Fig. 3.
Secreted aspartic protease 1 (SAP1) and SAP2 cleave MucD. a, c Protease activity of purified GFP (green fluorescent protein), SAP1, SAP2 (a), and SAP1D63/136A (c) protein. PepA, the aspartic protease inhibitor, pepstatin A. Data represent means and s.e.m of three biological replicates. Asterisks indicate significant differences (Student’s two-tailed t test; **P < 0.01). b, d In vitro growth of Pto (OD600 = 0.05) in minimal medium supplemented with purified recombinant proteins or boiled (b) recombinant proteins at 9 h after culturing. Data represent means and s.e.m. of four biological replicates. Asterisks indicate significant differences (Student’s two-tailed t ; *P < 0.05, **P < 0.01). e Proteins extracted from bacterial culture medium incubated with SAP1, boiled SAP1 (SAP1(b)), or without SAP were visualized (Coomassie Brilliant Blue (CBB) staining). f MucD-His incubated with SAP1-GST, SAP1D63/136A-GST, and SAP2-GST were visualized by immunoblotting using an anti-GST or anti-His antibody. Three independent experiments were performed with similar results. g, h Pto ΔmucD expressing MucD-HA (hemagglutinin) (OD600 = 0.05) was infiltrated into leaves of 4-week-old Col, pUB::SAP1-RFP, and sap1 sap2 plants. MucD-HA in total protein at the indicated time points was detected by immunoblotting using an anti-HA antibody. CBB staining serves as protein loading control. Experiments were repeated at least three times with similar results