Cell Reports

Supplemental Information

Acetylation of an NB-LRR Plant Immune-Effector

Complex Suppresses Immunity

Jiyoung Lee, Andrew J. Manning, Donald Wolfgeher, Joanna Jelenska, Keri A.

Cavanaugh, Huaqin Xu, Sandra M. Fernandez, Richard W. Michelmore, Stephen J. Kron,

and Jean T. Greenberg

AvrB3:nYFP+RIN4:cYFP

-WLH+3AT

-WL+X-gal

AAvrB AvrB3 Z3 Z3Z3 Z3 Z3 Z3 AvrB3 AvrRpm1Psy

BaitPrey Z3Z3 Z3Z3 Z3 Z3

α-HA

α-GFP

α-HA

α-GFP Input

IP:HA

AvrB3-HAHopZ3-GFP

C/A-GFPAvrRpm1Psy-GFP

GFP

D

*

**

**5572

kDa

5572

28

28

+---+

++---

+-+--

+--+-

1" 2"72"55"

BD(HopZ3"

BD: α(myc"

AD: α(HA"*" *" *" *" *"*"

*"72"55"

3" 4" 5" 6" 7"B1.  BD(HopZ3"+"AD(AvrRpm1"2.  BD(HopZ3"+"AD(AvrRpm1Psy$3.  BD(HopZ3"+"AD(AvrB"4.  BD(HopZ3"+"AD(AvrB3"5.  BD(HopZ3"+"AD(HopI1Psy$6.  BD(HopZ3"+"AD(RIN4"7.  BD(AvrB"+"AD(RIPK"

α-RIN4

His

-Hop

Z3H

is-C

/A

α-HisHis-HopZ3

RIN4

10%

of i

nput

Pull-down

C

-

*"

*

E HopZ3-nYFP+BIK1-cYFP HopZ3-nYFP+PBS1-cYFP HopZ3-nYFP+PBL1-cYFP

HopZ3-nYFP+AvrB-cYFP HopZ3-nYFP+AvrRpm1-cYFP

Figure S1

55"

28"

kDa

kDa

Figure S2

BAvrB3-nYFP+RIN4-cYFP AvrB3-nYFP+RIPK-cYFP

AvrB3-nYFP+ cYFP

A AvrB3-GFP AvrB3_G2A-GFP

Col

Hop

Z3 L

ine

8

Hop

Z3 L

ine

9

α-HA

HopZ3-HA5572kDa

Coomassie

C

5572

5572

+-+--

Ac-RIPKAc-HopZ3

C

His-Z3 or C/AHis-MPK4

His-HopI1Psy

GST-RIPK14C-acetyl

HopZ3C/A

PBS1

A

Ac-HopZ3Ac-PBS1

His-Z3 or C/A

Ac-HopZ3Ac-BIK1

His-Z3 or C/AGST-BIK1

His-HopI1Psy

14C-acetylHis-PBS1

HopZ3C/A

RIPKHopI1Psy

MPK4

+--

+-+

-+-

-++

--+

HopZ3C/ABIK1

HopI1Psy

+-+-

+--+

-++-

-+-+

--+-

---+

B

+--+-

+---+

-++--

-+-+-

-+--+

--+--

---+-

----+

Coomassie

Figure S3

CoomassieCoomassie

14C-acetyl

kDa

5572

5572

36

36

kDa

5536

72

5536

72

kDa

y1""""

y2""

y3"

y4"

b5"

b8"

b18 2+"

b17 2+""""

983.94"

b22 2+"

"

y22 2+"

"

y20 2+"

" b21 2+"

"

y6"

b10"

b23 2+"

"

y8"

b3 -N

H3"

y14 -N

H30"

1653.72"b14"

200400

600800

10001200

14001600

1800m

/z

Relative Abundance

244.16

968.12

946.79 1259.53 1330.10

1280.56

1199.03 974.39

226.15 1072.54

821.91 1167.53

935.47 147.11

311.17 458.21

198.09 343.23

910.40 506.29

790.41 829.38 1102.50

1470.63 1400.67

1034.46 413.21

1541.67 706.37

1642.82 651.30

990.45

1829.82 283.17

1717.69 369.70

571.29

LRD"QG"M

oxND"VG"E"PN

V"MLH

13CAc""T""Q""AE""Y""V""P""K"

b5

b8

b17

b18

b22

b21

b10

b23

b3

b14

y22"

y1"

y2"

y3"

y4"

y20"

y6"

y8"

y14"

0

10

20

30

40

50

60

70

80

90

100

Figure S4

y8

y7

Yform

ylESGASSSG

AND

TPQAE

GS

P Mox PS S S A L L Q

F R

200400

600800

10001200

14001600

18002000

22002400

26002800

30003200

m/z

0 10 20 30 40 50 60 70 80 90

100

Relative Abundance

392.32

152.06

1187.57

1105.59676.42

330.06747.45

921.52563.32

834.50450.24

3154.62175.12

650660

670680

690700

710720

m/z

0 10 20 30 40 50 60 70 80 90

100

Relative Abundance

676.42681.33

y1

y3

y4

y5

y6

y7

y8

y10 y

11 -64

y5y

13-NH3-H3PO4 2+

y1

y3

y4

y5

y6

y10

y11

y13

Figure S5

Supplemental Figure Legends Figure S1. HopZ3 interacts with members of RPM1 immune complex and their family members; related to Figure 1. (A) Examples of protein-protein interactions found using yeast two hybrid analysis. Interactions between HopZ3 (Z3) and AvrB, AvrB3, AvrRpm1 and AvrRpm1Psy, RIN4, MPK4 and RIPK family proteins or AvrB3 and AvrRpm1Psy are shown. Mated yeast strains expressing the bait and prey constructs were grown on the selective minimal SD medium (SD- Leu-/Trp-/His- supplemented with 3-amintriazole (3-AT)) and assessed by β-galactosidase activity. The AvrB and RIN4 mating was used as the positive control (5,6). The HopZ3 and HopIPsy mating was used as the negative control. All photographs were taken after 4 days of growth. See also Table S1 for a summary of these and additional interaction tests. (B) Western blot analysis was performed with yeast cells expressing combinations of the HopZ3 or AvrB bait and AvrB, AvrB3, AvrRpm1, AvrRpm1Psy, RIN4 and RIPK prey proteins and detected with anti-myc and anti-HA antibodies, respectively. White asterisk indicates the prey proteins. Red asterisk indicates the expected AvrB size, which coincides with an anti-myc background band. (C) HopZ3 interacts with RIN4. Pull-down assays with Arabidopsis plant extracts. His-HopZ3 and His-HopZ3_C300A (C/A) immobilized on Ni-NTA were incubated with Arabidopsis total plant extracts. Proteins bound to the Ni-NTA agarose beads were washed, eluted, separated on SDS-PAGE, and immunoblotted with His (top) and RIN4 (bottom) antibodies. Empty Ni-NTA was a negative control (-). White asterisk indicates the expected size of RIN4. A band corresponding to RIN4 is observed in the immobilized His-HopZ3 and His-HopZ3_C300A samples. Signals for all samples are from one exposure of one continuous membrane. (D) AvrB3 interacts with HopZ3 as well as AvrRpm1Psy in plants. Co-immunnoprecipitation assays performed after transient co-expression of AvrB3-HA with GFP vector, HopZ3-, HopZ3_ C300A (C/A)- and AvrRpm1Psy-GFP in N. benthamiana. Proteins were immunoprecipitated from total plant extracts (input) using anti-HA matrix and detected with anti-HA and anti-full length GFP antibodies. White asterisks indicate expected sizes. (E) HopZ3 interacts with RIPK family proteins, and two effectors (AvrRpm1 and AvrB). BIFC analysis of HopZ3 and BIK1, PBS1, PBL1, AvrB and AvrRpm1 interactions in planta. HopZ3-nYFP and BIK1-,

PBS1-, PBL1-, AvrB or AvrRpm1-cYFP were co-expressed in N. benthamiana.

Figure S2. AvrB3 localization and protein-protein interactions in N. benthamiana;

related to Figure 2. (A) AvrB3-GFP, but not AvrB3_G2A-GFP, localizes to the plasma membrane when expressed in N. benthamiana; related to Figure 2. GFP fusion proteins were induced by spraying leaves with 30µM dexamethasone for 16h and visualized by confocal microscopy. (B) BIFC analysis to detect the interactions between AvrB3 and RIN4 or AvrB3 and RIPK in planta. As a negative control, co-expression of AvrB3-nYFP and empty vector pSPCE (c-YFP) did not result in any fluorescent signal. YFP fluorescence was examined in leaves by confocal microscopy 2 days after Agroinfiltration of indicated combinations. (C) HopZ3-HA protein levels in transgenic plants. HopZ3-HA is expressed in stable transgenic Arabidopsis lines. Wild-type Col and Dex:HopZ3-HA (lines 8 and 9) transgenic plants were induced with 30 µM dexamethasone for 16 h, and subjected to western-blot analysis with HA antibody.

Figure S3. HopZ3 acetylates PBS1 and BIK1, but not MPK4; related to Figure 3. Purified recombinant His-PBS1, GST-BIK1, His-HopI1Psy, GST-RIPK and His-MPK4 proteins were incubated with His-HopZ3 or His-HopZ3_C300A (C/A) mutant in the presence [14C] acetyl-CoA for 2 h at 30 oC. Proteins were separated by SDS-PAGE and subjected to autoradiography for 14 days. HopI1Psy and GST-RIPK were used as a negative and a positive control, respectively. HopZ3 acetylated PBS1 (A) and BIK1 (B) but not MPK4 (C). In (A-C) signals for all samples are from one exposure of one continuous gel. Experiments presented in this figure were performed twice with similar results. Figure S4. HopZ3 acetylates H221 in AvrB3 from in vitro acetylation; related to Table 1. (A) Spectra of AvrB3 acetylated peptide containing modified histidine. Q-Exactive MS-MS data for AvrB3 H221 peptide LRDQGMoxNDVGEPNVMLH13CAcTQAEYVPK m/z 967.47 Da(3+) [M+H] 2899.39 Da with a MS error of 7.3 ppm, scan 7951. The b-ions

and y-ions have been labeled, and the fragmentation ions containing the 13C heavy acetylation (+44 Da) are noted in red (b17 2+, b18 2+, b21 2+, b22 2+, b23 2+, y14 -NH3, y20 2+, and y22 2+). Figure S5. RIPK phosphorylates HopZ3 at site S86 in vitro; related to Figure 6. Q-Exactive MS-MS data showing phosphorylated HopZ3 S86 AA(68-97) peptideYformylESGASSSGANDTPQAEGSPMoxPSSSALLQFR m/z 790.33 (4+) [M] 3157.29 Da with a MS error of -5.5 ppm. The y-ions have been labeled, and the fragmentation ions containing the phosphorylation site (loss of 98Da H3PO4) are noted in red (y13-NH3(17 Da)-H3PO4(98 Da)2+),shown in zoomed window. This along with the y10 ion confirms the site of phosphorylation as S86.

Table S1. Analysis of protein-protein interactions using yeast two hybrid (Y2H) and BIFC; related to Figure 1. 1 Interactions in Y2H system: activation of the lacZ reporter gene indicating interaction is represented by plus (+, activation) and minus (-, no activation) signs with increasing plus signs, based on the intensity of blue staining on agar medium containing x-gal. 2 Interactions in BIFC system: N, no visualized YFP signal; Y, visualized YFP signal.

Yeast two-hybrid BIFC Bait Prey Interaction1 n-YFP c-YFP Interaction2 HopZ3 HopI1Psy - HopZ3 HopI1 Psy N HopZ3 AvrB3 ++ HopZ3 AvrB3 Y HopZ3 AvrRpm1Psy +++ HopZ3 AvrRpm1Psy Y HopZ3 AvrB ++ HopZ3 AvrB Y HopZ3 AvrRpm1 +++ HopZ3 AvrRpm1 Y HopZ3 RIN4 +++ HopZ3 RIN4 Y HopZ3 RIPK - HopZ3 RIPK Y HopZ3 BIK1 - HopZ3 BIK1 Y HopZ3 PBL1 - HopZ3 PBL1 Y HopZ3 PBS1 +++ HopZ3 PBS1 Y HopZ3 MPK4 ++++ HopZ3 MPK4 Y HopZ3 HopZ3 - HopZ3 HopZ3 N HopI1 Psy HopZ3 - HopI1 Psy HopZ3 N AvrB3 HopZ3 +++ AvrB3 HopZ3 Y AvrRpm1Psy HopZ3 ++++ AvrRpm1Psy HopZ3 Y AvrB HopZ3 ++++ AvrB HopZ3 Y AvrRpm1 HopZ3 ++++ AvrRpm1 HopZ3 Y RIN4 HopZ3 +++ RIN4 HopZ3 Y RIPK HopZ3 ++ RIPK HopZ3 Y BIK1 HopZ3 ++ BIK1 HopZ3 Y PBL1 HopZ3 +++ PBL1 HopZ3 Y PBS1 HopZ3 ++++ PBS1 HopZ3 Y MPK4 HopZ3 ++++ MPK4 HopZ3 Y AvrB3 AvrRpm1Psy ++++ AvrB3 AvrRpm1Psy Y AvrRpm1Psy AvrB3 ++++ AvrB3 AvrB3 Y AvrB3 AvrB3 ++++ AvrRpm1Psy AvrB3 Y AvrRpm1Psy AvrRpm1Psy ++++ AvrRpm1Psy AvrRpm1Psy Y AvrB3 RIN4 +++ AvrB3 RIN4 Y AvrB RIN4 +++++ AvrB3 RIPK Y RIN4 AvrB3 +++ AvrB3 pSPCE N RIN4 AvrB +++++

Table S2. Phosphorylation sites in HopZ3 substrates expressed in N. benthamiana;

related to Table 1.

Substrate phosphorylation sitesa Substrates Empty vector HopZ3 HopZ3_C300A

RIPK S88, S89, T90, T201, S451

T201, S426, S451

S86, S88, S89, T90, S451

RIN4 S47, S122, S141, S186, T188

S47, S141, S186, T188

S47, S141, S185, S186, T188

AvrRpm1Psy S27, T39

S27, T39

S27, T39

AvrB3 None detected None detected None detected a Phosphorylation results for in planta experiments are shown for each individual protein that was immunoprecipitated and analyzed by MS/MS.

Table S3. Background phosphorylation sites in HopZ3 substrates purified from E. coli; related to Table 1. HopZ3 substrates Phosphorylation sitesa

RIPK

S11, T22, S41, S42, S86, S88, S89, T90, T201, S221, T230, T248, S251, T252, T257, T268, T272, S418, S419, S426, T430, S433, S455

RIN4 S35, S47, S49, S178, S183

AvrRpm1Psy Not done

AvrB3 S86 a Phosphorylation was assessed for all samples containing the same recombinantly expressed and purified protein.

Table S4. Co-occurrence of HopZ3 with interacting or epistatic effectors in sequenced P. syringae strains; related to Figure 1. Top line is % amino acid identity to PsyB728a effector, bottom line is prevalence among strains of a given pathovar that contained HopZ3. Pathovar/strain1

HopZ3-interacting effectors

Effectors from PsyB728a for which cell death in N. benthamiana was suppressed by HopZ32

HopZ3 AvrB3 AvrRpm1 AvrPto1 HopAA1 HopM1 HopAE1

syringae (1212) (B64)

99% 98%

98% 97%3

99%

100%

99% 94%

99% 92%

99%

actinidiae (31 strains)

72% (31/31)

44% (28/31)

97% (28/31) 42% (2/31)

43% (31/31)

74% (15/31) ≥64% (16/313)

≥72% (19/31) (12/313)

74% (29/31)

japonica (MAFF301072 PT)

97% (1/1)

95% (1/13)

(0/1)

(0/1)

94% (1/1)

91% (1/13)

(0/1)

no pv designation (CC440, CC1458, CC1543, BRIP 34876, BRIP 34881)

≥98% (5/5)

≥97% (4/53)

(0/5)

(0/5)

≥93% (4/5)

92% (3/5) (2/53)

97% (1/5)

aptata (DSM50252)

98% (1/13)

(0/1)

(0/1)

(0/1)

93% (1/1)

≥93% (1/13)

(0/1)

theae (ICMP 3923, NCPPB 2598)

81% (2/23)

(0/2)

86% (2/2)

(0/2)

74% (2/2)

71% (2/2)

73% (2/2)

1 HopZ3 was used to search draft genomes of P. syringae available in Genbank and JGI using tblastn or pblast. For P. syringae pv actinidiea, strains with HopZ3 included M302091, KW41, CFBP 7286, CH2010-6, CRAFU8.43, ICMP 18800, TP1, TP6-1, KMP 18708, ICMP 19439, ICMP 19455, Shaanxi M7, NCIPB 3739. ICMP 18744, NCPPB 3871, ICMP 9853, PA459, ICMP 19079, ICMP19097, ICMP 190101, ICMP 18886, ICMP 19102, ICMP 19071, ICMP 19072, ICMP 18801, Shaanxi_M228, ICMP 18708, ICMP 19103, ICMP 19068, ICMP 9855, ICMP 19104 2 Previously determined, see (Vinatzer et al., 2006). Cell death was used as a proxy for immune activation (Vinatzer et al., 2006). 3 A portion of the effector may be present but no intact copy of the effector gene is present in the draft sequence.

Table S5. Bacteria and Yeast Strains; related to Experimental Procedures Table S6. Plasmids; related to Experimental Procedures

Table S7. Primers used in the experiments; related to Experimental Procedures. Primer name Primer sequence HopZ3 EcoRI-F GGG AAT TCA TGA ATA TCT CAG GTC CG HopZ3 XhoI-R CCC TCG AGA GGC TTG GCC CGG ACC CTG TTT GC RIPK-DONR-F AAA AAG CAG GCT TCA TGG CGG TGA AGA AGA AA RIPK-DONR-R AGA AAG CTG GGT CGT ACC GTT CCC CAC CTG C RIPK BamHI-F GGG GAT CCA TGG CGG TGA AGA AGA AA RIPK SalI-R GGG TCG ACG TAC CGT TCC CCA CCT GC RIPK_K122R-F CCT GTT GCT GTT AGA TTG CTT GAT RIPK_K122R-R ATC AAG CAA TCT AAC AGC AAC AGG AvrB BamHI-F GGG GGA TCC ATG GGC TGC GTC TCG TCA AA AvrB XhoI-R GCA GCT CGA GTC AAA GCA ATC AGA ATC TAG AvrB DONR-F AAA AAG CAG GCT TCA TGG GCT GCG TCT CGT CA AvrB DONR-R AGA AAG CTG GGT CAA AGC AAT CAG AAT CTA G AvrB3 EcoRI-F GGG AAT TCA TGG GTT GCG TAT CGT CA AvrB3 SalI-R AGC TTG TCG ACG GTT ATG TTC GAA GAA CCC AvrB3_G2A-DONR-F AAA AAG CAG GCT CCT TAA AGG TGA GCC CGA TGG CTT

GC RIN4 BamHI-F CCC GGA TCC ATG GCA CGT TCG AAT GTA RIN4 XhoI-R CCC GTC GAC GTT TTC CTC CAA AGC CAA RIN4 DONR-F GGG GAC AAG TTT GTA CAA AAA AGC AGG CTG GAT GGC

ACG TTC GAA TGT AC RIN4 DONR-R GGG GAC CAC TTT GTA CAA GAA AGC TGG GTT TTT TCC

AAA GCC AAA G PBL1 DONR-F AAA AAG CAG GCT TCATG GGT TCT TGT CTC AGT PBL1 DONR-R AGA AAG CTG GGT CCA ATC CAA CGG TTT TTT TGT T BIK1 DONR-F AAA AAG CAG GCT TCATG GGT TCT TGC TTC AGT T BIK1 DONR-R AGA AAG CTG GGT CCA CAA GGT GCC TGC CAA A AvrRpm1Psydel-DONR-F1

AAA AAG CAG GCT TCC CAA GAA TAC GGC GCA CGG G

AvrRpm1Psy del- R1 TTC AAG CAA AAG GTC TTT TTC GGA TGG AGA TAA AG GTC

AvrRpm1Psy del- F2 TTA TCT CCA TCC GAA AAA GAC CTT TTG CTT GAA G AvrRpm1Psy del-DONR-R2

AGA AAG CTG GGT CTC AGC GCA AGG CCC GAT GAG

AvrB3 del-DONR-F1 AAA AAG CAG GCT TCT TCG ATC ATA ACG TCG ACC C AvrB3del-DONR-R1 AGA AAG CTG GGT CGT AAC GCT GCA ACG ACG GGC TCA

CCT TTT TTA ACT AvrB3 del-DONR-F2 AAA AAG CAG GCT TCA AAA AGG TGA GCC CGT CGT TGC

AGC GTT ACT GG AvrB3 del-DONR-R2 AGA AAG CTG GGT CCA GGA TGT TCT GAT GGA CCG

Supplemental Data Supplemental Data S1. Amino acid sequence alignments of orthologues of HopZ3 substrates RIPK, RIN4, AvrB3 and AvrRpm1Psy; related to Table 1. Alignment of RIPK paralogous proteins: Residues highlighted in black are conserved. Residues highlighted in grey have similar properties. Residues labeled in red boxes were found to be acetylated by HopZ3. RIPK(NP_178651), BIK1 (NP_181496), PBS1 (NP_196820), PBL1 (NP_191105). Alignment of RIN4 orthologous proteins: Residues highlighted in black are conserved. Residues highlighted in grey have similar properties. Residues labeled in red boxes were found to be acetylated by HopZ3. AtRIN4 (NP_189143), LeRIN4 (XP_010326285), LsRIN4-1 (ACZ64220), LsRIN4-2 (ACZ64227), GmRIN4a (NP_001235221), GmRIN4b (NP_001235221), GmRIN4c (NP_001235235), GmRIN4d(NP_001235252). Alignment of AvrB orthologous proteins: Residues highlighted in black are conserved. Residues highlighted in grey have similar properties. Residues labeled in red boxes were found to be acetylated by HopZ3. AvrB3 PsyB728a (AF232005), AvrB1pgyrace04 (AAA25726), AvrB2pph1448a (AAA88428), AvrB4-1pph1448a (AAX12108), AvrB4-2pph1448a (AAZ35340). Alignment of AvrRpm1 orthologous protein: Residues highlighted in black are conserved. Residues highlighted in grey have similar properties. Residues labeled in red boxes were found to be acetylated by HopZ3. Gene bank accession number, AvrRpm1Psy (AAY35802), AvrRpm1M2 (X67808), AvrRpm1M6 (AF359557), AvrRpm1 ppi HRI 1691(AJ222647), AvrRpm1act (KAK41039).

Supplemental Experimental Procedures Yeast two-hybrid assays

Two yeast two hybrid (Y2H) screens were performed. One used HopZ3 as bait, and identified MPK4 protein from a mixed Arabidopsis and N. benthamiana protein prey library. Briefly, a cDNA library from Arabidopsis and N. benthamiana was created in pDONR207 and recombined into the pLaw11 vector to create an AD-fusion library in the yeast strain AH109. Two hybrid interactions were screened by mating of yeast strain Y187 carrying the pLaw10 -HopZ3 bait construct with the AD fusion library at 30°C over

night following standard Y2H protocols (Clontech, Mountain View). pLAW10 and pLAW11 are Gateway (Invitrogen) compatible modifications of the yeast two-hybrid AD and BD vectors, pGBKT7 and pGADT7 respectively (Clontech). Positive interactions were verified on SC-Trp-Leu-His medium supplemented with 2.0 mM 3AT (3-aminotriazole) or SC-Trp-Leu with X-Gal (5-bromo-4-chloro-3-indolyl-D-glactopyranoside). The second set of Y2H experiments were part of large scale screens in which pathogen effectors were tested pairwise for interactions with each other or with known plant immune signaling proteins (http://charge.ucdavis.edu/). Over 300 pathogen effectors and 100 plant proteins were tested in pairwise combinations. This identified AvrRpm1Psy, AvrB3, RIN4 and PBS1 as interacting with HopZ3. Additionally, AvrRpm1Psy and AvrB3 were identified as interacting proteins. Subsequent directed Y2H assays were done with PBS1-family members, several of which were found to interact with HopZ3. Interactions of various combinations were screened and verified on SC-Trp-Leu-His medium supplemented with 2.0 mM 3AT (3-aminotriazole) or SC-Trp-Leu with X-Gal (5-bromo-4-chloro-3-indolyl-D-galactopyranoside).

In planta pull-down assay Total proteins were extracted from Arabidopsis Col WT plants with lysis buffer

(50 mM HEPES pH7.5, 50 mM NaCl, 0.2% Triton X-100). His-HopZ3 and His-HopZ3 _C300A bound to the Ni-NTA agarose beads were incubated with Arabidopsis lysates for 2 h at 4 °C. After washing the beads three times with lysis buffer, proteins were eluted by boiling with Laemmli loading buffer. The samples were analyzed by western blotting with anti-RIN4 (1:5000) and anti-His antibodies (1:5000).

In vitro and in vivo acetylation assay for LC-MS/MS

For in vitro acetylation mapping, reactions were performed with 13C-acetyl-CoA (Acetyl-1,2-13C coenzyme A lithium salt, Sigma) to differentiate between background 12C-acetylation that occurred in E. coli during production of recombinant protein and HopZ3-mediated acetylation in vitro. Substrates were mixed with either HopZ3, the catalytically inactive HopZ3_C300A, or as substrate alone in the presence of 13C-acetyl-

CoA to distinguish any background acetylation that could be chemically caused by the presence of 13C-acetyl-CoA. 1 µg of purified His-HopZ3 or His-HopZ3_C300A were mixed with 3 µg bead-bound substrate to which the acetylation reaction cocktail (50 mM

HEPES (pH 8.0), 10% glycerol, 5 µM IP6 and 50 µM of 13C-acetyl-CoA (Sigma)) was added in a total volume of 20 µl. Subsequently beads were washed twice with washing buffer (50 mM HEPES pH 8.0, 50 mM NaCl, 10% glycerol), boiled in Laemmli loading buffer and processed for LC-MS/MS analysis (see below). Data from the mass spectrometry of treated samples were analyzed for the presence of 13C-acetylated peptides in the substrate (RIPK, RIN4, AvrB3). For in planta acetylation mapping, Dex-HopZ3- and Dex-HopZ3_C300A-HA constructs were transiently co-expressed with Dex-RIPK-GFP in N. benthamiana. For other substrates, Dex-HopZ3-and Dex-HopZ3_C300A-GFP constructs were transiently co-expressed with Dex-RIN4-, Dex-AvrB3 and Dex-AvrRpm1Psy-HA in N. benthamiana. Plants were treated with 30 µM dexamethasone for 16 h to induce protein expression. Proteins were extracted with lysis buffer containing 50 mM Tris pH 8.0, 150 mM NaCl, 10% glycerol, 1% NP40, 0.5% sodium deoxycholate, 2 µM sodium butyrate (TOCRIS

bioscience) and 3 µM TrichostatinA (Sigma). Clarified total protein lysate was incubated for 3 h with anti-GFP or –HA tag magnetic beads (Medical and Biological Laboratories Co., LTD) at 4 °C. After washing the beads three times with the lysis buffer on the magnetic rack, proteins were eluted by boiling with Laemmli loading buffer.

Trypsin digestion of samples from SDS-PAGE gels

Protein bands to be analyzed were excised from 4-12 % MOPS buffer SDS-PAGE gels with sterile razor blade and chopped into ~1 mm3 pieces. Each sample was washed in water and destained using 100 mM ammonium bicarbonate pH 7.5 in 50% acetonitrile. A reduction step was performed by addition of 100 μl 50 mM ammonium bicarbonate pH 7.5 and 10 μl of 10 mM Tris (2-carboxyethyl)phosphine HCl at 37°C for 30 min. The proteins were alkylated by adding 100 μl 50 mM iodoacetamide and allowed to react in the dark at 20°C for 30 min. Gel samples were washed in water, then acetonitrile, and dried in a SpeedVac. Trypsin digestion was carried out overnight at

37°C with 1:50 enzyme-protein ratio of sequencing grade-modified trypsin (Promega) in 50 mM ammonium bicarbonate pH 7.5, and 20 mM CaCl2. Peptides were extracted with 5% formic acid and vacuum dried (Truman et al., 2012).

HPLC for Mass Spectrometry The peptide samples were loaded to a 0.25 μl C8 OptiPak trapping cartridge

custom-packed with Michrom Magic C8 (Optimize Technologies), washed, then switched in-line with a 20 cm by 75 μm C18 packed spray tip nano column packed with Michrom Magic C18AQ, for a 2-step gradient. Mobile phase A was water/acetonitrile/formic acid (98/2/0.2) and mobile phase B was acetonitrile/isopropanol/water/formic acid (80/10/10/0.2). Using a flow rate of 350 nl/min, a 90 min, 2-step LC gradient was run from 5% B to 50% B in 60 min, followed by 50%–95% B over the next 10 min, hold 10 min at 95% B, back to starting conditions and re-equilibrated.

LC-MS/MS Analysis

Samples were analyzed via electrospray tandem mass spectrometry (LC-MS/MS) on a Q-Exactive (Thermo scientific) mass spectrometer, using a 60,000 RP survey scan, m/z 375-1950, with lockmasses, followed by 15 HCD CID scans on only doubly and triply charged precursors between 375 Da and 1950 Da. Inclusion lists of expected acetylated or phosphorylated tryptic in-silico peptide ion masses were generated (in-house software) and were loaded into instrument method. Using the FASTA sequence file for RIPK, RIN4, AvrB3, and/or HopZ3, we modeled the following modifications: (formyl n-term, oxidation (M), acetyl (K, H, S, T), 13C heavy acetyl (K, H, S, T), phospho (S, T)), the produced m/z ions were filtered for 2+ and 3+ expected ions, then combined into a *.csv file ready to load into the QE instrumentation method. Due to a hard upper limit of 5000 inclusion ions allowed by the instrument, we generated 3 files (1: RIPK and HopZ3 ions, 2: RIN4 and HopZ3, and 3: AvrB3 and HopZ3 ions). Ions selected for MS/MS were placed on an exclusion list for 60 seconds.

Database Searching All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK;

version 2.3.02) and X! Tandem (The GPM, thegpm.org; version CYCLONE (2010.12.01.1)). Mascot was set up to search the 130416_Greenberg_db3 database (6626 entries) assuming the digestion enzyme trypsin. X! Tandem was set up to search the 130416_Greenberg_db3 database (6626 entries) also assuming trypsin. Mascot and X! Tandem were searched with a fragment ion mass tolerance of 0.60 Da and a parent ion tolerance of 20 PPM. Carbamidomethyl of cysteine was specified in Mascot and X! Tandem as a fixed modification. Oxidation of methionine, formyl of the n-terminus, acetyl of histidine, lysine, serine and threonine, heavy acetyl:13C(2) of histidine, lysine, serine and threonine, and phospho of serine and threonine were specified in Mascot and X! Tandem as variable modifications.

Criteria for Protein Identification

Scaffold (version Scaffold_4.2.1, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability. Peptide Probabilities from X! Tandem were assigned by the Peptide Prophet algorithm (Keller et al., 2002) with Scaffold delta-mass correction. Peptide Probabilities from Mascot (Ion Score Only) were assigned by the Scaffold Local FDR algorithm. Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii et al., 2003). Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. All acetylated and phosphorylated peptide spectra were manually validated. Manual validation included inspecting the *.raw file for the MS isotopic peak, validating the correct charge state and noting which isotope was selected for MSMS fragmentation. We then inspected the MSMS fragmentation data inspecting the *.raw scan with the scaffold labeled peaks and checked manually for any 13C-acetyl peptides that were wrongly identified as a 12C-acetyl peptide. This distinction

was enabled by inspection of the masses of MSMS fragmentation peaks and inspection of the MS Isotopic Peak pattern for the peptide.

Preliminary results using Scaffold as the output suggested the presence of 13C-acetylation sites on RIPK, RIN4, and AvrB3. Potential sites were manually checked using the primary MS data. For each site identified in the data file, a snapshot was taken of the spectra that corresponded to the greatest intensity of the peptide over the span of its detection. Peaks were then measured for their absolute intensity at the apex of each peak for all peaks within the isotopic distribution. Only peaks that were clearly within the isotopic distribution of the peptide in question were measured. The sum of all peaks within a distribution was then used as an indicator of the peptide quantity, which was then directly compared to the same summation of the corresponding peptide from the samples treated with either the catalytically inactive mutant or the substrate alone. This produced the list of sites of acetylation that resulted specifically from HopZ3 activity in Table 1, column 1. We also report acetylation sites where there was similar background with 13C-acetyl-CoA alone or 13C-acetyl-CoA plus HopZ3_C300A, but where HopZ3 caused an increase in the level of 13C-acetylation (Table 1, column 2).

Supplemental References

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Lee, J., Teitzel, G.M., Munkvold, K., del Pozo, O., Martin, G.B., Michelmore, R.W., and Greenberg, J.T. (2012). Type III secretion and effectors shape the survival and growth pattern of Pseudomonas syringae on leaf surfaces. Plant Physiol 158, 1803-1818. Mackey, D., Belkhadir, Y., Alonso, J.M., Ecker, J.R., and Dangl, J.L. (2003). Arabidopsis RIN4 is a target of the type III virulence effector AvrRpt2 and modulates RPS2-mediated resistance. Cell 112, 379-389. Mackey, D., Holt, B.F., Wiig, A., and Dangl, J.L. (2002). RIN4 interacts with Pseudomonas syringae type III effector molecules and is required for RPM1-mediated resistance in Arabidopsis. Cell 108, 743-754. Nesvizhskii, A.I., Keller, A., Kolker, E., and Aebersold, R. (2003). A statistical model for identifying proteins by tandem mass spectrometry. Analytical Chem 75, 4646-4658. Truman, A.W., Kristjansdottir, K., Wolfgeher, D., Hasin, N., Polier, S., Zhang, H., Perrett, S., Prodromou, C., Jones, G.W., and Kron, S.J. (2012). CDK-dependent Hsp70 Phosphorylation controls G1 cyclin abundance and cell-cycle progression. Cell 151, 1308-1318. Vinatzer, B.A., Teitzel, G.M., Lee, M.W., Jelenska, J., Hotton, S., Fairfax, K., Jenrette, J., and Greenberg, J.T. (2006). The type III effector repertoire of Pseudomonas syringae pv. syringae B728a and its role in survival and disease on host and non-host plants. Mol Microbiol 62, 26-44. Walter, M., Chaban, C., Schutze, K., Batistic, O., Weckermann, K., Nake, C., Blazevic, D., Grefen, C., Schumacher, K., Oecking, C., et al. (2004). Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. The Plant J 40, 428-438.

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