This step-by-step protocol shows how to detect and functionally characterize the enzymatic activity of RING-type E3 ubiquitin ligases, both in vitro and in planta, through a site-directed mutagenesis approach. By introducing mutation in a RING domain through site-directed mutagenesis, the resulting E3-deficient mutant can be tested in parallel with the wild-type protein to link the enzymatic activity with functionality. Elucidating the biochemical and mechanical basis of RING-type E3 ubiquitin ligases can contribute greatly to our understanding of their biological significance in development, stress signaling, and maintenance of homeostasis.
With slight modification of the in vivo expression system, this protocol can be applied to the analysis of any RING-type E3 ligase regardless of its origin. Begin by identifying the conserved Cys and His amino acids in the RING domain and designing primers that carry the substitution codon of interest flanked by 15 base pairs on either side of the mutation site. It is critical to properly identify the RING domain, particularly its conserved cysteine and histidine residues.
Online tools such as PROSITE can be used to do so. Use the mutagenic primers in PCR amplification to introduce the desired mutation into the plasmid harboring the gene of interest, making sure to use a high-fidelity DNA polymerase, such as Pfu. After PCR, digest the E.coli-derived parental methylated and semi-methylated DNA by adding three microliters of Dpnl restriction enzyme directly to the PCR reaction and incubating it at 37 degrees Celsius for two hours.
Purify the mutagenized plasmids with a commercial DNA extraction kit, and elute the DNA in 50 microliters of water. Then, transform the DH5-alpha E.coli chemically competent cells with 0.5 microliters of the recovered mutagenized plasmid DNA according to the manufacturer's protocol. Clone the wild-type RING and mutated RING genes of interest into the pMAL-c2 vector to fuse these genes with the MBP epitope tag.
Then, set up the in vitro ubiquitination reaction in a total volume of 30 microliters, as described in the manuscript, and incubate the mixture at 30 degrees Celsius for two hours. Terminate the reaction by mixing the sample with 7.5 microliters of 5x SDS-PAGE loading buffer and boiling it for five minutes, then separate the proteins with 7.5%SDS-polyacrylamide gel electrophoresis. Transfer onto the PVDF membrane, and detect the ubiquitination with western blotting and anti-FLAG.
Stain the membrane with Coomassie blue to verify the equal loading of tested MBP-RING-type protein. Streak the Agrobacterium tumefaciens strains carrying the epitope-tagged gene of interest on LB medium containing the appropriate selection antibiotics. After two days of growth at 28 degrees Celsius, pick single colonies, and grow them in LB liquid medium with antibiotics at 28 degrees Celsius and 250 rpm for another 24 hours.
Transfer 100 microliters of agrobacterial culture to three milliliters of fresh LB with antibiotics, and incubate the culture for an additional four to six hours. Spin down the cells at 1, 800 times g for six minutes, discard the supernatant, and resuspend the cells with three milliliters of wash buffer. Repeat the wash two times, then resuspend the cells in the induction buffer, and incubate them for an additional 10 to 12 hours at 28 degrees Celsius.
After the incubation, centrifuge the cells at 1, 800 times g for six minutes, and discard the supernatant. Resuspend the cells with two milliliters of infiltration buffer, and determine the concentration of bacteria using the OD600 value. Agroinfiltrate four-week-old Nicotiana benthamiana leaves by gently pricking them with a needle, and hand-inject Agrobacterium with a syringe, then circle the infiltrated area with a marker.
After 36 hours, collect the infiltrated leaf tissue, and grind it to a fine powder with liquid nitrogen. Resuspend the tissue powder with 300 microliters of protein extraction buffer, and centrifuge it at 15, 000 times g for 15 minutes at four degrees Celsius. Transfer the supernatant to a fresh tube, add 5x SDS-PAGE loading buffer to a final concentration of 1x, and boil the sample for five minutes.
Then, perform western blot analysis to detect in planta ubiquitination. Streak the appropriate Agrobacterium tumefaciens strains carrying tagged genes of interest or empty vector, and inject the Agrobacterium on Nicotiana benthamiana leaves, as previously described. For the E3-dependent substrate protein degradation, follow the previously described protocol, and perform western blotting with appropriate antibodies to detect protein accumulation in plant cells.
For E3-dependent hypersensitive response-mediated cell death inhibition, monitor the agroinfiltrated leaves for cell death symptoms two to four days post-infiltration. A typical in vitro ubiquitination assay outcome contains a multiband smear starting at the molecular weight of the tested protein and progressing upwards. As expected, negative controls missing individual components or using MBP did not demonstrate the smeared signal.
Furthermore, the Coomassie blue staining of the PVDF membrane showed equal loading of MBP-RHA1B or MBP in all controls. 11 different E2s were tested to investigate how in vitro ubiquitination varies depending on the specific E2 to E3 combination. The detected ubiquitination activity ranged from no signal to a multiband smear starting at different molecular weights, indicating different ubiquitination patterns.
The RING-and K-mutant versions of RHA1B were tested for ubiquitination in vitro and in planta. The lack of enzymatic activity of the RING-mutant version is supported by its inability to either generate a multiband smear in vitro or promote poly-ubiquitination signal in planta. Although a marginal self-ubiquitination signal was detected in vitro for the K mutant, only background ubiquitination was detected in planta, suggesting that the K146 residue is also essential for E3 activity.
Unlike the wild-type RHA1B, the RING mutant lacking E3 ligase activity did not interfere with hypersensitive response cell death. Western blotting confirmed that the Gpa2 did not accumulate in the presence of wild-type RHA1B, but the RING mutant had no impact on protein stability. Given that a proper E2-E3 combination is necessary for the ubiquitination reaction, in vitro ubiquitination assays should be carried in parallel with multiple E2 enzymes representing different E2 classes to avoid false negative results.
An E3 ligase-deficient mutant facilitates the testing of enzyme-substrate interactions in vivo. Furthermore, this mutant often confers a dominant negative phenotype that can be utilized in functional knockout studies as an alternative approach to RNAi. Using this protocol, it has been recently found that RHA1B nematode effector interfere with plant immune signaling in an E3-dependent manner by targeting the plant Gpa2 immunoreceptor for ubiquitination and degradation.
