Materials and Reagents

1. Centrifuge tubes
   
2. Sterile 1 ml syringe (needle removed)
   
3. Sterile bacterial culture tubes
   
4. Plant materials
   Nicotiana benthamiana, tomato (cultivar Moneymaker)
   Note: seeds can be obtained from Yule Liu’s lab.
   
5. Bacteria strains
   
   1. Escherichia coli: DH5α, ccdb survival (Thermo Fisher Scientific, Invitrogen^TM, catalog number: A10460 )
      
   2. Agrobacterium tumefaciens: GV3101, GV2260 (alternative to GV3101)
      Note: All strains can be obtained from Yule Liu’s lab.
6. Plasmids
   
   1. pTRV1 (Dong et al., 2007): a T-DNA vector containing 2 x 35 s promoter, Nos terminator and full cDNA of TRV RNA1 (from Ppk20 strain).
      
   2.  pTRV2e (Sha et al., 2014): a T-DNA vector containing 2 x 35 s promoter, Nos terminator and cDNA clone of TRV RNA2, of which the sub-genomic promoter of coat protein from Pea early brown virus (PEBV) (Wang et al., 1997) and a ligation independent cloning (LIC) cassette are inserted immediately downstream of the TRV CP gene.
      
   3. pTRV2e-GFP: GFP gene was inserted at LIC cassette into pTRV2e. This construct can be used in a control assay to show successful exogenous expression.
      
   4. The pTRV1 (Arabidopsis, ABRC, catalog number: CD3-1039 ) and pTRV2e (Arabidopsis, ABRC, catalog number: CD3-1866 ) vectors can be ordered at the Arabidopsis Biological Resource Center (ABRC, http://www.arabidopsis.org/).
7. Culture Media
   
   1. Liquid Luria-Bertani (LB) medium
      Solid LB medium plate with 1.5% agar
      Note: LB medium is autoclaved at 120 °C for 20 min before appropriate antibiotics are added.
8. Antibiotics
   
   1. Kanamycin (Sangon Biotech, USP Grade)
      
   2. Rifampicin (Sangon Biotech, USP Grade)
      
   3. Gentamicin (Sangon Biotech, USP Grade)
9. PCR reagents
   
   1. EasyTaq DNA polymerase (Beijing TransGen Biotech, catalog number: AP112 )
      
   2. EasyPfu DNA polymerase (Beijing TransGen Biotech, catalog number: AP211 )
      
   3. dNTP Mix (Roche Diagnostics, catalog number: 04729706103 )
10. Infiltration reagents
    
    1. Dimethyl sulfoxide, DMSO (AMRESCO, ACS grade)
       
    2. MgCl₂ (Beijing Chemical Works, Analytical pure) (see Recipes)
       
    3. 2-(N-Morpholino) ethanesulfonic acid, MES (AMRESCO, Regent Grade) (see Recipes)
       
    4. Acetosyringone (3, 5-Dimethoxy-4-hydroxyacetophenone) (AS) [Sigma-Aldrich, Purity (HPLC)] (see Recipes)
       
    5. Infiltration buffer (see Recipes)

Equipment

1. Plant growth chamber (24 °C, 16 h/8 h light/dark photoperiod, 40-80% humidity)
   
2. Centrifuge
   
3. PCR instrument
   
4. 37 °C and 28 °C incubators with shaking
   

Procedure