Reconstitution of the plant ubiquitination cascade in bacteria using a synthetic biology approach

Abstract

Ubiquitination modulates nearly all aspects of plant life. Here, we reconstituted the Arabidopsis thaliana ubiquitination cascade in Escherichia coli using a synthetic biology approach. In this system, plant proteins are expressed and then immediately participate in ubiquitination reactions within E. coli cells. Additionally, the purification of individual ubiquitination components prior to setting up the ubiquitination reactions is omitted. To establish the reconstituted system, we co-expressed Arabidopsis ubiquitin (Ub) and ubiquitination substrates with E1, E2 and E3 enzymes in E. coli using the Duet expression vectors. The functionality of the system was evaluated by examining the auto-ubiquitination of a RING (really interesting new gene)-type E3 ligase AIP2 and the ubiquitination of its substrate ABI3. Our results demonstrated the fidelity and specificity of this system. In addition, we applied this system to assess a subset of Arabidopsis E2s in Ub chain formation using E2 conjugation assays. Affinity-tagged Ub allowed efficient purification of Ub conjugates in milligram quantities. Consistent with previous reports, distinct roles of various E2s in Ub chain assembly were also observed in this bacterial system. Therefore, this reconstituted system has multiple advantages, and it can be used to screen for targets of E3 ligases or to study plant ubiquitination in detail.

The Xanthomonas effector XopK harbours E3 ubiquitin‐ligase activity that is required for virulence

“…In this system, the Arabidopsis E1 AtUBA1 (Ubiquitin‐activating enzyme 1, AT2g30110) and E2 AtUBC8 (Ubiquitin carrier protein 1, AT5g41700) were cloned into the Duet expression vectors and AtUBQ10, which encodes ubiquitin polymers, was cloned into the pET expression vector for coexpression of the ubiquitination cascades in bacteria. The fidelity and specificity for ABSCISIC ACID INSENSITIVE 3 (ABI3) ubiquitination by its cognate really interesting new gene (RING)‐type E3 ligase ABI3‐INTERACTING PROTEIN 2 (AIP2) and AIP2 autoubiquitination had been verified in this reconstituted ubiquitination assay (Han et al ., ). We next took advantage of this reconstituted system to examine putative ubiquitin‐ligase activity of XopK towards OsSERK2‐KD.…”

“…The resulting construct was transformed into A. tumefaciens strain GV3101 and used for transient expression in Nicotiana benthamiana Domin leaves. XopK , XopK‐E2BS , XopK‐F2 and OsSERK2‐KD were PCR amplified and cloned into the pCDFDuet or pACYCDuet vector (Han et al ., ) as indicated to generate constructs for in vitro ubiquitination assays. Schematic descriptions of the constructs for in vitro ubiquitination assays are shown in the Supporting Information.…”

“…The in vitro ubiquitination assay was performed as described by Han et al (2017). The pCDFDuet and pACYCDuet plasmids carrying XopK variants or OsSERK2-KD as indicated, together with pET-28a-FLAG-UBQ (Han et al, 2017), were cotransformed into E. coli BL21 (DE3) competent cells.…”

“…The in vitro ubiquitination assay was performed as described by Han et al (2017). The pCDFDuet and pACYCDuet plasmids carrying XopK variants or OsSERK2-KD as indicated, together with pET-28a-FLAG-UBQ (Han et al, 2017), were cotransformed into E. coli BL21 (DE3) competent cells. BL21 (DE3) strains containing the expression vectors were cultured to reach an OD 600 of 0.4-0.6 and induced by IPTG (isopropyl b-D-1thiogalactopyranoside) at 28°C for 12 h. The strains were then stored at 4°C overnight.…”

Identification of critical cysteine sites in brassinosteroid‐insensitive 1 and novel signaling regulators using a transient expression system

“…For PDI11-GFP-KDEL, PDI11 was subcloned into the plant expression vector fused to the GFP-KDEL tag . BRI1 cytosolic domain (BRI1CD) was cloned into the modified pET-28a vector and fused to the 6 9 His and FLAG tags at its N-terminus (Lin et al, 2013;Han et al, 2017). PCRK1/2 were subcloned into a glutathione S-transferase (GST) fusion protein expression vector pGEX4T-1.…”