX-5′-CCTGGATTGCAGAGGAAGATAGTGGGAATTC- 3′-Black Hole Quencher 1 to detect TYLCV. A TaqMan assay on COX (cytochrome oxidase 1) was used as a control as described (8). For this, primers COX F: 5′-CGTCGCATTCCAGATTA- TCCA-3′ and COX R: 5′-CAACTACGGATATATAAGRRC- CRRAACTG-3′ were used together with the probe COX-Sol 1511T: Yakima-Yellow-5′-AGGGCATTCCATCCAGCGTAA- GCA-3′-Black Hole Quencher 1. TaqMan PCR was performed on the ABI PRISM 7700 sequence detector (Applied Biosystems) with TaqMan Universal Master Mix (Applied Biosystems) using the following PCR conditions: 2 min at 50 °C followed by 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. ToSRV was detected by PCR using the primers 5′-AAGGC-
GACGTCTTTGGAAGG-3′ and 5′-CTCAGCGGCCTTGTT- ATATT-3′ amplifying a fragment of 863 bp starting at position 2016 and ending at position 284 in the DNA A component within the ORFs encoding the replicase and the coat protein, respectively. The following PCR conditions were used: 2 min at 95 °C, followed by 35 cycles of 15 s at 95 °C, 30 s at 60 °C, and 1 min at 72 °C, with a final cycle of 7 min at 72 °C.
Southern Blotting. siRNAs were dephosphorylated with calf intes- tinal alkaline phosphatase (Promega) and subsequently radio- labeled with [γ-32P]ATP by T4 polynucleotide kinase according to the manufacturer’s instructions (Invitrogen). Six sequence fragments representing the entire genome of
TYLCV were amplified from the TYLCV amplicon as template using the primers 5′-TAATATTACCGGATGG-3′ and 5′-TCA- GGGCTTCGATACATTC-3′ (V2), 5′-TTACGCCTTATTGG-3′ and 5′-CCATGGAGACCTAATAG-3′ (C2), 5′-TCTCGTGG- AGTTCTCTGC-3′ and 5′-TACGGATGGCCGCTTTAATG-3′ (C1), 5′-TGTTCCCCGTGGATGTG-3′ and 5′-TTAATTTGA- TATTGAATC-3′ (V1), 5′-TAAAATTTATATTTTATATCATG-3′ and 5′-GCGTGTAGACCTAGAC-3′ (C3), and 5′-CCGCGC- AGCGGAAGA-3′ and 5′-ATGGGGAACCACATCTC-3′ (C4). Primer sets C2 and C1 amplified the replicase sequence from the start codon of C2 to the start codon of C3, and from the stop codon of C4 to the start codon of C2, respectively. DNA con- centration of products was determined using a NanoDrop (Thermo Fisher Scientific). Equimolar amounts of purified PCR products were calculated and next resolved on a 1% agarose gel and subsequently blotted to Hybond-N membrane (Amersham Biosciences) by top-down blotting. Filters were subsequently hybridized overnight to γ-32P–labeled siRNAs in Church buffer at 48 °C as described by Sambrook et al. (9). Filters were washed in 2× SSC with 0.1% SDS at room temperature and subsequently exposed to a phosphor screen (Bio-Rad). Radioactive hybrid- ization signals were visualized by phosphoimaging (Molecular Dynamics Typhoon PhosphorImager; Amersham Biosciences).
Bisulfite Reaction, PCR, and Cloning. Primer sets encompassing specified TYLCV genome regions were designed using the BiSearch program (10) and resulted in two primer sets:
Butterbach et al. www.pnas.org/cgi/content/short/1400894111 1 of 6
5′-TGGTCCCCAAGTATTTTGTC-3′ and 5′-TCGCTTGTTT- GTGCCTTGGA-3′ (V1) and 5′-CGGTGTATCGGTGTCT- TATT-3′ and 5′-CTCGTAAGTTTCCTCAACGG-3′ (V2). For ToSRV, primer set 5′-CAATTAAAGTAAAGTGATTGT-3′ and 5′-CTCAGCGGCCTTGTTATATT-3′ was used. Bisulfite modification was carried out on DNA isolated using
the Qiagen Bisulfite Kit according to the manufacturer’s proce- dures. PCR amplification was carried out using GoTaq (Promega),
and PCR products were purified and cloned into the pGEM-T Easy Vector System (Promega) for further analysis.
Sequence Analysis. Sequence analysis was done according to Sanger- dideoxy sequencing (Eurofins Genomics; MWG Operon). The re- trieved sequences were aligned to the TYLCV(Alm) (GenBank accession no. AJ489258) or ToSRV1164 reference sequences using BioEdit software (11) for the identification of transformed cytosines.
1. Verlaan MG, et al. (2011) Chromosomal rearrangements between tomato and Solanum chilense hamper mapping and breeding of the TYLCV resistance gene Ty-1. Plant J 68(6):1093–1103.
2. Verlaan MG, et al. (2013) The tomato yellow leaf curl virus resistance genes Ty-1 and Ty-3 are allelic and code for DFDGD-class RNA-dependent RNA polymerases. PLoS Genet 9(3):e1003399.
3. Kalloo G, Banerjee MK (1990) Transfer of tomato leaf curl virus resistance from Lycopersicon hirsutum f. glabratum to L. esculentum. Plant Breeding 105:156–159.
4. Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544. 5. Borodina TA, Lehrach H, Soldatov AV (2003) DNA purification on homemade silica
spin-columns. Anal Biochem 321(1):135–137. 6. Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh
leaf tissue. Phytochem Bull 19(1):11–15.
7. Powell ME, et al. (2012) First record of the Q biotype of the sweetpotato whitefly, Bemisia tabaci, intercepted in the UK. Eur J Plant Pathol 133(4):1–5.
8. van Gent-Pelzer MP, Krijger M, Bonants PJ (2010) Improved real-time PCR assay for detection of the quarantine potato pathogen, Synchytrium endobioticum, in zonal centrifuge extracts from soil and in plants. Eur J Plant Pathol 126(1):129–133.
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Fig. S1. Systemic leaves of plants that were mock inoculated or infected with TYLCV after local agroinfiltration with an infectious TYLCV clone, 21 dpi, as described in Table S1.
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Line, no. Virus Ct-TYLCV Ct-COX S*
MM 1 TYLCV 18.35 21.07 3 MM 2 TYLCV 18.76 21.88 3 MM 3 TYLCV 19.16 21.41 3 MM 4 TYLCV 19.43 21.08 3 MM 5 TYLCV 19.71 21.49 3 Ty-1 1 TYLCV 37.60 21.82 1 Ty-1 2 TYLCV No product 21.98 1 Ty-1 3 TYLCV 22.50 22.44 1 Ty-1 4 TYLCV 23.30 22.20 1 Ty-1 5 TYLCV 36.03 22.24 1 Ty-3 1 TYLCV 21.59 21.55 2 Ty-3 2 TYLCV 21.92 21.93 2 Ty-3 3 TYLCV 38.11 22.36 1 Ty-3 4 TYLCV No product 21.95 1 Ty-3 5 TYLCV 21.85 21.72 2 MM 1 Mock No product 21.85 —
MM 2 Mock No product 21.85 —
Ty-1 1 Mock 39.17 22.36 —
Ty-3 1 Mock 39.52 22.19 —
Results of TaqMan PCR for TYLCV and COX as cycle thresholds (Ct). Symp- toms of tomato plants are at 21 dpi. *Symptoms: 1, no symptoms; 2, yellowing on lower leaves, size as mock infected plants; 3, plants are clearly stunted and yellowing.
Table S2. Control experiment for bisulfite conversion efficiency for TYLCV and ToSRV using bacterial derived and therefore unmethylated virus DNA for bisulfite conversion and subsequent PCR, cloning, and sequencing of clones
Virus amplicon used
TYLCV 13 13 ToSRV 5 5
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Line, no. Virus Ct-TYLCV Ct-COX
MM 1 CMV 29.02 21.25 MM 2 CMV 29.45 20.84 MM 3 CMV 30.41 20.80 MM 4 CMV 30.01 21.36 MM 5 CMV 29.65 22.53 MM 1 TYLCV 11.95 20.73 MM 2 TYLCV 11.66 20.21 MM 3 TYLCV 11.69 20.29 MM 4 TYLCV 14.67 20.47 MM 1 CMV+TYLCV 11.90 20.04 MM 2 CMV+TYLCV 11.66 20.03 MM 3 CMV+TYLCV 11.90 20.93 MM 4 CMV+TYLCV 18.08 19.94 Ty-1 1 CMV 28.93 21.61 Ty-1 2 CMV 29.95 22.06 Ty-1 3 CMV 27.92 21.87 Ty-1 4 CMV 30.08 21.55 Ty-1 5 CMV 28.70 20.88 Ty-1 1 TYLCV 27.36 19.91 Ty-1 2 TYLCV 28.91 19.76 Ty-1 3 TYLCV 29.52 19.74 Ty-1 4 TYLCV 18.97 20.68 Ty-1 5 TYLCV 28.87 22.00 Ty-1 1 CMV+TYLCV 18.20 20.25 Ty-1 2 CMV+TYLCV 25.79 19.43 Ty-1 3 CMV+TYLCV 19.71 20.40 Ty-1 4 CMV+TYLCV 21.07 19.83 Ty-3 1 CMV 27.83 20.92 Ty-3 2 CMV 25.23 20.76 Ty-3 3 CMV 29.41 21.92 Ty-3 4 CMV 26.62 21.99 Ty-3 1 TYLCV 20.00 20.49 Ty-3 2 TYLCV 27.74 21.50 Ty-3 3 TYLCV 27.79 No product Ty-3 4 TYLCV 14.38 21.91 Ty-3 5 TYLCV 18.83 20.29 Ty-3 1 CMV+TYLCV 19.21 21.46 Ty-3 2 CMV+TYLCV 17.36 21.67 Ty-3 3 CMV+TYLCV 27.27 20.45 Ty-3 4 CMV+TYLCV 14.80 20.17 MM 1 Mock 35.17 21.83 Ty-1 1 Mock 37.01 22.75 Ty-3 1 Mock 31.91 22.39
Results of TaqMan PCR for TYLCV and COX on tomato plants at 14 d postinoculation.
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