Fig. 5. Characterization of the enzyme specific for CAPE production (ESCAPE) by CNYDase activity and interaction with PR1-eYFP.

a Gel blot of biotinylated proteins from Arabidopsis wild-type (WT) and xcp1 extracts with or without the addition of biotin–CNYD–CHO. The biotinylated proteins were detected with streptavidin–HRP. Coomassie Blue staining shows total protein loaded. b Proteolytic activity of purified XCP1-His on three fluorogenic protease substrates (CNYD, CNAD, and ANAD) with or without adding biotin–CNYD–CHO. The proteolytic activity of purified XCP1-His was measured by RFU of the cleaved fluorophore after 10 h incubation with the substrate. c CNYDase activity of purified XCP1-His supplemented with ZnCl₂, MgCl₂, CaCl₂, E-64, PMSF, or EDTA for 1 h before substrate incubation. RFU of the cleaved fluorophore was measured after 10 h incubation with the substrate. d The CNYDase activity-pH profile of purified XCP1-His, with or without adding CaCl₂. RFU of the cleaved fluorophore was measured after 10 h incubation with the substrate. e The enzyme kinetics of purified XCP1-His for CNYD substrate. The V_max and K_m of XCP1-His proteolytic activity were determined by 10 h incubation of 0.2 µg purified XCP1-His with different concentrations of CNYD substrate at 22 °C. f Immunoblots of the protein extract from Arabidopsis PR1-eYFP transgenic plants with the addition of the Ni-NTA purified proteins from the Nicotiana benthamiana overexpressing P19, P19 plus XCP1-His or P19 plus XCP1^C161A-His gene. PR1-eYFP was detected with anti-GFP antibodies. Coomassie Blue staining shows total protein loaded. g Immunoblot of the immobilized PR1-eYFP with the addition of the Ni-NTA purified proteins from the Nicotiana benthamiana overexpressing P19, P19 plus XCP1-His or P19 plus XCP1^C161A-His gene. The protein extract from Arabidopsis PR1-eYFP transgenic plants was immunoprecipitated with anti-PR1 (IP: anti-PR1) before incubation with XCP1-His. a, f, g Experiments were repeated three times with similar results. b–d Each proteolytic activity assay for purified XCP1-His was performed by incubating 1 µg protein with 25 µM substrate. f, g the sizes of intact PR1-eYFP and AtCAPE9-eYFP were estimated to be ~44.7 and ~27.0 kDa, respectively, detected by the anti-GFP. The Coomassie Blue staining shows total protein loaded. b–e Values are means ± SD of n = 3 independent experiments. Each experimental result was obtained by using five Nicotiana benthamiana plants overexpressing XCP1-His transiently. P values were calculated by one-tailed unpaired t-test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).