Transient expression in N. benthamiana and extraction of SBT3.8

1. Plate agrobacteria (A. tumefaciens C58C1) containing the subtilase (SBT3.8) expression construct in pART27 or the P19 silencing suppressor on LB plates with appropriate antibiotics  (rifampicin, tetracyclin and spectinomycin for C58C1/pART27; kanamycin instead of spectinomycin for C58C1/p19). 
2. Grow for 2 days at 28 °C.
3. Add 10 ml infiltration buffer to resuspend the colonies and wash them off the plates.
4. Mix the bacterial suspensions at an OD₆₀₀ ratio of 0.7:0.1 for C58C1/pART27:C58C1/p19. 
5. Add acetosyringone to a final concentration of 150 µM and incubate at room temperature for 3 to 4 hrs.
6. Use a 1 ml plastic syringe without needle to gently infiltrate the bacterial suspension into the abaxial side of N. benthamiana leaves. Use six-week-old plants grown at 25 °C with 12 hrs photoperiod for agro-infiltration.
7. Harvest infiltrated leaves and cut into pieces of 3-5 cm².
8. Wash three times in ice cold _ddH₂O.
9. Transfer plant material into 200 ml ice cold extraction buffer and infiltrate twice for 1 min at 70 mbar in a desiccator. 
10. Carefully blot the leaf pieces dry, be careful not to damage them. Place the leaf pieces into a syringe barrel plugged with glass wool.
11. Place the syringe barrel into a 50 ml Falcon tube. 
12. Centrifuge for 7 min at 1.500 g and 4 °C.
13. Collect the wash fluid and spin for 10 min at 15.000 g and 4 °C to remove leaf debris.
14. Collect the supernatant containing the apoplastic proteins and store at -20 °C.

Solutions:

• 150 mM acetosyringone in DMSO
• infiltration buffer: 10 mM MES pH 5.6, 10 mM MgCl₂
• extraction buffer: 50 mM NaH₂PO₄/Na₂HPO₄  pH 7.0, 300 mM NaCl