Li Wang, Fabio C. Rinaldi, Pallavi Singh, Erin L. Doyle, Zoe E. Dubrow, Tuan Tu Tran, Alvaro L. Pérez-Quintero, Boris Szurek, and Adam J.

Size: px

Start display at page:

Download "Li Wang, Fabio C. Rinaldi, Pallavi Singh, Erin L. Doyle, Zoe E. Dubrow, Tuan Tu Tran, Alvaro L. Pérez-Quintero, Boris Szurek, and Adam J."

Louise Barber
6 years ago
Views:

1 TAL effectors drive transcription bidirectionally in plants Li Wang, Fabio C. Rinaldi, Pallavi Singh, Erin L. Doyle, Zoe E. Dubrow, Tuan Tu Tran, Alvaro L. Pérez-Quintero, Boris Szurek, and Adam J. Bogdanove SUPPLEMENTARY INFORMATION Figure S1. Determination of apparent binding affinity (K Dapp ) of select dtales for their corresponding EBEs: representative electrophoretic mobility shift assay (EMSA) data. Related to Figure 1 and Figure 2. Different concentrations of each dtale protein from the pairs dt1456/dt1457, dt1383/dt1384 and dt436/dt1296 were incubated in the presence of DNA duplexes tagged with biotin. DNA, and protein-dna complexes (asterisks), were visualized by chemiluminescence using streptavidin-hrp conjugate. EMSA data for the forward strand dtales are shown on the left and for reverse dtales on the right. The apparent dissociation constants (K Dapp ) are shown with their respective standard deviations calculated using two independent measurements from two independent protein purifications. 1

2 Figure S2. Map of the bidirectional luciferase reporter plasmid pps1 used to assess divergent transcription in plants. Related to Figure 4 and Figure 5. A KpnI site between the firefly and the renilla luciferase coding sequences allows insertion of test promoters. A Gateway cassette (ThermoFisher Scientific, Waltham, MA) allows introduction of a TAL effector coding sequence from entry vector ptal2 (Cermak et al., 2011) for translational fusion to a C-terminal FLAG epitope and expression under the 35S promoter. The vector was constructed by using binary vector pgwb511 (Nakagawa et al., 2007) as a backbone and incorporating the luc2 and Rluc luciferase coding sequences of vectors pgl2 and prl2, respectively (Promega Corporation, Madison, WI) (see Methods). FFLuc, firefly luciferase; RLuc, renilla luciferase; SmR, spectinomycin resistance gene; LB, left border repeat of the T-DNA; RB, right border repeat of the T-DNA. 2

release 1.1, http://snp-seek.irri.org/_snp.zul; Alexandrov et al.

3 a. b. Figure S3. Conservation of the Tal3c EBEs in the Os09g39810 promoter. a, A JBrowse view of single nucleotide polymorphisms (SNPs) and insertions and deletions (indels) relative to the Nipponbare reference across 3,380 rice genomes (Full 3k RG SNP & indel Datasets release 1.1, Alexandrov et al., 2015) in a roughly 675 bp region of DNA upstream of the Os09g39810 start codon encompassing the Tal3c forward and reverse strand EBEs. b, A closer view, across roughly 180 bp. The SNP in the Tal3c reverse EBE (top strand in this view) is a G to A (not shown) at position 12. The RVD at this position in Tal3c, NN, has a similar preference for A and G, so this SNP would not be expected to impact binding. 98.8% of the genomes (3,341) have 3

4 the A allele, while 1.2% (39) appear to be homozygous for the G allele, or heterozygous (G/A). The latter include genomes from the subpopulations admix, aro, ind1a, ind1b, ind2, ind3, indx, temp, and trop. Table S1. RVD and target EBE sequences for Tal2g, Tal3c, and the dtales used in this study. TAL effector RVD sequence a EBE (5-3 ) b Target locus Target strand Source Tal2g NN NN ND N* NS NS YG NI SN HD HD NG GGCCCGTAGCCTCTCCT Os01g (Cernadas et HD NS NN HD NG al., 2014) Tal3c NN HD NI HD NN NG NG HD NN HD N* NN AAACAACCCCCGCCCC Os09g HD HD HD NG Tal3c NN HD NI HD NN NG NG HD NN HD N* NN ACACCTACACTGCACT Os09g HD HD HD NG dt436 HD HD NI NI NI NI HD NN NG NI NI NG CCAAAACGTAATTTAGAAT Os01g (Cernadas et NG NG NI NN NI NI NG al., 2014) dt437 HD NG NN NI NN HD NI HD NI HD NN HD CTGAGCACACGCTGCACGTAAT Os01g (Cernadas et NG NN HD NI HD NN NG NI NI NG al., 2014) dt1294 NG NI HD NH NG NH HD NI NH HD NH NG TACGTGCAGCGTGTGCTCAGAA Os01g NH NG NH HD NG HD NI NH NI NI dt1296 NG HD NG NI NI NI NG NG NI HD NH NG TCTAAATTACGTTTTGGAT Os01g NG NG NG NH NH NI NG dt1381 HD NI HD NI HD NI HD HD NI NG NI NI CACACACCATAAGGGCAT Os11g (Streubel et al., NH NH NH HD NI NG 2013) dt1382 NH HD HD HD NG NG NI NG NH NH NG NH GCCCTTATGGTGTGTGAC Os11g (Streubel et al., NG NH NG NH NI HD 2013) dt1383 HD NI NG NH NG NH NG NH HD HD NG NG CATGTGTGCCTTTTCATT Os11g NG NG HD NI NG NG dt1384 NH NI NI NI NI NH NH HD NI HD NI HD GAAAAGGCACACATGACC Os11g NI NG NH NI HD HD dt1452 NI NH NH HD NG HD NI NH NI NG HD NG AGGCTCAGATCTAAACTCCT Os01g NI NI NI HD NG HD HD NG dt1453 NI NH NH NI NH NG NG NG NI NH NI NG AGGAGTTTAGATCTGAGCCT Os01g HD NG NH NI NH HD HD NG dt1454 HD HD NH NG NH HD HD NG HD NG NG NH CCGTGCCTCTTGAAAGTTC Os01g NI NI NI NH NG NG HD dt1455 NH NI NI HD NG NG NG HD NI NI NH NI GAACTTTCAAGAGGCACGG Os01g NH NH HD NI HD NH NH dt1456 NH HD NG NG NI NH NH HD HD HD NI NI GCTTAGGCCCAAACTACA Os01g NI HD NG NI HD NI 4

5 dt1457 NG NH NG NI NH NG NG NG NH NH NH HD HD NG NI NI NH HD dt1458 HD NH NI NI HD NH NH NG NH HD HD NG NG NI NH NH dt1459 HD HD NG NI NI NH NH HD NI HD HD NH NG NG HD NH dt1460 NH NG NI HD NH HD NI HD NI NH HD HD NI HD HD NH HD HD dt1461 NH NH HD NH NH NG NH NH HD NG NH NG NH HD NH NG NI HD dt1462 HD NI HD NI HD NG NI HD NG NH NG NI NH HD HD dt1463 NH NH HD NG NI HD NI NH NG NI NH NG NH NG NH dt1464 NH HD NI NI NI NH HD HD NI NG NI NI NG NG NI NH NH dt1465 HD HD NG NI NI NG NG NI NG NH NH HD NG NG NG NH HD dt1466 HD NI HD HD NH NI NH NG HD NH NI NG HD NI NH NI NH HD dt1467 NH HD NG HD NG NH NI NG HD NH NI HD NG HD NH NH NG NH dt1647 NN HD NN NI NG NN HD NN NG NG NG HD HD HD NI HD HD NG HD a An asterisk means a missing amino acid at that position. b The 5 T is omitted. TGTAGTTTGGGCCTAAGC Os01g CGAACGGTGCCTTAGG Os01g CCTAAGGCACCGTTCG Os01g GTACGCACAGCCACCGCC Os01g GGCGGTGGCTGTGCGTAC Os01g CACACTACTGTAGCC Os01g GGCTACAGTAGTGTG Os01g GCAAAGCCATAATTAGG Os01g CCTAATTATGGCTTTGC Os01g CACCGAGTCGATCAGAGC Os01g GCTCTGATCGACTCGGTG Os01g GCGATGCGTTTCCCACCTC Os09g Table S2. Selected candidate targets of Xoc BLS256 TAL effectors harboring a predicted EBE in the reverse orientation. Related to Figure 5. TAL effector Target locus a EBE position b Score/score ratio q (Xoc vs mock) c Gain of function test (Xag) d Loss of function test (Xoc) e Tal2d Os01g / Tal2d Os08g / Tal3a Os06g / Tal3a Os03g / Tal3c Os09g / E Tal5a Os07g / NT Tal5a Os01g / E-06 - NT 5

6 Tal7 Os08g / NT Tal7 Os06g / NT Tal8 Os02g / NT Tal8 Os05g / NT a Locus IDs are based on MSU7; the prefix LOC_ is omitted. b Distance in bases from the 5 end of the EBE to the translational start site of the gene. c Calculated for the comparison of transcript abundance in Xoc- vs. mock-inoculated leaves across all time points (Cernadas et al., 2014). d Induction by Xag strain EB08 expressing the cloned TAL effector from a plasmid, assessed by qpcr of RNA isolated from leaves 48 h after inoculation, relative to EB08 carrying the control vector pac99; + means induced, - means not induced, and NT means not tested. e Dependence of induction by Xoc BLS256 on the TAL effector, assessed by qpcr of RNA isolated from leaves 48 h after inoculation with the wildtype strain, relative to the TAL effector knockout mutant derivative; + means induction depends on the TAL effector, - means it does not, and NT means not tested. Table S3. Primers used in this study. Primer(s) Locus or use Sequence Reference B1311 OsSULTR3;6 5 -TGTCGATACGCGCTGCAGATA-3 B1312 (Os01g52130) 5 -AACTGTTACTAGCTCGTGGTACT-3 B1271 B1272 B1275 B1276 B1393 B1394 B1986 B1987 B1970 B1971 B1980 B1981 Actin (Os01g64630) OsSWEET14 (Os11g31190) Os01g27210 Os02g43760 Os03g05370 Os05g GCATGCGTCCCATGGTTTCAGTAGC-3 5 -CTGACCGACTCAGACGACCTACACTG-3 5 -GCGTGCATGCGTACATGGGTCG-3 5 -AGCATGTCTGCAGCTTCACTAGCTCTG-3 5 -GCCGATGAGCAGCAACTATATA-3 5 -TGTTGGAGAAGTGGCTGAGGT-3 5 -GACTCAATGAACTTCAACGTCATGGCTC-3 5 -GGTCTATGATCTAAAACCCCTGCAAAGAG-3 5 -CCAACTACGACCAACGGAAGATTGAG-3 5 -GAAGGCCTCGACTTGATAGCTAACCTC-3 5 -GCATCGAGAGCTACCTCAACGAGTAC-3 5 -AGGATCTTGCGGCGCTGGGCTTC-3 (Cernadas et al., 2014) 6

7 B1976 B1977 B1984 B1985 B1385 B1386 B1982 B1983 B2000 B2001 Os06g11210 Os06g47950 Os07g34510 Os08g09230 Os09g GATGAGGATCAGAACAACTCGGTTGCTG-3 5 -GACATCTTGCACGCAGGCATATACTAAC-3 5 -AGATGATTGAGATGGGGATGACTCCTAC-3 5 -CTGCCTTCATCATTTGCCTAATCTCCTC-3 5 -TCTGGTGGAATCATGTATCTA-3 5 -CAGTGGTCTACTGCCATCA-3 5 -GCTTTGATGGAGCTGACTGTAATGGTG-3 5 -AGCTGAATGGTACAGTTCAATGTAGTCCA-3 5 -ACATCTACGCCCGTCAACTA-3 5 -TGGTGTGGTCTCGGAGCATCTC-3 B RACE for OsSULTR3;6 5 -GATTACGCCAAGCTTGGTACGCGATCGGT CTCTCGATCC-3 B2069 B2070 B2071 B2072 B2073 B2074 B2191 B2192 B2253 B2254 B2255 B2256 B2257 Introducing KpnI site into pps1 OsSULTR3;6 promoter fragment Bs3 promoter fragment Os09g39810 promoter fragment 5 -ACCATGGAAGACGCCAAAAAC-3 5 -ACCATGACTTCGAAAGTTTATGATC-3 5 -CGGGGTACCTCATAGTCAAGCTAACGAAAC TTAT-3 5 -GCCGGTACCTGGGGTACGCGATCGGTCT-3 5 -CGGGGTACCTCATAGTCAAGCTAACGAAAC TTAT-3 5 -CGGGGTACCGAAATATATGTGCAACTAGGA CTACT-3 5 -CGGGGTACCAGTTTTGTACAACACATAAAG TTTTTTTAC-3 5 -CGGGGTACCTAGTTATATATATGTCGATCGA TCAAGCA-3 Renilla firefly CDS sequence 5 -AAAACCGCGGATCTAGTAACATAGATGACAC CG AAAAGGCGCGCCTGAAGGCTCTCAAGGGCAT CGGTC-3 Firefly luciferase CDS or CDS plus vector sequences 5 -ATGGAAGACGCCAAAAACATAAAGAAAG-3 5 -AAAAGGCGCGCCACTAGTGTTAACACGCGTGAC GTATGGAAGACGCCAAAAACATAAAGAAAG AAAAGGCGCGCCCATTTTGCTGCCGGTCACTG GATTTTG -3 (Cernadas et al., 2014) 7

8 B2249 B2250 Tal3c reverse EBE scramble 5 -AGGGTTGGATTAGTATAGTAATGAAGGG-3 5 -ACACTAGTTCTTCCCTTTTTCTCCCG-3 B2251 B2252 Tal3c forward EBE scramble 5 -ACCCCCAGCGCCGACTCCCATTCATTC-3 5 -TTGTGGGGAAAAGGAAGCAGAGGGAGAG-3 B2266 B2267 Introducing ptal1 fragment into expression vector 5 -TATATGGATCCAGATCTACGCACGCTCGGCTA CAGTC-3 5 -TAATAGTCGACTAACGCGATGGGACGTGC GTTCG-3 Table S4. DNA probes used in electrophoretic mobility shift assays. Related to Figure S1. Probe Sequence a 1456/ CAAACAAACGAATGCTTAGGCCCAAACTACAACTTTCTCGTTTT-3 3 -GTTTGTTTGCTTACGAATCCGGGTTTGATGTTGAAAGAGCAAAA-5 - Biotin 1383/ CATGTCAGCAGCTGGTCATGTGTGCCTTTTCATTCCCTTCTTCCTTC-3 3 -GTACAGTCGTCGACCAGTACACACGGAAAAGTAAGGGAAGAAGGAAG-5 - Biotin 436/ AAGAAATGAGCCAATCCAAAACGTAATTTAGAATGGCCCGTAGCCTC-3 3 -TTCTTTACTCGGTTAGGTTTTGCATTAAATCTTACCGGGCATCGGAG-5 - Biotin a Target sequences for the forward and reverse dtales are represented in blue and orange, respectively. REFERENCES Alexandrov, N., Tai, S., Wang, W., Mansueto, L., Palis, K., Fuentes, R.R., Ulat, V.J., Chebotarov, D., Zhang, G., Li, Z., et al. (2015). SNP-Seek database of SNPs derived from 3000 rice genomes. Nucleic Acids Res. 43:D Cermak, T., Doyle, E.L., Christian, M., Wang, L., Zhang, Y., Schmidt, C., Baller, J.A., Somia, N.V., Bogdanove, A.J., and Voytas, D.F. (2011). Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 39:e82. Cernadas, R.A., Doyle, E.L., Nino-Liu, D.O., Wilkins, K.E., Bancroft, T., Wang, L., Schmidt, C.L., Caldo, R., Yang, B., White, F.F., et al. (2014). Code-assisted discovery of TAL effector targets in bacterial leaf streak of rice reveals contrast with bacterial blight and a novel susceptibility gene. PLoS Path. 10:e

9 Nakagawa, T., Kurose, T., Hino, T., Tanaka, K., Kawamukai, M., Niwa, Y., Toyooka, K., Matsuoka, K., Jinbo, T., and Kimura, T. (2007). Development of series of gateway binary vectors, pgwbs, for realizing efficient construction of fusion genes for plant transformation. J. Biosci. Bioeng. 104: Streubel, J., Pesce, C., Hutin, M., Koebnik, R., Boch, J., and Szurek, B. (2013). Five phylogenetically close rice SWEET genes confer TAL effector-mediated susceptibility to Xanthomonas oryzae pv. oryzae. New Phytol. 200:

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION High-efficiency TALEN-based gene editing produces disease-resistance rice Ting Li 1, Bo Liu 1, Martin H. Spalding 1, Donald P. Weeks 2, Bing Yang 1 * 1 Department of Genetics,