Supplemental Figure S1. Co-localization of GH3-2 and putative disease resistance QTLs. LOD, logarithm of odds.

Transcription

1 C922 Resistance to M. grisea isolate F1814 LOD: 2.2 C922 Resistance to Xoo strain JL691 LOD: 1.6 RG101 G393 WRKY13 GH3-2 RG101 G393 WRKY13 GH3-2 RM212 C567 RM212 Chr LOD Chr LOD Supplemental Figure S1. Co-localization of GH3-2 and putative disease resistance QTLs. LOD, logarithm of odds.

2 A ATG GH3-2 TGA T (Minghui 63)/C (Zhenshan 97) cdna Fragment (2248 bp) for transformation KpnI BamHI RB GUS Ubi TEVL GH3-2 NOS Hpt LB Clone D176U in vector pu1301 B EcoRI AscI AvrII SacI SpeI HindIII LB Hpt 35S AdhI 442 bp Waxy-a 442 bp OCS GUS RB GH3-2 Intron I GH3-2 cdna of rice cdna Clone D175R in vector pds1301 Supplemental Figure S2. Schematic diagrams of GH3-2 gene and the transformation constructs of GH3-2. RB and LB, right and left T-DNA border; GUS, β-glucuronidase gene; Hpt, hygromycin phosphotransferase gene; Ubi, maize ubiquitin gene promoter; 35S, cauliflower mosaic virus 35S promoter; TEVL, 5 -nontranslated region of the tobacco etch virus; NOS, napoline synthase polyadenylation signal; OCS, octopine synthase polyadenylation signal. A, OsGH3-2 gene (upper part) and overexpression construct of GH3-2, D176U (lower part). The coding region (black boxes) of GH3-2 is interrupted by two introns (thick lines). The positions of 5 - and 3 -untranslated regions (hatched boxes), translation start codon (ATG), translation stop codon (TGA). The numbers indicate the base pairs of each substructure. The arrow indicates a single nucleotide difference between Minghui 63 and Zhenshan 97 causing a NdeI digestion site in Zhenshan 97 but not in Minghui 63. B, RNA interference construct D175R of GH3-2.

3 Minghui0 63 (-2135) ACACCGATCGTAAATTTCTCAGTGAAAAAAAAATGCAATGTAAAATCAGTTA Zhenshan 97 (-1756) ACACCGATCGTAAATTTCTCAGTGAAAAAAAAATGCAATGTAAAATCAGTTA Minghui0 63 (-2083) AAACATGTGGCCAAAAGATCAGACTCTGCTAATTACAGCCTTACAGGGGGGC Zhenshan 97 (-1704) AAACATGTGGCCAAAAGATCAGACTCTGCTAATTACAGCCTTACAGGGGGGC Minghui0 63 (-2031) TCAGAACCAAACAAAACCATCTCTAGCCACCAAAATTGACTCGCGAGAGGTT Zhenshan 97 (-1652) TCAGAACCAAACAAAACCATCTCTAGCCACCAAAATTGACTCACGAGAGGTT Minghui0 63 (-1979) AAAACTTGATTGAAAGGAACAACTCGATCGACAGATCGATCCGATTGATCGA Zhenshan 97 (-1600) AAAACTTGATTGAAAGGAACAACTCGATCGACAGATCGATCCGATTGATCGA Minghui0 63 (-1927) TCGTATCCTAGCTATTAGCTAGCTAGCTACTACTTCATGAGACGCGTTAATT Zhenshan 97 (-1548) TCGTATCCTAGCTATTAGCTAGCTAGCTACTACTTCATGAGACGCGTTAATT Minghui0 63 (-1875) GAACCGATGTTAACGAAAATAATTGGGTGTACTAGCTAGCACAAGTCAAGAC Zhenshan 97 (-1496) GAACCGATGTTAACGAAAATAATTGGGTGTACTAGCTAGCACAAGTCAAGAC Minghui0 63 (-1823) ACGGGAAATAGTGATCGTTTCTACGCCTCGACCCGATCGATGGATGATTCGA Zhenshan 97 (-1444) ACGGGAAATAGTGATCGTTTCTACGCCTCGACCCGATCGATGGATGATTCGA Minghui0 63 (-1771) TGGACGACACGTAAAACACGTTTGGCTCTTCGCGATCACAGAAAGGGGATAG Zhenshan 97 (-1392) TGGACGACACGTAAAACACGTTTGGCTCTTCGCGATCACAGAAAGGGGATAG Minghui0 63 (-1719) ATAGGCCGACACGCGCGGTACAAGCTGGATTAGTACATTATTGTCTCCACAT Zhenshan 97 (-1340) ATAGGCCGACACGCGCGGTACAAGCTGGATTAGTACATTATTGTCTCCACAT Minghui0 63 (-1667) GTATATATGATAATATGATTATTGCTGTAGCCCGTAGGTCTATCCAGCCGTA Zhenshan 97 (-1288) GTATATATGATAATATGATTATTGCTGTAGCCCGTAGGTCTATCCAGCCGTA Minghui0 63 (-1615) CAAGTACCACTATGTATATATACGTACGTACCCTGTGGATCAACCACAAATT Zhenshan 97 (-1236) CAAGTACCACTATGTATATATACGTACGTACCCTGTGGATCAACCACAAATT Minghui0 63 (-1563) ATTTTTGTGACGTGCTAATTTTATAAAGAGGTTTTGGAAGGATTTGCTAATT Zhenshan 97 (-1184) ATTTTTGTGACGTGCTAATTTTATAAAGAGGTTTTGGAAGGATTTGCTAATT Minghui0 63 (-1511) ATATTCTAACGAGCTTATATGGAAGATGAGTCGACAAATCCAGCCAACACGT Zhenshan 97 (-1132) ATATTCTAACGAGCTTATATGGAAGATGAGTCGACAAATGCAGCCAACACGT Minghui0 63 (-1459) TCACATGATTAATTACGTCCAGCCCGGCAGTCTATATTTCGACGTGAAGTTA Zhenshan 97 (-1080) TCACATGATTAATTACGTCCAGCCCGGCAGTCTATATTTCGACGTGAAGTTA Minghui0 63 (-1407) ATTAGCTATCAGAAGATCCTATTTGTGGGTCGTGTGTGATTAGTAAACTGT- Zhenshan 97 (-1028) ATTAGCTAGCAGAAGATCCTATTTGTGGGTCGTGTGTGATTAGTAAACTGTC Minghui0 63 (-1356) AT Zhenshan 97 (-976) TGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTATGTATGTATATGTA-- Minghui0 63 (-1354) ATATATATATATATATATATGACACTCATATGGGGCACCATGGTATCTAATG Zhenshan 97 (-) Minghui0 63 (-1302) TACAAAATCACTCAAATTTTTCAAAATTTTCAAATTGTGAGTATATACCTAG Zhenshan 97 (-) Minghui0 63 (-1250) TTATAATAATTCAATTCATATCTATACCTATCAACAAAACAACTCAAATTTA Zhenshan 97 (-) Minghui0 63 (-1198) ACTTTATTTCACAATCCATGATTACATACCTAACTAATTATATGTATGGATG Zhenshan 97 (-)

4 Minghui0 63 (-1146) CTATATGCCTAGAATCAAAATCTAACAAAAACAAGTTCAAATTCAGTACAAA Zhenshan 97 (-) Minghui0 63 (-1094) CATGCTTTGAACTAGTTAGTAAAGTAGTTAATGTTAATAACTAAGTAATTGA Zhenshan 97 (-) Minghui0 63 (-1042) AAAAGTTTCATACTCATTTGAATTGGGTATATACTCAATTTCAACAATGGTG Zhenshan 97 (-) Minghui0 63 (-990) CCCGGGCACCATGGAGCACCAAACAAATTTAATATATATATATATATATATA Zhenshan 97 (-) Minghui0 63 (-938) TATCTATGTATGTATGTAATACTGAACACATTTTAAATGGATTTTTAGTTAG Zhenshan 97 (-926) TATGTAATACTGAACACATTTTAAATGGATTTTTAGTTAG Minghui0 63 (-886) AGCCGTGTTAGATTTTAAATGGATATTTTGGAACACTAGCCCGTCGTCAGTC Zhenshan 97 (-886) AGCCGTGTTAGATTTTAAATGGATATTTTGGAACACTAGCCCGTCGTCAGTC Minghui0 63 (-834) ATCACTTTACTAATTTGAACTGATGTCGATCGTGAAAAATTTTAAACCGATG Zhenshan 97 (-834) ATCACTTTACTAATTTGAACTGATGTCGATCGTGAAAAATTTTAAACCGATG Minghui0 63 (-782) AAAGGGTAGAATTCCTAGCATAAAGAAACTTTTCAAACTTACATTACATTAC Zhenshan 97 (-782) AAAGGGTAGAATTCCTAGCATAAAGAAACTTTTCAAACTTACATTACATTAC Minghui0 63 (-730) TTTAACTTGCAAATAAAAAATTCTACAATGCTAAAATGTCAAATATTCTTGT Zhenshan 97 (-730) TTTAACTTGCAAATAAAAAATTCTACAATGCTAAAATGTCAAATATTCTTGT Minghui0 63 (-678) GGAAATTCTCTTGTCCGTCAATGCCCCGCTTGTTCCATGCATGCATCTCCAG Zhenshan 97 (-678) GGAAATTCTCTTGTCCGTCAATGCCCCGCTTGTTCCATGCATGCATCTCCAG Minghui0 63 (-626) CTGCCGGCTCACTTGTATTAAGGAAGAAAAATCCCATTTCTCCTTTCAGTCC Zhenshan 97 (-626) CTGCCGGCTCACTTGTATTAAGGAAGAAAAATCCCATTTCTCCTTTCAGTCC Minghui0 63 (-574) CTGTCCACTTACCATTTCCTTAATACCCTCACACTAGCACATAATTCTCTCT Zhenshan 97 (-574) CTGTCCACTTACCATTTCCTTAATACCCTCACACTAGCACATAATTCTCTCT Minghui0 63 (-522) GATGATCTGGTCAGGTGTGGCGCTGCACACTACTGTGTCGACACATACCCAA Zhenshan 97 (-522) GATGATCTGGTCAGGTGTGGCGCTGCACACTACTGTGTCGACACATACCCAA Minghui0 63 (-470) GCAGCAACAAAACACCCCCTCTCCTGGATTTCCATTTTCTTTTCGTCCAACC Zhenshan 97 (-470) GCAGCAACAAAACACCCCCTCTCCTGGATTTCCATTTTCTTTTCGTCCAACC Minghui0 63 (-418) TTGATAATACATTTTTATAAACCTATTCATGTCGTATATTACTTATTAATTT Zhenshan 97 (-418) TTGATAATACATTTTTATAAACCTATTCATGTCGTATATTACTTATTAATTT Minghui0 63 (-366) CTCCATCAGATGAGAGATTGCCGTTGATTAATCCGGAGACGAACAACTACAC Zhenshan 97 (-366) CTCCATCAGATGAGAAATTGCCGTTGATTAATCCGGAGACGAACAACTACAC Minghui0 63 (-314) AACCCGACCGGTCGGGTAATTAAAACCAATTTAGCTCTCGTTCGTCAGCGCC Zhenshan 97 (-314) AACCCGACCGGTCGGGTAATTAAAACCAATTTAGCTCTCGTTCGTCAGCGCC Minghui0 63 (-262) GATGGCTAAGCTCGCTGCCGGGGCGCGCCGGCCGCGCGTCCCGTCGCGGGGC Zhenshan 97 (-262) GATGGCTAAGCTCGCTGCCGGGGCGCGCCGGCCGCGCGTCCCGTCGCGGGGC Minghui0 63 (-210) CCGGGCGTCCGACGTGGCCGACCAGGCGGGCCCACGTGCCCCCTCCTCGCTC Zhenshan 97 (-210) CCGGGCGTCCGACGTGGCCGACCAGGCGGGCCCACGTGCCCCCTCCTCGCTC Minghui0 63 (-158) GGGCAGTGACGCCCGCGTGGGCCACGCCCTGCCTCCCCAGTCCCCACCCTCA Zhenshan 97 (-158) GGGCAGTGACGCCCGCGTGGGCCACGCCCTGCCTCCCCAGTCCCCACCCTCA

5 Minghui0 63 (-106) CCGGCCCGCCTCGCTCGCCCGCGCGCGCGCGCGCGACGTGCATGGCGCGCGG Zhenshan 97 (-106) CCGGCCCGCCTCGCTCGCCCGCGCGCGCGCGCGCGACGTGCATGGCGCGCGG Minghui0 63 Zhenshan 97 Minghui0 63 Zhenshan 97 (-54) CCTCCTCCCCCCCTCCCGCCGCTATATATACCCCTCCCTTGCAACCGCCTCC (-54) CCTCCTCCCCCCCTCCCGCCGCTATATATACCCCTCCCTTGCAACCGCCTCC (-2) TC (-2) TC Supplemental Figure S3. Sequence comparison of promoter regions of GH3-2 alleles in Minghui 63 and Zhenshan 97. The underlined nucleotides are the predicted cis-elements from database of Plant Cis-acting Regulatory DNA Elements ( The TGTCTC, TGACG, CATATG, and ACTTTA are cis-elements correlating to auxin responsiveness in plants (Terzaghi and Cashmore, 1995; Xu et al., 1997; Baumann et al., 1999; Goda et al., 2004). The GAAAAA, TGAC, and TGTCA are cis-elements involved in disease defense in plants (Eulgem et al., 1999; Park et al., 2004; Luo et al., 2005). References Baumann K, De Paolis A, Costantino P, Gualberti G (1999) The DNA binding site of the Dof protein NtBBF1 is essential for tissue-specific and auxin-regulated expression of the rolb oncogene in plants. Plant Cell 11: Eulgem T, Rushton PJ, Schmelzer E, Hahlbrock K, Somssich IE (1999) Early nuclear events in plant defence signalling: rapid gene activation by WRKY transcription factors. EMBO J 18: Goda H, Sawa S, Asami T, Fujioka S, Shimada Y, Yoshida S (2004) Comprehensive comparison of auxin-regulated and brassinosteroid-regulated genes in Arabidopsis. Plant Physiol 134: Luo H, Song F, Goodman RM, Zheng Z (2005) Up-regulation of OsBIHD1, a rice gene encoding BELL homeodomain transcriptional factor, in disease resistance responses. Plant Biol (Stuttg) 7: Park HC, Kim ML, Kang YH, Jeon JM, Yoo JH, Kim MC, Park CY, Jeong JC, Moon BC, Lee JH, Yoon HW, Lee SH, Chung WS, Lim CO, Lee SY, Hong JC, Cho MJ (2004) Pathogen- and NaCl-induced expression of the SCaM-4 promoter is mediated in part by a GT-1 box that interacts with a GT-1-like transcription factor. Plant Physiol 135: Terzaghi WB, Cashmore AR (1995) Light-regulated transcription. Annu Rev Plant Biol 46: Xu N, Hagen G, Guilfoyle T (1997) Multiple auxin response modules in the soybean SAUR 15A promoter. Plant Sci 126:

6 A Lesion area (%) GH3-2 a a a a a a a a a a a rrna WT T 0 transgenic plants D176UM a a B Lesion area (%) a a a a a a a a a a a a a GH3-2 rrna WT T 1 family D176UM14 C Bacteria [ln (cfu/leaf)] 10 Xoo strain PXO Mudanjiang 8 (wild type) D176UM11 (GH3-2- overexpressing) D176UM34 (GH3-2- overexpressing) Days after inoculation Supplemental Figure S4. Enhanced resistance of GH3-2 overexpressing plants (D176UM) to Xoo strain PXO61 was associated with increased GH3-2 expression. The a indicates a significant difference (P < 0.01) from wild type (WT). Data represent mean (5 replicates for lesion area and 3 replicates for bacterial growth) ± SD. A, GH3-2 overexpressing T 0 plants showed enhanced resistance. B, Enhanced resistance of GH3-2 overexpressing T 1 plants was associated with increased GH3-2 expression. C, GH3-2 overexpressing T 1 plants showed lower bacterial growth rate in rice leaves. cfu, colony-forming unit; 0 d, 2 h after bacterial inoculation.

7 Relative expression level WT D175RZ Supplemental Figure S5. The expression levels of GH3-2 in GH3-2- RNAi T 0 plants (D175R). Zhonghua 11 is wild type (WT). D175RZ7 was a negative transgenic plant.

8 Relative expression level GH3-2 expression Lesion area (%) PXO347 infection WT D175RZ6 T 1 family Relative expression level GH3-2 expression Lesion area (%) PXO347 infection WT D175RZ16 T 1 family Supplemental Figure S6. The GH3-2 expression level and performance to Xoo strain PXO347 of two GH3-2-RNAi T 1 families. Zhonghua 11 is wild type (WT). D175RZ6-13, D175RZ16-11 and D175RZ16-12 were negative transgenic plants.

9 GH3-2 GH3-8 (1) MAPAAVAAAEAGSKAAAVAGKAVAACERDAEKLEFIEEITRGFDAVQERVLAAILARNN (1) ---MAVMTDVSTTGTALRTPAAGAVKEGDVEKLRFIDEMTTNVDAVQERVLGEILGRNA GH3-2 (60) GAEYLRRHGMEGRTDREAFKARVPVVTYEDLRPEIERIANGDRSNIISSHPITEFLTSS GH3-8 (57) GTEYLTKCGLDGATDRAAFRAKVPVVSYDDLQPYIQRIANGDRSPILSTHPVSEFLTSS GH3-2 (119) GTSAGERKLMPTIEDELDRRQMLYSLLMPVMNLYVPGLDKGKGLYFLFIKSETKTPGGL GH3-8 (116) GTSAGERKLMPTIMDELDRRQLLYSLLMPVMNLYVPGLDKGKGLYFLFVKSETKTPGGL GH3-2 (178) PARPVLTSYYKSDHFKHRPFDPYNVYTSPTAAILCTDAFQSMYAQMLCGLVARAEVLRV GH3-8 (175) TARPVLTSYYKSDHFKNRPYDPYHNYTSPTAAILCADAFQSMYAQMVCGLCQRNDVLRL GH3-2 (237) GAVFASGLLRAIRFLQLHWRELAHDIRTGTLSAKVTEPSIRDAVAEVLAAPDAELAAFV GH3-8 (234) GAVFASGLLRAIRFLQLNWEQLADDIESGELTPRVTDPSVREAVAAILLP-DPELAKLI GH3-2 (296) EAECGKDKWEGIITRMWPNTKYLDVIVTGAMAQYIPTLKFYSGGLPMACTMYASSECYF GH3-8 (292) RAECSKGDWAGIITRVWPNTKYLDVIVTGAMAQYIPTLEFYSGGLPMACTMYASSECYF GH3-2 (355) GLNLRPMCDPSEVSYTIMPNMGYFELMPHDPDAPPLPRDAPPPRLVDLADAEVGREYEL GH3-8 (351) GLNLRPMCDPSEVSYTIMPNMGYFEFLPVDETG---AASGDATQLVDLARVEVGREYEL GH3-2 (414) VITTYAGLCRYRVGDILQVTGFHNAAPQFRFVRRKNVLLSIDSDKTDEAELQAAVERAS GH3-8 (407) VITTYAGLNRYRVGDVLRVTGFHNAAPQFRFVRRKNVLLSIESDKTDEAELQRAVERAS GH3-2 (473) ALLSPYGASIVEYTSQADATTIPGHYVVYWELMVREGGAWPPPAEEEGRGVFERCCLEM GH3-8 (466) ALLRPHGASVVEYTSQACTKRIPGHYVIYWELLTKGAGATVVDADTLG-----RCCLEM GH3-2 (532) EEALNAVYRQGRNGE-AIGPLEIRVVRAGTFEEVMDYAISRGASINQYKAPRCVSFGPI GH3-8 (520) EEALNTVYRQSRVADGSIGPLEIRVVRPGTFEELMDYAISRGASINQYKVPRCVTFPPI GH3-2 (590) IELLNSRVISKHFSPACPKYSPHKK-- GH3-8 (579) VELLDSRVVSSHFSPALPHWTPARRSE Supplemental Figure S7. Sequence comparison of rice GH3-2 and GH3-8.

10 Wild type D176UM Supplemental Figure S8. GH3-2-overexpressing plants (T 2 ) D176UM showed dwarf morphology. Mudanjiang 8 is wild type.

11 25 D176UM11 (GH3-2-overexpressing) Mudanjiang 8 (wild type) Relative expression level ck ck ck ck ck ck ck PR1a PR1b PR10 PAL1 PAD4 LOX AOS2 Time after inoculation of Xoo (h) Supplemental Figure S9. Expression of defense-responsive genes related to SA and JA signaling in GH3-2 overexpressing line. Plants were inoculated with Xoo strain PXO61 at the booting stage. Bars represent means (3 replicates) ± SD. ck, before infection.

12 1.2 GH IAA 3.5 IAA-Asp Relative expression level Concentration (ng/g FW) Concentration (ng/g FW) Minghui 63 Zhenshan 97 0 Minghui 63 Zhenshan Minghui 63 Zhenshan 97 Supplemental Figure S10. IAA and IAA-Asp concentrations and GH3-2 expression in Minghui 63 and Zhenshan 97 at four-leaf stage. Bars represent mean (3 replicates) ± SD. FW, fresh weight.

13 Supplemental Table S1. Performance of EXPA1-, EXPA5- and EXPA10-overexpressing T 1 families after inoculation with Xoc strain RH3 at booting stage Rice material PCR a Lesion length (cm) b P value Zhonghua 11 (wild type) 0.51 ± 0.07 EXPA1-overexpressing plants OVEXPA ± OVEXPA ± OVEXPA ± EXPA5-overexpressing plants OVEXPA ± OVEXPA ± OVEXPA ± EXPA10-overexpressing plants OVEXPA ± OVEXPA ± OVEXPA ± a PCR primers used for identification of positive transgenic plants were GUSF/R (Supplemental Table 1). b Data represents mean (10 lesions) ± standard deviation.

14 Supplemental Table S2. Performance of EXPA1-, EXPA5- and EXPA10-overexpressing T 2 families after inoculation with M. grisea isolate CHL358 at 4-leaf stage Rice material Disease Index a Resistance/susceptibility CO39 (susceptible control) 84.4 Highly susceptible Zhonghua 11 (wild type) 31.1 Moderately susceptible EXPA1-overexpressing plants OVEXPA Moderately susceptible OVEXPA Moderately susceptible OVEXPA1 (negative) 30.0 Moderately resistant EXPA5-overexpressing plants OVEXPA Moderately susceptible OVEXPA Moderately susceptible OVEXPA5 (negative) 32.2 Moderately susceptible EXPA10-overexpressing plants OVEXPA Moderately susceptible OVEXPA Moderately susceptible OVEXPA10 (negative) 31.1 Moderately susceptible a Disease index was calculated with the individual leaf ratings using the following formula: disease index = [sum of numerical ratings from all leaves/(number of leaves assessed maximum lesion rating)] 100. Each data was from 17 to 30 plants.

15 Supplemental Table S3. Primers used for PCR amplification Gene Primer name Forward primer (5-3 ) Reverse primer (5-3 ) Use (GenBank accession number) GH3-2 (GU001814) pei5p11a(4)/ pei5p11a(3) CGGCAAGCAACGCCAACTAA GCTCATCTTCTATCGTCGGCA Amplifying a fragment of the coding region for RNAi-vector EXPA1 (AK069548) EXPA5 (AK073572) EXPA10 (AK066414) construction Amplifying the entire coding region YHBD3.2-FNd ei/yhbd3.2-r CATATGATGGCTCCGGCGGCG GT a AAGCTTGGACCGTGTAGTGT AGTGAGTG b P3-2F2036 TTAGTAAACTGTCTGTGTGTG DNA sequencing pg3-2f3450 ATCACCGAGTTCCTCACCAG DNA sequencing pg3-2f4127 ATGCGTTTGTAGCCTTGGT DNA sequencing pg3-2f5411 TGCGGGAAGGACAAGTGGG DNA sequencing pg3-2r5514 CCCACCGCTGTAGAACTTGA DNA sequencing EI5P11aMF AAGCAGACATGGGAGAGTGG DNA sequencing EI5P11aF/R EI5P11aF/ EI5P11aMF OsEXPA1-979F /1042R OsEXPA5-609F /708R OsEXPA F/877R GTGCGTGTGTAATTTCTCGTGT TT GTGCGTGTGTAATTTCTCGTGT TT GTAACTACCACTGGACAGCA TGATCT AAGCAGACATGGGAGAGTG G Quantitative PCR Amplifying 3 -end untranslated region for generating a PCR-based cleave amplification polymorphic sequence marker after digestion of the PCR products using NdeI CCTGCTTTTTTCAATGCGAAT AAAGCATGCCGATCATCGA Quantitative PCR TCTTCAAGGCCGGCATTGT TGACCAACTACAACGTGGTCC C AGGTTGAAGTAGGAGTGCCC GT GCCAGTGTATGTTTTGCCGAA G Quantitative PCR Quantitative PCR

16 PR1aF/R CGTCTTCATCACCTGCAAC TACTC CATGCATAAACACGTAGCATA GCA Quantitative PCR PR1bF/R GGCAACTTCGTCGGACAGA CCGTGGACCTGTTTACATT Quantitative PCR TTCA PR10F/R CCCTGCCGAATACGCCTAA CTCAAACGCCACGAGAATTT Quantitative PCR G PALF/R GGGCAACCCAGTGACCAA CGATTGCCTCGTCGGTCTT Quantitative PCR PR1a (AJ278436) PR1b/PR-1 (U89895) PR10/PBZ1 (D38170) PAL1 (X16099) PAD4 (CX118864) AOS2 (AY062258) LOX (D14000) NIT1 (AK104033) AAO3 (AK065990) GUS (AF234297) Cloning vector pmcg161 (AY572837) Actin (X15865) a The underlined nucleotides are the digestion site of NdeI. b The underlined nucleotides are the digestion site of HindIII. PAD4F/R GCCAGCTCCCCTACGACTTC CGTGTGCGGTGTAGGTTGTT Quantitative PCR AOS2F/R CAATACGTGTACTGGTCGAATG G AAGGTGTCGTACCGGAGGAA Quantitative PCR LOXF/R GCATCCCCAACAGCACATC AATAAAGATTTGGGAGTGAC Quantitative PCR ATATTGG NIT1F/R CACCCCGGCCACTCTAGATA GATAGCCGCCAACAAAAGCT Quantitative PCR AAO3F/R TTCACCAGCTGAAGGCACAA GCTCATCCAAGCCATTAGCAA Quantitative PCR GUSF/R CCAGGCAGTTTTAACGATCAGT TCGC GAGTGAAGATCCCTTTCTTGT TACC pmcgf/r GGCTCACCAAACCTTAAACAA CTGAGCTACACATGCTCAGGT T Identifying positive overexpression transgenic plants Identifying positive RNAi transgenic plants Actin-F/R TGTATGCCAGTGGTCGTACCA CCAGCAAGGTCGAGACGAA Quantitative PCR