Fig. 4

AvrPtoB mediates CYP707A1 degradation by 26S proteasome. a AvrPtoB targets CYP707A1 for degradation. N. benthamiana leaves were co-infiltrated with 35S:CYP707A1-T7 and Dex:AvrPtoB-FLAG. Plants were infiltrated with 3 μM Dex at 48 hpi to induce the expression of AvrPtoB-FLAG. Leaf extracts were sampled for immunoblotting after mock or Dex treatment at indicated times. Mock is 10 mM MgCl₂. b The proteasome inhibitor MG132 prevents CYP707A1 from degradation. 35S:CYP707A1-T7 was transiently expressed with 35S:GFP-FLAG or 35S:AvrPtoB-FLAG in N. benthamiana. MG132 (100 μM) was used to inhibit 26S proteasome-mediated protein degradation at 36 hpi. Samples were harvested at 8 h after MG132 treatment for immunoblotting. c Pst harbored AvrPtoB degrades CYP707A1. 35S:CYP707A1-T7 stable transgenic plants were inoculated with Mock (10 mM MgCl₂), Pst or Pst (ΔavrPtoB) at a concentration of 2.5 × 10³ CFU/mL, respectively. Infected leaves were sampled for immunoblotting at 12 hpi. d AvrPtoB ubiquitinates CYP707A1 in vivo. 35S:CYP707A1-FLAG was transiently expressed with 35S:AvrPtoB-T7 or 35S:EV-T7 in N. benthamiana. MG132 (100 μM) was used to inhibit 26S proteasome-mediated protein degradation at 36 hpi. Samples were harvested at 8 h after MG132 treatment. Plant leaves were immunoprecipitated with anti-FLAG beads, and the proteins were immunoblotted by anti-FLAG and anti-T7 antibodies