vector AvrRpt2 vector AvrRpt2 AvrRpt2 3.5 hours 6 hours

Transcription

1 A 30 kd RIN4 ACP2 17 kd vector AvrRpt2 vector AvrRpt2 AvrRpt2 3.5 hours 6 hours B ACP3 7 kd vector AvrRpt2 vector AvrRpt2 total membrane Figure S1. Accumulation of ACP2 and ACP3 upon cleavage of native RIN4 by AvrRpt2. (A) rpm1 rps2 plants were infiltrated with 5x10 7 CFU/ml of Pto DC3000 carrying empty vector or a plasmid expressing avrrpt2. Infiltrated leaves were sampled at the indicated times and total protein was blotted with anti-rin4 sera. The predicted size of ACP2 is 16 kd. (B) Extracts from plants treated similar to those in (A) were collected after 6 hours, separated into total and membrane fractions, and blotted with anti-rin4 sera. The predicted size of ACP3 is 7 kd.

2 A w.t. RIN4. N-NOI CCC RIN4FL N-NOI N-NOI N-NOI N-NOI B Col C hrcc growth (log CFU/cm 2 ) day 0 Col-0 RIN4 FL Figure S2. Residues between the NOI domains are not essential for PTI suppression by RIN4. (A) Schematic diagram of RIN4 derivatives as in figure 1. (B) and (C) Callose induced by flg22 infiltration (B) and growth of hrcc (C) in Col-0 and lines of Col-0 inducibly expressing the indicated RIN4 derivatives as in figure 1. **P<0.005, *P<0.05 [two-tailed t test for comparison with Col-0 plants].

3 T S M T S M RIN4FL ACP NOI CCC>AAA Figure S3. Membrane association of RIN4 derivatives depends on the C-terminal cysteines. Total protein extracts (T) of Col-0 leaves expressing indicated derivatives of RIN4 were separated into soluble (S) and microsomal (M) fractions and subjected to anti-t7 western blotting. The microsomal fractions are overloaded by approximately 5-fold relative to the total and soluble fractions.

4 Figure S4. Alignment of NOI domains. The indicated sequences were aligned by ClustalW using MEGA4. Sequences are shown in same order as in the tree in figure 3. The bold line separates the two primary clades apparent in figure 3. Red and orange squares denote the N-terminal NOIs from RIN4 homologs from other species and Arabidosis NOI proteins with a structure similar to RIN4 (NOI10 and NOI11), respectively, and blue and purple diamonds denote the C-terminal NOIs from the same proteins. Black triangles denote the NOIs from Arabidopsis proteins with only a single NOI domain. For other Arabidopsis proteins containing NOIs, the AGI numbers are shown. RIN4 homologs are from the following plant species: Arabidopsis lyrata, Brassica juncea, Medicago truncatula, Lactuca saligna, Solanum lycopersicum, Populus trichocarpa, Vitis vinifera, Oryza sativa, Zea mays, Brachypodium distachyon, and Physcomitrella patens.

5 A w.t. RIN4. N-NOI CCC D RIN4FL N-NOI 1 64 α-t B Col-0 RIN4FL C * hrcc growth (log CFU/cm 2 ) day 0 Col-0 RIN4FL Figure S5. A small N-terminal deletion disrupts the PTI suppressing activity of RIN4. (A) Schematic diagram of RIN4 derivatives as in figure 1. (B) and (C) Callose induced by flg22 infiltration (B) and growth of hrcc (C) in Col-0 and lines of Col-0 inducibly expressing the indicated RIN4 derivatives as in figure 1. *P<0.005 [two-tailed t test for comparison with Col-0 P<0.05 [two tailed t test for comparison with Col-0 plants expressing 1 31]. (D) Anti-T7 western blot showing similar expression levels of 1 31 and 1 64.

6 A rpm1rps2rin4 RPM1-myc rpm1rps2rin4 RPM1-myc Col-0 RIN4FL Col-0 RIN4FL DC3000 (AvrRpm1) DC3000 (AvrB) B DC3000 (AvrRpm1) DC3000 (AvrB) Conductance (µs/cm) Figure S retains the ability to support RPM1 function. (A) Plant lines expressing the indicated derivatives of RIN4 under control of the native promoter in the rpm1 rps2 rin4 RPM1-myc background were infiltrated with 5X10 7 CFU/ml of Pto expressing either AvrRpm1 (left) or AvrB (right). Photographs of the macroscopic HR in representative leaves were taken 20 hours after infiltration. (B) Infiltrations were conducted as in (A) and cell death was measured as an increase in conductance of the water in which the infiltrated leaf discs floated. The infiltrated plants included wild-type Col-0 (diamonds), non-transgenic rpm1 rps2 rin4 RPM1-myc (asterisks), and rpm1 rps2 rin4 RPM1-myc expressing RIN4FL (squares), 1 31 (triangles), and 1 64 (Xs).

7 Figure S7. Alignment of the C-termini of RIN4 homologs and NOI containing proteins. The C-terminal 16 amino acids from the indicated proteins were aligned by ClustalW. RIN4 is At3G For other Arabidopsis proteins containing NOIs, the AGI numbers are shown. RIN4 homologs are from the following plant species: Arabidopsis lyrata, Brassica juncea, Medicago truncatula, Lactuca saligna, Solanum lycopersicum, Populus trichocarpa, Vitis vinifera, Oryza sativa, Zea mays, Brachypodium distachyon, and Physcomitrella patens.

8 Experiment CCC>AAA RIN4FL RIN4FL CCC>AAA Experiment CCC>AAA RIN4FL RIN4FL CCC>AAA Experiment CCC>AAA RIN4FL Figure S8. Expression of RIN4 derivatives in plants screened for use in figure 5A. Anti-T7 western blots were used to assess levels of expression of RIN4 derivatives in rpm1 rps2 rin4 plants. Each lane is a sample from an individual plant. Levels of expression vary due to post-transcriptional gene silencing. Plants with dashed lines through them were excluded from the callose assays in figure 5A.

9 Supplemental Figure 9. Expression of RIN4 derivatives in plants screened for use in figures 5B and Supplemental Figure 10. Anti-T7 immunoblots were used to assess levels of expression of RIN4 derivatives in rpm1 rps2 rin4 plants. Each lane is a sample from an individual plant. Levels of expression vary due to post-transcriptional gene silencing. Plants with black lines through them were excluded from the growth analysis shown in 5B. Plants included in luminol assay in Supplemental Figure 10B are indicated with blue stars.

10 A rpm1 rps2 rin4 peak value rpm1 rps2 rin4 Luminescence (RLU) peak value RIN4FL CCC>AAA fls2 B Time (Minutes) 1 relative luminescence (arbitrary units) flg22 flg22 H 2 O flg22 H 2 O flg22 H 2 O flg22 H 2 O RIN4FL CCC>AAA fls2 rpm1/rps2/rin4 Supplemental Figure 10. Flg22-induced ROS accumulation in plants expressing RIN4 derivatives. (A) Representative samples of ROS accumulation from leaves of indicated plants treated with flg22. Horizontal lines represent the peak value for representative traces. (B) Data were pooled from 5 independent experiments to determine the effect of RIN4 on flg22-induced ROS. Data were included from plants expressing similar, low levels of RIN4FL and derivatives (see Supplemental Figure 9). In 4 out of 5 independent experiments, both RIN4FL and the C-terminal derivatives did not significantly (P<0.05) suppress flg22-induced ROS generation. Peak luminescence values (as outlined in Supplemental Figure 10A) were normalized to peak values from rpm1 rps2 rin4 plants, which were set to 1. P- values from two-tailed t test compared to rpm1 rps2 rin4: RINFL = 0.135, = 0.169, CCC>AAA =

11 12 relative gene expression (arbitrary units) * ** * * myb51 flg22 H 2 O flg22 H 2 O flg22 H 2 O flg22 H 2 O flg22 H 2 O flg22 H 2 O flg22 H 2 O flg22 H 2 O flg22 flg22 FRK1 MYB51 FRK1 MYB51 FRK1 MYB51 FRK1 MYB NOI RIN4FL FRK1 MYB51 Col-0 fls2 Figure S11. Flg22-induced expression of FRK1 and MYB51 in plants expressing RIN4 derivatives. Control plants or plants inducibly expressing RIN4 derivatives were infiltrated with flg22 or water and amounts of FRK1 and MYB51 transcript were measured after 2 hours by quantitative real-time PCR. Expression of FRK1 and MYB51 were normalized relative to actin. Data was pooled from 3 independent experiments with expression of each gene normalized relative to flg22-treated Col-0. Bars represent standard error. *P<0.01, **P=0.02 [two-tailed t test for comparison of flg22- versus waterinfiltrated samples in each plant type].

12 RIN4 Derivative Expression Level Membrane Localization Symptom Induction Callose Suppression Bacterial Growth RIN4FL +++/+* + m/ns* + /+* +/-* 1Δ ns-m - - 1Δ m Δ m Δ m Δ m Δ m + + ΔΔNOI ns-m Δ211 +/±* - s/ns-m* +/++* +++/++* 149Δ m-s Δ s CCC>AAA +/±* - vs/ns-m* +/++* +++/+++* 1Δ m - ++ ACP2 + - m-s + ++ ACP3 + + m - + Figure S12. Summary of results obtained from RIN4 structure:function analysis. Expression levels, plasma membrane localization, symptom induction, suppression of flg22-induced callose deposition, and promotion of hrcc bacterial growth are shown for Dex-inducible, T7-tagged RIN4 derivatives. Results are from Col-0 and, where tested, from rpm1 rps2 rin4 plants (rpm1 rps2 rin4 results after / and marked with *). Expression level refers to the accumulation of RIN4 protein as measured by anti-t7 western blot analysis and membrane localization is based on blotting of membrane fractions. Symptom induction is determined on the following scale (ns: no symptoms, m: mild chlorotic symptoms, s: strong cell death symptoms, vs: very severe cell death symptoms).

13 A B C D E Figure S13. Structural model of RIN4. (A) Spacefill model showing that the two halves of RIN4FL fold independently. The N-terminal two thirds (AA 1 to 152) is shown in white and the C-terminal one third (AA 153 to 211) is shown in blue. (B) View of spacefill model highlighting surface exposed regions. Surface exposed regions in N-NOI (AA 6 to 42) are shown in white with the opaque background highlighting unexposed regions. Exposed residues (AA 148 to 187) are shown in blue with an opaque background highlighting unexposed region. The C-terminal cysteines (AA 203 to 205) are in cyan and Met1 of RIN4 is in green. Of interest is the observation that the N-NOI, and the C-terminal cysteines are juxtaposed in the model. Landmark residues K8 and A180 are highlighted for reference. (C) View of spacefill model highlighting surface exposed residues in the AvrB binding site (Desveaux et al, 2007). Asp153, Trp154, Asp155, Glu156, Asn157 are buried residues (red in the opaque background), whereas residues Asn158 (yellow) through Ile168 (purple) are surface exposed. Phe169 is also exposed but is on the opposite face of the protein (not shown). Exposed residues 159 to 167 are shown in blue. Ile168 is the start of the C-terminal α-helix. (D) and (E) Views of spacefill model showing the AvrRpt2 cleavage sites comprising Gly10 (green) and Asn 11(yellow) in the N-NOI (blue) (D) and Gly152 (green) and Asp153 (yellow) in the (blue) (E). Gly10 and Asn11 in N-NOI are surface exposed residues while Gly152 and Asp153 in are located in a solvent exposed pocket.

14 A B C D Figure S14. RIN4 is modeled as globular in additional structural predictions. RIN4FL was modeled using freely available web-based algorithms with the highest rankings in the CASP8 and CASP9 experiments. Structures of T7-RIN4 determined using (A) HHPred (CASP9 rank: 6), (B) Quark (Ab initio prediction server, CASP9 free modeling (FM) server rank: 1), (C) Raptor (Single template threading, CASP8 Rank: 2), (D) 2nd best Raptor model and (E) Robetta (Baker lab server, CASP9 rank: 3). E

15 Figure S15. Validation of structural model of RIN4FL by Ramachandran plot. The plot shows the ψ / φ torsion angles for all residues of RIN4FL except the glycines (18 total), prolines (18 total), and the two terminal amino acid residues. The red, yellow, cream, and white areas of the plot represent the core/favored, allowed, generously allowed, and disallowed regions, respectively. Of the analyzed residues, 96.7 % (178/184) are in allowed conformations, whereas 3.3% (6/184: Asp42, Asn46, Ser47, Arg61, Lys63, and Lys144) are in disallowed conformations. The amino acids comprising the T7 tag (AA 1 to 11), all of which are in allowed regions, are omitted from the plot so residues numbers correspond to those of untagged RIN4.

16 A B D C Supplemental Figure 16. Ribbon diagrams of predicted structures of RIN4FL, 1D31, and 1D64. (A) Secondary structural elements of RIN4FL (the same model as in Figure 4) are shown as: α helices (red), β sheets (yellow) and β turns (blue). The N-terminal two thirds and the C-terminal one third of the protein are separated by the dotted line. Landmark residues (Met1, Gly152 and Cys 203) are indicated. (B) Predicted structural model of The N-terminal two thirds of the protein is predicted to be unstructured. (C) Blowup of the C-terminus of 1 31 from (B). The structure in the C-terminus consists of the (AA 148 to 187) including the C-terminal helix (Lys171, Val172 and Arg173). (D) Predicted structural model of Partially restructured N-terminus and internal regions include the 2nd helix (absent in 1 31 model and present in the RIN4FL model). The C-terminal helix (Lys171, Val 172 and Arg173) is also structured.