Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

1. Site-directed mutagenesis (Figure 1)

1. Identify the conserved Cys and His amino acids in the RING domain (Figure 1A) and design primers carrying the substitution codon of interest flanked by 15 base pairs on either side of the mutation site (Figure 1B).
2. Introduce the desired mutation by PCR-based amplification of the plasmid harboring the gene of interest using mutagenic primers and high-fidelity DNA polymerase containing Pfu in 50 µL of total PCR reaction volume as shown in Table 1 and Table 2 according to the manufacturer's protocol.
3. Digest the Escherichia coli-derived parental methylated and semi-methylated DNA by adding 3 µL of DpnI restriction enzyme directly to the PCR reaction (step 1.2) and incubating at 37 °C for 2 h.
   NOTE: Methylation is a posttranscriptional protein modification that is added to the plasmid produced and isolated from bacteria. New copies of PCR-generated plasmid lack methylation, therefore, the new copies will remain intact during DpnI treatment.
4. Purify the mutagenized plasmids using a commercial DNA extraction kit based on spin column technology and elute the DNA with 50 µL of water.
5. Transform DH5α E. coli chemically competent cells with 0.5 µL of the recovered mutagenized plasmid DNA according to the manufacturer's protocol. In brief, incubate competent cells with DNA on ice for 30 min, then heat-shock them for 20 s at 42 °C, and place tubes again on ice for 2 min. Incubate cells with 500 µL of LB media at 37 °C for 1 h at 250 rpm and then spread them on selective pates.
6. Verify the desired mutation by Sanger sequencing the DNA plasmids isolated from E. coli.

2. Recombinant protein purification and in vitro ubiquitination assay

1. Clone the wild type RING and mutated RING genes of interest into the pMAL-c2 vector (follow the manufacturer's protocol; Table 3) to fuse these genes with the MBP epitope tag that permits one-step purification using amylose resin. Introduce the resulting constructs into the E. coli BL21 strain as described in step 1.5.
2. Grow the E. coli strain BL21 harboring the desired construct in 50 mL LB liquid medium at 37 °C for 2–3 h until it reaches the logarithmic phase (OD₆₀₀ of 0.4–0.6).
3. Add IPTG to a final concentration of 0.1–1 mM to induce the expression of MBP-tagged recombinant protein of interest and incubate the E. coli culture for 2–3 h at 28 °C. Place the culture on ice after incubation.
   NOTE: Perform steps 2.4–2.13 on ice to protect proteins from degradation.
4. To check for the induction efficiency, collect 1.5 mL of induced cells, spin them down at 13,000 x g for 2 min, remove the supernatant, and resuspend the cells in 20 µL of 2x SDS-PAGE loading buffer (24 mM Tris-HCl pH 6.8, 0.8% SDS, 10% (v/v) glycerol, 4 mM DTT, 0.04% (w/v) bromophenol blue).
5. Boil the samples for 5 min and run them on a 10% SDS-PAGE gel. To visually evaluate accumulation of MBP-fusion protein (molecular weight of protein of interest + 42.5 kDa MBP), stain the gel for 20 min by agitating with Coomassie staining buffer (50% methanol, 10% acetic acid, 0.1% Coomassie blue) and destaining overnight with the destaining buffer (20% methanol, 10% acetic acid).
6. Harvest the remaining E. coli cells by centrifugation at 1,350 x g for 6 min, discard the supernatant, and resuspend the cell pellet with 5 mL of column buffer (20 mM Tris HCl, 200 mM NaCl, 1 mM EDTA, bacterial protease inhibitor).
   NOTE: This is a good place to stop the protocol overnight. The frozen cells can be stored up to 1 week at -20 °C.
7. Break down E. coli cells by placing the tube containing the bacteria in an ice-water bath and applying 10 sonication cycles: 10 s sonication at 30% amp followed by 20 s breaks.
8. Centrifuge sample at 13,000 x g at 4 °C for 10 min and save the supernatant (crude extract).
9. Prepare 500 µL of amylose resin in a 15 mL tube. Wash the resin by adding 10 mL of cold column buffer and centrifuging at 1,800 x g, 4 °C for 5 min. Do this 2x.
10. Add 5 mL of crude extract to the tube with the amylose resin and incubate overnight at 4 °C.
11. Centrifuge at 1,800 x g at 4 °C for 5 min and discard the supernatant.
12. Add 10 mL of column buffer to the resin pellet and incubate for 20 min. Then centrifuge at 1,800 x g at 4 °C for 5 min. Repeat this step 2x.
13. Elute the fusion protein with 0.5 mL of column buffer containing 10 mM maltose by incubating a sample for 2 h at 4 °C. Centrifuge at 1,800 x g at 4 °C for 5 min and collect the eluted protein. Repeat this step 2x.
14. Dialyze 1 mL of the protein fraction against the cold PBS. Aliquot protein into single-use tubes (10–20 µL) to avoid freeze-thawing and store at -80 °C until needed.
15. Measure the protein concentration using the Bradford assay⁹.
16. Set up the in vitro ubiquitination reaction in a total volume of 30 µL by mixing up 40 ng of E1 (e.g., AtUBA1), 100 ng of E2 (e.g., AtUBC8, SlUBC1/4/6/7/12/13/17/20/22/27/32), 1 µg MBP-RING type protein, and 2 µg FLAG-Ub (or HA-Ub) in the ubiquitination buffer (50 mM Tris-HCl pH 7.5, 2 mM ATP, 5 mM MgCl₂, 30 mM creatine phosphate, 50 µg/mL creatine phosphokinase). Incubate the mixture at 30 °C for 2 h.
    NOTE: Premake 20x ubiquitination buffer and store it up to 6 months at -20 °C in small aliquots for a single use. The creatine phosphokinase easily loses its enzymatic activity when the buffer is thawed and frozen repeatedly.
17. Terminate the reaction by mixing the 30 µL samples with 7.5 µL of 5x SDS-PAGE loading buffer (60 mM Tris-HCl pH 6.8, 2% SDS, 25% (v/v) glycerol, 10 mM DTT, 0.1% (w/v) bromophenol blue) and boiling for 5 min.
18. Separate the proteins with 7.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), then transfer onto the PDVF membrane, and detect ubiquitination by Western blotting using the anti-FLAG (or anti-HA).
19. Stain the PVDF membrane with Coomassie blue to verify the equal loading of tested MBP-RING-type protein.

3. Agrobacterium-mediated transient protein expression in Nicotiana benthamiana leaves and in planta ubiquitination assay

1. Streak appropriate Agrobacterium tumefaciens strains carrying the epitope-tagged gene of interest (e.g., HA-RHA1B, HA-RHA1B^C135S, HA-RHA1B^K146R, HA-Ub) and empty vector as a control on the LB medium containing the appropriate selection antibiotics.
2. After 2 days of growth at 28 °C, pick up single colonies and grow them in LB liquid medium with the appropriate antibiotics at 28 °C/250 rpm for another 24 h.
3. Transfer 100 µL of agrobacterial culture to 3 mL fresh LB with the appropriate antibiotics and incubate the culture for an additional 4–6 h at 28 °C with rotation (250 rpm) to the late exponential growth phase.
4. Spin down agrobacterial cells at 1,800 x g for 6 min, discard the supernatant, and resuspend cells with 3 mL of wash buffer (50 mM MES pH 5.6, 28 mM glucose, 2 mM NaH₂PO₄). Repeat this step 2x.
5. After the second wash, resuspend the cells in the induction buffer (50 mM MES pH 5.6, 28 mM glucose, 2 mM NaH₂PO_4, 200 µM acetosyringone, 37 mM NH₄Cl, 5 mM MgSO₄.7H₂O, 4 mM KCl, 18 µM FeSO₄.7H₂O, 2 mM CaCl₂). Incubate the cells with induction buffer for an additional 10–12 h at 28 °C.
   NOTE: Acetosyringone induces T-DNA transfer.
6. Centrifuge the cells at 1,800 x g for 6 min, discard the supernatant, and resuspend the cells with 2 mL of infiltration buffer (10 mM MES pH 5.5, 200 µM acetosyringone).
   NOTE: If Agrobacteria aggregate after incubation with induction buffer, let the aggregated cells sink to the bottom of the tube by leaving it on the bench for a few minutes, and transfer the clear Agrobacterium suspension to a new tube before proceeding with step 3.6.
7. Measure the concentration of bacteria using the OD₆₀₀ value (the optical density at absorbance of 600 nm). Adjust OD₆₀₀ values to the desired ones.
   NOTE: Usually an OD₆₀₀ value between 0.2–0.4 works best for a single agrobacterial stain expression. If a combination of different agrobacterial strains is applied, the total OD₆₀₀ values of Agrobacterial strains should not exceed 1.
8. Agroinfiltrate 4-week-old N. bethamiana leaves by gently pricking them with a needle, followed by hand-injecting Agrobacterium with a syringe without the needle. Circle the infiltrated leaf area with the marker (usually 1–2 cm in diameter).
9. Collect the infiltrated leaf tissues 36 h post-infiltration. Grind the tissue to a fine powder with liquid nitrogen.
10. Resuspended tissue powder with 300 μL of protein extraction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 2 mM DTT, 10% glycerol, 1% polyvinylpolypyrrolidone, 1 mM PMSF, plant protease inhibitor cocktail) and centrifuge at 15,000 x g for 15 min at 4 ˚C.
11. Transfer the supernatant to a new tube. Add 5x SDS-PAGE loading buffer to a final concentration of 1x and boil for 5 min.
12. Separate crude proteins on 10% SDS-PAGE gels, transfer onto PVDF membranes, and probe with anti-HA to detect in planta ubiquitination.

4. Establishing the link between enzymatic activity and function in planta

NOTE: For example, RHA1B promotes degradation of resistant protein Gpa2 to suppress HR cell death. This step shows how to verify that those virulent activities of RHA1B are E3-dependent.

1. Streak appropriate Agrobacterium tumefaciens strains carrying tagged genes of interest (in this example HA-RHA1B, HA-RHA1B^C135S, HA-RHA1B^K146R, myc-Gpa2, RBP1) and empty vector as a control. Follow steps 3.1–3.8 for Agrobacterium preparation and injection on N. bethamiana leaves.
2. For the E3-dependent substrate protein degradation, follow steps 3.9–3.12 and perform the Western blotting using appropriate antibodies to detect the protein accumulation in plant cells (e.g., anti-HA and anti-MYC).
3. For the E3-dependent hypersensitive response (HR) mediated cell death inhibition, monitor the agroinfiltrated leaves for HR cell death symptoms 2–4 days post-infiltration.