Figure S1. Electron micrographs showing the differences between OsWRKY89 transgenic and the control plants.

Shown are scanning electron micrography images of culm surfaces (the second internode from the tassel) of XS-C (A, B) and overexpressing line S13 (C,D), adaxial leaf surfaces (the leaf closest to the tassel) of XS-C (E, F) and S1 (G, H), andadaxial leaf surfaces (the second leaf from inside of three-week old seedlings) of ZH-C (I to K) and RNAi line D2 (L to N). (Scale bars = 20 m in (A, C, E, G), 10 m in (F, H, I, L), and 5m in others.

Figure 2S. RT-PCR analysis of some of up-regulated genes in the OsWRKY89over-expression plants

Analysis of gene expression profiles: Leaves of 25-day-old plants were harvested. Total RNA wasisolated from control and overexpressing transgenic S1 and S13 plants with Trizol reagent (Invitrogen) following the manufacturer’s instruction. Total RNA was used for synthesis of cDNA and biotin-labeled cRNA according to the manufacturer’s protocol. The labeled cRNA was used forhybridizationsto the GeneChip rice genome array (Affimetrix, Santa Clara, CA) on the Affymetrix GeneChip instrument system at China Agricultural University’s microarray facility. MAS (5.0 specific terms) program was used for statistical algorithms. Up- or down-regulated genes wereidentified based on ratios of >2 or <0.5, indicatingat least 2-fold changes compared with thecontrol.

To understand how OsWRKY89 regulates responses to biotic and abiotic stresses, we performed a transcriptome analysis of two OsWRKY89 overexpressing lines of T3 progeny in comparison to control plants with Affymetrix rice GeneChips. Some of the up-regulated genes were selected and amplified by RT-PCR. The mRNA from S13 and the control plants was reverse-transcribed and then used as templates for PCR. The amplification cycles were 28. M: DNA ladder; G actin: amplification of actin from gDNA; R actin: amplification of actin from un-transcribed mRNA.

The primers used are following: Actin (XM469569), 5’-ggaactggtatggtcaaggc-3’ and 5’-agtctcatggatacccgcag-3’; AP (putative acid phosphatase, AAU90121.1), 5’-tctgggtgttcgacgtcgatgag-3’ and 5’-agctgtagtagccttgcgagagg-3’;CER (epicuticular wax biosynthesis and pollen fertility, AK068166), 5’-tcgaagagtgggtacaaggtc-3’ and 5’-accttgctgctgtaactgcatgac-3’; CHS (chalcone synthase, CAE05340.2), 5’-tggtcaagagcacgttccacgag-3’ and 5’-ctagctgttacgtggcactgcac-3’; CS (putative cycloartenol synthase, AK068026.1), 5’-tgaaagagatccagtgcctctg-3’ and 5’-atcatgctcctatatgttgcctg -3’; Cys Prot (cysteine protease CP1, AAP97431.1), 5’-agctcatcgactgcgacaccgac-3’ and 5’-agttcctgcctgaggcctcgatg-3’; FACR (fatty acyl CoA reductase, OSJNBb0002N06.11), 5’-acatgattctacggtacaagctg-3’ and 5’-tgctttcgccttctggtcattg-3’; bHLH (BAB64701.1), 5’-agcgacaagcaagcatggagcag-3’ and 5’-cggtactagtgcttaaagctttgc-3’; hydroxylase (XP_469101.1), 5’-tcgacctgaactctgaaatcac-3’ and 5’-cgtctgcagcttagcgaaatct-3’; IFR (isoflavone reductase, NP_908373.1), 5’-acgacgagctggtgagcatgtg-3’ and 5’-tggatttccgggtagagctgag-3’; LS (putative linalool synthase, AK110925.1), 5’-acctagctgcatgagatcatgct-3’ and 5’-tgtacgatccgtccaatccttct-3’; Pir7b (BAD87247.1), 5’-gtccagctgaggacttgacactg-3’ and 5’-agtcatacttgctacctatcttagc-3’;POD (peroxidase 15 precursor, CAH69258.1), 5’-gacgctcgtcgacggcttcaag-3’ and 5’-gatgagctggtcgtcggagaag-3’; and WI (wound induced, XP_474028.1), 5’-cagcgggaagctcaagaagagatcg-3’ and 5’-attggtactccagcagctgatgtac-3’.

Figure S3. Staining of lignin.

Wiesner’s staining of the transverse culm sections ofOsWRKY89 overexpressing S8 (D) and control XS-C (A) plants, and magnified sections (B, C,E,F) showing the increased level of lignin in the walls of sclerenchyma cells.Scale bars = 50 m in (A, D), 20 m in others.