Supplementary information Supplementary Figure 1. Comparison of BPH resistance in RH and Taichung Native 1.

Transcription

1 Supplementary information Supplementary Figure 1. Comparison of BPH resistance in RH and Taichung Native 1.

2 a, Representative image of RH and Taichung Native 1 (TN1) infested with BPH. b, Seedling mortality rate of RH and TN1 infested with BPH. Data were collected 10 days post infestation (dpi). Error bars indicate s.e.m. of the mean of 3 replicates (about 25 seedlings each). c, Dynamic changes of the BPH populations on TN1 (solid circles) and RH (open circles) in the feeding preference experiment. d, Dynamic changes of the BPH populations on single seedling of TN1 and RH infested with 90 BPHs in a closed container. e, Measurement of honeydew excreted from BPH on RH and TN1. f, BPH survival rate on RH and TN1 in a non-choice feeding experiment. Error bars indicate s.e.m. of the mean by the Student s t-test (b, c, d, f, n = 3; e, n = 10, *P<0.05, ** P < 0.01).

3 Supplementary Figure 2. Sequence analysis of Lectin receptor kinases. a, Phylogenetic tree of the Lectin receptor kinase family. The four OsLecRK proteins all belong to the G-type LecRK subfamily. b, Amino acid sequence alignment of the four OsLecRK proteins. Identical and similar amino acids are shaded in black and in grey, respectively. Various predicted domains are underlined. PAN-AP, Plant PAN/APPLE-like domain, predicted to possess protein- and/or carbohydrate-binding functions; S-TKC, serine/threonine kinase catalytic domain. The lectin and transmembrane domains are boxed in red and green, respectively.

4 Supplementary Figure 3. Arrangement of the OsLecRKs gene cluster in RH and a, A >8-kb insertion was identified upstream of the predicted start site of OsLecRK3 in No amplification product could be obtained using 4 pairs of primers specific to OsLecRK4, indicating that OsLecRK4 may be missing or highly diverged in b, Diagrams showing the amino acid substitutions in OsLecRK1 OsLecRK3 between RH and A 7 nucleotides deletion (position ) in OsLecRK2 leads to premature termination in

5 Supplementary Figure 4. Identification of four new BPH-resistant germplasms. a, Seedlings of BP348-e-4-2, Sarinah, Lv-7 and Lv-4 are resistant to BPH as RH, while 02428, C418, 9311 and Nipponbare (NIP) are susceptible to BPH. Pictures were taken 10 dpi. b, Mapping of the BPH resistance gene in the four new accessions to the same region as in RH (Fig. 2a). PVE, percentage of phenotypic variance explained.

6 Supplementary Figure 5. RNA levels of OsLecRKs in transgenic lines. Measurement of seedling mortality rates (shown on left Y-axis) and relative gene expression (shown on right Y-axis) levels in Kitaake plants individually transformed with RH genomic sequences of OsLecRK1, OsLecRK2, OsLecRK3 or OsLecRK4 (gl1, gl2, gl3 and gl4, respectively) or co-transformed with OsLecRK1+OsLecRK2 (gl1+gl2), or OsLecRK1+OsLecRK2+OsLecRK3 (gl1+gl2+gl3). RNA samples used for the assay were prepared using 14-day old seedlings (24 h post-infestation with BPH). Error bars indicate s.e.m. of the mean of 3 replicates for individual transgenic line. L1, L2 and L3 represent the relative transcript levels of OsLecRK1, OsLecRK2 and OsLecRK3, respectively. ND: not detectable.

7 Supplementary Figure 6. RNA levels of the closest potential off targets (Os06g41560 and Os08g13420) in two representative RNAi transgenic lines. RNA samples used for the assay were prepared using 14-day old seedlings (24 h post-infestation with BPH). Means of three independent samples assayed by quantitative RT-PCR are shown with bars representing s.e.m. Asterisk indicates a significant difference at P < 0.05 (n = 3), by the Student s t-test.

8 Supplementary Figure 7. Induction of OsLecRKs expression by BPH infestation. Expression of OsLecRK3 was essentially undetectable in RNA samples used for the assay were prepared using 14-day old seedlings. The expression values were presented relative to that in non-infested RH plants. Error bars indicate s.e.m. of the mean. Asterisk indicates a significant difference at P < 0.05, based on 3 replicates by the Student s t-test. ND: not detectable.

9 Supplementary Figure 8. Histochemical staining and activity assay of GUS reporter lines. a-c, The left panels show leaf cross sections of OsLecRK1::GUS, OsLecRK2::GUS and OsLecRK3::GUS reporter transgenic lines respectively. The right panels show enlarged view of the vascular tissues. Scale bar, 50 μm. X, xylem; P, phloem. d, Measurement of GUS activity of OsLecRK1::GUS, OsLecRK2::GUS and OsLecRK3::GUS reporter transgenic lines pro- and 24h after BPH infestation. ** P < 0.01, based on 3 replicates by the Student s t-test.

10 Supplementary Figure 9. Subcellular localization of GFP fusions of OsLecRK1 OsLecRK3. GFP or OsLecRK-GFP fusion constructs (represented by L1 L3, respectively) were cotransformed into rice leaf sheath protoplasts with the membrane marker aquaporin PIP2a fused to mrfp. Scale bar, 5 μm.

11 Supplementary Figure 10. BPH resistance of a Bph3-containing near-isogenic line (R1266) at the mature stage.

12 Supplementary Figure 11. Evaluation of white back planthopper resistance in R1266. a, Acquired WBPH resistance in R1266 at the seedling stage. Picture was taken 15 dpi. b, Seedling mortality rate. Data are means ± s.e.m. Letters indicate a significant difference at P < 0.01 (n = 3) by the Student s t-test.

13 Supplementary Table 1. Plant materials used for origin analyses of Bph3 Sample No. Accession name Accession ID Status Origin Type 1 DZ78 IRGC 8555 Landrace Bangladesh AUS 2 Mehr IRGC Landrace Iran AUS 3 Kalamkati IRGC Landrace India AUS 4 Phudugey IRGC Landrace Bhutan AUS 5 Jhona 349 IRGC 6307 Landrace India AUS 6 Haginomae Mochi IRGC 2540 Elite Japan IND 7 IR 36 IRGC Improve Brazil IND 8 JC 91 IRGC 9177 Elite India IND 9 Gie 57 IRGC 8231 Landrace Vietnam IND 10 TD2 IRGC 9148 Elite Thailand IND 11 JC101 IRGC 9060 Elite India ARO 12 JC111 IRGC 9062 Elite India ARO 13 Bico Branco IRGC Elite Brazil ARO 14 Kitrana 508 IRGC Elite Madagascar ARO 15 Firooz RA 4952 Landrace Iran ARO 16 Lemont IRGC Elite TX, USA TRJ 17 Canella De Ferro IRGC Elite Brazil TRJ 18 Maintmolotsy IRGC Elite Madagascar TRJ 19 NPE 844 IRGC Landrace Pakistan TRJ 20 Chodongji IRGC Landrace South Korea TEJ 21 Darmali IRGC Landrace Nepal TEJ 22 Leung Pratew IRGC Landrace Thailand TEJ 23 BADAL 89 IRGC 6513 Landrace Bangladesh III 24 HAISHA CAMAN IRGC Landrace Bangladesh IV 25 KUI SALI IRGC Landrace Bangladesh V 26 DX-06 DX-06 wild rice Jiang Xi, China rufipogon 27 Yuan3-9 Yuan3-9 wild rice Yun nan, China rufipogon 28 P25 P25 wild rice Guang rufipogon dong,china 29 P46 P46 wild rice Hai nan, China rufipogon 30 P61 P61 wild rice Guang Xi China rufipogon 31 - IRGC wild rice India rufipogon 32 - IRGC wild rice PNG rufipogon 33 - IRGC wild rice Sri Lanka rufipogon 34 - IRGC wild rice India rufipogon 35 - IRGC wild rice Myanmar rufipogon 36 - IRGC wild rice Thailand rufipogon 37 PADI PADIAN IRGC wild rice Indonesia rufipogon

14 38 DAL AHAN IRGC wild rice Bangladesh rufipogon 39 - IRGC wild rice Laos rufipogon 40 Nepal Nepal wild rice Nepal rufipogon 41 HK 47 IRGC wild rice India nivara 42 - IRGC wild rice Nepal nivara 43 - IRGC wild rice Myanmar nivara 44 - IRGC wild rice Bangladesh nivara 45 - IRGC wild rice Cambodia nivara 46 CA IRGC wild rice Laos nivara 47 - IRGC wild rice Thailand nivara 48 - IRGC wild rice Sri Lanka nivara 49 - IRGC wild rice India nivara 50 - IRGC wild rice India nivara

15 Supplementary Table 2. Primers used in this study I. Primers for high resolution mapping of Bph3 Primers Name Forward Primer Reverse Primer RM8213 AGCCCAGTGATACAAAGATG GCGAGGAGATACCAAGAAAG A4 AAGCAGCATAAACTGATTGA TCATCTTCTGAAAAAGCAAT RH7845 AGTATGTCACTTTGGGCTTAT ACAATACATAGAGAGTTAGGTTCA RM5953 AAACTTTCTGTGATGGTATC ATCCTTGTCTAGAATTGACA RH784 TTCTTGGCTGATTGGGGAGTAT ATTTGGAATGGAGCAGAGTGGA RH078 GCCGATTTGGTCTAACTTCATT CAACCAGCAAGGAAACAATAAA W4 GTTTCAACCGAATAGCACGAG CGGAATAACTCACATCGGCA RM16533 TTTGCTTAGTCGGCAGATGTCC CATAAGAACGTACCTCCACTGATTCC RHD3 GGTAAGGTTGGGCGGTAG AGTGAAGGGTGAGGGTGG RHD9 GTCCATCCGAAGGTGAAAGT CCGAACATCGAGGAATACAA RH7 CTTGCGTTCCGTAGGAGAAG TGAGTGTAACCCGAAGTGGC WH2 CCCACCACACCAGAGATAAA ACACAACACCCGCATACAA RHC10 CAATACGGGAGATTTGGAGT TTGGGAAGCATACGAGTGA W1 TCCTAATCAGCCAATAAATCA GCAATCTAGTGCACGAACATA II. Primers for gene cloning, and sequencing Primers Name Forward Primer Reverse Primer P1 AGTCTTCGAGTTCAACTTGCT TCAAGCATTTGAGTTACCTT P2 TTAGGAACTTACATTGCTGT ACCAGCAGGTCGACCCTGCCA P3 TGCACTTTATACCATGGCA CAGCCTGAGGCCAATCCAC P4 ATGGCACCTCCACTCTTCTTG TGAATTGTGTTCCATTCTTCA TP1-1 GATCATCAATGAACTTATAATTT TP1-2 CCTGTGAACCATTAAGTATGA TP1-3 AGCCCAATATGGATCGTATA TP2-1 TATGGTCAAGCCAGCTAGAAT TP2-2 ACGTATTCAGTCACCGTCTTC TP2-3 TCAGTCACTGGAGGGTTCAG TP3-1 TCTAATATGCACCATACCTA TP3-2 TCGTAGGCAGCCAATTTGCT TP3-3 CAAGAATGTTATTCAAATAG TP4-1 AAGGTATGGCCAAGGTTTA TP4-2 TTGTGTTGGGAGTTAATCCT TP4-3 TACTCATTAGCGCGTGATT OsLecRK1 ATTGTTTACTCCAATAACGGT TTATGGGACAGAGTGAGTATTT OsLecRK2 CTTTCGCAGGGTGGCAAATAGGGT CCTTCGCTGCTCACTAGGACCGTGTA OsLecRK3 GCCCGCTCGCTACCGCT CGGTCCTGCACATTACTGTAG OsLecRK4 CCCAGATCAACATGGGTTCTGT GGAGGTAACAGGAGGTACCAC Ⅲ. Primers for promoter cloning, complementation, RNAi and qpcr Primers Name Forward Primer* Reverse Primer* Promoter cloning OsLecRK1-P ATCCATGGCACATCTCTGCCCTCACCTGC ATGGATCCGAGGGGATCACGGGGAGTAAT OsLecRK2-P GCGGAATTCTTAGTTCACATTTCCTTATCA ATAGTCGACGTCAAAGTCGCAACCGTCC OsLecRK3-P GCGGAATTC ACATGATCCTTGCTGGTCT ATAGTCGAC CCTGGCTGTCACTACTCACT Construct for complementation pcambia1305-oslecrk1 pcambia1305-oslecrk2 pcambia1305-oslecrk3 ACCCGGGGATCCTCTAGAATTGTTTACTC CAATAACGGT ACCCGGGGATCCTCTAGACTTTCGCAGGG TGGCAAATAGGGT ACCCGGGGATCCTCTAGAGCCCGCTCGCT ACCGCT AAGCTTGCATGCCTGCAGTTATGGGACAG AGTGAGTATTT CTGCAGGTCGACTCTAGACCTTCGCTGCTC ACTAGGACCGTGTA CTGCAGGTCGACTCTAGACGGTCCTGCAC ATTACTGTAG

16 pcambia1305-oslecrk RK2(BamHI) 1305-RK2(KpnI/XbaI) 1305-RK3(KpnI /ApaI) ACGAATTCGAGCTCGGTACCCAGATCAAC ATGGGTTCTGT GCTCGGTACCCGGGGATCCCTTTCGCAGG GTGGCAAATAGGGT GAATTCGAGCTCGGTACCGGGCCCCTTTC GCAGGGTGGCAAATAGG GAATTCGAGCTCGGTACCCTTGCCATGAC GAAGAGATG AGGTCGACTCTAGAGGATCCGGAGGTAAC AGGAGGTACCAC GAGCGGGCTCTAGAGGATCCCCTTCGCTG CTCACTAGGACCGTGTA TATTATGGGTCTGTCTAGACCTGCACATTA CTGTAGA CCACCCTGCGAAAGGGGCCCGATGCATCT AACTGAATG RNAi cloning RNAi 1 ATGTCGACATGGCACCTATCCTGTTCTTG GCACTAGTTGTGAACCATTAGTCAGCGT RNAi 2 TTGGATCCTGGCACCTATCCTGTTCTTG GCTCTAGATGTGAACCATTAGTCAGCGT qpcr primers OsLecRK-1E TTGGCGTGTCTGGAAGGTAT TCCAGACCGCATTAGTTCCT OsLecRK-2E TGAGTGCCCTGAATAACAT CCCAACGCCTCCTGAC OsLecRK-3E CTTGGAGAACGGCATTTGT TGTCAGGAGAACCCAGCAT OsLecRK-4E ATCTGGGTCAAGCGTAG CCAATGGCAGGTTTCT Os06g41560 GCAGCGGATGCTCGTCTTC AGTCGGCTATCTTTGGGTTGTTC Os08g13420 TAGACCTCAATGGCACCCT GCGTAGAGTCGCAATAAGC IV. Primers for subcellular localization Primers Name Forward Primer* Reverse Primer* OsLecRK1-GFP ATCACCATCACGCCATGGTCATGGTTGCT CTGCTACTC GCTCACCATCACTAGTACTGGAAGTGAGC TGATGAAGG OsLecRK2-GFP TATCCATGGTAGCAATGGCACCTATCCTG CGAACTAGTGATGCGAGTGAACTGATATA GG OsLecRK3-GFP TATCCATGGTGGCTCATCTCCTGTTCT CGAACTAGTGCAAGTGAACTGATATAGGA *Restriction sites/recombination sites are underlined