Supplemental Data. Hu et al. Plant Cell (2017) /tpc

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1 Supplemental Figure 1. DNA gel blot analysis of homozygous transgenic plants. (Supports Figure 1.) Rice genomic DNA was digested with the restriction enzymes EcoRⅠ and BamHⅠ. Lanes in the photo show representative samples from T2 homozygous transgenic plants (as indicated). All of these offspring had a single-copy insertion. WT was used as a negative control. 1

2 Supplemental Figure 2. Protein expression of BPH14 and the derived fragments in transgenic and WT plants. (Supports Figure 1.) Protein samples were isolated from WT and the indicated transgenic plants. BPH14 and the derived fragments proteins fused with 4 HA tag under the control of the UBI promoter were detected by anti-ha antibody. 2

3 Supplemental Figure 3. BPH survival rate on BPH14- and CC, NB, CN domain-expressing transgenic plants. (Supports Figure 1.) The condition of third instar nymphs (alive or dead) was recorded daily for 7 days on the WT and transgenic plants. BPH survival rate was calculated as the percentage of surviving nymphs divided by the total number of nymphs released at the start of the experiment. Data represent the means (10 replicates with each repeat having 10 BPH insects per rice line used for analysis ) ± SD. Data were subjected to analysis of variance (ANOVA) as detailed in the Methods and asterisks indicate significant differences between transgenic and WT plants (*, P 0.05; **, P 0.01). 3

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5 Supplemental Figure 4. Expression of the CC domain, NB domain, CN domain and FL BPH14 in WT and transgenic plants. (Supports Figure 1.) (A) Protein expression of BPH14 and the CC, NB, CN domains in transgenic and WT plants. Protein samples were isolated from BPH14- and the CC, NB, CN domain-native expressing transgenic and WT plants. BPH14 and the CC, NB, CN domain proteins fused with 4 Myc tag under the control of the native BPH14 promoter were detected by anti-myc antibody. (B) and (C) Expression of BPH14 and the CC, NB, CN domains in transgenic and WT plants. BPH14 and the CC, NB, CN domain transcripts were detected by RNA gel blot analysis (B) and qrt-pcr (C). The rrna bands served as equal loading controls. For qrt-pcr, the rice Actin1 gene was used as a reference control. Data represent the means (three biologically independent experiments for gene expression) ± SD. 5

6 Supplemental Figure 5. BPH-resistance test of BPH14- and CC, NB, CN domain-expressing transgenic and WT rice. (Supports Figure 1.) (A) BPH-resistance phenotypes of BPH14- and the CC, NB, CN domain-expressing transgenic and susceptible WT rice. (B) BPH-resistance scores of the same lines. Scores are inversely related to BPH resistance. Data represent the means (three biologically independent experiments with each experiment having 15 seedlings per rice line used for analysis) ± SD. Data were subjected to analysis of variance (ANOVA) as detailed in the Methods and asterisks indicate significant differences between transgenic and WT plants (*, P 0.05; **, P 0.01). 6

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8 Supplemental Figure 6. Characterization of insect resistance in BPH14- and CC, NB, CN domain-expressing transgenic rice. (Supports Figure 1.) (A) Settling of BPH in a host choice test. Numbers of BPH nymphs that settled on WT and transgenic plants were recorded at 3, 6, 24, 48 and 72 h after infestation. Data represent the means (10 replicates with each repeat having 40 BPH insects per rice line used for analysis) ± SD. (B) BPH honeydew excretion. The amount of honeydew excreted by BPH nymphs on the WT and transgenic plants after infestation for 2 days. Data represent the means (30 replicates with each repeat having one BPH insect per rice line used for analysis) ± SD. (C) BPH weight gain. Weight gain of BPH insects after feeding on the WT and transgenic plants for 2 days. Data represent the means (30 replicates with each repeat having one BPH insect per rice line used for analysis) ± SD. (D) BPH survival rate. The condition of third instar nymphs (alive or dead) was recorded daily for 7 days on the WT and transgenic plants. BPH survival rate was calculated as the percentage of surviving nymphs divided by the total number of nymphs released at the start of the experiment. Data represent the means (10 replicates with each repeat having 10 BPH insects per rice line used for analysis) ± SD. Data were subjected to analysis of variance (ANOVA) as detailed in the Methods and asterisks indicate significant differences between transgenic and WT plants (*, P 0.05; **, P 0.01). 8

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10 Supplemental Figure 7. Phenotypes of CC, NB, CN domain- and BPH14-transgenic plants. (Supports Figure 1.) (A-C) Grain length, width and thickness of wild-type Kasalath and transgenic plants. (D) Seed germination of wild-type Kasalath and transgenic plants. (E) 7-days-old seminal root length of wild-type Kasalath and transgenic plants. (F) The fourth leaf (from the base leaf of the plant) of rice seedlings in four-leaf stage of wild-type Kasalath and transgenic plants. (G) Plant height of four-leaf stage rice seedlings of wild-type Kasalath and transgenic plants. Bars = 1 cm in (A-D), 3 cm in (E-G). Statistical analyses of the data in (A-G) are presented in Supplemental Data Set 1. WT, wild-type Kasalath plants; CC22-3, NB10-9, CN12-12, FL12-1, the transgenic plants overexpressing the CC, NB, CN domain and BPH14, respectively; CC8-4, NB35-2, CN3-18, FL16-2, the transgenic plants native expressing the CC, NB, CN domain and BPH14, respectively. 10

11 Supplemental Figure 8. ROS generation in the CC, NB and CN domain- or FL BPH14-overexpressing rice protoplast lines. (Supports Figure 4.) Relative luminescence units indicate relative amounts of H 2 O 2 production in rice protoplasts at the indicated times. Data represent the means (three technical replicates from one biological replicate) ± SD. Three biologically independent experiments yielded similar results. CK, control check, rice protoplasts transfected with empty vector; WT, rice protoplasts without transfection. CK and WT were incubated with 200 μg/ml chitin as a positive control. ROS generation in the CC, NB and CN domain- or FL BPH14-overexpressing rice protoplast lines significantly differs from levels in CK and WT protoplasts from the second timepoint onwards. Data were subjected to analysis of variance (ANOVA) as detailed in the Methods

12 Supplemental Figure 9. Expression analysis of plant callose-related genes in BPH14- and CC, NB, CN domain-expressing transgenic and WT rice. (Supports Figure 4.) Four callose hydrolase-encoding genes (1, 3-glucanase genes), GNS5, GNS6, GNS8 and GNS9, were analyzed. The rice Actin1 gene was used as a reference control. Expression of genes was quantified relative to the value obtained from 0 h susceptible samples. Data represent the means (three biologically independent experiments for gene expression) ± SD. Data were subjected to analysis of variance (ANOVA) as detailed in the Methods and asterisks show significant differences between transgenic and WT plants at indicated time points after the start of BPH feeding (*, P 0.05; **, P 0.01). 12

13 Supplemental Figure 10. Accumulation of bait and prey proteins in yeast. (Supports Figure 6 and Figure 7.) (A) Protein gel blots of protein extracts from yeast diploids expressing pgbkt7-, BPH14, the CC, NB, CN domains (upper panel), and pgadt7-, BPH14, the CC, NB, CN domains (lower panel), detected using anti-myc and anti-ha antibodies, respectively. (B) Protein gel blots of protein extracts from yeast diploids expressing pgbkt7-, BPH14, the CC, NB, CN domains (upper panel) and pgadt7-wrky46 (lower panel), detected using anti-myc and anti-ha antibodies, respectively. (C) Protein gel blots of protein extracts from yeast diploids expressing pgbkt7-, BPH14, the CC, NB, CN domains (upper panel) and pgadt7-wrky72 (lower panel), detected using anti-myc and anti-ha antibodies, respectively. Staining of total yeast proteins with Coomassie brilliant blue (CBB) was used to show equal loading. 13

14 Supplemental Figure 11. Screening of several rice WRKY proteins with BPH14 identified two different BPH14 interactor clones (WRKY46 and WRKY72). (Supports Figure 7.) Analysis of BPH14 and several rice WRKY proteins interactions in yeast. Yeast diploids carrying the indicated constructs were grown on DDO plates and transferred to TDO/X-α-Gal/AbA plates with 20 μg/ml X-α-Gal and 200 ng/ml AbA for 3 days. 14

15 Supplemental Figure 12. Verification of negative control proteins tested in the BiFC experiments. (Supports Figure 7.) Protein gel blots of protein extracts from rice protoplasts co-expressing pusyne-tagged FL BPH14 protein and the CC, NB, and CN domains (upper panel) and pusyce-tagged WRKY74 (lower panel), detected using anti-myc and anti-ha antibodies, respectively

16 Supplemental Figure 13. Expression analysis of WRKY46 and WRKY72 in BPH14- and CC, NB, CN domain-expressing transgenic and WT rice. (Supports Figure 7.) Expression of WRKY46 and WRKY72 genes was quantified relative to the value obtained from 0 h susceptible samples. The rice Actin1 gene was used as a reference control. Data represent the means (three biologically independent experiments for gene expression) ± SD. Data were subjected to analysis of variance (ANOVA) as detailed in the Methods and asterisks show significant differences between transgenic and WT plants at indicated time points after the start of BPH feeding (*, P 0.05; **, P 0.01). 16

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18 Supplemental Figure 14. Putative promoter sequences and DNA probes of RLCK281 and LOC_Os01g (Supports Figure 9.) (A) Putative promoter sequences (2-kb upstream of the translational start codon) of genes that function downstream of RLCK281 and LOC_Os01g (B) DNA probes derived from RLCK281 and LOC_Os01g promoters used in EMSAs. W-box RLCK281 and W-box LOC_Os01g are shown in bold letters. The mw-box RLCK281 and mw-box LOC_Os01g contained mutated binding sites with the TGAC and GTCA sequences mutated into TGAA and TTCA. Asterisks represent the mutated bases in the W-box elements. 18

19 Supplemental Figure 15. Verification of DNA bait and prey proteins in yeast. (Supports Figure 9.) (A) Verification of DNA baits in the yeast genome. The inserts of baits (4 W-box RLCK281, 4 W-box LOC_Os01g , p53dbs) were amplified by yeast colony PCR. (B) Verification of prey proteins in yeast. SV40 NLS-GAL4 AD-HA fused to WRKY46, WRKY72 and p53 were detected using anti-ha antibody. 19

20 Supplemental Figure 16. Competitive binding assays of W-box RLCK281 and W-box LOC_Os01g probe to 6 His-WRKY46 and 6 His-WRKY72. (Supports Figure 9.) Results of competitive binding assays using biotin-labeled W-box RLCK281 (at 10 nmol), W-box LOC_Os01g (at 10 nmol) probe and indicated amounts (in nmol) of non-labeled mw-box RLCK281 or W-box RLCK281, mw-box LOC_Os01g or W-box LOC_Os01g to 6 His-WRKY46 (left panel) and 6 His-WRKY72 (right panel). The free and bound probes in EMSAs were separated in a 6% nondenaturing polyacrylamide gel. Positions of the bound and free probes are indicated on the left. Protein amounts are in nanograms (ng). Each EMSA experiment was repeated three times with similar results. 20

21 Supplemental Table 1. ANOVA tables. Sum Sq = Sum of squares; df = degrees of freedom; Mean Sq = Mean Squares Figure 1C Genotype E -19 Residual Total Figure 1D upper left Genotype Time E -08 Genotype:Time Residual Total Figure 1D upper right Genotype Time E -06 Genotype:Time Residual Total Figure 1D lower left Genotype Time E -07 Genotype:Time Residual Total Figure 1D lower right Genotype Time E

22 Genotype:Time Residual Total Figure 1E Genotype E -77 Residual Total Figure 1F Genotype E -15 Residual Total Figure 2A Genotype Residual Total Figure 3A EDS1 Genotype E -31 Time E -18 Genotype:Time E -23 Residual Total NPR1 Genotype E -61 Time E -64 Genotype:Time E

23 Residual Total PAL1 Genotype E -37 Time E -52 Genotype:Time E -36 Residual Total ICS1 Genotype E -30 Time E -27 Genotype:Time E -25 Residual Total AOS2 Genotype E -14 Time E -60 Genotype:Time E -23 Residual Total LOX Genotype E -14 Time E -60 Genotype:Time E -23 Residual Total Figure 3B 23

24 SA Genotype E -16 Time E -08 Genotype:Time E -12 Residual Total JA Genotype Time E -23 Genotype:Time E -08 Residual Total JA-Ile Genotype E -17 Time E -47 Genotype:Time E -32 Residual Total Figure 4A Treatment E -10 Residual Total Figure 4B Genotype E -07 Residual Total

25 Figure 4C PR1b Genotype E -29 Time E -48 Genotype:Time E -28 Residual Total PR4 Genotype E -29 Time E -51 Genotype:Time E -29 Residual Total PR5 Genotype E -32 Time E -41 Genotype:Time E -31 Residual Total PR10 Genotype E -18 Time E -34 Genotype:Time E -23 Residual Total Figure 5B Genotype E

26 Residual Total Figure 5D Genotype Residual Total Figure 9D Genotype E -04 Treatment E -17 Genotype:Treatment E -04 Residual E -04 Total Figure 9E Genotype E E -07 Treatment E -21 Genotype:Treatment E E -07 Residual Total Figure 9F Genotype E -10 Treatment E -27 Genotype:Treatment E -10 Residual Total Figure 9G Genotype E -05 Treatment E

27 Genotype:Treatment E -05 Residual E -0.5 Total Figure 9H left Treatment E -38 Residual Total Figure 9H right Treatment E -38 Residual Total Figure 9I left Treatment E -20 Residual Total Figure 9I right Treatment E -11 Residual Total Supplemental Figure 3 Genotype E -64 Time E -98 Genotype:Time E -13 Residual Total Supplemental Figure 5 27

28 Genotype E -08 Residual Total Supplemental Figure 6A upper left Genotype Time E -12 Genotype:Time Residual Total Supplemental Figure 6A upper right Genotype Time E -09 Genotype:Time Residual Total Supplemental Figure 6A lower left Genotype Time E -05 Genotype:Time Residual Total Supplemental Figure 6A lower right Genotype Time E -06 Genotype:Time Residual Total Supplemental Figure 6B 28

29 Genotype E -93 Residual Total Supplemental Figure 6C Genotype E -41 Residual Total Supplemental Figure 6D Genotype E -26 Time E -78 Genotype:Time E -06 Residual Total Supplemental Figure 7A Genotype Residual Total Supplemental Figure 7B Genotype E -17 Residual Total Supplemental Figure 7C Genotype E -12 Residual Total

30 Supplemental Figure 7D Genotype Residual E -04 Total Supplemental Figure 7E Genotype Residual Total Supplemental Figure 7F The fourth leaf length Genotype Residual Total the fourth leaf width Genotype Residual Total Supplemental Figure 7G Genotype Residual Total Supplemental Figure 8 Treatment E E E -289 Time E E -197 Treatment:Time E E -131 Residual Total E

31 Supplemental Figure 9 GNS5 Genotype E -42 Time E -62 Genotype:Time E -49 Residual Total GNS6 Genotype E -17 Time E -53 Genotype:Time E -20 Residual Total GNS8 Genotype E -09 Time E -30 Genotype:Time E -29 Residual Total GNS9 Genotype E -39 Time E -30 Genotype:Time E -28 Residual Total Supplemental Figure 13 WRKY46 31

32 Genotype E -19 Time E -31 Genotype:Time E -25 Residual Total WRKY72 Genotype E -25 Time E -58 Genotype:Time E -29 Residual Total