SUPPLEMENTARY INFORMATION

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1 doi: /nature10962 Supplementary Figure 1. Expression of AvrAC-FLAG in protoplasts. Total protein extracted from protoplasts described in Fig. 1a was subjected to anti-flag immunoblot to detect AvrAC-FLAG expression. Supplementary Figure 2. Bacterial growth assay on WT (Col-0) and AvrAC transgenic plants inoculated with Pst, Pst (avrrpt2), and Pst (avrpphb). Plants were pre-treated with oestradiol and infiltrated with the indicated bacteria. Bacterial population was determined at the indicated times (mean+s.d.; n>6, ** P<0.01, Students t-test). 1

2 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 3. Amounts of Xcc8004 bacteria and Pst type III secretion activity in the leaf are not affected by avrac. a. Leaves pretreated with Xcc8004 and Xcc8004 avrac bacteria allow similar Pst type III secretion activity. Leaves were infiltrated with Xcc8004 or Xcc8004 avrac one day before infiltration of the indicated Pst strains carrying the type III efffector gene avrpto fused to a cya reporter gene (calmodulin-dependent adenylate cyclase domain) 38. The AvrPto-Cya secretion activity was measured 9 hr later and presented as amount of camp production (mean+s.d.; n>6). b. Xcc8004 and Xcc8004 avrac bacterial numbers in leaves at the time of Cya reporter assay (mean+s.d.; n>6). Together with Fig. 1f, these results indicated that the differential HR induction was not caused by different type III secretion activity in Pst bacteria nor different amounts of Xcc bacteria in the plant. Supplementary Figure 4. MAPK activation by MKK5 DD in the presence of AvrAC. Protoplasts transfected with MKK5 DD and AvrAC-HA constructs were subjected to immunoblot with an anti-perk antibody. 2

3 RESEARCH Supplementary Figure 5. AvrAC induces a phosphatase-resistant modification on BIK1. BIK1-HA co-expressed with AvrAC-FLAG in protoplasts (a) or His-BIK1 co-expressed with GST-AvrAC in E. coli (b) were affinity purified, treated withλprotein phosphatase (PPase), separated by SDS-PAGE, and subjected to anti-ha immunoblot or Coomassie Brilliant Blue staining. c. Coomassie Brilliant Blue straining of total protein profile of E. coli cells co-expressing His-BIK1 and GST or GST-AvrAC. Supplementary Figure 6. AvrAC isolated from E. coli contains multiple UMP modifications on serines and threonines. a, GST-AvrAC was purified from E. coli, and its mass was measured by mass spectrometry. represents removal of the N-terminal methionine residue from the full length GST-AvrAC protein. Adjacent components in the M series or M series protein peaks 3

4 RESEARCH SUPPLEMENTARY INFORMATION differ by 306 Da in mass, indicating multiple UMP adds-on. b, List of UMP modified peptides in GST-AvrAC identified by tandem mass (MS/MS) spectrometry. The three Ser residues on which the single UMP modification site could not be pinpointed are grouped in parenthesis. Supplementary Figure 7. AvrAC uridylylates PBL1 in vitro. GST-AvrAC was incubated with PBS1 and His-PBL1 in the presence of 32 P-UTP. The resulting products were subjected to SDS-PAGE and autoradiography. Supplementary Figure 8. a and b, AvrAC uridylylates conserved serine and threonine residues in the activation loop of BIK1 and RIPK in E. coli. His-BIK1 (a) or His-RIPK (b) was co-expressed with GST-AvrAC in E. coli, affinity-purified, and subjected to tandem mass (MS/MS) spectrometric analyses. The b- and y- ions are marked in the spectrum and illustrated along the peptide sequence above the spectrum. This unambiguously identified Ser236 and Thr237 of BIK1 4

5 RESEARCH and Ser251 and Thr252 of RIPK as UMP-modified amino acid residues. The symbol represents neutral loss of an UMP group with nominal mass of 324 Da. Supplementary Figure 9. Relative amounts of UMP-modified BIK1 and RIPK in protoplasts. The LC-MS/MS raw data files for Fig. 3b and Fig. 3c were used to extract XICs (the extracted ion chromatograms) of the BIK1 and RIPK1 peptides corresponding to kinase activation loop. The peak areas of the corresponding peptides in the XICs are shown in black color. a. XIC of BIK1 peptide DGPMGDLSYVSTR. b. XIC of BIK1 peptide DGPMGDLSYVSumpTumpR. c. XIC of RIPK1 peptide DGPEGDDTHVSTR. d. XIC of RIPK1 peptide DGPEGDDTHVSumpTumpR. e. Calculated peak areas from the XICs in graph a, b, c, and d. 5

6 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 10. Amino acid residues in the activation loop, but not ATP-binding site of BIK1 is required for AvrAC-mediated modification. BIK1-HA constructs carrying a Lys105 to Glu substitution (K105E) in the activation loop or the SYST/AAAA mutations, in which Ser233, Tyr234, Ser236, and Thr237 were simultaneously substituted with alanine 5, were co-expressed with AvrAC-FLAG in protoplasts. The total protein was subjected to anti-ha immunoblot. Supplementary Figure 11. Alignment of AvrAC with the C-terminal subdomains of well-characterized Fic domain proteins. Identical amino acid residues are shaded in black, whereas conserved amino acid substitutions are shaded grey. The catalytic His residue is highlighted in red. The Fic catalytic motif is underlined. 6

7 RESEARCH Supplementary Figure 12. AvrAC NTD and LRR are responsible for the interaction with BIK1. The indicated AvrAC-FLAG deletion constructs were co-expressed with BIK-HA and subjected to co-ip assays. Supplementary Figure 13. His469 is required for modification of BIK1 in protoplasts. The indicated AvrAC-FLAG constructs were co-expressed with BIK1-HA in protoplasts, and anti-ha immunoblot was used to detect BIK1 modification. 7

8 RESEARCH SUPPLEMENTARY INFORMATION Supplementary Figure 14. Expression of AvrAC mutant proteins in protoplasts. Protein samples from protoplasts described in Fig. 4d were subjected to anti-flag immunoblot. Supplementary Figure 15. The AvrAC(H469A) mutant is less active in inhibiting flg22-induced FRK1::LUC expression. a. Protoplasts were transfected with the indicated AvrAC-FLAG constructs, and the flg22-induced expression of FRK1::LUC was determined (mean+s.d.; n>3). b. Amount of the AvrAC protein in protoplasts. 8

9 RESEARCH Supplementary Figure 16. BIK1 is not required for flg22-induced MAPK activation. WT (Col-0), bik1, pbl1, and bik1/pbl1 seedlings were treated with flg22 for indicated times and MAPK phosphorylation was determined using a specific anti-perk antibody. 9