Spheroplast Preparation and Protease Assay

Spheroplast preparation and protease susceptibility assay were performed as described [22] (link) with modifications. A. tumefaciens cells were grown in liquid AB-MES medium (pH 5.5) [56] (link) at 25°C for 6 hr. Cells were harvested by centrifugation at 10,000× g for 15 min at 4°C and washed once with 50 mM Tris-HCl (pH 7.5). To prepare spheroplasts, cell pellets were resuspended gently in buffer containing 50 mM Tris-HCl (pH 7.5), 20% sucrose, 2 mM EDTA, 0.2 mM DTT, and 1 mg/ml lysozyme and incubated on ice for 1 hr. After lysozyme treatment, spheroplasts were treated with Streptomyces griseus protease (Sigma) at a final concentration of 12.5 or 25 µg/ml for 10 min on ice in the presence of 10 mM MgSO₄. The reaction was stopped by adding an equal amount of 2× SDS loading buffer and incubated at 96°C for 20 min before SDS-PAGE.