Flagellin fragmentation by SBT5.2-H6 or SBT1.7-H6

Aliquots (200 µL) of apoplastic fluids from agroinfiltrated leaves were diluted in buffer A (20 mM sodium phosphate buffer (pH 7.4), 500 mM NaCl, and 10 mM imidazole in MilliQ water) to a volume of 500 µL. Each sample then was loaded onto a Ni-Sepharose-packed-tip column equilibrated with 1 mL of Buffer A. The tip columns were prepared as follows: A chelating Sepharose-packed column (HiTrap chelating HP 1 mL; Cytiva) was washed with 5 mL of water, and 0.5 mL of water containing 0.1 M NiSO₄ was loaded onto the column. After washing with 5 mL of water, the column was equilibrated with 10 mL of buffer A. Ni-Sepharose beads were recovered from the dismantled column and suspended in 1.5 mL of Buffer A. To prepare protease-immobilized beads, a small amount of cotton was packed into a 200-µL micropipette tip, onto which 50 µL of suspended Ni-Sepharose beads were loaded. These micropipette-tip columns were loaded with the apoplast samples, then washed with 1 mL of Buffer A, and the Ni-Sepharose beads carrying immobilized proteases were collected from the micropipette-tip columns. Beads harboring immobilized SBT5.2-H₆, SBT1.7-H₆ or SAP2-H₆ were used for the proteolysis assay. The assays were performed in a 200-µL reaction mixture containing 25 mM MES-KOH (pH 5.7), 2 µM flagellin or 500 nM synthetic peptides, and 10 µL of protease immobilized beads. The reaction mixtures were rotated for 1 h at room temperature. Reactions were terminated by the addition of 1/10 volume of 10% TFA. For flagellin proteolysis assay, aliquots (50 μL) of the quenched reaction mixtures were desalted using a GL-Tip SDB (GL Science) according to the manufacturer’s instructions. After vacuum concentration, the samples were dissolved in 20 µL of 2% acetonitrile (containing 0.1% TFA) and 3.33-µL aliquots were analyzed by nano-LC-MS/MS. For peptide proteolysis assay, aliquots (5 μL) of the quenched reaction mixtures then were analyzed by nano-LC-MS/MS.