Materials and Reagents

1. Plant material
   Soybean (Glycine max Merr.) plants of V2 (2^nd trifoliate), V3 (3^rd trifoliate), or higher stages of growth were used. Plants at VC (two leaf stage)/V1 (1^st trifoliate) stages can also be used. Cultivars for various R loci: Rpg1-b (Harosoy), Rpg2 (Merit, Norchief), Rpg3 (Flambeau), Rpg4 (Flambeau), rpg (Essex).
   
   
2. Bacterial strains
   
   1. P. syringae pv. glycinea expressing avr gene of interest via the broad host range plasmids pDSK519 or pDSK600.
      
   2. P. syringae pv. glycinea expressing empty pDSK519/600 plasmids as control.
   
   
3. Media and buffers
   
   1. 10 mM MgCl₂ (sterile)
      
   2. King’s B medium (plates and liquid media) (see Recipes)
      
   
   
4. Other reagents
   
   1. Tryptone (Teknova, catalog number: T9012 )
      
   2. K₂HPO₄ (Fisher BioReagents, catalog number: BP363 )
      
   3. Glycerol (Affymetrix, catalog number: 16374 )
      
   4. Agar (Affymetrix, catalog number: 10654 )
      
   5. Silwet L-77 (Momentive, New Smyrna Beach)
      
   6. Antibiotics (Gold Biotechnology)
      
      1. Rifampicin (50 mg/ml) (R-120)
         
      2. Kanamycin (50 mg/ml) (K-120)
         
      3. Spectinomycin (100 mg/ml) (S-140)
         
      4. Sreptomycin (300 mg/ml) (S150)

Equipment

1. High-speed floor centrifuge (Thermo Fisher Scientific, Sorvall RC 6 plus)
   
2. Spectrophotometer (Thermo Fisher Scientific, model: BioMate 5 )
   
3. Pressure pump (Gast Manufacturing, model: DOA-P704-AA )
   
4. Airbrush (Badger Air-brush, model: 250-2 )
   
5. Test tubes (Thermo Fisher Scientific)
   
6. Microcentrifuge tubes (Thermo Fisher Scientific)
   
7. Glass rods (Thermo Fisher Scientific)
   
8. Pellet pestles (Sigma-Aldrich)
   
9. Cork borer (1 cm diameter, Thermo Fisher Scientific)
   

Procedure