Materials and Reagents

1. Ralstonia solanacearum UY031 strain transformed with the reporter plasmid pRCG-Ppslux, which bears the chloroplast promoter PpsbA and the entire LuxCDABE operon (Figures 1 and 2)
   Note: Plasmid and strain available under material transfer agreement (MTA) from Marc Valls’ laboratory.
   
2. Four-week-old Solanum tuberosum (S. tuberosum) and Solanum commersonii (S. commersonii) plants
   Note: They were grown in a TREF universal potting soil mix, in a greenhouse, with 12 h light and temperatures maintained between 22 to 25 °C (50 to 60% relative humidity -RH) for three weeks and then transferred for an additional week into a growth chamber at 27 °C and 65% RH with a photoperiod of 12 h of light.
   
3. Gentamicin (75 µg/ml in solid and 5 µg/ml in liquid cultures) (Sigma-Aldrich, catalog number: G1264 )
   
4. Bacto-peptone (Bacto^TM, catalog number: 211677 )
   
5. Yeast extract (Bacto^TM, catalog number: 210929 )
   
6. Tryptone (Bacto^TM, catalog number: 211699 )
   
7. Casamino acids (Bacto^TM, catalog number: 223050 )
   
8. Dextrose Glucose (Difco^TM, catalog number: 215510 )
   
9. Bacto-agar (Bacto^TM, catalog number: 214030 )
   
10. Triphenyltetrazolium chloride (TTC) (Difco^TM, catalog number: 231121 )
    
11. Sodium phosphate dibasic heptahydrated (Na₂HPO_4^.7H₂O) (Sigma-Aldrich, catalog number: S9390 )
    
12. Potassium phosphate (KH₂PO₄) (Sigma-Aldrich, catalog number: P5655 )
    
13. Sodium chloride (NaCl) (VWR International, catalog number: 443824T )
    
14. Ammonium chloride (NH₄Cl) (VWR International, catalog number: 0621 )
    
15. Magnesium sulfate (MgSO₄) (VWR International, catalog number: 0338 )
    
16. Calcium chloride (CaCl₂) (VWR International, catalog number: 1.02391.1000 )
    
17. L-glutamate (Sigma-Aldrich, catalog number: G1626-100G )
    
18. Rich B medium (see Recipes)
    
19. Boucher’s minimal medium (MM) (see Recipes)
    
20. Luria and Bertani broth (LB) (see Recipes)
    
21. 1 M magnesium sulfate (MgSO₄) (see Recipes)
    
22. 20% glucose (see Recipes)
    
23. 1 M calcium chloride (see Recipes)
    
24. 20 mM L-glutamate (see Recipes)
    
25. To prepare 1 L of 1x MM (see Recipes)
    

Equipment

1. LAS4000 Chemiluminescence and Fluorescence Imaging System (Fujifilm Life Science)
   
2. Luminometer Berthold FB 12 (Titertek-Berthold, catalog number: 11010102 )
   
3. Spectrophotometer (Shimadzu Kyoto, model: UV-1603 visible spectrophotometer)
   
4. Incubator that can be set at 30 °C containing shaker for Erlenmeyer and tubes
   
5. Tabletop centrifuge (Eppendorf, model: 5418R )
   
6. Culture tubes (overflow volume 22 ml) (VWR International, catalog number: 47729-580 )
   
7. Sterile 150 ml flasks
   
8. 2 ml Eppendorf tubes
   
9. Razor blade
   
   
   Figure 1. Vector map of pRCG-Pps-lux, designed to generate the luminescent strain for detection of bacterial colonization and growth in planta
   
   
   Figure 2. Visualization of Ralstonia solanacearum transformed with the pRCG-Pps-lux plasmid under white light A or chemiluminiscence B using the LAS4000 light imager
   

Procedure