Supporting Online Material

Transcription

1 Supporting Online Material 1. Materials and Methods 2. Supporting online figures 3. Supporting online references 1. Material and Methods Plasmid constructs All AvrPphB and protease inactive AvrPphB() constructs were generated by PCR amplification of the 28 kda form of AvrPphB, which lacks the amino-terminal 62 amino acids, using previously described plasmid clones as templates (S1, S2). The PBS1 gene was amplified from a cdna construct (S3) for transient expression assays, and from genomic DNA for complementation assays. For mammalian expression, AvrPphB was inserted into a derivative of the pcdna3 vector (Invitrogen) containing a C-terminal FLAG epitope, and PBS1 was inserted into a pcdna3 derivative containing an HA epitope. An AU1 tag was inserted into the pcdna3-pbs1-ha construct between amino acid residues 19 and 20 of PBS1, which is after the PBS1 myristoylation site. Bacterial expression constructs of AvrPphB-His6 were generated by cloning wild-type and mutant AvrPphB into pet21a (Novagen). The bacterial expression construct pgex-kg-pbs1-his6 was constructed by inserting PBS1 into the pgex- KG vector (Pharmacia). Arabidopsis kinases At3g17410 and At5g02800 were amplified from an Arabidopsis cdna library and cloned into the pcdna3-ha vector. RPS5 coding sequence was amplified from a genomic RPS5 clone in the pgem-t vector (Promega) and cloned into the pef4-myc vector (Invitrogen). For dexamethasone-inducible expression in planta, wild-type and

2 mutant forms of AvrPphB and PBS1-HA were cloned into the vector pta7002 (S4). For complementation assays, a genomic fragment spanning from 875 bp 5' of the PBS1 start codon to 229 bp 3' of the stop codon was amplified by PCR and cloned into the pgreen0229 vector (S5). All point mutations as well as the AU1 tag insertion constructs were generated using a QuikChange Site-Directed Mutagenesis Kit (Stratagene). Plant material Nicotiana tabacum cultivar xanthi, Nicotiana benthamiana, Arabidopsis thaliana accessions Columbia (Col-0) and Landsberg-erecta (Ler), and the Col-0 mutant lines rps5-2, pbs1-1 and rar1-20 were grown under a 9-hour photoperiod at 24ûC. Agrobacterium transient expression in planta Agrobacterium tumefaciens GV3101 strains carrying the various dexamethasone-inducible constructs were grown and prepared for transient expression as described in (S6). Agrobacterium cultures was resuspended in infiltration medium at OD 600 = 0.4. For experiments requiring coexpression of AvrPphB and PBS1, suspensions were mixed in a 1:1 ratio, maintaining an OD 600 = 0.4. Bacterial suspensions were infiltrated into expanding leaves of 4-week old tobacco and N. benthamiana or 5-week old Arabidopsis plants. Plants were sprayed with 50 µm dexamethasone 40 hours after injection to induce expression. Samples were collected for protein extractions 4 hours after dexamethasone application for tobacco and N. benthamiana and 24 hours after application for Arabidopsis. Samples were flash-frozen in liquid nitrogen and ground in an extraction buffer (10 mm Tris-HCl, ph 7, 0.33 M sucrose, 1mM EDTA, plant Protease Inhibitor Cocktail (Sigma)). Extracts were centrifuged briefly to clear debris, and protein in the

3 supernatant quantified. Approximately 50 µg of proteins per sample were separated on a 10% SDS-PAGE and transferred to a nitrocellulose membrane. The resulting blots were probed with Anti-HA-Peroxidase High Affinity monoclonal antibody (Roche) or polyclonal anti-avrpphb antibody. Immunoprecipitation Following transient expression in N. benthamiana, 8 leaf-discs were collected and ground in lysis buffer (50 mm Tris, ph 7.5, 150 mm NaCl, 0.1% NP-40 and Plant Proteinase Inhibitor Cocktail (Sigma)). Protein concentrations were determined and normalized to 2 mg/ml. 100 µl of rat monoclonal Anti-HA matrix slurry (50 µl resin) (Roche) were added to 2 mg of protein in a 1.5 ml tube and mixed gently. The mixtures were incubated at 4ûC overnight with rotation endover-end. The resins were centrifuged at 16,000 g for 10 seconds and the pellets washed three times with 1 ml of lysis buffer. The immunocomplexes were resuspended in 50 µl of 1x SDS loading buffer, boiled for 5 minutes and 15 µl separated in a 10% SDS-PAGE gel. Proteins were transferred to a nitrocellulose membrane and probed with anti-ha-peroxidase high affinity monoclonal antibody (Roche). The blot was stripped and re-probed with anti-avrpphb polyclonal antibody. Protein expression and purification Recombinant AvrPphB-His6 and GST-PBS1-His6 mutants were expressed in E. coli BL21(DE3). E. coli clones were grown in LB medium containing 100 µg/ml ampicillin to a density of OD 600 =0.6. Protein expression was induced overnight at room temperature with 0.4 mm isopropyl b-d-thiogalactopyranoside (IPTG). Cells were lysed in the buffer containing 20

4 mm Tris (ph 8.0), 200 mm NaCl, 20 mm imidazole, and 5% glycerol supplemented with 1 mm phenylmethylsulfonylfluoride (PMSF) and 10 mm ß-mercaptoethanol. Both AvrPphB-His6 and GST-PBS1-His6 proteins were purified by Ni + -agarose affinity chromatography and eluted with 250 mm imidazole in the same buffer. Protein concentrations were estimated by Coomassie blue staining of SDS-PAGE gels using BSA standards. PBS1 and RPS5 cleavage assays in mammalian cells and in vitro For cleavage assay in mammalian cells, 3 µg of pcdna3-pbs1-ha plasmids were cotransfected with 2 µg of pcdna3-avrpphb-flag (wild type or ) constructs into 6 x 10 6 HEK293T cells. The cells were lysed in 200 µl of SDS sample buffer, 15 µl of which were loaded onto a SDS-PAGE gel followed by anti-ha, or anti-au1, or anti-flag immuno-blot. For RPS5 cleavage experiments, 1 x 10 7 HEK293T cells were co-transfected with 6 µg of pef4- RPS5-myc constructs and 4 µg of pcdna3-avrpphb-flag (wild type or ) constructs. The cells were lysed in 1 ml of the buffer containing 10 mm HEPES (ph 7.5), 50 mm NaCl, 1% Triton, 2 mm EDTA and a protease inhibitor mixture. The cell lysates were then subjected to anti-myc immunoprecipitation followed by anti-myc Western blot analysis. For in vitro cleavage assays, the mammalian expression constructs for PBS1, At3g17410, At5g02800 and RPS5 described above were in vitro transcribed and translated in the presence of 35 S-methionine using the TnT wheat germ extract system (Promega) following the manufactures' protocol. 5 µl of the transcription/translation reactions were added as substrates into a 40 µl cleavage reaction containing 50 mm Tris-HCl, 50 mm NaCl, 4 mm dithiotreitol, and 2 µg of purified recombinant AvrPphB-His6 protein (wild type or ). The reaction was carried out

5 by incubation at 30ûC for 1 hour and stopped by the addition of SDS sample buffer. One fifth of the reaction mixture was analyzed by SDS-PAGE, followed by autoradiography. Cleavage assays on purified recombinant PBS1 proteins were performed under the same condition as that used for the TnT cleavage assay except that purified GST-PBS1(G252R)-His6 proteins were used as the substrates. Cleavage products were visualized on SDS PAGE gels by Coomassie Blue staining, as well as analyzed by anti-gst and anti-his immunoblotting. Edman sequencing of PBS1 cleavage product Protein samples were separated on an SDS-PAGE gel and transferred to a PVDF membrane (Bio Rad ) in CAPS buffer. Following Coomassie staining and extensive destaining in 50% methanol of the membrane, protein bands to be sequenced were excised and subjected to Edman sequencing performed by the Macromolecular Structure Core Facility in the Biochemistry Department at Michigan State University. Kinase assays The kinase activity of PBS1 variants was measured using an in vitro autophosphorylation assay. HEK 293T cells were transfected with equal amounts of various pcdna3-pbs1-ha constructs. Cells were lysed in a buffer containing 50 mm Tris (ph 8.0), 150 mm NaCl, 1% NP-40, and a protease inhibitor mixture 24 hours after transfection. The cell lysates were then subjected to anti-ha immunoprecipitation. To measure the kinase activity, the immunoprecipitated PBS1 proteins on the protein A agarose were incubated in an in vitro kinase assay buffer containing 50 mm Tris, ph 7.2, 10 mm MnCl 2, 1 mm dithiothreitol (DTT), 20 µm ATP, 1 mg/ml BSA and 5 µci (g- 32 P)-ATP (New England Nuclear). The reaction was incubated at room tempreture for 30

6 min and quenched with SDS loading buffer. One-third of the kinase reaction was electrophoresed on a 12% SDS/PAGE gel. Radioactivity was analyzed by autoradiography and the level of PBS1 protein was measured by anti-ha Western blot. We also performed kinase assays on recombinant PBS1 protein purified from E. coli as previously described (S3). Complementation assays The function of various pbs1 mutant alleles was assayed by stable transformation of pbs1-1 mutant plants. Genomic clones (described above) were transformed into Arabidopsis using the floral dip method (S7). Transformed plants were selected by spraying soil grown seedlings with the herbicide Finale (glufosinate-ammonium; Aventis). Selected plants (T1 generation) were grown under 9 hour days for four weeks and then assayed for resistance to Pseudomonas syringae strain DC3000(avrPphB) using a hypersensitive resistance (HR) assay. Briefly, bacteria were grown overnight on King's medium B agar plates, resuspended in 10 mm MgCl 2 at on OD 600 of 0.075, and injected into the underside of leaves using a needleless syringe. Injections were performed in the late afternoon and HR-associated collapse of leaves scored hours later. For the PBS1-GDK clone, eleven T1 plants were scored by injecting 3 to 6 leaves per plant (38 leaves total); none displayed an HR. For the wild-type PBS1 clone, four T1 plants were scored and all injected leaves displayed an HR (26 leaves total). For the PBS1(K115N) allele, 5 T1 plants were scored and none of the injected leaves displayed an HR (26 leaves total). Leaves were removed from plants for photography at 20 hours after inoculation.

7 2. Supporting Online Figures Figure S1. Autophosphorylation activity of recombinant PBS1, PBS1-GDK, and PBS1(K115N). Figure S2. AvrPphB induces cleavage of PBS1 in mammalian cells. Figure S3. Recombinant AvrPphB cleaves PBS1 produced in wheat germ extract. Figure S4. Identification of residues in PBS1 required for cleavage by AvrPphB. Figure S5. Model depicting possible mechanism by which AvrPphB triggers the resistance response in Arabidopsis.

8 Figure S1 PBS1 GDK K115N Fig. S1. Autophosphorylation activity of recombinant PBS1, PBS1-GDK, and PBS1(K115N). Approximately 250 ng of purified recombinant protein was incubated in a kinase reaction buffer with γ- 32 P-ATP, then separated on an SDS-PAGE gel and viewed by autoradiography.

9 Figure S2 (AU1)-PBS1-HA G/R G/R G/R AvrPphB ( AU1)-PBS1-HA 28 kda α-ha (AU1)-PBS1-HA 32 kda α-au1 α-gapdh Fig. S2. AvrPphB induces cleavage of PBS1 in mammalian cells. Plasmids encoding (AU1)-PBS1-HA (wild type or G252R mutant) were co-transfected with AvrPphB-Flag constructs (wild type or ) into HEK 293T cells. Total cell lysates were analyzed by anti-ha (upper panel) and anti-au1 (lower panel) immunoblot. Total protein loading was controlled by analysis of the same gel using anti-gapdh antibody.

10 Figure S3 PBS1 G252R AvrPphB Input Input PBS1 Nonspecific 32 kda 28 kda Fig. S3. Recombinant AvrPphB cleaves PBS1 produced in wheat germ extract. PBS1 (wild type or the pbs1-2 allele G252R) was in vitro transcribed/translated in a wheat germ extract system containing 35 S-methionine. The labeled proteins were then incubated with purified recombinant AvrPphB proteins, and samples analyzed by autoradiography of SDS-PAGE gels.

11 Figure S4 PBS1 I219A L221A G241A D242A K243A AvrPphB Fig. S4. Identification of residues in PBS1 required for cleavage by AvrPphB. The indicated PBS1 variants were in vitro transcribed/translated in a wheat germ extract containing 35 S-methionine, and the labeled proteins used as substrates for cleavage by recombinant AvrPphB. Arrows indicate the cleavage products. Mutants I219A and L221A blocked cleavage and eliminated kinase activity (not shown), possibly indicating gross structural changes. All other mutants retained kinase activity.

12 Figure S5 Fig. S5. Model depicting possible mechanism by which AvrPphB triggers the resistance response in Arabidopsis. The AvrPphB protease cleaves the auto-phosphorylated PBS1 protein, and a PBS1 cleavage product binds to and activates the RPS5 protein. All three proteins are shown as being associated with a membrane because all contain putative myristoylation motifs and pellet with the membrane fraction during high-speed centrifugation (data not shown).

13 3. Supporting Online References S1. F. Shao, P. M. Merritt, Z. Bao, R. W. Innes, J. E. Dixon, Cell 109, 575 (2002). S2. M. T. Simonich, R. W. Innes, Mol Plant Microbe Interact 8, 637 (1995). S3. M. R. Swiderski, R. W. Innes, Plant J 26, 101 (2001). S4. T. Aoyama, N.-H. Chua, Plant J. 11, 605 (1997). S5. R. P. Hellens, E. A. Edwards, N. R. Leyland, S. Bean, P. M. Mullineaux, Plant Mol Biol 42, 819 (2000). S6. Z. Nimchuk et al., Cell 101, 353 (2000). S7. S. J. Clough, A. F. Bent, Plant J 16, 735 (1998).