In vivo ubiquitination assay by agroinfiltration.

The ubiquitination/proteasome system is involved in nearly all plant signaling processes. Many signaling components are degraded by the 26S proteasome upon ubiquitination by specific E3 ubiquitin ligase. However, due to technical limitations, only a few pairs of E3 ligase-substrate interactions have been directly demonstrated in plants. The method described here provides an efficient way to detect E3-mediated protein ubiquitination in vivo by agroinfiltration in Nicotiana benthamiana. This assay allows for fast and reliable detection of the specific interaction between the substrate and the E3 ligase, the effect of E3 ligase on substrate ubiquitination and degradation, and the effects of proteasome inhibitor, such as MG132, on substrate stability.