Fig. 2

SDE1 inhibits PLCP activity in vitro and in plant cells. a Proteolytic activity of papain measured by digestion of a fluorescent casein substrate in the presence of E-64, purified SDE1 protein, or BSA (as a negative control). Fluorescence was measured at 485/530 nm excitation/emission. Mean ± standard deviation (n = 3) is shown. Asterisks (*) indicate statistically significant differences based on the two-tailed Student’s t-test. p < 0.01 = **, p < 0.001 = ***. b Inhibitory effect of SDE1 on the protease activity of papain examined by activity-based protein profiling (ABPP). Active papain was labeled by DCG-04 in the presence of 10 μM E-64 or 1.6 μM purified SDE1 protein and detected using streptavidin conjugated with horseradish peroxidase (HRP). c SDE1 inhibits the activity of CsRD21a. CsRD21a-Flag (with its N-terminal secretion signal) was expressed in N. benthamiana. Active protease in the apoplastic fluid was labeled via ABPP. ImageJ analysis of the signal intensity revealed approximately 9%, 62%, and 96% reduction of CsRD21a activity in the presence of 0.8, 1.6, or 3.2 μM purified SDE1 protein, respectively. d SDE1 inhibits PLCP activity in citrus. Total protein extracts from Navel orange (C. sinensis) leaves were labeled via ABPP in the presence of 120 nM purified SDE1 protein. Active proteases were enriched using streptavidin beads and detected using streptavidin-HRP conjugates. e Transgenic grapefruit (Duncan) seedlings expressing SDE1 exhibit reduced protease activity. Five individual lines were analyzed by ABPP. SDE1-10 does not have significant SDE1 protein accumulation and served as a negative control