Figure 7.

Interaction with 14-3-3 proteins increases HopQ1 stability in plants. A, The presence of Ser-51 affects HopQ1 steady-state level in planta. Wild-type HopQ1 or HopQ1-S51A proteins carrying C-terminal 3xHA epitopes were transiently coexpressed with GFP in N. benthamiana leaves. Crude protein extracts (20 μg of proteins per lane) were isolated from leaves 48 h after agroinfiltration. HopQ1 variants were detected by immunoblot analysis using an antibody specific to HA. As an expression control, the level of GFP was checked using anti-GFP antibody. The experiment was repeated twice. B, Modification of the 14-3-3 binding motif does not change HopQ1 stability in P. syringae. C-terminally His-tagged HopQ1 variants were expressed in P. syringae pv tabaci DAPP-PG677. Crude protein extracts prepared from overnight bacterial cultures were fractionated on 12.5% SDS-PAGE and subjected to immunoblot analysis using a specific anti-His antibody. Equal protein loading is shown by Ponceau Red staining. The experiment was repeated twice. C, R18, the inhibitor of 14-3-3 binding, affects HopQ1 stability in plant extracts. Wild-type HopQ1 protein carrying a C-terminal Flag epitope was transiently expressed in N. benthamiana leaves. Crude protein extract was supplemented with recombinant 14-3-3a-Strep II protein isolated from E. coli or R18 peptide at various concentrations, as indicated. HopQ1 was detected by immunoblot analysis using specific primary antibodies. The level of 14-3-3a was monitored using Strep-Tactin AP conjugate. Equal protein loading is shown by Ponceau Red staining. D, Application of 14-3-3a reverts the effect of R18 on HopQ1 stability. Wild-type HopQ1 protein carrying a C-terminal Flag epitope was transiently expressed in N. benthamiana leaves. Crude protein extract was supplemented with R18 and increasing amounts of recombinant 14-3-3a-Strep II protein isolated from E. coli, as indicated. HopQ1 was detected by immunoblot analysis using specific primary antibodies. The level of 14-3-3a was monitored using Strep-Tactin AP conjugate. Equal protein loading is shown by Ponceau Red staining. E, Assembled in vitro complex of HopQ1-6xHis and 14-3-3a-Strep II was incubated with bean crude protein extract in the absence or presence of R18. HopQ1 and 14-3-3a were detected by immunoblot analyses using anti-His antibodies or Strep-Tactin AP conjugate, respectively. Equal protein loading is shown by Ponceau Red staining. F, In vitro phosphorylated HopQ1-6xHis was added to bean crude extract and incubated in the presence of 14-3-3a or BSA. HopQ1 and 14-3-3a were detected by immunoblot analysis using anti-His antibodies or Strep-Tactin AP conjugate, respectively. Equal protein loading is shown by Ponceau Red staining.