Light triggers PILS-dependent reduction in nuclear auxin signalling for growth transition

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1 In the format provided y the authors and unedited. SUPPLEMENTARY INFORMATION VOLUME: 3 ARTICLE NUMBER: Light triggers PILS-dependent reduction in nuclear auxin signalling for growth transition Chloé Béziat 1,2, Elke Barez 1,2, Mugurel I. Feraru 1, Doris Lucyshyn 1 and Jürgen Kleine-Vehn 1,2 * The phytohormone auxin induces or represses growth depending on its concentration and the underlying tissue type. However, it remains unknown how auxin signalling is modulated to allow tissues transiting etween repression and promotion of growth. Here we used apical hook development as a model for growth transitions in plants. A PIN-FORMED (PIN)-dependent intercellular auxin transport module defines an auxin maximum that is causal for growth repression during the formation of the apical hook. Our data illustrate that growth transition for apical hook opening is largely independent of this PIN module, ut requires the PIN-LIKES (PILS) putative auxin carriers at the endoplasmic reticulum. PILS proteins reduce nuclear auxin signalling in the apical hook, leading to the de-repression of growth and the onset of hook opening. We also show that the phytochrome (phy) B-reliant light-signalling pathway directly regulates PILS gene activity, therey enaling light perception to repress nuclear auxin signalling and to control growth. We propose a novel mechanism, in which PILS proteins allow external signals to alter tissue sensitivity to auxin, defining differential growth rates. T1 Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences (BOKU), Muthgasse 18, 119 Vienna, Austria. 2 Department of Plant Systems Biology, VIB and Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, 952 Gent, Belgium. * juergen.kleine-vehn@oku.ac.at NATURE PLANTS DOI: 1.138/nplants Macmillan Pulishers Limited, part of Springer Nature. All rights reserved.

2 a 2 ns pin pin4-3 ns Hours after light exposure 2 1 ns pin3 pin Hours after light exposure Supplementary Figure 1. Intercellular auxin transport and hook opening. a,, Kinetics of apical hook opening during light exposure of pin3, pin4 single (a) and pin3 pin4 doule mutants () compared to seedlings (germinated in dark). Please note that pin3 pin4 doule mutants do not form closed apical hooks. For comparative reasons, the initial angle of opening was aligned (n 12). The opening kinetics (K) was statistically evaluated as descried in Supplementary Fig. 2. We did not detect statistically significant differences etween and pin mutants (ns).

3 a Y plateau K Y=(Y - Plateau)*exp(-K*X) + Plateau Y=(18-5)*exp(-K*X) Y plateau K pils2 pils Hours after light exposure constants: Y = 18 (angle during the maintenance phase) plateau= 5 (angle at full opening) variales: K=? (defines the speed of the opening) Y plateau X K Y= IF( X<X, Y, Plateau+(Y -Plateau)*exp(-K*(X-X ))) Y= IF( X<X, 18, 5+(18-5)*exp(-K*(X-X ))) 2 Y plateau 5 X 1 pils2 pils5 K 15 constants: Y = 18 (angle during the maintenance phase) plateau= 5 (angle at full opening) variales: X =? (defines the end of maintenance phase) K=? (defines the speed of opening) Supplementary Figure 2. Statistical analysis of the apical hook kinetics. a, Apical hook opening in light. Seedlings germinated in the dark were, in the early maintenance phase, exposed to light. The apical hook opening kinetics were monitored from the start of the light exposure onwards as shown in Fig. 4f and 3a as well as in Supplementary Fig.1a,;Fig. 8c and 11. Using non-linear regression, the data points were fitted to a model, representing a single phase exponential decay. The variales (K), defining the opening speed, of and pils2 pils5 seedlings have een statistically compared using an Extra sum of squares F test., Apical hook development in dark. Seedlings were kept in the dark. The apical hook developmental kinetics were monitored from 12 hours after germination onwards as shown in Fig. 2; 6a,,c as well as in Supplementary Fig. 5a,; 6d; 7; 8; 9a. Using non-linear regression, the data points were fitted to a model, representing a plateau phase, followed y a single phase exponential decay. The variales (X ) and (K), defining the end of the maintenance phase and opening speed respectively, of and pils2 pils5 seedlings have een statistically compared using an Extra sum of squares F test. Left panels depict schematic overviews of the used model and the preset constants and variales.right panels depict the apical hook kinetics of and pils2 pils5 seedlings in light (a) or in dark () along with the respective regression curves. Graphpad ( was used for all the analysis.

4 a 3 FORMATION 3 MAINTENANCE 3 OPENING Distance from the hook outer side (µm) 1 2 Distance from the hook outer side (µm) 1 2 Distance from the hook outer side (µm) Distance from the hook outer side (µm) 1 2 Distance from the hook outer side (µm) 1 2 Distance from the hook outer side (µm) Supplementary Figure 3. The activity of ppils2 and ppils5 is asymmetric during the 3 sequential phases of hook development. a, Quantification of GUS expression during formation, maintenance and opening phases in seedlings expressing ppils2::gus (a) or ppils5::gus (). The quantification was performed from the outer to the inner side of the hook along the arrow indicated in the pictures illustrating the staining in the different phases. Both lines showed increased expression towards the inner side of apical hooks. Arrow represents 15 µm.

5 DMSO NPA ppils2::gus DMSO NPA *** 2 1 *** S U pp IL S5 ::G S U S U ns pp IL S2 ::G ppils5::gus ppils3::gus 3 pp IL S3 ::G a Supplementary Figure 4. Intercellular auxin transport regulates PILS expression in the hook zone. a, Expressions patterns of 3 days dark grown seedlings expressing ppils2::gus, ppils3::gus, ppils5::gus grown on medium supplemented with NPA (5 µm) or DMSO control. Scale ar, 1 µm. The white rectangle represents the region of interest (ROI) where the quantification was performed., Quantification of mean grey values in the ROI of respective lines grown on NPA or DMSO (n=1). Two-way ANOVA followed y Bonferroni post-hoc test was performed comparing DMSO and NPA for each transgenic line ***p<.1. ns : non significant.

6 a pils2 ** pils5 ns pils2pils5 *** ** pils3 ns pils2 pils3 ns pils2 pils3 pils Supplementary Figure 5. PILS functional redundancy during apical hook opening. a,, Kinetics of apical hook development in, pils2, pils5, and pils2pils5 dark-grown seedlings (n 12) (a) and in, pils3, pils2pils3, pils2pils3pils5 dark-grown seedlings (n 12) (). Errors ars represent standard error of the mean. For the statistics X (end of maintenance phase) and K (speed of opening) were compared (see Supplementary Fig. 2) to (a) or pils2pils3pils5 (). Statistical significance is indicated y *p<,5 **p<.1 ***p<.1; ns: non significant.

7 a AT1G7652 ATG TGA 5 3 PILS3.5 k pils3-1 Promoter UTR Exon Intron c pils3-1 pils3-1 PILS3 Tuulin Expression fold change pils3-1 *** PILS3 expression d ppils3::pils3:gfp in pils ns Supplementary Figure 6. pils3 knockdown mutant characterization and complementation. a, PILS3 gene contains 8 exons. The analysed T-DNA insertion is depicted in the figure (arrow head). Scale ar, 5p., RT-PCR of PILS3 expression level in pils3-1 compared to (upper and), the expression level of the tuulin as a control gene is also presented in the same lane (lower and). Note that PILS3 expression is knocked-down in pils3-1 compared to. c, Quantitative RT-PCR of PILS3 expression in and pils3-1 normalized to EIF1A expression. Statistical significance was evaluated with the student T-test; the p value is indicated y ***p<.1. d, Kinetics of apical hook development in and ppils3::pils3:gfp in pils3 dark grown seedlings (n 12). For the statistics X (end of maintenance phase) and K (speed of opening) were compared to (see Supplementary Fig. 2) (p>,4). Errors ars represent standard error of the mean. Both parameters were statistically not significant (ns).

8 a 4h 6h PILS3OX *** pils3 *** 2 PILS3OX 15h pils h 4h 6h d pils3 4h 6h ** ** f PILS3OX 8 6 ** ** h 8 15 pdr5::rfp in PILS3OX e pdr5::gfp in pils3 1 6 c Supplementary Figure 7. PILS3 is required for apical hook development. a, Representative pictures of apical hooks in dark-grown wild type (), PILS3OX and pils3 seedlings during formation (15 hours), maintenance (4 hours) and early opening (6 hours). Scale ars, 3 µm., Kinetics of apical hook development in, PILS3OX and pils3 dark-grown seedlings (n 12). For the statistics X (end of maintenance phase) and K (speed of opening) were compared to (see Supplementary Fig. 2 ). Significant results for oth parameters are indicated ***p<.1. c, e, DR5 promoter activity in pils3 (c) as well as in PILS3OX (e) compared to dark-grown seedlings at 15h (formation), 4h (maintenance) and 6h (opening) time points after germination. Colour code (lack to green or red) depicts (low to high) pdr5rev::gfp/rfp1er signal intensity. Scale ars, 2 µm. d, f, Quantification of DR5 promoter activity in pils3 (d) as well as in PILS3OX (f) compared to dark-grown seedlings at 15, 4 and 6 hours after germination (n 5). The region of interest (ROI) was delimited to the inner side of the hook. Error ars represent standard error of the mean. Statistical significance was evaluated with two-way ANOVA followed y Bonferroni post-hoc test y comparing the 2 mutant lines at the different time point; the p value is indicated y **p<.1 PILS3OX: p35s::gfp:pils3

9 a PILS2 OX PILS3 OX PILS5 OX c PILS2 OX *** PILS2 OX *** Hours after light exposure Supplementary Figure 8. PILS2 overexpressor shows faster hook opening. a, Representative pictures of GFP signal in, PILS2 OX, PILS3 OX, PILS5 OX dark grown seedlings, Scale ar, 2 µm., Kinetic of apical hook development in and PILS2 OX dark grown seedlings (n 12). For the statistics X (end of maintenance phase) and K (speed of opening) (see Supplementary Fig. 2 ) were compared to. Statistical significance are indicated for oth parameters y ***p<.1. c, Kinetic of apical hook opening during light exposure in and PILS2 OX dark grown seedlings (n 12). For the statistics K (speed of opening) was compared to (see Supplementary Fig. 2a). Significant results are indicated ***p<.1. Error ars represent standard error of the mean. PILS2 OX : p35s::gfp:pils2; PILS3 OX : p35s::gfp:pils3; PILS5 OX : p35s::pils5:gfp

10 a 25 2 PILS5 OX *** ACC *** PILS5 OX ACC*** PILS3 OX PILS5 OX c *** *** ACC 2 1 *** ACC PILS3 OX PILS5 OX Supplementary Figure 9. Ethylene dependent hook exaggeration mediated y PIN is unchanged in PILS OX. a, Kinetics of apical hook development in and PILS5 OX grown on control medium or supplemented with ethylene precursor (ACC 5 µm). For the statistics X (end of maintenance phase) and K (speed of opening) (see Supplementary Fig. 2) were compared to. Statistical significance is indicated for oth parameters y ***p<.1., Representative pictures of, PILS3 OX, PILS5 OX 4 DAG on control medium or supplemented with ACC 5 µm. Scale ar, 15 µm. c, Apical hook angle measurement 4 DAG in the respective lines and treatments (n=25). Two-way ANOVA followed y Bonferroni post-hoc test was performed comparing the treatment and control of the respective lines. ***p<.1. PILS3 OX : p35s::gfp:pils3 PILS5 OX : p35s::pils5:gfp

11 a 1h 2h 1h 2h 3h VV VV V V ppils5::gus h h 3h ppils5::gus 3 1 ppils5::gus 2 a 1 15 c 5 Hours after light exposure Supplementary Figure 1. Light regulates PILS5 expression in a complex manner. a,, Expression patterns of ppils5::gus in apical hook maintenance phase of dark-grown seedlings or after 1, 2 or 3 hours of light exposure. Scale ars, 2 µm (a), 1 µm (). c, GUS quantification of ppils5 in dark or after 1, 2 or 3 hours of light exposure (n 8). The region of quantification was the apical hook region (). Error ars represent standard error of the mean. Statistical significance was evaluated y one way ANOVA followed y multiple comparisons Tukey s test p<,5. Distinct letters indicate statistically significant differences.

12 5h h a pils3 PILS3 OX 2 pils3*** Hours after light exposure 2 PILS3 OX *** Hours after light exposure Supplementary Figure 11. PILS3 defines growth transition in light. a, Representative pictures of apical hooks darkgrown or after eing exposed 5 hours to light in wild type (), pils3 and PILS 3O X seedlings. Scale ars, 2 µm., Kinetics of apical hook opening during light exposure in pils3 and PILS 3OX darkgrown seedlings compared to (n 12). For the statistics K (speed of opening) (see Supplementary Fig. 2a) were compared to. Statistical significance is indicated y ***p<.1. Error ars represent standard error of the mean. PILS3 OX : p35s::gfp:pils3.

13 a > ppils2 CCGAGATCAGAATTTTGGATCCGGTACCCGCCGATTCATGCTTTTTAATCTTCTTAACCAATTACGTACAGACACGAGTTA GTATGTCTGGTTTTGGGTTCCACGTGGATCTGTTTGGTTTGCGGCAGCTATTTTATTCCCACGTGCGATTACTGATCAAAT CTCAAGCCAAGCGTTCCCTTTTGTCGTCTTGTTCTGATTTAATATTAAACCGGAAATGACCGATCATTCAGCCGCTAGTGT TTGGTTTGTATATTTTGACGCATTAATGGGCTTATAATTGGGCTTCTTTTTTAGACTCGTGTTGTCGCTAGCCCAATGGAT AATCTTTGACAGTATCGTTCAACCACAGATTATCTTCTTCGCACGATGTGATAAATACAAAATTTTCAAATC > ppils3: TTCCAATAGTACAAATCAAGATTTTCAATATATGTCAAGTTTGTTATTTTCATTCACGGACATTAAAACGAGTGGAGCATT TCAATTTGACATATCTTTGTCCTTTTCTTTTTGTTACAGATATCTTTTGTTCACGTTCTTTGAATCATCATATTCATAGCT ATGAGATCATAGAAATATCAAATACTGTTAATTAACCTCTTTAAAAATACATATTCACAAGTCTCTTGTGTGAGTAGAAAT CCTATACAAACAAGTTTATTCATGCTCTAATAAGATAGATAAATAAGAAGATAGCTCATGACATGGCCTTCATTAGGCTTG TTGCATATACAATAAGCTTACATATAAGGAATTAAGGATGGTCTAATATCTCTTGCTTCACATGAGAAGAGTTATAGGATT CGAGTCACATAAGAGATTTTTATTGTAGTGTGTATAATATATCCCACTTCTACAATTATCCAGTCTATTGTGTATTTTTCA TTAATAAATCACTAAAATTTGCACTTTGACAATATATACTTAAATATTTGTATCTCGTATATATATATATACATATTTTTA GCACTGTATGATAATTCTAATTATTCGCTAATTGTTTTTTTTTGGTAAGAATTAGTCGACTATTTTGATATTGGGGTTGTT ACATTTATATCTTGCATTGAATTTTATGGACGGACACTGGTCGCAGCCTCACACACTAATTTGAATTTTCCGTTATCATTA ATATTCGATCGTTTCGTTGGTACGCTTCCATCACACAATTAATTTAAGCCACCACACTATTATCTTATTCTCATTCAGTTT TGTTTCCATTTCAAATTTCTTACGACGCATATGAAAATGTACATTTTTATAACAATTCCCCTTAAAAATAGATGACAAAAA GAAAAATGTTTTTTTTAAAAAAATATATAAAATAAAATAAATAATAAATCTTCAACAATCGATGAACACTTTGCAATATCT CCACACCTTGGGCAAGAACCAGAAAAAAACATGGGGCACGTTTCGTAGTCGATGTGACGAAAGGCATGACGACGGTTACCA TATAAATTCGTCTTCTCAGATAAAGCCTTATTATTCTCAATAGTGTTATACGACAATATACGAAACAAACAAAGACCCCGC TTGAGTTGAGTTTTCCAAAAATTGTGGAACTGTTTCCTCTTTCTTTTTGTACATAAACTAGGTTACAAAAAATTGGAAAAT ATACAAAATCAAAGGACATAAACTTACGAGATATATCATAACTCATAAGTTGTGAAAGTTGGAATTCAAAAAGGAGCTAAC TATGAATCTATGATACGTTATTTTGTTGCTTATACGGAACAAAACAATTCCAAAAACAAAGAAGATGTAAAAGTCTGACCC GGATCATGCGGACACCAAGTTGTACATACAAAATATGTAGGATGAAAATATGAAATTCTCTTCCCATATGCATAGAATAAT AATTTACCTTAGTTAAAAAGATTAGTTTCTAAGTTTGCCCCAAATAAAGAGATTAGTTTCTTGAAAAAAGCATTTT >ppils5: ACACCTACACGATGACACCTACACGATTACACTGTTGCAGGCTGCTCCCACTACAAGCATGCTTACTCTAGAT TATAGCTTAGTTTTCCAAGTAAACAAAAGCAACTAGATTCAAACCAACTTCTTTCACATGTAACTACTTGTTC TCTCTTTGACTTCTTCAGTATGGTTTGAAAGCAAAATAGGCATTATTCAACAGTCTGCTTTCTAATTAATCCT TTGAGATTGCAGTTTACGAAAATTATCCAAGGGATTCATAATTTTAGTTGAAGATATAAAGCAATTTCCTTAG CAGATTACAATTACCTTTAGCTGTTACTTGTCTGTTTAGCTTCGATTGGTCTAGTAAACAGTTTTGGTTTGGA TTAATCATATTTCTTATTCTCTGTTACCGTTTTGTTGGATCTCAAATTTCTTTGATGATTCTAGCAAGTGGTT GAGTTTAATTGATCTGTATTTTATCCATTAAGCTGTTTTGCAGTAGCAGGTGAAAAACAAACGAATTCACTGT GTTGAATTTCATTCACCGGAATAATCAGAGGCTACGACGGCGAAGGATGACGATGGAAATCTTTTGATTACTC ATCATACACGTGTTGATAACATGTGGCTTTGAAAGGAATTATCTGATCAAATCTTCTTCTTCAATATTGAGTT TGAGTTGGGATTTAGTTTGTTTTCGTAACTGGTTATGTTGTAGGGCTTTTTTAGCCCATGTTTGTAATGCTTA CCTCAATAAAATATGAACTTGTGAGAAAAAAAAAAAGATTTGGGTTTTAGTTAAAAGAGGAGAAAGAAATGTT TAAAATGATGAGCTAAATTTTAAATAAGTTGTAAGATTAAGCAACAAATGAGACTTGTAGAAATTGCAAAAGT TGGGACCTAAATCTTGAGAAGCCACTAGAATAAGGACCTTATTTTATTAATGTTGTGGCCCTTGAGAAAAATA ATATTGGGTAAGAAAATAAACAACTTATAGTTTTCAAAAACACAAAAACACAAAACCCTCAATAGTTGGAAGA GGGCTCCCCAAGACCGTATTCGCGTCCAGAATCGTAACATTTTCGATAAGATTAAAAAAACCTGCAAAATCTG GTCCGCACGTTTTTAGCAACACAACAAAGTATGACGAGAAGTTCCACGATTTTTGCAGGTATAGTACATTCGC TTTTCGCCACCTCATTGCCTTGGATTCATACACTGGATCGGTTTAATCTTCTTCTCTGTTTTTCCATGTCAAC TATTATATAGTCATTGTATTTGTATTTATTTTCTATATATTGCACCGAAAAAAGCAAGAGAGGGATATGGTTC ACATGAACTATGTAGGAGGTGAATAAATTAGACACATGTATTCTTAATTTCAGGATCACATGAATTGTGATAG TTCGGTGCTCAGTAGAACTTATAAAAAAGCACAACACTTATTCTTTTTGTCGGATATGATAATCCGCTTTTGA TTTTTTTTAAATATATGGAGTTTTCGATAGTCCAAAAAAAAGTTTCATGCTTAGGCAGACAAAAACTTTAATC TTTGTGTGTGGACGACATATTTGTTTTAACTTGCTAGTGAATTTTTCTTTAATCTACCTTTGTTTTGTATATA TATCTAACCATTATAATTATGTATATGTTAGCTGATTCCAAACGATATCATAAGTTCTAATGAGTAATGATTG GTCAATTTATCTTGTTGTAAATTGTTCAATATACGTGACGTGGTCCACTTCTATCCTCCAAGACTCCAGCTAC ACAAAGACACAGTAACACAAACGCATACGCATATGCATGTGTCGTGTCTTTGTGTGTGTATGTTGTATATATT AATATATTATCTAAACTATACCATTTTCATGAAATTTATAATATATTTTCTCAAATGATTTAAGAAGATAATG ATATGTTTACGAGAGTAAAAAAATTAATGATCTTCCAGTAATCAATAAATATAGTAAATTGATATCAGATCTT GTTGCCAACTATAGAATTGAACAAAATACAAAACTGTCATCGCCATGTTATATTATCGTAAAGC putative PIF5 inding sites predicted y Supplementary Figure 12. Predicted PIF5 inding motifs in PILS2, PILS3 and PILS5 promoters. a, G oxes predicted in silico positions are depicted in red letters in PILS2, PILS3 and PILS5 promoters sequences. The underlined sites were considered for ChIP. Proaility of inding was in silico defined for ppils2 (p>95%), as well as for ppils3 and ppils5 (p>85%).

14 Supplementary Tale 1. Primers used in this study. Genotyping primers pils2-2 FW: ATTGCTCAAGGTGAATCCAT pils2-2 RW: AGACCAATCACGGTTAAACA Salk_LB1-3:ATTTTGCCGATTTCGGAAC pils5-2 FW: CCCTTGTTTGGATCATGGTA pils5-2 RW: TCTTACTGCACCGAAAATGA; Salk_LB1-3:ATTTTGCCGATTTCGGAAC pils3-1 FW: AAAGGCATGACGACGGTTAC pils3-1 RW: AAGAGCGTCTCCAAAATTTGG; Salk_LB1-3: ATTTTGCCGATTTCGGAAC pif5 FW: CCATCCATTCAGAGGCTTC RW: ATCGTTCGTGGCTTCTCAG; Salk_LB1-3:ATTTTGCCGATTTCGGAAC RT PCR primers PILS3 FW: AAGCTTTTGGAGCTGTTCA PILS3 RW: CTAAGCTACAAGCCACATGA Tuulin FW: ACTCGTTGGGAGGAGGAACT Tuulin RW: ACACCAGACATAGTAGCAGAAATCAAG q PCR primers PILS3 FW: AGGCGACCATGCAAGTGTTG PILS3 RW: GTGGTACAGCTAGATGACAGTGAG EIF1A FW: CTGGAGGTTTTGAGGCTGGTAT EIF1A RW: CCAAGGGTGAAAGCAAGAAGA Primers used for Chromatin immunoprecipitation PILS2 G ox FW: GTTTGGTTTGCGGCAGCTAT RW: CTAGCGGCTGAATGATCGGT PILS3 G ox FW: AGTCTCTTGTGTGAGTAGAAATCCT RW: CAACAAGCCTAATGAAGGCCA WAG2 G ox FW: GGCCCACAGATATGTGATTAG RW: TCAAAATATTATGAGGGATTCGC FT 3 UTR FW: CATTTTATGATACGAGTAACGAACGGTG RW: CACTCTCATTTTCCTCCCCCTCTC Cloning primers

15 ppils1-attb4_fw: GGGGACAACTTTGTATAGAAAAGTTGCGGATTCAAGCTGATGCAATGCCTG ppils1-attb1_rw: GGGGACTGCTTTTTTGTACAAACTTGCCTTCGCTTTCAATCTGATACC ppils3-attb4_fw: GGGGACAACTTTGTATAGAAAAGTTGCGGAAGCTAATTCTCTGAGACATAGC ppils3-attb1_rw: GGGGACTGCTTTTTTGTACAAACTTGCCTTTTTACTATCAACGCGAGAATC ppils4-attb4_fw: GGGGACAACTTTGTATAGAAAAGTTGCGGTGAAGCTTTTGGAGCTGTTCAT ppils4_attb1_rw: GGGGACTGCTTTTTTGTACAAACTTGCCTCTACTTCCAAAAGTTAGAATC ppils6_attb4_fw: GGGGACAACTTTGTATAGAAAAGTTGCGCCATCGAGAGATTTAATTTGATC ppils6_attb1_rw: GGGGACTGCTTTTTTGTACAAACTTGCTCTGAGAATTTTGAGGGTGGAC ppils7_attb4_fw: GGGGACAACTTTGTATAGAAAAGTTGCGCTGCGGACAACAAAAAAGTGTTAG ppils7_attb1_rw: GGGGACTGCTTTTTTGTACAAACTTGCCCCTAATCTCTTCTTTTTCTTTTCT