Improvement of multiple agronomic traits by a disease resistance gene via cell wall reinforcement

Transcription

1 In the formt provided y the uthors nd unedited. SUPPLEMENTARY INFORMATION VOLUME: 3 ARTICLE NUMBER: 179 Improvement of multiple gronomic trits y disese resistnce gene vi cell wll reinforcement Keming Hu, Jino Co, Jie Zhng, Fn Xi, Yinggen Ke, Hito Zhng, Weny Xie, Hongo Liu, Ying Cui, Yinglong Co, Xinli Sun, Jinghu Xio, Xinghu Li, Qinglu Zhng nd Shiping Wng* The Ntionl mjor Key Lortory disese of resistnce Crop Genetic gene Improvement, X4 confers Ntionl rce-speci Centre of Plnt c durle Gene Reserch resistnce (Wuhn), ginst Huzhong Xnthomons Agriculturl University, oryzewuhn pv. oryze, 437, Chin. *e-mil: swng@mil.hzu.edu.cn NATURE PLANTS DOI: 1.138/nplnts Mcmilln Pulishers Limited, prt of Springer Nture. All rights reserved.

2 X3/X2 fmily WAK TRKd TRK TRKe TRK TRKf TRKg TRKc TRKh TRKi TRKj Teqing X4-88 Su11 (WAK) X4 2/15B-29/IRK M19-1 IRBB4 47 k Minghui 3 MWAK MRKd MRK X3/X2 (or MRK) X3/X2 fmily MRKc Supplementry Figure 1. The physicl mp of X4 region in rice line IRBB4 nd its reltionship to the physicl mps of rice vrieties Teqing nd Minghui 3. The mpping informtion of X4 in rice line IRBB4 nd X3/X2 fmily in rice vrieties Minghui 3 nd Teqing re from previous pulictions (Sun et l. Theor. Appl. Genet. 1:83-87, 23; Sun et l. Plnt J. 37: , 24; Sun et l. Plnt Physiol. 14:998-18, 2). The mpping of IRK, WAK, nd MWAK is from present study. X4-88, Su11, 2/15B-29, nd M19-1 re moleculr mrkers.

3 WAK (XA4) wk (x4) IRBB4 IR4 Teqing IR24 Minghui 3 Zhenshn 97 Mudnjing 8 Zhonghu 11 Nipponre Glcturonn-inding domin (1) MAAAAALSAVAPGQRPADCPSECGGVDIPYPFGVDNCSWPGPDDFTIICKYS----RPYY (1) MAAAAALSAVAPGQRPADCPSECGGVDIPYPFGVDNCSWPGPDDFTIICKYS----RPYY (1) MAAAAALSAVAPGQRPADCPSECGGVDIPYPFGVDNCSWPGPDDFTIICKYS----RPYY (1) MAAAAALSAVAPGQRPADCPSECGGVDIPYPFGVDNCSWPGPDDFTIICKYS----RPYY (1) MAAAAALSAVAPGQRPADCPSECGDVDIPYPFGVDNCSWPGPDDFDVSCNHSFSPPRPYY (1) MAAAAALSAVAPGQRPADCPSECGGVDIPYPFGVDNCSWPGPDDFTIICKYS----RPYY (1) MAAAAALSAVAPGQRPADCPSECGGVDIPYPFGVDNCSWPGPDDFTIICKYS----RPYY (1) MAAAAALSAVAPGQRPADCPSECGGVDIPYPFGVDNCSWPGPDDFTIICKYS----RPYY (1) MAAAAALSAVAPGQRPADCPSECGGVDIPYPFGVDNCSWPGPDDFTIICKYS----RPYY IRBB4 IR4 Teqing IR24 Minghui 3 Zhenshn 97 Mudnjing 8 Zhonghu 11 Nipponre (57) RGAEIVNISVEAGEMRVYSPVVSQCYNSSNTTDSDGFEFLRLNITNTPFLVAPERNEFTA (57) RGAEIVNISVEAGEMRVYSPVVSQCYNSSNTTDSDGFEFLRLNITNTPFLVAPERNEFTA (57) RGAEIVNISVEAGEMRVYSPVVSQCYNSSNTTDSDGFEFLRLNITNTPFLVAPERNEFTA (57) RGAEIVNISVEAGEMRVYSPVVSQCYNSSNTTDSDGFEFLRLNITNTPFLVAPERNEFTA (1) RDAEIVSISVEAGEMRVYSPVVSQCYNSSNTTDSDGFEFLQLNITNTPFLVAPERNEFTA (57) RGAEIVNISVEAGEMRVYSPVVSQCYNSSNTTDSDGFEFLRLNITNTPFLVAPERNEFTA (57) RGAEIVNISVEAGEMRVYSPVVSQCYNSSNTTDSDGFEFLRLNITNTPFLVAPERNEFAA (57) RGAEIVNISVEAGEMRVYSPVVSQCYNSSNTTDSDRFELLQLNITNTPFLVAPERNEFTA (57) RGAEIVNISVEAGEMRVYSPVVSQCYNSSNTTDSDGFEFLQLNITNTPFLVAPERNEFTA IRBB4 IR4 Teqing IR24 Minghui 3 Zhenshn 97 Mudnjing 8 Zhonghu 11 Nipponre (117) IGCATLAWLWGRDDGSYLTGCISTCASLATAAKDRDPCTGLGCCQVPSIPANLSVLNISL (117) IGCATLAWLWGRDDGSYLTGCISTCASLATAAKDRDPCTGLGCCQVPSIPANLSVLNISL (117) IGCATLAWLWGRDDGSYLTGCISTCASLATAAKDRDPCTGLGCCQVPSIPANLSVLNISL (117) IGCATLAWLWGRDDGSYLTGCISTCASLATAAKDREPCTGLGCCQVPSIPANLSVLNISL (1) IGCATLAWLWGRDDGSYLTGCISTCASLATAAKDREPCTGLGCCQVPSIPANLSVLNISL (117) IGCATLAWLWGRDDGSYLTGCISTCASLATAAKDREPCTGLGCCQVPSIPANLSVLNISL (117) IGCATLAWLWGRDDGSYLTGCISTCASLATAAKDREPCTGLGCCQVPSIPANLSVLNISL (117) IGCATLGWLQGRDDGNYLTGCISTCASLETATDDGEPCTGLGCCQVPSIPPNLGILNISL (117) IGCATLAWLWGRDDGSYLTGCISTCASLATAAKDREPCTGLGCCQVPSIPANLSVLNISL IRBB4 IR4 Teqing IR24 Minghui 3 Zhenshn 97 Mudnjing 8 Zhonghu 11 Nipponre (177) GTGIANVAWEESPCSYAFVAEKHWYNFNRQDFERDGKSFEHRDEKMVVPTVLDWAIRKNG (177) GTGIANVAWEESPCSYAFVAEKHWYNFNRQDFERDGKSFEHRDEKMVVPTVLDWAIRKNG (177) GTGIANVAWEESPCSYAFVAEKHWYNFNRQDFERDGKSFEHRDEKMVVPTVLDWAIRKNG (177) GTGIANVAWEESPCSYAFVAEKHWYNFNRQDFERDGKSFEHRDEKMVVPTVLDWAIRKNG (181) GTGIANVAWEESPCSYAFVAEKHWYNFNRQDFGPRSKSFEHRDGKMVVPTVLDWAIRKSG (177) GTGIANVAWEESPCSYAFVAEKHWYNFNRQDFERDGKSFEHRDEKMVVPTVLDWAIKKDG (177) GTGIANVAWEESPCSYAFVAEKHWYNFNRQDFERDGKSFEHRDEKMVVPTVLDWAIRKNG (177) GTGIANVAWEESPCSYAFVAEKHWYNFNRQDFERDGKSFEHRDEKMVVPTVLDWAIRKNG (177) GTGIANVAWEESPCSYAFVAEKHWYNFNRQDFERDGKSFEHRDEKMVVPTVLDWAIRKNG

4 IRBB4 IR4 Teqing IR24 Minghui 3 Zhenshn 97 Mudnjing 8 Zhonghu 11 Nipponre IRBB4 IR4 Teqing IR24 Minghui 3 Zhenshn 97 Mudnjing 8 Zhonghu 11 Nipponre IRBB4 IR4 Teqing IR24 Minghui 3 Zhenshn 97 Mudnjing 8 Zhonghu 11 Nipponre Clcium-inding (237) SCPSTGQGAPACKSEHSECVNATNGKGYLCNCSRGYAGNPYRDDGCKNINECKEPSITCY (237) SCPSTGQGAPACKSEHSECVNATNGKGYLCNCSRGYAGNPYRDDGCKNINECKEPSITCY (237) SCPSTGQGAPACKSEHSECVNATNGKGYLCNCSRGYAGNPYRDDGCKNINECKEPSITCY (237) SCPSTGQGAPACKSEHSECVNATNGKGYLCNCSRGYAGNPYRDDGCKNINECKEPSITCY (241) SCPSTGQGAPACKSEHSECVNATNGKGYLCNCSRGYAGNPYRDDGCKNINECKEPSITCY (237) SCPSTGQGAPESKSEHSECVNATNGMGYLCNCSRGYAGNPYRVDGCKNINECKEPSITCY (237) SCPSTGQGAPACKSEHSECVNATNGKGYLCNCSRGYAGNPYRDDGCKNINDCKEPSITCY (237) SCPSTGQGAPACKSEHSECVNATNGKGYLCNCSRGYAGNPYRDDGCKNINECKEPSITCY (237) SCPSTGQGAPACKSEHSECVNATNGKGYLCNCSRGYAGNPYRDDGCKNINECKEPSITCY epiderml growth fctor domin Trnsmemrne helix (297) GGSTCQDTDGSYECKCQFGYRGDGRKNESQKGRCQPIIPAAIANAIAIVCIVIVLLGLFW (297) GGSTCQDTDGSYECKCQFGYRGDGRKNESQKGRCQPIIPAAIANAIAIVCIVIVLLGLFW (297) GGSTCQDTDGSYECKCQFGYRGDGRKNESQKGRCQPIIPAAIANAIAIVCIVIVLLGLFW (297) GGSTCQDTDGSYECKCQFGYRGDGRKNESQKGRCQPIIPAAIANAIAIVCIVIVLLGLFW (31) GGSTCQDTDGSYECKCQFGYTGDGRKNESQKGRCRPIIPATIANAIAIVCIVIVLLGLFW (297) GGSTCQDTDGSYECKCQFGYRGDGRKNESQKGRCQPIIPAAIANAIAIVCIVIVLLGLFW (297) GGSTCQDTDGSYECKCQFGYRGDGRKNESQKGRCQPIIPAAIANAIAIVCIVIVLLGLFW (297) GGSTCQDTDGSYECKCQFGYRGDGRKNESQKGRCQPIIPAAIANAIAIVCIVIVLLGLFW (297) GGSTCQDTDGSYECKCQFGYRGDGRKNESQKGRCQPIIPAAIANAIAIVCIVIVLLGLFW Serine/threonine kinse domin (357) LPKRWKRRVFFDNNGGRLLKDMDIIVFTEKELNKITNKKRTKIGEGAFGEVYKGNHNNQP (357) LPKRWKRRVFFDNNGGRLLKDMDIIVFTEKELNKITNKKRTKIGEGAFGEVYKGNHNNQP (357) LPKRWKRRVFFDNNGGRLLKDMDIIVFTEKELNKITNKKRTKIGEGAFGEVYKGNHNNQP (357) LPKRWKRRVFFDNNGGRLLKDMDIIVFTEKELNKITNKKRTKIGEGAFGEVYKGNHNNQP (31) LPKRWKRRVFFDNNGGRLLKDMDIIVFTEKELNKITNKKCTKIGEGAFGEVYKGNHNNQP (357) LPKRWKRRVFFDNNGGRLLKDMDIIVFTEKGLNKITSKKRTKIGEGAFGEVYKGNHNNQP (357) LPKRWKRRVFFDNNGGRLLKDMDIIVFTEKELNKITNKKRTKIGEGAFGEVYKGNHNNQP (357) LPKRWKRRVFFDNNGGRLLKDMDIIVFTEKELNKITNKKRTKIGEGAFGEVYKGNHNNQP (357) LPKRWKRRVFFDNNGGRLLKDMDIIVFTEKELNKITNKKRTRIGEGAFGEVYKGNHNNQP IRBB4 IR4 Teqing IR24 Minghui 3 Zhenshn 97 Mudnjing 8 Zhonghu 11 Nipponre (417) VAVKYSIAKNMTQTHYKDVVESINQNVFQTVFRQSKVPPSTPGQNAVVNEIKVQLQIRHP (417) VAVKYSIAKNMTQTHYKDVVESINQNVFQTVFRQSKVPPSTPGQNAVVNEIKVQLQIRHP (417) VAVKYSIAKNMTQTHYKDVVESINQNVFQTVFRQSKVPPSTPGQNAVVNEIKVQLQIRHP (417) VAVKYSIAKNMTQTHYKDVVESINQNVFQTVFRQSKVPPSTPGQNAVVNEIKVQLQIRHP (421) VAVKYSITKNMTQTHYKD-VESVNQNVFQTVFRQSKVPPSTPGQNAVVNEIKAQLQIRHP (417) VAVKYSIAKNMTQTHYKDVVESINQNVFQTVFRQSKVPPSTPGQNAVVNEIKVQLQIRHP (417) VAVKYSIAKNTTQTHYKGVVESINQNVFQTVFRQSKVPPSTPGQNAVVNEIKVQLQIRHP (417) VAVKYSIAKNMTQTHYKDVVESINQNVFQTVFRQSKVPPSTPGQNAVVNEIKVQLQIRHP (417) VAVKYSIAKNMTQTHYKDVVESINQNVFQTVFRQSKVPPSTPGQNAVVNEIKVQLQIRHP

5 IRBB4 IR4 Teqing IR24 Minghui 3 Zhenshn 97 Mudnjing 8 Zhonghu 11 Nipponre (477) NIVRLIGCCMETEVPMLVFEFIPNGSLETVLHGIDRCSLSLQQRLDIAIGSAEALAYMHW (477) NIVRLIGCCMETEVPMLVFEFIPNGSLETVLHGIDRCSLSLQQRLDIAIGSAEALAYMHW (477) NIVRLIGCCMETEVPMLVFEFIPNGSLETVLHGIDRCSLSLQQRLDIAIGSAEALAYMHW (477) NIVRLIGCCMETEVPMLVFEFIPNGSLETVLHGIDRCSLSLQQRLDIAIGSAEALAYMHW (48) NIVRLIGCCMETEVPMLVFEFIPNGSLETVLHGIDRCRLSLHQRLDIAIGSAEALAYMHW (477) NIVRLIGCCMETEVPMLVFEFIPNGSLETVLHGIDRCSLSLQQRLDIAIGSAEALAYMHW (477) NIVRLIGCCIETEVPMLVFEFIPNGSLETVLHGIDRCSLSLQQRLDIAIGSAEALAYMHW (477) NIVRLIGCCMETEVPMLVFEFIPNGSLETVLHGIDRCSLSLQQRLDIAIGSAEALAYMHW (477) NIVRLIGCCMETEVPMLVFEFIPNGSLETVLHGIDRCSLSLQQRLDIAIGSAEALAYMHW IRBB4 IR4 Teqing IR24 Minghui 3 Zhenshn 97 Mudnjing 8 Zhonghu 11 Nipponre (537) HGHHQIIHGDIKPGNILLGDNLMPKVSDFGSSELTLKVKRAGKWNVYADMNYIDPVYIKT (537) HGHHQIIHGDIKPGNILLGDNLMPKVSDFGSSELTLKVKRAGKWNVYADMNYIDPVYIKT (537) HGHHQIIHGDIKPGNILLGDNLMPKVSDFGSSELTLKVKRAGKWNVYADMNYIDPVYIKT (537) HGHHQIIHGDIKPGNILLGDNLMPKVSDFGSSELTLKVKRAGKWNVYADMNYIDPVYIKT (54) HGHHQIVHGDIKPGNILLGDNLMPKVSDFGSSELTLKVKRARKWNVYADMNYIDPVYIET (537) HGHHQIIHGDIKPGNILLGDDLMPKVSDFGSSELTLKVKRAGKWNVYADMNYIDPVYIKT (537) HGHHQIIHGDIKPGNILLGDNLVPKVSDFGSSELTLKVKRAGKWNVYADMNHIDPVYIKT (537) HGHHQIIHGDIKPGNILLGDNLMPKVSDFGSSELTLKVKRAGKWNVYADMNYIDPVYIKT (537) HGHHQIIHGDIKPGNILLGDNLMPKVSDFGSSELTLKVKRAGKWNVYADMNYIDPVYIKT IRBB4 IR4 Teqing IR24 Minghui 3 Zhenshn 97 Mudnjing 8 Zhonghu 11 Nipponre (597) GDFTDKSDVYSFGVVLLELITRKKAKYDDRSLPVEFVSHYEDEDTRRKMYDQDMLPTEAS (597) GDFTDKSDVYSFGVVLLELITRKKAKYDDRSLPVEFVSHYEDEDTRRKMYDQDMLPTEAS (597) GDFTDKSDVYSFGVVLLELITRKKAKYDDRSLPVEFVSHYEDEDTRRKMYDQDMLPTEAS (597) GDFTDKSDVYSFGVVLLELITRKKAKYDDRSLPVEFVSHYEDEDTRRKMYDQDMLPTEAS () GDFTDKSDVYSFGVVLLELITRKKAKYDDRSLPVEFVNHYEDEDTRRKMYDQDMLSTETL (597) GDFTDKSDVYSFGVVLLELITRKKAKYDDRSLPVEFASHYEDEDTRRKMYDQDMLPTEAS (597) GDFTDKSDVYSFGVVLLELITRKKAKYDDRSLPVEFVSHYEDEDTRRKMYDQDMLPTEAS (597) GDFTDKSDVYSFGVVLLELITRKKAKYDDRSLPVEFVSHYEDEDTRRKMYDQDMLPTEAS (597) GDFTDKSDVYSFGVVLLELITRKKAKYDDRSLPVEFVSHYEDEDTRRKMYDQDMLPTEAS IRBB4 IR4 Teqing IR24 Minghui 3 Zhenshn 97 Mudnjing 8 Zhonghu 11 Nipponre (57) HPHCMECLDRMADIVLRCLENEVGKRPTMAEVLEELKKLLPLLTTTPVELV (57) HPHCMECLDRMADIVLRCLENEVGKRPTMAEVLEELKKLLPLLTTTPVELV (57) HPHCMECLDRMADIVLRCLENEVGKRPTMAEVLEELKKLLPLLTTTPVELV (57) HPHCMECLDRMADIVLRCLENEVGKRPTMAEVLEELKKLLPLLTTTPVELV () HPHCMECLDRMADIVLRCLENEVGKRPTMVEVLEELKKLMPLLTTTPVELV (57) HPHCMECLDRMADIVLRCLENEVGKRPTMAEVLEELKKLLPLLTTTPVELV (57) HPHCMECLDRMADIVLRCLENEVGKRPTMAEVLEELKKLLPLLSTTPVELV (57) HPHCMECLDRMADIVLRCLENEVGKRPTMAEVLEELKKLLPLLTTTPVELV (57) HPHCMECLDRMADIVLRCLENEVGKRPTMAEVLEELKKLLPLLTTTPVELV

6 Supplementry Figure 2. Comprison of mino cid sequences encoded y WAK (X4) nd recessive wk (x4) from different rice lines nd vrieties. Blck shdings indicte mino cid sustitution, deletion, or insertion compred with reference sequence from IRBB4. The conservtive mino cid sustitution is outlined in red. The predicted glcturonninding domin, clcium-inding epiderml growth fctor domin, trnsmemrne helix, nd serine/threonine kinse domin of WAK (XA4) protein re highlighted with purple, pink, light lue, nd lue lines, respectively. The predicted protein kinse ATP-inding region nd kinse ctive site re outlined in yellow nd ornge, respectively.

7 IRBB4 54 p +1 ATG WAK (X4) IR4 ATG Teqing 9 ATG IR24 Minghui ATG ATG wk (x4) Zhenshn 97 Mudnjing 8 Zhonghu ATG ATG ATG Nipponre 4 ATG insertion sustitution deletion Supplementry Figure 3. Sequence comprison of the promoter regions of WAK (X4) nd recessive wk (x4) from different rice lines nd vrieties. Nucleotide sustitution, deletion, or insertion is identified using the sequence from IRBB4 s the reference. Arrowheds with figure indicte numers of nucleotides inserted or deleted, while rrowheds without figure represent single-nucleotide insertion/deletion. Arrow indictes four-se nucleotide deletion t 9 p (reltive to the trnsltion strt codon) promoter regions. ATG, trnsltion strt codon.

8 WAK (X4) gene (5887 nt) nd flnking frgments (13 nt) used for trnsformtion WAK (X4) gene ATG TGA Coding region Untrnslted region Intron Intergenic region Supplementry Figure 4. Structures of WAK (X4) gene nd DNA frgments used for trnsformtion. The DNA frgment ws from rice vriety Teqing cteril rtificil chromosome clone 25P4 digested using restriction enzyme BmHI. ATG, trnsltion strt codon; TGA, trnsltion stop codon. The figures indicte the nucleotides of ech sustructure.

9 IRBB4 (X4) K E B S Sp M Supplementry Figure 5. Resistnce IRBB4 crries single copy of X4 y DNA gel lot nlysis. Totl DNA ws digested with restriction enzymes KpnI (K), EcoRI (E), BmHI (B), ScI (S), or SpeI (Sp). M, DNA/EcoT14 DNA ldder.

10 Lesion length (cm) Reltive Expression level Correltion nlysis: r =.39, n = 14, P <.5 Xoo infection X4 expression X4-RNAi (T genertion) IR24 RNAi Lesion length (cm) Reltive expression level Correltion nlysis: r =.81, n = 14, P <.1 r =.893, n = 1, P <.1 Xoo infection Xoo infection RNAi X4 expression X4-RNAi3 T 1 fmily IR X4 expression X4-RNAi9 T 1 fmily IR24 Correltion nlysis: r =.735, n = 9, P <.5 r =.711, n = 1, P <.5 Lesion length (cm) Reltive expression level RNAi Xoo infection X4 expression X4-RNAi11 T 1 fmily IR X4-RNAi13 T 1 fmily Supplementry Figure. Reduced resistnce to Xoo ws correlted with suppression of X4 nd the existence of RNAi frgment in X4-RNAi plnts. Plnts were inoculted with Xoo strin PXO1 t the ooting stge. Dt represent men (3 to 5 replictes for lesion length nd 3 replictes for gene expression) ± stndrd devition. or, significnt difference etween trnsgenic plnts nd wild-type () IRBB4 t P <.1 or P <.5. IR24, susceptile control; RNAi, frgment of the RNAi construct mplified using PCR primers PMCG-F nd PMCG-R (Supplementl Tle )., Reduced resistnce to Xoo ws correlted with RNAi-medited suppression of X4 in X4-RNAi T plnts., Reduced resistnce to Xoo ws correlted with RNAi-medited suppression of X4 in X4-RNAi T 1 fmilies X4 expression Xoo infection IR24

11 Protein size 55KD 43KD 35KD Supplementry Figure 7. XA4 kinse domin showed utophosphoryltion. Left imge, Coomssie rillint lue-stined gel showing expression of the recominnt XA4 kinse domin (XA with GST tg; rrow-pointed nd); right imge, utordiogrph of the left gel showing in vitro utophosphoryltion of recominnt XA (rrow-pointed nd).

12 GFP Bright Merged XA4-GFP After plsmolysis with 5% (w/v) sucrose for 5 min GFP GFP RFP Bright Merged GFP ZmWAK-GFP XA4-GFP Supplementry Figure 8. Anlysis of sucellulr locliztion of XA4 protein y trnsient expression., The XA4-green fluorescent protein (GFP) fusion protein loclized in cell wll of onion epiderml cells. Brs indicte 5 μm., the XA4-GFP coloclized with the endoplsmic reticulum mrker HDEL (histidine, sprtic cid, glutmic cid, leucine)-red fluorescent protein (RFP) in rice protoplsts. The HDEL is retention signl of endoplsmic reticulum proteins (Npier et l. J. Cell Sci. :21-271, 215). The mize WAK resistnce protein (ZmWAK) is memrne protein (Zuo et l. Nt. Genet. 47: , 215). Trnsient expression of ZmWAK- GFP in rice protoplsts s control showed tht ZmWAK lso coloclized with the endoplsmic reticulum mrker nd in ddition, it ppers to e prtly ssocited with plsm memrne. Brs indicte 1 μm.

13 GFP & RFP merged Plsmolysis with 5% sucrose GFP PI stining Merged OsPAD4-GFP GFP ZmWAK-GFP XA4-GFP Supplementry Figure 9. Anlysis of sucellulr locliztion of XA4 protein in rice y stle trnsformtion. The root tips of two-lef trnsgenic rice plnts were used for nlysis. The cell wll ws stined with propidium iodide (PI) (Deng et l. Plnt Physiol. 158:87-889, 2). Mize ZmWAK-GFP nd rice OsPAD4-GFP re controls. OsPAD4 is rice plsm memrne protein (Ke et l. Plnt J. 78:19-31, 214). ZmWAK loclized in plsm memrne nd cytoplsm, nd OsPAD4 loclized in plsm memrne in trnsgenic rice plnts. Brs indicte 2 μm.

14 With nti-xa4 ntiody XV XV PC 5 µm PC PC.5 µm CW PC c MC XV PC d SC e PC SC EC SC 5 µm d e MC PC CW MC Ch PC CW SC SC.5 µm.5 µm f Without nti-xa4 ntiody (control) g PC XV g PC PC CW CW PC 5 µm.5 µm h SC i i CW CW EC 5 µm SC.5 µm SC Supplementry Figure 1. Gold prticle-leled nti-xa4 ntiody loclized in cell wll (CW) of lef tissue under trnsmission electron microscope. Lef smples were collected from rice line IRBB4 t the four-lef stge. Arrows point to representtive gold prticles. Ch, chloroplst; EC, epidermis cell; MC, mesophyll cell; PC, vsculr prenchym cell (upper) nd phloem prenchym cell (middle nd lower); SC, sclerenchym cell; XV, xylem vessel., Vsculr tissue., Enlrged region outlined in. c, Vsculr nd sclerenchym tissues. d, e, Enlrged regions outlined in c. f, Vsculr tissue. g, Enlrged region outlined in f. h, Sclerenchym tissue. i, Enlrged region outlined in h.

15 Breking force (N) Lef Culm (internode) First Second X4-RNAi11(+) X4-RNAi11( ) X4-RNAi13(+) X4-RNAi13( ) Crystl cellulose content (mg/g) Lef Culm (internode) First Second c XV XV XV XV XV XV XV XV XV 1 μm 1 μm 1 μm Wild type (IRBB4, X4) X4-RNAi13(+) X4-RNAi13( ) d Reltive expression level Culm CesA4 CesA7 CesA Culm CesA1 CesA3 CesA8 Supplementry Figure 11. X4 influenced mechnicl strength, cellulose content, nd relted gene expression. Smples were collected from flg leves nd the first nd second internodes of culms. Dt represent men (5 plnts for reking force nd 3 replictes for cellulose content) ± stndrd devition. or, significnt difference etween positive trnsgenic plnts (+) nd its segregting wild-type silings ( ) t P <.1 or P <.5., X4-RNAi plnts required smller force to rek lef nd culm compred to controls. N, newton., X4-RNAi plnts hd lower level of cellulose content (milligrms per grm of totl cell wll residues) thn controls in lef nd culm. c, The X4-RNAi plnts showed ovious decresed cellulose (light lue color) content in the cell wll of lef cells round xylem vessels (XV) compred to their wild-type silings nd IRBB4. Lef smples were collected from rice plnts t the four-lef stge. d, X4-RNAi plnts showed suppressed expression of CesA genes in the first internode of culm.

16 Breking force (N) Lef Culm ces4 Wild type 1 µm ces4 1 µm c XV Wild type PC PC ces4 XV PC PC 2 µm 2 µm PC PC XV CW CW XV CW CW PC 1 µm PC 1 µm Supplementry Figure. Muttion of CesA4 influenced mechnicl strength, cellulose content, nd cell-wll thickness of rice plnt., wild-type Zhonghu 11., The ces4 mutnt exhiited decresed mechnicl strength in flg lef nd the first internode of culm. N, newton., The ces4 mutnt showed decresed cellulose (light lue color) level in flg lef. Arrows indicte remrkly decresed level of celluose in the cell wll of sclerenchym cells in the ces4 mutnt. c, The ces4 mutnt hd reduced thickness of cell wll (CW). PC, vsculr prenchym cell; XV, xylem vessel.

17 2 Culm Breking force (N) ZH EXPA1-oe EXPA5-oe Supplementry Figure 13. Reduced force ws required to rek the first internode of culm in EXPA1-overexpressing (oe) nd EXPA5-oe plnts. Dt represent men (5 plnts) ± stndrd devition., wild-type Zhonghu 11; N, newton;, significnt difference etween trnsgenic plnts (+) nd their segregting wild-type silings ( ) t P <.1.

18 1 Reltive expression level Flg lef Lef Sheth Culm X4 (IRBB4) Flg lef Lef Sheth Culm x4 (IR24) Supplementry Figure 14. X4 hd tissue-specific expression pttern. Dt represent men (3 replictes) ± stndrd devition.

19 Plnt height (cm) EXPA1-oe + EXPA5-oe EXPA1-oe EXPA5-oe Length of internode (cm) First EXPA1- EXPA5- oe oe Culm (internode) Second EXPA1- EXPA5- oe oe EXPA1- EXPA5- oe oe Third c Cell l ength (μm) EXPA1-oe + EXPA5-oe + Ⅴ+ EXPA1-oe EXPA5-oe Supplementry Figure 15. Constitutive expression of EXPA1 nd EXPA5 resulted in incresed plnt height. Dt represent men (5 to 1 plnts for plnt height nd internode length, 3 plnts for cell length) ± stndrd devition. or, significnt difference etween positive trnsgenic plnts (+) nd their segregting wild-type silings ( ) t P <.1 or P <.5., wild-type Zhonghu 11., EXPA1-oe nd EXPA5-oe plnts showed incresed plnt height., EXPA1-oe nd EXPA5-oe plnts showed incresed length of culm internodes compred with controls t the grin-filling stge. c, EXPA1-oe nd EXPA5-oe plnts showed incresed cell length in the first internode of culm compred with controls. Arrow indictes the line of cells for sttisticl nlysis. Scle rs, 1 μm.

20 Lesion length (cm) 9 3 ces4 PXO1 infection Bcterium [log (cfu/lef)] ces Dys fter PXO1 infection 15 9 PXO99 infection 15 9 PXO347 infection Lesion length (cm) ces4 mutnt PXO341 infection ces4 mutnt ces4 mutnt PXO1 infection ces4 mutnt Supplementry Figure 1. Compromised resistnce to Xoo of the ces4 mutnt. Plnts were inoculted with different Xoo strins (PXO1, PXO99, PXO341, PXO1, nd PXO347) t the ooting stge. Dt represent men ( 5 leves for lesion length nd 3 replictes for cteril growth) ± stndrd devition. or, significnt difference etween mutnt nd wild-type () Zhonghu 11 t P <.1 or P <.5., The ces4 mutnt exhiited incresed susceptiility (longer lesion nd higher cterium growth rte) to PXO1 compred to. cfu, colony-forming unit., The ces4 mutnt exhiited incresed susceptiility to other Xoo strins compred to.

21 Lesion length (cm) Correltion nlysis: r =.84, n = 8, P <.1 8 PXO1 infection CesA4-RNAi Correltion nlysis: r =.57, n = 13, P <.5 PXO1 infection CesA7-RNAi Correltion nlysis: r =.77, n = 13, P <.1 PXO1 infection CesA9-RNAi Reltive expression level CesA4 expression CesA7 expression CesA9 expression RNAi CesA4-RNAi 1. CesA7 expression CesA4 expression CesA9 expression CesA7-RNAi 2.1 CesA9 expression CesA4 expression CesA7 expression CesA9-RNAi Supplementry Figure 17. Incresed susceptiility to Xoo ws correlted with suppression of trget CesA gene nd the existence of RNAi frgment in CesA-RNAi plnts. Trnsgenic plnts of T genertion were inoculted with Xoo strin PXO1 t the ooting stge. The red rs indicte negtively trnsgenic plnts. Dt represent men (3 to 5 replictes for lesion length nd 3 replictes for gene expression) ± stndrd devition. or, significnt difference etween trnsgenic plnts nd wild-type () Zhonghu 11 t P <.1 or P <.5. RNAi, frgment of the RNAi construct mplified using PCR primers PMCG-F nd PMCG-R (Supplementry Tle ).

22 Reltive expression level PXO1 (incomptile) 5 4 CesA4 3 2 * * CesA7 CesA9 * * PXO99 (comptile) ck Time fter Xoo inocultion (h) Reltive expression level IRBB4 (incomptile) IR24 (comptile) CesA4 CesA7 * * CesA9 * * ck Time fter PXO1 inocultion (h) Supplementry Figure 18. Incomptile (resistnce) rection rpidly induced the expression of CesA genes. Rice lines IRBB4 crrying X4 nd susceptile IR24 were inoculted with Xoo strin PXO1 or PXO99 t the ooting stge. Dt represent men (3 replictes) ± stndrd devition. or, significnt difference etween ck (efore infection) nd infected plnts t P <.1 or P <.5; sterisk, significnt difference etween incomptile nd comptile rection sujected to the sme tretment t P <.1 () or P <.5 (*)., The expression of CesA genes ws rpidly induced y incomptile PXO1 ut not comptile PXO99 in IRBB4., The expression of CesA genes ws rpidly induced in incomptile rection (IRBB4 versus PXO1) ut not comptile rection (IR24 versus PXO1).

23 Reltive expression level * * X4-crrying1(+) X4-crrying1( ) X4-crrying7(+) X4-crrying7( ) * * * * * * * * * * ck Time fter PXO1 inocultion (h) * * Supplementry Figure 19. Xoo strin PXO1 (incomptile) rpidly induced X4 expression. Plnts were inoculted with Xoo t the ooting stge. Dt represent men (3 replictes) stndrd devition. or, significnt difference etween ck (efore infection) nd infected plnts t P <.1 or P <.5; sterisk, significnt difference etween trnsgenic plnts (+) nd their wild-type silings ( ) sujected to the sme tretment t P <.1 () or P <.5 (*).

24 Concentrtion (µg g -1 FW) SA * ck 4 8 Time fter Xoo inocultion (hour) IRBB4 (X4) IR24 (wild type) X4-crrying1(+) X4-crrying1( ) X4-crrying7(+) X4-crrying7( ) Supplementry Figure 2. The levels of slicylic cid (SA) did not show mrked difference in trnsgenic plnts compred with their wild-type silings. Plnts were inoculted with Xoo strin PXO1 t the ooting stge. Dte represent men (three replictes) ± stndrd devition. or, significnt difference etween ck (efore infection) nd infected plnts t P <.1 or P <.5; sterisk, significnt difference etween IRBB4 nd IR24 or etween trnsgenic plnts (+) nd their wild-type silings ( ) sujected to the sme tretment t P <.1 () or P <.5 (*).

25 Concentrtion (ng g -1 FW) * * * 24 Skurnetin 18 Wild type (Zhonghu 11) JA * * * * ces4 JA-Ile Momilctone A 9 3 * * ck Time fter Xoo inocultion (hour) Supplementry Figure 21. The impirment of rice disese resistnce ginst Xoo y ces4 mutnt ws ssocited with suppressed levels of jsmonic cid-isoleucine (JA- Ile), skurnetin, nd momilctone A. Plnts were inoculted with Xoo strin PXO1 t the ooting stge. Dt represent men (three replictes) ± stndrd devition. or, significnt difference etween ck (efore infection) nd infected plnts t P <.1 or P <.5; sterisk, significnt difference etween wide type nd ces4 mutnt sujected to the sme tretment t P <.1 () or P <.5 (*).

26 XA4 OsWAK1 ZmWAK ZmWAK-RLK1 AtRFO1 Glcturonn-inding domin (1) MAAAAALSAVAPGQRP (1) MHGHCKSLTPTTLEEMYCTPHAVQYSY..KNVMQL.ILST..VVLLLWLLHAPAAADAAL (1) MSSLLLRVLLIQL..VEC...AI (1) MAAHQPHLSVLLLVLL..HVV.T (1) MKRRRLFFCVLLSILTLFINGPLVTT.QS.PSST Trnsmemrne helix Single peptide XA4 (17) ---ADCPSECGGVDIPYPFGV-DNCSWPGPDDFTIICKYS RPYYRGAEI OsWAK1 (1) TTV.G...K..D...L...IG.H.A.ES---.DVV.NE.FS PP..HTGNI.. ZmWAK (25) ----G.LTQ...E...GT...RK.---.R.K.INGSAGEEIPVLLPTTR.QNIRV ZmWAK-RLK1 (24) AH.EPPL.S.YNTSAHGE PPL.STYN.SM AtRFO1 (35) ----S.NRI..RIE..F...IGRRDCFLN-.WYEVVCNSTTSGKS---LAPFL.KINR.L XA4 (2) VNISVEAG-EMRVYSPVVSQCYNSSNTTDSDGFEFLRLNITNTPFLVAPERNEFTAIGCA OsWAK1 (111) KE T..AD...S.S-AP..GAS-.EL.-A...L.QS-...N ZmWAK (78) L.L..SPLP.A..LL..AW..F.AAGGVTGIYSGDVDF.PE-GVYRISNTQ..LFVL..D ZmWAK-RLK1 (54) CSSFWCG.V.I.YPFYLANAIADY.GSYY.C.YTD.SVSCELEVEGSPTTPTIRL.GG AtRFO1 (87).S.TLRSSIDSSYGVVHIKSPVT..GCSQRPVKPLPLNLTGKGSPFFITDS.RLVSV..D XA4 (1) TLAWLWGR----DDGSYLTGCISTCASLATAAKD--RDPCTGLGCCQVPSIPANLSVLNI OsWAK1 (1).V.F.D..----NN...S...T..G.VEA..QN--GE...P..TT.H. ZmWAK (137).Y.FTK.VRVHNVNARFPYRYFTG.ITVSVDE.DPRDGA.A...R.D-..PGITDTSM ZmWAK-RLK1 (114) DYTVKNIS YLYDQQTISL.DRD-----VL.G.G.P.VRHNVSFDETWL AtRFO1 (147) NR.LITDIES Q.TG.E.SCDGD.SRLDKI.G.YT...AKIPADRPQ.IGV XA4 (175) SLG-TGIANVAWEESPCSYAFVAEKHWYNFNRQDFERDGKS--FEHRDEKMVVPTVLDWA OsWAK1 (22).WNDQ.FL.FTPIGT...Q.D...S...GPV.SKDFITNSTWDKS...N.. ZmWAK (19) TFS-STRANQTFC..D...IV..GN.T.KAS.LVSHTPDNRLPLDWW--TM.LR... ZmWAK-RLK1 (157) H.HNASAFDNLTFFFG.HWGPRNTPPEFADYNISCAGFNTPTISGG.SFVFKTGDLDEQE AtRFO1 (197) D.ESS.GNTTQRGNCKVAFLTNETYSPA.VTEPEQFYTNGFTVI.LGWYFDTSDSR.TNP XA4 (232) IRKNG--SCPSTGQGAP------ACKSEHSECVNATNGKGYLCNCSRGYAGNPYRD--DG OsWAK1 (28)..N..--..S..TGL V.AN.N...TS..V...K..P...ATGA.. ZmWAK (253)..D.NGD.MT.ISCAQAPNEPDYG.R.K...T.S...P..F.K.AH..D...VQSDGE ZmWAK-RLK1 (217) EQELALHCDEVFSVPVR------RDALQAIVSNFSLTRD..GEVLRQ.FELEWN.TSE.Q AtRFO1 (257) VGCVN-LTETGIYTS S.VC.YGYFSGFGYSNC.CN--QI..R...LP--G. Clcium-inding epiderml growth fctor domin XA4 (282) CKN-INECK EPSITCYGGSTCQDTDGSYECKCQFGYRGDGRKNESQKGRCQ OsWAK1 (332).T.-...DLRRAEPAKY.KLYP..S..K.H..E.D.K...R.WH...KLDKGCRAIIP ZmWAK (313).T.-...Q DPK.HN.SS..K.I...G.Y.Q.N.FR QQCDPLIP ZmWAK-RLK1 (271).GRCE GSGSG.WCAYSQKREFLG.L.SG.KV.SPFCKP.RSK.KE AtRFO1 (3).ID-.D..E EGKGLSSC.EL..VNVP..WR.EN GVGKIKPLF Trnsmemrne helix XA4 (332) PIIPAAIANAIAIVCIVIVLLGLFWLPKRWKRRVFFDNNGGRLLK DMDIIV OsWAK1 (391) AV..V.TLL.SAFLAAL..YIRRER..RQ.KGL..K...NI.RN VLN.KI ZmWAK (359) MAAV.LLTTFA.V.LGCVAIVL.QT.NN.KR----.NR...K..N AQG.TT ZmWAK-RLK1 (31) GP.VG.V.V.FLCLV.LTCF.ACRHGSLPF.SENKPGTRIESF.QKN------ESIHPKR AtRFO1 (347).GLVLGFTLLFLVLG.WGLIKFVKKRR.IIRK.M..KR...L...QQLTTRGGNVESSKI

27 Serine/threonine kinse domin XA4 (383) FTEKELNKITNKK--RTKIGEGAFGEVYKGNHN-NQPVAVKYSIAKNMTQTHYKDVVESI OsWAK1 (443) YS.D...M.TNY--SNML.N.C..G...ITDEK.E...RFNPRDEERS ZmWAK (4) Y.KR..K...GY--SKRL.G.H..N..E.TIVDGRK...CPLRTRVSSHR C ZmWAK-RLK1 (37) Y.YADVKRM.KSF--AV.L.Q.G..A...SLHDGRQ...MLKDTQGDGEE AtRFO1 (47).SS...E.A.DNFNMNRVL.Q.GQ.T...MLVDGRI...R.KVLDEDKVE XA4 (44) NQNVFQTVFRQSKVPPSTPGQNAVVNEIKVQLQIR-HPNIVRLIGCCMETEVPMLVFEFI OsWAK1 (492) RDD..R..TS.SS.Q-.D.LL..V...L..D..R..L... ZmWAK (457) HWKNLIRPR.VPLPQQRVEEDGSFM...RF.FEVSR.K.L.Q.L...L..DI.I...V ZmWAK-RLK1 (42) FM..VASISRTS-.V.V.T.L.F.LQGSKRA.IY.YM AtRFO1 (459) EFI..VG.LS..N-.R...K.M...LQ...I..Y.H. XA4 (499) PNGSLETVLHG--IDR-CSLSLQQRLDIAIGSAEALAYMH-WHGHHQIIHGDIKPGNILL OsWAK1 (531)...H...--AG.NMHIP.LA...V...SNI..NS.V...V.SA...I ZmWAK (517) A...DI..S--AKKP.T...PE...I...-SLDNQKRV...S... ZmWAK-RLK1 (45)...RYAFTGDMNSENL.TWERLF...T.RG.E.L.-RGCNTR.V.F...H... AtRFO1 (49)...D.FKR..H--DSDDYTMTWDV..R..VEI.G...L.-SAASTPVY.R.V.TT... XA4 (555) GDNLMPKVSDFGSSELTLKVKRAG-KWNVYADMNYIDPVYIKTGD--FTDKSDVYSFGVV OsWAK1 (589)...ME...A.K.MSVA.YN--..S.FG.L...TS ZmWAK (574) D.D.N...K.LAIHSYY--VRA.A..IG.M..L.M..EH--..LEC... ZmWAK-RLK1 (515) DQDFC..I...LAK.C.NKES.ISIAGARGTIG..A.EVYSKQFGIISS...Y.MM AtRFO1 (553) DEKYRA...T.RSINVDQTHL-TTL.AGTFG.L..E.FQ.SQ--... XA4 () LLELITRKKAKYDDRS------LPVEFVSHYEDEDTRRKMYDQ--DMLPTEASHPHCMEC OsWAK1 (45)...R...GT R.Q.DK..K.D.M...--.L.SDD.-Q...L.. ZmWAK (3)...RR.SWYEQDQQGNKI..I...KCFK.HGSGCA...SRL.FSGEDTQSRCNKR. ZmWAK-RLK1 (575) V..MVGARDRNTSAD DHS.Q.FPQWLYEHLD.Y---CVGASEINGETT.L AtRFO1 (1).V...GE.PFSVM.P ENRGLVSHFNEAMKQNRVL.IVDSRIKEGCTL.Q XA4 (4) LDRMADIVLRCLENEVGKRPTMAEVLEELKKLLPLLTTTPVELV OsWAK1 (9)..K...AVQ..R.N.DE...D...RESAK.HNTYPTRVDGEGKPDR ZmWAK (9)..MIGMLAV...KEDKRE...V...RVKV..LG.HI ZmWAK-RLK1 (23) VRK.IVVG.W.IQVIPTD...TR.V.M.EGSTSN.ELP.RV.LS AtRFO1 (1) VLAV.KLAR...SLKGK...N.R..SI..ERIRSSPEDLELHIEEEDEEECAMEINMDDS XA OsWAK ZmWAK ZmWAK-RLK AtRFO1 (721) WSVDMTAPASLFDLSPKLDVEPLVPQRTW Supplementry Figure 22. Comprison of mino cid sequences of XA4 nd other WAK proteins known to e involved in host pthogen interctions. The dot indictes n identicl mino cid residue compred with XA4 sequence. The dsh (-) indictes lck of n mino cid residue. The predicted domins nd motif of XA4 re highlighted with purple, pink, light lue, nd lue lines. The conservtive mino cid residue (D) tht is sustituted y E in recessive x4 is outlined in red. The predicted protein kinse ATP-inding region nd kinse ctive site of XA4 re outlined in yellow nd ornge, respectively. The predicted signl peptides nd trnsmemrne helixes of ll the proteins re shown in red nd light lue colors, respectively. No signl peptides hs een detected in XA4 nd OsWAK1. Two trnsmemrne helixes hve een detected in AtRFO1 nd the mino terminl one prtilly overlps with the signl peptide.

28 Incomptile Xoo X4 X4 JAR2/GH3-3 Incresing JA-Ile Incresing phytolexins CesAs Incresing cellulose content EXPAs Reducing cell wll loosening Reinforcement of cell wll Resistnce to Xoo Incresed mechnicl strength Reduced plnt height Supplementry Figure 23. A proposed model of reinforcing cell wll contriutes to X4-regulted disese resistnce, mechnicl strength, nd plnt height. Solid line indictes direct ction nd dotted line indictes multiple steps of ction my e required.

29 Supplementry Tle 1. Response of trnsgenic plnts crrying IRK, TRK or TRKe-TRK to Xoo strin PXO1 t the ooting (pnicle developmentl) stge Rice mteril Lesion length (cm) Trnsgenic mrker gene P vlue c T plnts of Zhenshn 97 (ZS97) ckground trnsformed with IRK construct IRK1 (ZS97) IRK2 (ZS97) IRK3 (ZS97) IRK4 (ZS97) IRK5 (ZS97) IRK (ZS97) IRK7 (ZS97) IRK8 (ZS97) IRK9 (ZS97) IRK1 (ZS97) IRK11 (ZS97) IRK (ZS97) IRK13 (ZS97) IRK14 (ZS97) IRK15 (ZS97) IRK1 (ZS97) IRK17 (ZS97) IRK18 (ZS97) IRK19 (ZS97) IRK2 (ZS97) IRK21 (ZS97) IRK22 (ZS97) IRK23 (ZS97) IRK24 (ZS97) IRK25 (ZS97) IRK2 (ZS97) IRK27 (ZS97) IRK28 (ZS97) IRK29 (ZS97)

30 IRK3 (ZS97) IRK31 (ZS97) IRK32 (ZS97) Zhenshn 97 (wild type) IRBB4 (IRK) T plnts of Mudnjing 8 (MDJ8) ckground trnsformed with IRK construct IRK1 (MDJ8) IRK2 (MDJ8) IRK3 (MDJ8) IRK4 (MDJ8) IRK5 (MDJ8) IRK (MDJ8) IRK7 (MDJ8) IRK8 (MDJ8) IRK9 (MDJ8) IRK1 (MDJ8) IRK11 (MDJ8) IRK (MDJ8) IRK13 (MDJ8) IRK14 (MDJ8) IRK15 (MDJ8) IRK1 (MDJ8) IRK17 (MDJ8) IRK18 (MDJ8) IRK19 (MDJ8) IRK2 (MDJ8) IRK21 (MDJ8) IRK22 (MDJ8) IRK23 (MDJ8) IRK24 (MDJ8) IRK25 (MDJ8) IRK2 (MDJ8)

31 IRK27 (MDJ8) IRK28 (MDJ8) IRK29 (MDJ8) IRK3 (MDJ8) IRK31 (MDJ8) IRK32 (MDJ8) IRK33 (MDJ8) IRK34 (MDJ8) IRK35 (MDJ8) IRK3 (MDJ8) IRK37 (MDJ8) IRK38 (MDJ8) IRK39 (MDJ8) IRK4 (MDJ8) IRK41 (MDJ8) IRK42 (MDJ8) IRK43 (MDJ8) IRK44 (MDJ8) IRK45 (MDJ8) IRK4 (MDJ8) IRK47 (MDJ8) IRK48 (MDJ8) IRK49 (MDJ8) IRK5 (MDJ8) IRK51 (MDJ8) IRK52 (MDJ8) Mudnjing 8 (wild type) IRBB4 (IRK) T plnts of Mudnjing 8 ckground trnsformed with TRK construct TRK TRK TRK TRK

32 TRK TRK TRK TRK TRK TRK TRK TRK TRK TRK TRK TRK TRK TRK TRK TRK TRK TRK TRK TRK TRK TRK TRK TRK TRK TRK TRK TRK TRK TRK TRK TRK TRK TRK

33 TRK TRK TRK TRK TRK TRK TRK TRK TRK TRK Mudnjing 8 (wild type) Teqing (donor of trnsgene TRK) T plnts of Zhonghu 15 ckground trnsformed with TRKe-TRK in the sme construct TRKe-TRK TRKe-TRK TRKe-TRK TRKe-TRK TRKe-TRK TRKe-TRK TRKe-TRK7 + TRKe-TRK TRKe-TRK TRKe-TRK TRKe-TRK TRKe-TRK TRKe-TRK TRKe-TRK TRKe-TRK TRKe-TRK TRKe-TRK TRKe-TRK18 +

34 TRKe-TRK TRKe-TRK TRKe-TRK TRKe-TRK TRKe-TRK TRKe-TRK TRKe-TRK TRKe-TRK TRKe-TRK TRKe-TRK TRKe-TRK TRKe-TRK TRKe-TRK TRKe-TRK TRKe-TRK TRKe-TRK Zhonghu15 (wild type) Teqing (donor of trnsgenes TRKe nd TRK) Three to six uppermost fully expnded leves of ech plnt were inoculted. Lesion length ws mesured t two weeks fter inocultion. Dt represent men (3 to replictes) stndrd devition. The positive trnsgenic plnts (+) were determined y detecting the -glucuronidse (GUS) gene using primers GUS2F nd GUS2R (Supplementl Tle ). c Ech P vlue ws clculted in comprison with wild-type.

35 Supplementry Tle 2. Response of trnsgenic plnts crrying TRKd to Xoo strin PXO1 t the ooting stge Cndidte genes of X4 Recipient Numer of trnsgenic plnts Phenotype TRKd Zhonghu 11 2 Susceptile

36 Supplementry Tle 3. Response of trnsgenic plnts crrying WAK (X4) or X4-RNA interference (RNAi) construct to Xoo strin PXO1 t the ooting stge Rice mteril Lesion length (cm) Trnsgenic mrker gene P vlue c T plnts trnsformed with WAK (X4) construct (D174OI) WAK WAK WAK WAK WAK WAK WAK WAK WAK IR24 (wild type) IRBB4 (WAK or X4).54.1 T plnts trnsformed with X4-RNAi construct (D173RI) X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi

37 X4-RNAi X4-RNAi X4-RNAi18 2. X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi X4-RNAi

38 X4-RNAi X4-RNAi X4-RNAi IR24 (susceptile control) IRBB4 (wild type).54.1 Three to six uppermost fully expnded leves of ech plnt were inoculted. Lesion length ws mesured t two weeks fter inocultion. Dt represent men (3 to replictes) stndrd devition. The positive trnsgenic plnts (+) were determined y detecting the -glucuronidse (GUS) gene using primers GUS2F nd GUS2R (Supplementl Tle 5) for plnts trnsformed with WAK (X4) construct or detecting the RNAi construct using primers PMCGF nd PMCGR (Supplementl Tle ) for plnts trnsformed with WAK (X4)-RNAi construct. c Ech P vlue ws clculted in comprison with either wild-type IR24 for WAK plnts or IRBB4 for WAK-RNAi plnts.

39 Supplementry Tle 4. Yield trits in rice lines with nd without X4 under nturl condition Rice mteril Numer of full grins per pnicle Numer of null grins per pnicle Numer of grins per pnicle Seed setting rte (%) Numer of pnicles per plnt 1-grin weight (g) Yield per plnt (g) IRBB4 (X4) 9. ± 24.* 41. ± 13.9* 17.7 ± ± ± ± ± 4.8 IR ± ± ± ± ± ± ± 5.2 Ech dt represents men (3 plnts) stndrd devition. Asterisks indicte tht significnt difference ws detected etween IRBB4 nd IR24 t P <.1 () or P <.5 (*).

40 Supplementry Tle 5. Sequence similrity/identity (%) mong WAKs involved in host pthogen interctions XA4 OsWAK1 qhsr/zmwak Htn1/ZmWAK-RLK1 AtRFO1 XA4 1/1 54.2/4.5 4./ / /24.1 OsWAK1 1/1 44.1/ / /2.1 qhsr/zmwak 1/1 31.2/ /22.3 Htn1/ZmWAK-RLK1 1/1 31.8/19.9 AtRFO1 1/1 The XA4 nd OsWAK1 re from rice, the qhsr/zmwak nd Htn1/ZmWAK-RLK1 re from mize, nd the AtRFO1 is from Aridopsis.

41 Supplementry Tle. Primers used for PCR mplifiction Gene nd vector Primer nme Forwrd primer (5-3 ) Reverse primer (5-3 ) Use (GenBnk ccession numer) X4 nd x4 (KU7135 nd KU713) WAK-1F/1R AGACTAGTGGTACC TAG AGGCGATGGCAGGAA AAGAGCTCGGATCC CC CGATGAGGCGGACAA Amplifying X4 cdna frgme for generting RNAi construct WAK-WA1/NUP AATGGGTAGGGGATGTCC Provided y the Kit Amplifying X4 5 -UTR A WAK-WA2/UPM CCACCGAGATGTTCACGA Provided y the Kit Amplifying X4 5 -UTR T WAK-K1R/UPM GCTTGTCTTTGAGTTCATC Provided y the Kit Amplifying X4 3 -UTR CCT WAK-7F/NUP TAGAAGCCTCCCGGTAGA Provided y the Kit Amplifying X4 3 -UTR ATTT WAK-WS1/E3R GAGATGCGCGTCTACTCT CC CGCACTCACTATGTTCG CTCT Confirming the 1 st intron of X4 cdna WAK-8F/K1F TGGTAGTTCCGACGGTGC TTG GGCTTCTATCGTCATAC TTGGC Confirming the 2 nd intron of X4 cdna WAK-9F/K1F AGGAACCTTCTATTACTT GTTACGG GGCTTCTATCGTCATAC TTGGC Confirming the 3 rd intron of X4 cdna WAK-P 359 /P stop ATAGGATCC c AAGAGATGG AAGCGGAGAGTGTTCT ATAGTCGAC d CTACACGA GTTCGACCGGCGTCGTG Construction of XA expression vector WAK-2F/Fullr -BmHI ATAGGATCC c ATGGCAGCA ATAGGATCC c CACGAGTT Construction of XA4 for

42 Cloning vector pmcg11 (AY572837) GUS (AF234297) Actin (X1585) CesA1 (AK98978) CesA3 (AK7351) CesA4 (AK1475) CesA7 (AK72259) WAK-K1R/K1F PMCG-F/R GUS-2F/2R Actin-F/R CesA1-F/R CesA3-F/R AY57-F/R AY57-F/TosRS CesA4-F/R DSCesA4-F/R CesA7-F/R GCCGCCGCACTTTC CGACCGGCGTCGTG sucellulr locliztion vector GCTTGTCTTTGAGTTCATC GGCTTCTATCGTCATACT Quntittive PCR CCT TGGC GGCTCACCAAACCTTAAA CTGAGCTACACATGCTC PCR identifiction of positive CAA AGGTT RNAi trnsgenic plnts CCAGGCAGTTTTAACGAT CAGTTCGC TGCTATGTACGTCGCCATC CAG TTGACTTGCACGATCGATA CG GCATTTTTGCTACTGGCAT CC ATGTTCATCTTTGTTCCTC CAG ATGTTCATCTTTGTTCCTC CAG GTTCGATGGCATTGATCGC A ACTAGTGGTACC GAGGG ACGCACCACCTGC CCGGATGGATGATTCTTGT TG GAGTGAAGATCCCTTTC TTGTTACCG AATGAGTAACCACGCTC CGTCA TCCCACATAAACTGGAC CCTG TCCCTGGAACAAAGCAA AGAG GTGGCAACCTAAACTTC ATT GAAGGGGGGTGTTAAATATA TATAC CCACATAAACCGGACCT TGGA GAGCTCGGATCC GCTCA TATGCATCTGTCAAGT CCCCCAAAACACTTTTA TCCC PCR identifiction of positive trnsgenic plnts Quntittive PCR Quntittive PCR Quntittive PCR Genotyping for CesA4 Confirming Tos17 insertion in ces4 mutnt Quntittive PCR Amplifying CesA4 cdna frgment for generting RNAi construct Quntittive PCR

43 CesA8 (AK7235) CesA9 (AK117) DSCesA7-F/R CesA8-F/R CesA9-F/R DSCesA9-F/R ACTAGTGGTACC GCTGGA CGACTTCGAGGAGG TGTTGAAGGTGCTGGATT CGA AGGCCATCCATGTCATCA GCT ACTAGTGGTACC TTGACG ACCTGGAGCACG EXPA1 (AK9548) EXPA1-F/R CCTGCTTTTTTCAATGCGA AT EXPA5 EXPA5-F/R AAGGCTGTGGCTTGATTG (AK73572) ACA EXPA1 (AK41) JAR2/GH RT-F/R AAGCGCAAGTACCTGCTA (AK725) TTCAA AOS2 AOS2-F/R CAATACGTGTACTGGTCG (AY2258) AATGG The underlined nucleotides re the digestion site of SpeI nd KpnI. GAGCTCGGATCC CCGC GAGCAGCATGTACT TGAGAGTGGAGGCAAC AAACG TTGAACCCCGTTAGGAT GTCC GAGCTCGGATCC CGAC ACCTTCCTCGAC AAAGCATGCCGATCATC GA TTAGGCCCAATTTTGCTA TTTTG EXPA1-F/R CCAGTACCGCCGGTACGT TGCAAAGTAGACTAAAG ATAGCAGCAA GTAGATGAACTGCAGCG CCTTC AAGGTGTCGTACCGGAG GAA Amplifying CesA7 cdna frgment for generting RNAi construct Quntittive PCR Quntittive PCR Amplifying CesA9 cdna frgment for generting RNAi construct Quntittive PCR Quntittive PCR Quntittive PCR Quntittive PCR Quntittive PCR The underlined nucleotides re the digestion site of ScI nd BmHI. c The underlined nucleotides re the digestion site of BmHI. d The underlined nucleotides re the digestion site of SlI.