Figure S1. Immunoblotting showing proper expression of tagged R and AVR proteins in N. benthamiana.

Transcription

1 SUPPLEMENTARY FIGURES Figure S1. Immunoblotting showing proper expression of tagged R and AVR proteins in N. benthamiana. YFP:, :CFP, HA: and (A) and HA, CFP and YFPtagged and AVR1-CO39 (B) were expressed in N. benthamiana leaves by agro-infiltration and leaf samples were harvested 24 hours after inoculation. Proteins were extracted and analyzed by immunoblotting using either anti GFP ( -GFP) or anti HA ( -HA) antibodies. Figure S2. Cell death assays in N. benthamiana. A) constructs induce cell death in N. benthamiana., and :CFP fusion proteins were transiently expressed in N. benthamiana leaves by agro-infiltration. Cell death response was observed 3 days after inoculation. Agro-bacteria allowing expression of the GFP protein are used as a negative controls. Identical results were obtained in at least 3 independent experiments. B) constructs repress -mediated cell death. Indicated combinations of constructs were expressed in N. benthamiana leaves as described in A). Inhibition of cell death was observed 3 days after inoculation. C) _H3 and AVR1-CO39 do not induce cell death in the presence of and in N. benthamiana. and YFP: proteins were expressed in N. benthamiana leaves together with, _H3, AVR1-CO39 or GFP. The picture and the scan of red fluorescence were made 3 days after inoculation. Figure S3. specifically represses -mediated cell death but not cell death induced by the autoactive CC-NB-LRR Orin1 or the L6MHV mutant. The constitutively active rice CC-NB-LRR Orin1 (A) and the autoactive MHD motif mutant L6 MHV of the flax rust resistance protein L6 (B and C) were

2 transiently expressed in N. benthamiana (A and C) or N. tabacum (B) by agroinfiltration with or without. Cell death response was observed 3 days after inoculation. GFP was used as a negative control. Identical results were obtained in 3 independent experiments. Proper expression of L6 MHV, and was verified by immunoblotting (D). Figure S4. Cell-death induction by and and cell-death repression by in rice protoplasts. induces a cell death response that is repressed by and recognition requires both and. Protoplasts from Oc cells were transfected with a plasmid for constitutive LUC expression in combination with plasmids allowing expression of,, or the Venus protein. LUC activity was determined in protein extract from protoplast samples harvested 40 hours after transfection. Average values and standard deviations were calculated from 3 replicate samples and values were normalized with respect to the average value of the Venus sample without. The experiment was repeated 3 times with equivalent results. Values presenting significant differences are indicated (p<0.01 in Student s T-test). Figure S5. RT-PCR shows silencing of and in rice protoplasts. Rice protoplast of the resistant rice variety Sasanichiki or of the rga4 mutant line Sas1493 were co-tranfected with the Luciferase reporter gene and RNAi constructs directed against ( RNAi-1 or RNAi-2) or ( RNAi-1 or RNAi-2) or the empty vector. or levels were measured by semi-quantitative RT-PCR using specific primers for, and actin. Figure S6. Complete alignment of CC-NB-LRRs. MLA10, Rx, and protein sequences were aligned using CLUSTALX (Larkin et al., 2007). Alignment was visualized using GeneDoc

3 ( Motifs conserved in NB-LRR proteins are displayed (red boxes). RATX1 domain is highlighted and its VHD motif is shown (blue box). Figure S7. LHV and LAH mutants repress and recognize AVR- Pia. A) The indicated combinations of YFP:, YFP:rga5 LHV, YFP:rga5 LAH and constructs were expressed in N. benthamiana. The picture (left panel) and the scan of red fluorescence (right panel) were made 3 days after inoculation B) Transient expression of the indicated constructs was performed as in A) including the construct. Observation were made as described in A). Figure S8. Mutation of the VHD sequence does not lead to cell death induction and does not impair function. A), B), C) Indicated combinations of constructs were expressed in N. benthamiana. was used as a positive control for cell death induction. YFP: and the P-loop mutant YFP:rga5 K/R were used as controls for proper function. D) Immunoblotting showing proper expression of the constructs. Figure S9. Cell death activity of epitope-tagged and in rice protoplasts. Constructs were expressed together with Luciferase in rice protoplasts derived from Oc cells. LUC activity was determined in protein extract from protoplast samples harvested 40 hours after transfection. Average values and standard deviations were calculated from 3 replicate samples and values were normalized with respect to the average value of the - sample. The experiment was repeated 3 times with equivalent results. Average values significantly different from the average value of the corresponding - samples are indicated (p<0.01 in Student s T-test).

4 Figure S10. Full panel of figure 5B showing detection of GFP after IP-GFP. Enlarged picture showing the full input and IP-GFP panel of figure 4B. The GFP protein is properly expressed and immunoprecipitated. GFP fusion proteins are not degraded. Figure S11. Details of the constructs used in the yeast two-hybrid assays. A) and B) For yeast two hybrid analysis, constructs for expression of CC 1-176, NB-ARC and LRR domains (A) and for CC 1-177, NBS , LRR and RATX domains (B) have been generated. C) For yeast two hybrid analysis, 3 different additional CC domain constructs were generated for and based on homologies of the, and MLA10 N-termini. Alignment of MLA10, and N-terminal sequences including the CC and part of the NB domains. EDVID and P-loop motifs are displayed. Arrow 1 marks the end of the MLA10 CC domain construct characterized by structure analysis (Maekawa et al., 2011) and of and Arrow 2 indicates the end of the MLA10 CC domain construct shown to possess cell death activity in planta and of and Arrow 3 indicates the end of the MLA10 construct that forms homo-complexes in the yeast two-hybrid assay and the corresponding and constructs. Figure S12. Yeast two-hybrid assays with distinct domains of and. A) and B) Interactions between the indicated combinations of CC, NB-ARC and LRR domains from and, as well as the RATX1 domain, were tested by yeast two-hybrid assays. All combinations of bait and prey constructs were transformed into the yeast strain AH109 and protein-protein interactions were evaluated by blue coloration and growth of yeast on basal medium lacking His, Trp, Leu and Ade (-HTLA) and supplemented with 5-Bromo-4-Chloro-3-

5 indolyla-d-galactopyranoside (X-a-gal). Pictures were taken after 3 days of growth. C) Immunoblotting showing proper expression of fusion proteins in yeast. Anti- Myc ( -Myc) and anti-ha ( -HA) antibodies were used to detect the proteins fused to BD and AD respectively. Figure S13. The CC domains of and form homo and heterocomplexes. A) Interaction between CC domains of and of different length ( 1-130, 1-164, 1-229, 1-133, and 1-228; see Supplementary Figure S9 C for details) was tested by yeast two-hybrid. Yeast cells expressing GAL4-AD and GAL4-BD fusions of the indicated fragments were spotted on a non selective synthetic media lacking Trp and Leu (-TL) to monitor proper growth, and on a selective media additionally lacking His (-HTL) to assay for interactions. Pictures were taken after 3 days of growth. B) Immunoblotting showing proper expression of fusion proteins in yeast. Anti- Myc ( -Myc) and anti-ha ( -HA) antibodies were used to detect the proteins fused to BD and AD respectively. Figure S14. Immunoblotting showing expression of deletion constructs. Indicated constructs were transiently expressed in N. benthamiana leaves. Proteins were extracted 24 hours after infiltration and immunoblotting was performed using anti-gfp antibodies ( -GFP). Longer exposure was used to detect some of the constructs expressed at a lower level. Rubisco ponceau staining shows equal protein loading and stars indicate cleaved YFP. Figure S15. Expression of :GFP and Venus: in rice protoplasts.

6 Protoplasts isolated from rice Oc suspension culture were transformed with the 10 Myc:GFP, :GFP and Venus: constructs. Transfected cells were incubated at 30 C for and protein extracts were prepared from protoplasts harvested 16 hours after transfection. Immunoprecipitation using anti-gfp (IP: - GFP) antibodies was performed to enrich for recombinant proteins and samples were analyzed by immunoblotting using anti-gfp (IB: -GFP) antibodies.

7 A 140 kda 115 kda B AVR1-CO39:CFP YFP:AVR1-CO39 :CFP YFP: Size (kda) 43 -GFP α-gfp α-ha HA: :HA HA:AVR1-CO39 AVR1-CO39:HA Size (kda) Rubisco Rubisco -HA Figure S1. Immunoblotting showing proper expression of tagged R and AVR proteins in N. benthamiana

8 A B GFP YFP: :CFP HA: GFP C + YFP: + AVR1-CO39 _H3 + YFP: + AVR1-CO39 _H3 GFP GFP Figure S2. Cell death assays in N. benthamiana

9 A Orin1 Orin1 GFP B C YFP: YFP: L6 MHV :YFP L6 MHV :YFP YFP: L6 MHV :YFP L6 MHV :YFP YFP: D -GFP YFP: Mock L6 MHV :YFP + YFP: L6 MHV :YFP Size (kda) HA Mock Size (kda) Rubisco Rubisco Figure S3. specifically represses -mediated cell death but not cell death induced by the autoactive CC-NB-LRR Orin1 or the L6 MHV mutant

10 A B Relative luciferase activity a b c a Relative luciferase activity Venus Venus - + C D Relative luciferase activity Venus - + Relative luciferase activity Venus - P< Figure S4. Cell-death induction by and and cell-death repression by in rice protoplasts

11 Sasanishiki (+ +) Sas1493 (rga4+ +) Empty RNAi-1 RNAi-2 Empty RNAi-1 RNAi-2 Empty RNAi-1 RNAi-2 actin actin actin Figure S5. RT-PCR showing silencing of and in rice protoplasts

12 EDVID GRExE P-Loop P-Loop RNBS-A Walker B RNBS-B GLPL RNBS-D MHD VHD RATX1 Figure S6. Complete alignment of CC-NB-LRRs

13 A YFP:rga5 LHV YFP:rga5 LHV YFP: YFP:rga5 LAH YFP: YFP:rga5 LAH B YFP: YFP:rga5 LHV YFP: YFP:rga5 LHV YFP: YFP:rga5 LAH YFP: YFP:rga5 LAH Figure S7. LHV and LAH mutants are functional to repress and to recognize

14 A C YFP: YFP: YFP:rga5 K/R YFP:rga5 D510V YFP:rga5 D510V YFP: B D YFP:rga5 D510V α-gfp 140 kda 115 kda Figure S8. Mutations of VHD sequence does not lead to cell death induction and does not impair function

15 Relative luciferase activity P<0.01 P<0.05 P< :T7 :GFP 10xMyc: Venus: Figure S9. Cell death activity of epitope-tagged and in rice protoplasts

16 :CFP YFP: HA: GFP Size (kda) 140 α-gfp Input α-ha 115 Rubisco 140 α-gfp IP GFP α-ha 115 Figure S10. and form hetero-and homo-complexes in planta

17 A B CC MEAALLSGFIKAILPRLFSLVDDKHKLHKGVKGDIDFLIKELRMIVGAIDDDLSL DHPAAAAVQTLCMEDLRELAHGIEDCIDGVLYRAARDQQQSPVRRAVQAPK KLQRNLQLAQQLQRLKRMAAEANQRKQRYTAAAPGQHGQVYSSAAAQVD EPWPSCSSASDPRIHEADLVG CC MDAPASFSLGAMGPLLRKLDSLLVAPEIRLPKPLKEGIELLKEDLEEIGVSLVE HSVVDSPTHKARFWMDEVRDLSYHIEDCIDTMFSMRSGGDDGKPRSERRH KVGRAKIDGFSKKPKPCTRMARIAELRALVREASERLERYQLGDVCGSSSPV VFTADGRARPLHHGVSANLVG NB-ARC VDADREELLEQLAERQPEQLKVIAIVGFCGLGKTALAAEAYNRETGGGRFER HAWVCAGHRSAREVLGELLRRLDADGRSFHGDSDAGQLCVDIRQQLEKNR YFIVIDDIQTEDQWKSIKSAFPTDKDIGSRIVVTTTIQSVANACCSANGYLHKM SRLDKNCSKQLLSKKACPERYSHYKQPDSAAILKKCDGQPLALVTIGEFLQA NGWPTGPNCEDLCNRLHYHLENDKTLERMWRVLVRNYTSLPGHALKACLL YFGMFPSDHPIRRKSLLRRWLAEGFVEPLSS SSNIDSTAAFNVLMDRNIIEPINVSNNDKVKTCQTYGMMREFISHMSISQNFV TFFCDDKFVPKYVRRLSLHGDTVVNGDNFNGIDLSLVRSLAVFGEAGTTVLD FSKYQLLRVLDLEKCDDLKDDHLKEICNLVL NB-ARC VDEFKTKLNRWLSDEEGPHLKVAAIVGPAGIGKTALATELYRDHRWQFECR AFVRASRKPDMQRLLGGILSQVQRRQRSSDAYADSTVQSLIDNLREHLQDR RYLIIIDGLWETAVWNIANSAFPDVNSFSRILITADIEQVALECCGYKYDYIMR MEPLGSLDSKKVFFNKVFGSEDQCPPELKEVSNTILEKCGGLPLAIISIAGLLG SQPENPVLWDYVTKYLCSSLGTNPTLKDVVKETLNLSYNSLPHPFKTCLLYL GMYPDGHIMLKADLMKQWSAEGFVSANEA KDTEEIVDKYFDELVNRGILEPVEINKNGKVLSCTLHHAVHDLVMPKFNDDKF TMSVDYSQTITGPSTMVRRLSLHFSSTRYATKPAGIILSRVRSLAFFGLLNCM PCIGEFKL LRR LKYLSLGGNISKLPKDIAK LKDLEALDVRRSKVKIMPVEVFG LPCLIHLLGKFKLSDKVKQKTEVQEFLLKGKSN LQTLAGFASNGSEGFLHLMRYMNK LRKLKIWCTSSAGSTDWTDLREAIQQFILDEKEANIGTRSLSLHFSGCSEDAI NSLKEPCY LSSLKLHGNFPQLPQFVTS LRGLKELCLSSTKFTTGL LEALSNLSYLQYLKLVADELEKFIIKVQGFPR LLRLCIVLQYPTFPVIEEGA LPFLVTLQLLCKDLHGLSDIQIECFKHLQEVTLH SGVTPATRQEWVKAAKEHPNRPKVLLLKSVDTAESEHTDVDSVMEAVKSET TEYSIAPEGPEQVNNKMQLDHGLESSSVLNKQNNFADQSSSKDQLHYSFNN MGLSDVSCCE RATX1 LRR LRVLILEFWGSHGEQRSLNLIPVCR LFQLRYLKTSGDVVVQLPAQISG LQYLETLEIDARVSAVPFDLVH LPNLLHLQLQDETKLPDGIGCMRS LRTLQYFDLGNNSVDN LRGLGELTNLQDLHLSYSAPSSNEGLMINLNAITSS LSRLSNLKSLILSPGAISMVIFFDISSIISVVPVF LQRLELLPPICIFCRLPKSIGQ LHKLCILKVSVRELLTTDIDN LTGLPSLTVLSLYAQTAPEGRFIFKDGT LPVLKYFKFGCGELCLAFMAGAMPN LQRLKLVFN IRKSEKYRHTLFGIEHLVSLQDIATRIGVDTSTGESDRRAAESAFKETVNKHP RCLRSSLQWVVSTEEESHPLEKQHHKREKGSSAGHGVLEKESVEDSEKNT DRVQTLLSPQLSNMESVVESALTGQRTK IVVKVHMPCGKSRAKAMALAASVNGVDSVEITGEDKDRLVVVGRGIDPVR LVALLREKCGLAELLMVELVE KEKTQLAGGKKGAYKKHPTYNLSPFDYVEYPPSAPIMQDINPCSTM* C EDVID 1 2 P-loop 3 Figure S11. Details of the constructs used in the yeast two-hybrid assays

18 A BD B AD EV EV CC CC AD NB-ARC LRR BD NB-ARC LRR RATX1 RATX1 -HTLA + X-α-gal -HTLA + X-α-gal C Size (kda) α-ha α-myc Figure S12. Yeast two-hybrid assays with distinct domains of and

19 A B AD BD -WL -HWL α-myc α-ha Size (kda) Size (kda) Figure S13. The CC domains of and form homo and hetero-complexes

20 YFP:CC YFP:CC-NB YFP:NB YFP:NB-ARC YFP:LRR-Cter YFP:Cter YFP:RATX Size (kda) 75 YFP: YFP:CC-NB-ARC YFP:NB-ARC-LRR-Cter YFP: Cter YFP: RATX Size (kda) α-gfp * Over Exposure * α-gfp * Rubisco Rubisco Figure S14. Immunoblotting showing expression of deletion constructs

21 Size (kda) IP : α-gfp IB : α-gfp Figure S15. Expression of :GFP and Venus: in rice protoplasts