G2A:sYFP K115N:sYFPA B

nPBS1:sYFP cPBS1:sYFPC D

Figure S1. Localization of PBS1 derivatives. A, The G2A mutation does not cause PBS1 dissociation from the plasma membrane. B, The PBS1 kinase inactive mutant, K115N, still localizes to the plasma membrane. The chlorophyll signal is shown in magenta. C, The engineered N-terminal PBS1 cleavage product, nPBS1, localizes to the plasma membrane. D, The engineered C-terminal PBS1 cleavage product, cPBS1, localizes to the cytoplasm and nucleus. All constructs were fused to sYFP and transiently expressed in N. benthamiana using a dexamethasone-inducible promoter. Confocal laser scanning microscopy was performed at 5 hours post induction. Panel A shows a 3D projection of a Z stack, while panels C-D show single optical sections.

0

1

2

3

4

5

6

7

8

Day0 Day3

PBS1G2AC3/6A-4

G2AC3/6A-5

Figure S2. Mutation of the PBS1 S-acylation site does not affect the susceptibility of Arabidopsis to P. syringae strain DC3000 lacking avrPphB. The G2AC3/6A PBS1 derivative under control of the native PBS1 promoter was transformed into the pbs1-1 mutant and T3 homozygous transgenic plants were syringe-infiltrated with strain DC3000 at a concentration of 5×104 CFU/ml. Results shown represent the mean (n = 3) and standard deviation. Two independent transgenic lines were tested. This experiment was repeat-ed twice with similar results.

Bac

teria

l gro

wth

[log

(cfu

cm

-2)]

A B

C

pbs1-3 pbs1-4 pbs1-5 pbs1-6

EAR5 Col-0

EAR5 Col-0

Figure S3. Isolation of new pbs1 alleles using an estradiol (ED)-inducible AvrPphB expression system. A, Induction of AvrPphB inhibits growth of EAR5 seedlings. Seeds were germinated on 1/2 MS solid media containing 20 μM estradiol and photographed 10 days after sowing. B, EAR5 transgenic plants behave like wild type Col-0 in the absence of estradiol induction. The indicated P. syringae strains were syringe infiltrated at 105 cfu/ml. Data represent mean (n=3) and standard deviation. Different letters above the bars denote a significant difference as determined by a two-tailed Student’s t-test (P < 0.01). This experiment was repeated twice with similar results. C, Cell death induction in transgenic Arabidopsis plants homozygous for ED:AvrPphB and RPS5:HPB transgenes (EAR5). Three week-old plants were sprayed with 20 μM estradiol and photographed 48 hours later. D, Screening for new pbs1 alleles that block AvrPphB-induced cell death. EAR5 was mutagenized using EMS and M2 generation plants were grown on ½ MS solid media containing 20 μM estradiol. Photographs were taken 10 days after sowing.

876543210B

acte

rial g

row

th [l

og(c

fu c

m-2)]

EAR5 Col

Day 0 Day 3DC3000(e.v.)

EAR5 Col

DC3000(avrPphB)

a a

b b

D

AT1G07570

AT2G28930

AT5G02290

AT3G55450

AT1G14370

AT2G02800

AT1G26970

AT1G69790

AT2G07180

AT5G01020

AT5G56460

AT1G61590

AT2G26290

AT2G05940

AT5G35580

AT4G17660

AT5G47070

AT1G20650

AT1G76370

AT3G07070

AT3G24790

AT4G13190

AT3G20530

AT1G07870

AT2G28590

AT5G02800

AT5G13160

AT5G18610

PpPBS1

AT1G24030

100

100

100

100

100

100

99

100

99

78

84

98

67

63

52

78

95

89

72

100

50

99

48

46

100

79

44

(PBS1)(PBL27)

(PBL7)

A

(PBL5)(PBL6)

(PBL22)

(PBL23)

(PBL3)

(PBL28)

(PBL24)

(PBL25)(PBL26)

(PBL21)(PBL19)

(PBL20)(PBL13)(PBL14)(PBL12)(PBL15)(PBL16)(PBL8)(PBL17)(PBL18)(PBL4)

(PBL2)

(PBL1)(PBL11)(PBL10)(PBL9)

PBL5 PBL6 PBL7 PBL9 PBL23 PBL27PBS1+RPS5

AvrPphB

C98S

B

C

Figure S4. PBS1 paralogs cannot substitute for PBS1 in activation of RPS5 in N. benthamiana transient assays. A, Phylogenetic tree showing relationship of PBS1 to Arabidopsis paralogs, and to a moss ortholog (PpPBS1). This tree was constructed using the Neighbor-Joining method implemented within MEGA5.2 using full-length protein sequences. Boostrap values were calculated from 1000 iterations. Underlined proteins were used in this study. B, Immunoblot of PBS1 and PBLs after co-expression in N. benthamiana together with active AvrPphB wt (+) or inactive AvrPphB mutant C98S (-). C, PBS1 and the indicated PBLs were co-expressed in N. benthamiana together with RPS5 and AvrPphB wt (+) or the inactive AvrPphB mutant C98S (-). Pictures were taken 16h after induction of protein expression by dexamethasone.

PBS1 PBL5 PBL6 PBL7 PBL9 PBL23 PBL27AvrPphB - + - + - + - + - + - + - +C98S + - + - + - + - + - + - + -

70

55

35

25

25

kDa

α-HA

α-AvrPphB

nPpPBS1PpPBS1cRPS5

nPpPBS1PBS1cRPS5

nPBS1PpPBS1cRPS5

nPBS1PBS1cRPS5

Figure S5. The moss PBS1 N-terminal cleavage product (nPpPBS1) can substitute for the PBS1 N-terminal cleavage product. The indicated engi-neered cleavage products of Arabidopsis and moss PBS1 were co-expressed with RPS5 in N. benthamiana. Picture was taken 16h after induc-tion of protein expression by dexamethasone. Co-expression of the moss PBS1 N-terminal cleavage product with the Arabidopsis PBS1 C-terminal cleavage product (lower left) induced HR, whereas the reciprocal combination (upper right) did not.

PBS1WT NARAP WT SEMPH

- + - + - + - + AvrPphB

PBL27

A

B

(PBS1)(PBL27)

Pp1s116_115V6.1

(PBS1)(PBL27)

Pp1s116_115V6.1

(PBS1)(PBL27)

Pp1s116_115V6.1

(PBS1)(PBL27)

Pp1s116_115V6.1

(PBS1)(PBL27)

Pp1s116_115V6.1

(PBS1)(PBL27)

Pp1s116_115V6.1

Figure S6. Mutation of the C-terminal SEMPH motif in PBS1 does not affect cleavage by AvrPphB. A, Amino acid sequence alignment for the kinase domains of PBS1, PBL27 and PpPBS1. The green bar denotes the SEMPH motif in PBS1 and the red bar denotes the GDK motif that precedes the cleavage site. B, Immunoblot for PBS1 wt, PBS1NARAP, PBL27 wt and PBL27SEMPH after co-expression with active AvrPphB (+) or inactive AvrPphB mutant C98S (-) in N. benthamiana.

Arabidopsis thalianaNP_196820 243 KSHVSTRVMGTYGYCAPEYAMTGQLTVKSDVYSFGVVFLELITGRKAIDSEMPHGEQNLVAWARPLFNDRRKFIKLADPR 322 Capsella rubella EOA20310 341 KSHVSTRVMGTYGYCAPEYAMTGQLTVKSDVYSFGVVFLELITGRKAIDSEMPHGEQNLVAWARPLFNDRRKFIKLADPK 420 Theobroma cacao EOX99706 242 KSHVSTRVMGTYGYCAPEYAMTGQLTVKSDVYSFGVVFLELITGRKAIDSTRPHGEQNLVTWARPLFNDRRKFSKLADPR 321 Ricinus communis XP_002514864 244 KSHVSTRVMGTYGYCAPEYAMTGQLTVKSDVYSFGVVFLELITGRKAIDSTRPHGEQNLVTWARPLFNDRRKFSKLADPQ 323 Populus trichocarpa XP_002304664 243 KSHVSTRVMGTYGYCAPEYAMTGQLTVKSDVYSFGVVFLELITGRKAIDSSRPHGEQNLVTWTRPLFNDRRKFSKLADPR 322 Prunus persica EMJ26931 247 KSHVSTRVMGTYGYCAPEYAMTGQLTVKSDVYSFGVVFLELITGRKSIDSNRPHGEQNLITWARPLFNDRRKFSKLADPR 326 Glycine max NP_001235164 236 KSHVSTRVMGTYGYCAPEYAMTGQLTVKSDVYSFGVVFLELITGRKAIDSTQPQGEQNLVTWARPLFNDRRKFSKLADPR 315 Cicer arietinum XP_004497430 242 KSHVSTRVMGTYGYCAPEYAMTGQLTVKSDVYSFGVVFLELITGRKAIDGTRPHGEQNLVTWARPLFNDRRKFPKLADPR 321 Cucumis sativus XP_004140546 240 KSHVSTRVMGTYGYCAPEYAMTGQLTVKSDVYSFGVVFLELITGRRAIDSTRPQGEQNLVTWARPFFNDRRRFSKLADPQ 319 Fragaria vesca XP_004310112 248 KSHVSTRVMGTYGYCAPEYAMTGQLTVKSDVYSFGVVFLELITGRKAIDSTRSHGEQNLVTWARPLFNDRRKFSKLADPR 327 Vitis vinifera XP_002265076 242 KSHVSTRVMGTYGYCAPEYAMTGQLTVKSDVYSFGVVFLELITGRKAIDSTLPHGEQNLVTWARPLFNDRRKFAKLADPR 321 Glycine max XP_003556585 252 KSHVSTRVMGTYGYCAPEYAMTGQLTVKSDVYSFGVVFLELITGRKAIDSTRPHGEQNLVTWARPLFSDRRKFPKLADPQ 331 Solanum lycopersicumXP_004239305 240 KSHVSTRVMGTYGYCAPEYAMTGQLTVKSDVYSFGVVFLELITGRKAIDSTKPQGEQNLVAWARPLFNDRRKFAKLADPS 319 Zea mays NP_001149465 272 KSHVSTRVMGTYGYCAPEYAMTGQLTVKSDVYSFGVVLLELITGRRAIDSTRPHGEQNLVSWARPLFNDRRKLPKMADPR 351 Setaria italic XP_004952572 268 KSHVSTRVMGTYGYCAPEYAMTGQLTVKSDVYSFGVVLLELITGRRAIDSTRPHGEQNLVSWARPLFNDRRKLPKMADPR 347 Sorghum bicolor XP_002452162 272 KSHVSTRVMGTYGYCAPEYAMTGQLTVKSDVYSFGVVLLELITGRRAIDSTRPHGEQNLVSWARPLFNDRRKLPKMADPR 351 Oryza sativa NP_001046943 279 KSHVSTRVMGTYGYCAPEYAMTGQLTVKSDVYSFGVVLLELITGRRAIDSTRPHGEQNLVSWARPLFNDRRKLPKMADPR 358 Figure S7. Alignment of the PBS1 SEMPH loop region from PBS1 orthologs from the NCBI non-redundant protein database. The SEMPH motif is boxed. Amino acids that differ from the Arabidopsis PBS1 sequence are highlighted in green. All others are identical, illustrating the high conservation of PBS1 among plant species.

Supplemental Table S1. Primers used in this study

Primer Sequences (5’-3’) Purpose

SHP89 TACAAAAGAAGGCTTGACAGCAC G103R mutagenesis

SHP90 AAGCCTTCTTTTGTAGACACGTC G103R mutagenesis

SHP91 GACAAATTTCATGTCTCCACTAGAGT S244F mutagenesis

SHP92 GACATGAAATTTGTCTCCCGTTG S244F mutagenesis

SHP93 ATTACTGATCGCAAAGCTATAGACAG G286D mutagenesis

SHP94 TTTGCGATCAGTAATGAGCTCGAG G286D mutagenesis

SHP98 GGATTTGAACGTGTCTACAAAGGA G98E mutagenesis

SHP99 GACACGTTCAAATCCACCTTCGC G98E mutagenesis

UD197 GGGGACAAGTTTGTACAAAAAAGCAGGCTCAatgggttgtttcggatgc

attB1-PBL5

UD198 GGGGACAACTTTGTATAGAAAAGTTGGGTGtgaggacccttcacattcag

attB4-PBL5

UD195 GGGGACAAGTTTGTACAAAAAAGCAGGCTCAatgggatgtttcggacg

attB1-PBL6

UD196 GGGGACAACTTTGTATAGAAAAGTTGGGTGagttgcttgatctgagcatatct

attB4-PBL6

UD193 GGGGACAAGTTTGTACAAAAAAGCAGGCTCAatgggttggatcccgtg

attB1-PBL7

UD194 GGGGACAACTTTGTATAGAAAAGTTGGGTGgaccctctttgatctaggtgg

attB4-PBL7

UD185 GGGGACAAGTTTGTACAAAAAAGCAGGCTCAatggggatttgcttgagtg

attB1-PBL9

UD186 GGGGACAACTTTGTATAGAAAAGTTGGGTGaacatacagtggcgaggc

attB4-PBL9

UD199 GGGGACAAGTTTGTACAAAAAAGCAGGCTCAatgaaaatcaattgcttgttctg

attB1-PBL23

UD200 GGGGACAACTTTGTATAGAAAAGTTGGGTGaagtttggatctttcgtcttctt

attB4-PBL23

UD191 GGGGACAAGTTTGTACAAAAAAGCAGGCTCAatgagtgggtgtttgccttg

attB1-PBL27

UD192 GGGGACAACTTTGTATAGAAAAGTTGGGTGgtcatttgtactatcaaagctgcc

attB4-PBL27

UD237 GGGGACAACTTTTCTATACAAAGTTGCGATGGGTTGTTTCTCGTGTTT

attb4r PBS1 N-terminus

UD238 GGGGACCACTTTGTACAAGAAAGCTGGGTctaTTTGTCTCCCGTTGGTCC

attB2 PBS1 N-terminus

UD239 GGGGACAAGTTTGTACAAAAAAGCAGGCTCAatgTCTCATGTCTCCACTAGAGTTATGG

attB1 PBS1 C-terminus

UD240 GGGGACAACTTTGTATAGAAAAGTTGGGTGCCCGGTACTGTTGCTCTC

attB4 PBS1 C-terminus

UD233 GGGGACAACTTTTCTATACAAAGTTGCGatgagtgggtgtttgccttg attb4r PBL27 N-terminus

UD234 GGGGACCACTTTGTACAAGAAAGCTGGGTActatttatcgcccacgggac

attb2 PBL27 N-terminus

UD235 GGGGACAAGTTTGTACAAAAAAGCAGGCTCAatgacacatgtgtcaactcgtg

attb1 PBL27 C-terminus

UD236 GGGGACAACTTTGTATAGAAAAGTTGGGTGgtcatttgtactatcaaagctgcc

attb4 PBL27 C-terminus

UD241 GGGGACAACTTTTCTATACAAAGTTGCGatgggctgtttcccatg attB4r PpPBS1 N-terminus

UD242 GGGGACCACTTTGTACAAGAAAGCTGGGTActacttgtcccccaccgg

attB2 PpPBS1 N-terminus

UD51 GGGGACAAGTTTGTACAAAAAAGCAGGCTCAATGACGCACGTATCGACGC

attB1 PpPBS1 c-termins

UD90 GGGGACAACTTTGTATAGAAAAGTTGGGTGAGCCGTCCCCCTTTTCC

attB4 PpPBS1 c-terminus

UD243 CGGGTCGAAAAGCTATTGATagcgagatgcctcat PBL27 SEMPH

UD244 GACAAGGTTGTGCTCTCCatgaggcatctcgct PBL27 SEMPH

UD227 CTGGTCGCAAAGCTATAGACaatgctcgagcaccc PBS1 NARAP

UD228 CACCAGGTTCTGCTCTCCgggtgctcgagcatt PBS1 NARAP

Tony111 GGACAAGTTTGTACAAAAAAGCAGGCTCTATGGGTTGTTTCTCGTGTTTTG

attB1-PBS1f

Tony273 GGACAAGTTTGTACAAAAAAGCAGGCTCTATGGCTTGTTTCTCGTGTTTTG

attB1-G2APBS1f

Tony265 GGACAAGTTTGTACAAAAAAGCAGGCTCTATGGCTGCTTTCTCGGCTTTTGATTCGAGTG

attB1-G2C3/6APBS1f

Tony133 GGACAACTTTGTATAGAAAAGTTGGGTGCCCGGTACTGTTGCTCTCTG

attB4-PBS1r

Tony114 GGACAAGTTTGTACAAAAAAGCAGGCTCTATGTCTCATGTCTCCACTAGAG

attB1-PBS1Cf

Tony132 GGACAACTTTGTATAGAAAAGTTGGGTGTTTGTCTCCCGTTGGTCC

attB4-PBS1Nr

Tony266 GGACAAGTTTGTACAAAAAAGCAGGCTCTATGGCTTCGGCTACTGTTGATTTTGGGATCGGACGGATTCTTAGTGTCCTGGAAAACGAGGTGAGCAAGGGCGAGGAG

attB1-PBS1N20-

sYFPf

TONY162 GGACAACTTTGTATAGAAAAGTTGGGTGCTTGTACAGCTCGTCCATGCC

attB4-FPr

Tony209 GGACAAGTTTGTACAAAAAAGCAGGCTCTATGGGGTGTGCATCCTCTTC

attB1AvrPphBf

Tony134 GGACAAGTTTGTACAAAAAAGCAGGCTCTATGGCATGTGCATCCTCTTCAGGC

attB1-G2A AvrPphBf

Tony210 GGACAACTTTGTATAGAAAAGTTGGGTGCGAAACTCTAAACTC

attB2AvrPphBr