Fig. 2
Secreted aspartic protease 1 (SAP1) and SAP2 redundantly contribute to resistance against P. syringae. a Leaves of 4-week-old Col, sap1, sap2, and sap1 sap2 double mutant plants were infiltrated with Pto (OD600 = 0.0005), and bacterial titer was determined at 3 days post infection (dpi). Bars represent means and s.e.m. of four independent experiments with three biological replicates. Asterisks indicate significant differences from Col (Student’s two-tailed t test; *P < 0.05, **P < 0.01). b Leaves of 4-week-old pUB::SAP1-RFP (red fluorescent protein) and SAP2-RFP with or without signal peptide (ΔSP) were infiltrated with Pto (OD600 = 0.001), and bacterial titer was measured at 2 dpi. Bars represent means and s.e.m. of three independent experiments with at least three biological replicates. Asterisks indicate significant differences from Col (Student’s two-tailed t test; *P < 0.05, **P < 0.01). Protein expression determined by immunoblotting using an anti-RFP antibody. Ponceau S staining serves as protein loading control. c, i Relative expression of PR1 in leaves of 4-week-old Col, pUB::SAP1-RFP, pUB::SAP1ΔSP-RFP, pUB::SAP2-RFP, pUB::SAP2ΔSP-RFP, and pUB::SAP1D63/136A plants were determined by quantitative reverse transcription PCR (RT-qPCR). Bars represent means and s.e.m of three biological replicates. The vertical axis shows the log2 expression levels relative to Actin2. Col plants 24 h post infiltration (hpi) with flg22 serves as a positive control for activated PR1 expression. Asterisks indicate significant differences from Col (Student’s two-tailed t test; *P < 0.05, **P < 0.01). d, j Phenotype of 4-week-old leaves of Col, pUB::SAP1-RFP and pUB::SAP2-RFP, and pUB::SAP1D63/136A plants. Shoot fresh weight (4-week-old; e) and silique numbers (f) of Col, pUB::SAP1-RFP, pUB::SAP1ΔSP-RFP, pUB::SAP1D63/136A-RFP, pUB::SAP2-RFP, and pUB::SAP2ΔSP-RFP plants. Bars represent means and s.e.m. of three independent experiments each with four biological replicates (e) or means and s.e.m. of three independent experiments (f). Statistically significant differences are indicated by different letters (adjusted P < 0.01). g Protein expression of SAP1 and SAP1D63/136A in Col, pUB::SAP1-RFP and pUB::SAP1D63/136A-RFP plants by immunoblotting using an anti-RFP antibody. Rubisco large subunit (RbCL) serves as a loading control. Three independent experiments were performed with similar results. h Leaves of 4-week-old Col and pUB::SAP1D63/136A plants were infiltrated with Pto (OD600 = 0.001), and bacterial titer was measured at 2 dpi. Bars represent means and s.e.m, of four independent experiments with four biological replicates. n.s., not significant
