B. Transgenic plants with strong phenotype (%)

Transcription

1 A. TCTAGTTGTTGTTGTTATGGTCTAGTTGTTGTTGTTATGGTCTAATTT AAATATGGTCTAAAGAAGAAGAATATGGTCTAAAGAAGAAGAATATGG 2XP35S STTM165 5 GGGGGATGAAGctaCCTGGTCCGA3 3 CCCCCUACUUC---GGACCAGGCU5 mir165 HindIII mir nt GTTGTTGTTGTTATGGTCTAGTTGTTGTTGTTATGGTCTAATTT AAATATGGTCTAAAGAAGAAGAATATGGTCTAAAGAAGAAGAAT 88 nt spacer EcoRI mir166 T35S STTM166 5 GGGGAATGAAGctaCCTGGTCCGA3 3 CCCCUUACUUC---GGACCAGGCU5 mir166 B. Transgenic plants with strong phenotype (%) STTM165/ STTM165/ STTM165/ n=56 n=86 n=50 Supplemental Figure 1. The Optimal Length of the Spacer between the Two Small RNA Binding Sites in STTM. (A) Diagrams of the STTMs with different spacer lengths. Orange indicates the spacer region and the spacer sequence. Blue indicates the bulge sequences in the mirna binding sites. Red indicates the nucleotides that are different between mir165 and mir166. (B) Comparison of the efficacy of the STTMs with different spacer lengths.

2 STTM165/166-8nt STTM165/166-31nt STTM165/166-48nt STTM165/166-88nt STTM165/166-96nt A B C D E dg= dg= dg= dg= dg= Supplemental Figure 2. The Secondary Structures and Thermodynamic Stabilities of Various STTM165/166s with Different Spacer Lengths (8nt, 31nt, 48nt, 88nt, and 96nt). Green indicates mir165/166 binding sites or the shared portion (AUUUAAAU) of the spacer region. Black indicates the spacers or the six nucleotides of the 5 and 3 ends adjacent to the mirna binding sites. dg indicates the thermodynamic stability of the STTM165/166 and the unit is kcal/mol. The lower the dg, the more stable the STTM165/166. STTM165/ and STTM165/ are more stable than STTM165/ STTM165/ and STTM165/ are more stable than STTM165/ and STTM165/ The spacers of STTM165/ (C),-88 (D), and -96 (E) form a stable stem region, while those of STTM165/ (B) and STTM165/166-8 (C) do not.

3 STTM165/166-48mut STTM165/166-48mut spacer sequence: CCAACACGCUAACCGCACCCAUUUAAAUCGCGAGCGAUCGAGGAGGGA Supplemental Figure 3. Mutation in the Spacer Region Changed the Stem Structure and Reduced the Efficacy in the Functional Blockage of mir165/166. The spacer of STTM165/ was randomly mutated except the AUUUAAAU region, resulting in STTM165/166-48mut. Green indicates mir165/166 binding sites or the shared portion (AUUUAAAU) of the spacer region. Black indicates the spacer or the six nucleotides of the 5 and 3 ends adjacent to the mirna binding sites. Compared with STTM165/166-48, the stem structure was disrupted in STTM165/166-48mut and the proportion of STTM165/166-48mut transgenic plants with strong abnormal phenotype was reduced approximately from 30% to around 20%.

4 Vector MIM165 STTM165/166 spacer length 31nt 48nt mir165/166 U6 Supplemental Figure 4. Comparison of the Expression Levels of mir165/166 in STTM165/166 and MIM165 Transformants and Vector Control Plants. mir165/166 and U6 was assayed by RNA gel blotting analysis.

5 Relative Expression Levels 8nt 31nt 48nt STTM165/166 Spacer Length Supplemental Figure 5. Comparison of the Expression of mir165/166 Targets in STTM165/166 Transformants with Different Spacer Lengths. The expression levels were assayed by qrt-pcr. Bars show standard error.

6 A. STTM expression B. mir166 expression STTM165/166del-48 STTM165/166mut-48 STTM165/ STTM165/ STTM165/166del-48 STTM165/166mut-48 STTM165/ STTM165/ Supplemental Figure 6. Comparison of the Expression Levels of STTM and mir166 in STTM165/166del-48, STTM165/166mut-48, STTM165/ and STTM165/ Transformants. (A) qrt-pcr analysis of STTM expression in STTM165/166del-48, STTM165/166mut-48, STTM165/ and STTM165/ transgenic plants. Bars show standard error. (B) qrt-pcr analysis of mir166 expression in STTM165/166del-48, STTM165/166mut-48, STTM165/ and STTM165/ transgenic plants. Bars show standard error.

7 Forward Primer 2X35S promoter STTM-0 (Vector Control) HindIII EcoRI 35S terminator Reverse Primer Forward Primer 2X35S promoter STTM-31/48nt 35S terminator HindIII EcoRI Reverse Primer Supplemental Figure 7. Diagrams of the STTM-31/48 plasmids and Their Vector Control as Well as the Real-time Primer Locations. The arrow lines represent the locations of two real-time PCR primers (Forward Primer: STTM-common-real-PF: catttggagaggacagcccaag; Reverse Primer: STTM-common-real-PR: ctggtgatttcagcgtaccgaa) used for the quantification of STTM transcripts. Please note: The vector control, termed STTM-0, also contains the two real-time primer binding sites and thus gives a value for the real-time quantification of STTM transcripts. Dashed line indicates the empty vector. Blue line indicates the STTM. Green indicates the promoter or terminator.

8 A. Vector MIM160 STTM160/ B. Expression Level ARF17 expression Vector MIM160 STTM160/ Supplemental Figure 8. Comparison of the Phenotypes and the Expression Levels of the mir160 Target, ARF17, in Vector Control, MIM160, and STTM160/ (A) The representative phenotypes of vector control, MIM160 and STTM160/ Bar corresponds to 1 cm. (B) The ARF17 expression levels of the vector control, MIM160, and STTM160/ assayed by qrt-pcr. Bars show standard error.

9 A. 3.5 MIM 3.0 Expression level B. mir166 expression ACTIN MIM165 MIM165 STTM ACTIN STTM165/ STTM165/ Supplemental Figure 9. Comparison of the Expression Levels of MIM, STTM, and mir165/166 in MIM165 and STTM165/ Transformants. (A) Expression of MIM (i.e. IPS1 backbone) and STTM assayed by qrt- PCR. The expression levels of ACTIN in MIM165 and STTM165/ were compared and normalized to the same level. Bars show standard error. (B) Expression of mir165/166 was quantified by qrt-pcr. Bars show standard error.

10 pri-mir156a wild type STTM156/ pri-mir156b wild type STTM156/ mir156 wild type STTM156/ Supplemental Figure 10. The Expression Levels of mir156 Primary Transcripts (pri-mir156a, pri-mir156b) as Well as the Mature mir156 in STTM156/ Expression levels were assayed by qrt-pcr. Bars show standard error.

11 Table S1. Primers Used for Plasmid Construction in This Study Construct Primer name Sequences STTM165/ STTMSwa8ntlink-PF gccatttaaatggggaatgaagctacctggtccgagaattcggtacgctgaaatcaccag STTMSwa8ntlink-PR gccatttaaattcggaccaggtagcttcatcccccaagcttgggctgtcctctccaaatg STTM165/ STTMSwa31ntlink-PF gccatttaaatatggtctaggggaatgaagctacctggtccgagaattcggtacgctgaaatcaccag STTMSwa31ntlink-PR gccatttaaaggcaaattagaccattcggaccaggtagcttcatcccccaagcttgggctgtcctctccaaatg STTM165/ STTMSwa48ntlink-PF gccatttaaatatggtctaaagaagaagaatggggaatgaagctacctggtccgagaattcggtacgctgaaatcaccag STTMSwa48ntlink-PR gccatttaaattagaccataacaacaacaactcggaccaggtagcttcatcccccaagcttgggctgtcctctccaaatg T7-STTM165/ PF taatacgactcactataggaagcttgggggatgaagctacctggtccgagttgttgttgttatggtctaatt T7-STTM165/ T7-STTM165/ PR gaattctcggaccaggtagcttcattccccattcttcttctttagaccatattt STTM-Spacer-48nt-template GTTGTTGTTGTTATGGTCTAATTTAAATATGGTCTAAAGAAGAAGAAT STTM165/ STTMSwa88ntlink-PF gccatttaaatatggtctaaagaagaagaatatggtctaaagaagaagaatggggaatgaagctacctggtccgagaattcggtacgctgaaatcaccag STTMSwa88ntlink-PR gccatttaaattagaccataacaacaacaactagaccataacaacaacaactcggaccaggtagcttcatcccccaagcttgggctgtcctctccaaatg STTM165/ STTMSwa96ntlink-PF STTMSwa96ntlink-PR gccatttaaatatggtctaaagaagaagaatatggtctaaagaagaagaatatggggggaatgaagctacctggtccgagaattcggtacgctgaaatcaccag gccatttaaattagaccataacaacaacaactagaccataacaacaacaactagatcggaccaggtagcttcatcccccaagcttgggctgtcctctccaaatg STTM165del/ del-166-STTMSwa48ntlink-PF gccatttaaatatggtctaaagaagaagaatggggaatgaagctacctggtccgagaattcggtacgctgaaatcaccag 165del-166-STTMSwa48ntlink-PRgccATTTAAATaagcttgggctgtcctctccaaatg STTM166/ STTMSwa48ntlink-PF STTMSwa48ntlink-PR gccatttaaatatggtctaaagaagaagaatggggaatgaagctacctggtccgagaattcggtacgctgaaatcaccag gccatttaaattagaccataacaacaacaactcggaccaggtagcttcattccccaagcttgggctgtcctctccaaatg STTM165mut/ mut-166-STTMSwa48ntlink-PF gccatttaaatatggtctaaagaagaagaatggggaatgaagctacctggtccgagaattcggtacgctgaaatcaccag 165mut-166-STTMSwa48ntlink-PRgccATTTAAATtagaccataacaacaacaacTCGGACgAcGtaggTaCtTCCCggaagcttgggctgtcctctccaaatg STTM165/166mut mut-STTMSwa48ntlink-PFgccATTTAAATatggtctaaagaagaagaatccGGAAaGtAcctaCgTcGTCCGAgaattcggtacgctgaaatcaccag mut-STTMSwa48ntlink-PRgccATTTAAATtagaccataacaacaacaacTCGGACCAGGtagCTTCATCCCCCaagcttgggctgtcctctccaaatg STTM165/166-48mut TMSwa48ntlink-mut-PF gccatttaaatcgcgagcgatcgaggagggaggggaatgaagctacctggtccgagaattcggtacgctgaaatcaccag TMSwa48ntlink-mut-PR gccatttaaatgggtgcggttagcgtgttggtcggaccaggtagcttcatcccccaagcttgggctgtcctctccaaatg STTM160/ STTMSwa48ntlink-PF STTMSwa48ntlink-PR gccatttaaatatggtctaaagaagaagaattggcatacaggctagagccaggcagaattcggtacgctgaaatcaccag gccatttaaattagaccataacaacaacaactgcctggctctagcctgtatgccaaagcttgggctgtcctctccaaatg STTM156/ STTMSwa48ntlink-PF STTMSwa48ntlink-PR gccatttaaatatggtctaaagaagaagaatgtgctctctatctacttctgtcaagaattcggtacgctgaaatcaccag gccatttaaattagaccataacaacaacaactgacagaagataggagtgagcactaagcttgggctgtcctctccaaatg STTMD7(+)D8(+)-48 D7D8-STTMSwa48ntlink-PF D7D8-STTMSwa48ntlink-PR gccatttaaatatggtctaaagaagaagaataaggccttacactaaggtcaagaagaattcggtacgctgaaatcaccag gccatttaaattagaccataacaacaacaacttcttgaccttagtgtaagaccccaagcttgggctgtcctctccaaatg Origin Deletion Origin-del-PacI-PF Origin-del-PacI-PR TCCCTTAATTAAGTTTGCAAGCAGCAGATTACGCG TCCCTTAATTAAGAAAGGCGGACAGGTATCCGGTAAG

12 Supplemental Table 2. Synthetic mirnas and Small RNA Probes Used in This Study Probe/RNA Oligo/Probe name Sequences mir165/166 mir165-probe GGGGGATGAAGCCTGGTCCGA mir166-probe GGGGAATGAAGCCTGGTCCGA mir156/157 mir156-probe GTGCTCACTCTCTTCTGTCA mir157-probe GTGCTCTCTATCTTCTGTCAA D7(+)/D8(+) Synthetic mir165/166 D7(+)-probe GGGGTCTTACAAGGTCAAGAA D8(+)-probe AAGGCCTTACAAGGTCAAGAA mir165-rna UCGGACCAGGCUUCAUCCCCC mir166-rna UCGGACCAGGCUUCAUUCCCC mir160 mir160-probe ACGGACCGAGGGACATACGGT mir168 mir168-probe TTCCCGACCTGCACCAAGCGA

13 Supplemental Table 3. Real-time PCR Primers Used in This Study Gene Name Real-time PCR Primer Name sequences PHB PHV REV ATHB8 ATHB15 ARF17 Pri-MIR156A Pri-MIR156B MIM (IPS1) STTM ACTIN2 PHB-real-new-PF CCAGCAGGACTCCTTTCTAT PHB-real-new-PR TGCGCGAAATAGCGACTATG PHV-real-new-PF CCAGCTAATCTTCTCTCGAT PHV-real-new-PR TGCGTGAAACAGCTACGATA REV-real-new-PF CCTGCTGGATTGCTCTCAAT REV-real-new-PR GAAATGGCAAAGATGCCAACCG AT4G32880-ATHB-8-real-PF TTAGACCTTGCGAAGGAGGT AT4G32880-ATHB-8-real-PR AGCGGCCATTGTAGTTCTTT AT1G52150-ATHB-15-real-PF GTTCCCACAGATCATGCAAC AT1G52150-ATHB-15-real-PR TGAACACAAAGCAGATGCAA At-ARF17-real-PF CGAGTCAAGATGGCTATGGA At-ARF17-real-PR CATCCCATGTGATCTGAAGC Pri-MIR156A-F CATCTTGTAGATCTCTGAAGTTGGACT Pri-MIR156A-R GAGATTGAGACATAGAGAACGAAGACA Pri-MIR156B-F GAAGAGGGAGAGATGGTGATTG Pri-MIR156B-R GCAGAGATAGGCAACTGACAG IPS1-F1 GAAAGCGTTTTAAGATATGGAGC IPS1-R1 TACAACCCAAACATAATGAAGAA STTM-common-real-PF CATTTGGAGAGGACAGCCCAAG STTM-common-real-PR CTGGTGATTTCAGCGTACCGAA ACTIN2-real-PF CCAGTGGTCGTACAACCGGTAT ACTIN2-real-PR ACCCTCGTAGATTGGCACAGT