A Subset of Ubiquitin-Conjugating Enzymes Is Essential for Plant Immunity

Abstract

Of the three classes of enzymes involved in ubiquitination, ubiquitin-conjugating enzymes (E2) have been often incorrectly considered to play merely an auxiliary role in the process, and few E2 enzymes have been investigated in plants. To reveal the role of E2 in plant innate immunity, we identified and cloned 40 tomato genes encoding ubiquitin E2 proteins. Thioester assays indicated that the majority of the genes encode enzymatically active E2. Phylogenetic analysis classified the 40 tomato E2 enzymes into 13 groups, of which members of group III were found to interact and act specifically with AvrPtoB, a Pseudomonas syringae pv tomato effector that uses its ubiquitin ligase (E3) activity to suppress host immunity. Knocking down the expression of group III E2 genes in Nicotiana benthamiana diminished the AvrPtoB-promoted degradation of the Fen kinase and the AvrPtoB suppression of host immunity-associated programmed cell death. Importantly, silencing group III E2 genes also resulted in reduced pattern-triggered immunity (PTI). By contrast, programmed cell death induced by several effector-triggered immunity elicitors was not affected on group III-silenced plants. Functional characterization suggested redundancy among group III members for their role in the suppression of plant immunity by AvrPtoB and in PTI and identified UBIQUITIN-CONJUGATING11 (UBC11), UBC28, UBC29, UBC39, and UBC40 as playing a more significant role in PTI than other group III members. Our work builds a foundation for the further characterization of E2s in plant immunity and reveals that AvrPtoB has evolved a strategy for suppressing host immunity that is difficult for the plant to thwart.Ubiquitination as a major posttranslational modification of proteins in eukaryotes has emerged in recent years as an important regulatory mechanism underlying plant innate immunity Fu et al., 2012;Marino et al., 2012;Li et al., 2014). The ubiquitination process involves a consecutive, threestep enzymatic cascade that is catalyzed by three different classes of enzymes: ubiquitin-activating (E1), ubiquitin-conjugating (E2), and ubiquitin ligase (E3) enzymes. The first step of the process activates ubiquitin, a highly conserved 76-amino acid protein, in an ATP-dependent manner by attaching ubiquitin to an E1 enzyme. The activated ubiquitin is then transferred from the E1 to the Cys residue at the active site of an E2 conjugating enzyme. An E3 ligase then recruits the substrate protein to the E2 to transfer the ubiquitin molecule from E2 to the substrate. Through the action of E1, E2, and E3, ubiquitin is covalently attached usually to the Lys residue of a substrate through an isopeptide bond (Hershko and Ciechanover, 1998). Of the three enzymes involved in ubiquitination, E3 ubiquitin ligases have been the focus of many studies due to their key role in determining substrate specificity for the ubiquitination

Functional analysis of the seven in absentia ubiquitin ligase family in tomato

“…Significantly, the unique bacterial ubiquitin ligase AvrPtoB, which is a virulence effector encoded by Pseudomonas syringae pv. tomato (Abramovitch, Janjusevic, Stebbins, & Martin, ; Janjusevic, Abramovitch, Martin, & Stebbins, ), specially works with group III E2s for its in planta virulence activity (Zhou et al ., ), suggesting that a given E3 ubiquitin ligase may have preference to distinct E2 ubiquitin‐conjugating enzyme for its intrinsic activity. Thus, the weak ubiquitination rendered by SlSINA3/4/5 in the in vitro ubiquitination assay (Figure a) could be attributable to the intrinsic weak E3 activity or inappropriate E2 used in the assay.…”

“…It is logical to hypothesize that the most appropriate combination of E3 with E2 will lead to optimization of ubiquitination process. There are 34 distinct functional E2 found in tomato, and they are classified into 13 groups (Zhou et al ., ). Our results indicated that the SlSINAs only function cooperatively with Group III of tomato E2s (Figure a; Figure S2).…”

“…The tomato E2s used in this study are as follows: SlUBC1 (KY246895), SlUBC4 (KY246898), SlUBC6 (KY246900), SlUBC7 (KY246901), SlUBC12 (KY246906), SlUBC13 (KY246927), SlUBC17 (KY246911), SlUBC20 (KY246912), SlUBC22 (KY246914), SlUBC27 (KY246919), and SlUBC32 (KY246924). And the expression and purification of the tomato E2 recombinant proteins were performed as described previously (Zhou et al ., ).…”

MAC3A and MAC3B, Two Core Subunits of the MOS4-Associated Complex, Positively Influence miRNA Biogenesis

“…As previously reported (Wu et al, 2013), the HYL1-containing D-bodies 303 existed in most cells (~ 84%, Figure 6A, 6B and Supplemental Figure 6A Because MAC3A has considerable sequence difference from MAC3B, we tested if it is a 318 ubiquitin E3 ligase using MBP-MAC3A (Supplemental Figure 5A). We examined the E3 ligase 319 activity in the presence of ubiquitin, the ubiquitin-activating enzyme (E1) SlUBA and the 320 ubiquitin-conjugating enzyme (E2) UBC8 (Zhou et al, 2017). However, MBP-MAC3A 321 displayed only weak activity ( Figure 7B).…”

“…The in vitro ubiquitination assay was performed as described (Zhou et al, 2017) protein modification on MAC3A activity, the recombinant proteins were treated as previously 485 described with modifications (Wang et al, 2015). Briefly, 4 µg MBP, MBP-MAC3A, MBP-486…”