Protease activity assays using fluorogenic substrates

Activity of PLCPs present in isolated apoplastic fluids was monitored using 10 µM final concentration of the fluorogenic substrate Z-Phe-Arg-7-amido-4-methylcoumarin (Sigma-Aldrich, Taufkirchen, Germany). AMC released (RFU) over time was monitored at 460 nm using a Tecan Infinite M200 PRO plate reader (Tecan Group Ltd., Männedorf, Switzerland). As a control apoplastic fluids were preincubated with 10 µM E-64 (Sigma-Aldrich, Taufkirchen, Germany) for 10 min. The inhibitor control was used to normalize all measurements and obtain a read-out for specific PLCP activity. Percentage of PLCP activity was calculated by setting up the sample without inhibitor to 100% activity. Isolation and ion-exchange chromatography fractionation of apoplastic fluids was performed according to the method published by ref. ¹⁶. UmPID14, StrepPID14, and UhPID14 were obtained as synthetic peptides with >98% purity from the company GenScript (New Jersey, USA). cMIP was obtained as synthetic peptide with >98% purity from the company Biomatik (Wilmington, Delaware, USA). Peptides were solubilized in water to a desired concentration. For papain activity measurements, 1 mg/ml papain from Carica papaya (Merck Millipore, Burlington, Massachusetts, USA) was activated at room temperature for 30 min in 60 mM sodium acetate pH 6 containing 10 mM DTT. 1.25 µg/ml final concentration was used for the experiments. 10 µM final concentration of the fluorogenic substrate Z-Phe-Arg-AMC (Sigma-Aldrich, Taufkirchen, Germany) was used to monitor the activity of papain as described before for apoplastic fluids. Percentage of papain activity was calculated by setting up the sample without inhibitor to 100% activity. Percentage of papain inhibition was plotted against the inhibitor concentration. The activity of recombinant CP1 and CP2 proteins was monitored using 10 µM final concentration of the fluorogenic substrate Z-Leu-Arg-AMC (Sigma-Aldrich, Taufkirchen, Germany).