An Assay to Test the Capacity of Arabidopsis Plant Defensin Type1 Protein to Induce Cellular Zinc (Zn) Tolerance in Yeast

Materials and Reagents

1. Plastic foil (Saran Wrap)
   
2. Sterile square 12 cm x 12 cm Petri dishes (CML HealthCare, catalog number: BP 120SJ )
   
3. Sterile-R Filtropur S (0.2 µm) (SARSTEDT AG, catalog number: 83.1826.001 )
   
4. 20 ml sterile syringe without needle (Medline Scientific, catalog number: BS-20ES )
   
5. 50 ml sterile tubes (SARSTEDT AG, catalog number: 62.547.254 )
   
6. Autoclaved 1.5 ml Eppendorf tubes (SARSTEDT AG, catalog number: 72.690 )
   
7. Sterile inoculating loops (Greiner Bio One International GmbH, catalog number: 731175 )
   
8. Sterile pipet tips
   
9. Parental BY4741 S. cerevisiae strain (Winston et al., 1995; Brachmann et al., 1998) having the genotypes MATa, his3∆1, leu2∆0, met15∆0, ura3∆0) expressing: the pFL38H vector (Bonneaud et al., 1991), complementing the his3∆1 auxotrophy
   
10. Transformed BY4741 yeast harbouring both: the pFL38H vector, complementing the his3∆1 auxotrophy and the pYX212 vector (Ingenius, R&D systems) complementing the ura3∆0 mutation and expressing the entire coding sequences of Arabidopsis PDF1s, i.e., including the signal peptide (Shahzad et al., 2013)
    
11. Yeast nitrogen base (YNB) without amino acids and without ammonium sulfate (BD, Difco, catalog number: 233520 )
    
12. Ammonium nitrate (NH₄NO₃) (Sigma-Aldrich, catalog number: A9642 )
    
13. D(+)Glucose anhydrous (C₆H₁₂O₆) (EUROMEDEX, catalog number: UG3050 )
    
14. Succinic acid (C₄H₆O₄) (Sigma-Aldrich, catalog number: S7501 )
    
15. Potassium hydroxide (KOH) (Bio Basic Canada Inc., catalog number: PB0441 )
    
16. Agarose type D-5 (EUROMEDEX, catalog number: D5 )
    
17. Zinc sulphate heptahydrate (ZnSO₄^.7H₂O) (Sigma-Aldrich, catalog number: Z4750 )
    
18. L-leucine (C₆H₁₃NO₂) (Sigma-Aldrich, catalog number: L8912 )
    
19. L-methionine (C₅H₁₁NO₂S) (Sigma-Aldrich, catalog number: M9625 )
    
20. Sterile ultra-pure water
    
21. 0.5 M zinc sulfate
22. 10 N KOH (see Recipes)
    
23. 0.075 M L-leucine (see Recipes)
    
24. 0.1 M L-methionine (see Recipes)
    
25. Sterile liquid and solid selective yeast nitrogen base (YNB) media (see Recipes)
    

Equipment

1. Sterile 250 ml borosilicate glass Erlen flask
   
2. 1,000 ml bottle
   
3. Typical 20 µl pipet
   
4. Autoclave
   
5. Heating oven (Universal Memmert, model: 400 )
   
6. New Brunswick^TM Agitator (Eppendorf, model: Innova 44 )
   
7. A normal cell density meter (Thermo Fisher Scientific, model: 40 )
   
8. Laminar flow hood (ADS, model: VP120 )
   
9. Centrifuge (Eppendorf, model: 5804R )
   
10. Integral water purification system (Merck Millipore Corporation, model: Milli-Q+ )
    
11. Scanner (Epson, model: 124OU )
    

Procedure