(see Note 1) but are only recommended if further immunoblot
analysis is desired for the purpose of protein expression control.
For binary interactions, only two pair combinations of constructs with different fusion orientations are necessary (see
Note 1).
2. Restriction enzymes, DNA Polymerase, DNA ligase, DNA
primers, and reagents for polymerase chain reaction (PCR),
molecular cloning, and restriction enzyme analyses of tripartite
split-sfGFP-based constructs.
2.2 Agrobacterium
Culture and Infiltration
1. Chemical-competent Agrobacterium tumefaciens strain
EHA105 [25].
2. Agrobacterium EHA105 strain containing p19 silencing
suppressor [26].
3. Chemical-competent Escherichia coli DH5α cells
4. 2 M NaOH.
5. 1 M KOH and 5 M KOH.
6. 1 M MgSO 4 (autoclave or sterile infiltrate).
7. 1 M MgCl 2 (autoclave or sterile infiltrate).
8. 500 mM 2-(N-morpholino) methanesulfonic acid (MES).
Adjust to pH 5.7 with 5 M KOH and filter sterilize through a
0.45 μm filter.
9. Luria-Bertani (LB) medium: yeast extract 10 g/L, NaCl 10 g/
L, peptone 5 g/L (autoclave and store at room temperature).
For solid medium add 15 g/L agar, autoclave, and pour into
petri dishes.
10. Infiltration medium for N. benthamiana infiltration: 10 mM
MES (pH 5.7), 10 mM MgCl 2 , 150 μM Acetosyringone.
2.3 Plant Culture
1. N. benthamiana seeds.
2. Plant pots with 3-inch diameter.
3. Growing matrix in the proportion of peat moss: vermiculite:
perlite ¼ 8:1:3.
4. Water-soluble fertilizer NPK 20-20-20.
5. Ultra-pure H 2 O (e.g., Milli Q) autoclaved
6. Food grade industrial 95% ethanol (EtOH).
7. Seed sterilization solution: a diluted bleach solution (with
1:1 ¼ water:commercial bleach solution) containing 0.1% Triton X-100.
8. 1 M KOH
9. 0.5 MS agar plates: one-half-strength Murashige and Skoog
medium incl. MES buffer (M0254, Duchefa) adjusted to
pH 5.7 with 1 M KOH and supplemented with 1% sucrose
and 0.8% agar.
Tripartite Split-sfGFP Assay
327
analysis is desired for the purpose of protein expression control.
For binary interactions, only two pair combinations of constructs with different fusion orientations are necessary (see
Note 1).
2. Restriction enzymes, DNA Polymerase, DNA ligase, DNA
primers, and reagents for polymerase chain reaction (PCR),
molecular cloning, and restriction enzyme analyses of tripartite
split-sfGFP-based constructs.
2.2 Agrobacterium
Culture and Infiltration
1. Chemical-competent Agrobacterium tumefaciens strain
EHA105 [25].
2. Agrobacterium EHA105 strain containing p19 silencing
suppressor [26].
3. Chemical-competent Escherichia coli DH5α cells
4. 2 M NaOH.
5. 1 M KOH and 5 M KOH.
6. 1 M MgSO 4 (autoclave or sterile infiltrate).
7. 1 M MgCl 2 (autoclave or sterile infiltrate).
8. 500 mM 2-(N-morpholino) methanesulfonic acid (MES).
Adjust to pH 5.7 with 5 M KOH and filter sterilize through a
0.45 μm filter.
9. Luria-Bertani (LB) medium: yeast extract 10 g/L, NaCl 10 g/
L, peptone 5 g/L (autoclave and store at room temperature).
For solid medium add 15 g/L agar, autoclave, and pour into
petri dishes.
10. Infiltration medium for N. benthamiana infiltration: 10 mM
MES (pH 5.7), 10 mM MgCl 2 , 150 μM Acetosyringone.
2.3 Plant Culture
1. N. benthamiana seeds.
2. Plant pots with 3-inch diameter.
3. Growing matrix in the proportion of peat moss: vermiculite:
perlite ¼ 8:1:3.
4. Water-soluble fertilizer NPK 20-20-20.
5. Ultra-pure H 2 O (e.g., Milli Q) autoclaved
6. Food grade industrial 95% ethanol (EtOH).
7. Seed sterilization solution: a diluted bleach solution (with
1:1 ¼ water:commercial bleach solution) containing 0.1% Triton X-100.
8. 1 M KOH
9. 0.5 MS agar plates: one-half-strength Murashige and Skoog
medium incl. MES buffer (M0254, Duchefa) adjusted to
pH 5.7 with 1 M KOH and supplemented with 1% sucrose
and 0.8% agar.
Tripartite Split-sfGFP Assay
327