G2A:sYFP K115N:sYFP A B nPBS1:sYFP cPBS1:sYFP C 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.
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nPBS1:sYFP D cPBS1:sYFP - Plant Physiology...2013/11/13 · C nPBS1:sYFP D cPBS1:sYFP Figure S1. Localization of PBS1 derivatives. A, The G2A mutation does not cause PBS1 dissociation
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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.
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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.
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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.
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.
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.
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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.