Materials and Reagents

1. 6-leaf-stage Nicotiana benthamiana plants 
   Note: Nicotiana benthamiana can be obtained from our lab (Figure 1).
   
   
   Figure 1. 6-leaf-stage Nicotiana benthamiana plant
   
   
2. Bacteria strains 
   a.  Escherichia coli strains, such as DH5α 
   b.  Agrobacterium strains, such as GV3101
   Note: All strains were obtained from our lab.
3. pTRV1 and pTRV2-LIC based expression vectors (Dong et al., 2007)
   1. pTRV1: a T-DNA vector containing duplicated CaMV 35S promoter, NOS terminator and cDNA clone of TRV RNA1 of Ppk20 strain.
      
   2. pTRV2-LIC: a T-DNA vector containing  duplicated CaMV 35S promoter,  NOS terminator and cDNA clone of TRV RNA2, of which non-structural genes were replaced by a multiple cloning site (MCS).
      
   3. pTRV2-NbPDS: gene fragement of NbPDS was inserted at MCS into pTRV2-LIC. This construct was usually used as a control to show the successful gene silencing.
      
   4. pTRV1 (ABRC, catalog number: CD3-1039 ) and pTRV2-LIC (ABRC, catalog number: CD3-1042 ) could be ordered at  http://www.arabidopsis.org/abrc/catalog/vector_3.html.
      
4. Media for Agrobacteria  
   1. Liquid Luria-Bertani (LB) medium
      
   2. Solid LB plates with 0.12% agar
      Note: LB medium is autoclaved under 120 °C for 20 min.
5. Antibiotics 
   a.  Kanamycin
   b.  Rifampicin
   c.  Gentamicin
   
6. Easy Taq DNA polymerase (Beijing TransGen Biotech)
   
7. dNTP (Roche)  
   
8. TIANprep Mini Plasmid Kit (Beijing TransGen Biotech)
   
9. MgCl₂ (Sigma-Aldrich)
   
10. MES (AMRESCO)
    
11. Acetosyringone (Sigma-Aldrich)
    
12. DMSO (AMRESCO)
    
13. Infiltration buffer (see Recipes)
    

Equipment

1. Centrifuge tubes
   
2. Plant growth chamber (24 °C, 16 h photoperiod conditions, 50% huminity)
   
3. Sterile 1 ml syringe without needle
   
4. Sterile bacterial culture tubes
   
5. Centrifuge
   
6. PCR instrument
   
7. 37 °C and 28 °C incubators with shaking
   

Procedure