Quantification of Ethylene Production in Tomato Leaves Infected by Xanthomonas euvesicatoria

Materials and Reagents

1. 1% Ethylene gas can (SCOTT Specialty Gases, catalog number: 01-04-855 )
   
2. 1 L Tedlar^® PLV Gas Sampling Bag w/Thermogreen^® LB-2 Septa (Sigma-Aldrich, Supelco, catalog number: 24633 )
   
3. 1.5 ml Microtubes (Corning, Axygen^®, catalog number: MTC-150-C )
   
4. 1 ml Syringes Without Needles (BD, catalog number: 309659 )
   
5. 25 G Needle (BD, catalog number: 305122 )
   
6. 15 ml Polypropylene Centrifuge Tubes (Greiner Bio-One GmbH, catalog number: 188271 )
   
7. 16 mm diameter x 100 mm Glass tubes (VWR International, catalog number: 47729-576 )
   
8. 18 mm diameter x 150 mm Glass tubes (VWR International, catalog number: 47729-583 )
   
9. Suba-Seal^® septa (Sigma-Aldrich, catalog number: Z124613 )
   
10. Tomato plants (4-5 week-old) were grown in a greenhouse or growth chamber (16 h light, 25~28 °C)
    
11. Xanthomonas euvesicatoria (Xcv) strain 85-10 wild type and mutants (for example, type III effector deletion mutants)
    Note: Wild type strain is available upon request.
    
12. Magnesium chloride hexahydrate (Sigma-Aldrich, catalog number: M2393 )
    
13. Peptone (BD, catalog number: 211677 )
    
14. Yeast extract (BD, catalog number: 212750 )
    
15. Agar (BD, catalog number: 214530 )
    
16. Glycerol (Certified ACS) (Fisher Chemical, catalog number: G334 )
    
17. Sodium hydroxide (Sigma-Aldrich, catalog number: 221465 )
    
18. Distilled water
    
19. NYGA medium (see Recipes)
    
20. 10 mM MgCl₂ (see Recipes)
    

Equipment

1. 28 ºC Incubator (VWR International, catalog number: 414005-128 )
   
2. Vortexer (Scientific Indrustries, model: Vortex-Genie 2 )
   
3. Spectrophotometer (Amersham Biosciences, model: Ultrospec 3100 Pro )
   
4. Gas chromatograph (GC) (Shimadzu Corporation, model: GC-8A )
   

Procedure