JUL1, Ring-Type E3 Ubiquitin Ligase, Is Involved in Transcriptional Reprogramming for ERF15-Mediated Gene Regulation

Abstract

JAV1-associated ubiquitin ligase 1 (JUL1) is a RING-type E3 ubiquitin ligase that catalyzes ubiquitination of JAV1, a jasmonate signaling repressor, in Arabidopsis thaliana in response to herbivore attack. Here we present a new insight into the nature of JUL1 as a multi-targeting enzyme for not only JAV1 but also transcription factors (TFs) screened using in vitro and in vivo protein interaction assays. Reporter assays using protoplasts showed that the JUL1-interacting TFs (JiTFs), including ERF15, bZIP53 and ORA59, were involved in transcriptional activation of jasmonate-responsive PDF1.2 and abscisic acid-responsive GEA6. Likewise, assays using mutant plants suggested that the 3 JiTFs were indeed responsible for transcriptional regulation of PDF1.2 and/or GEA6, and ERF15 and ORA59 were substantially responsible for the anti-herbivore trait. In vitro protein ubiqutination assays showed that JUL1 catalyzed ubiqutination of JAV1 but not any of the TFs. This was in accord with the finding that JUL1 abolished JAV1's interference with ERF15 function, according to the reporter assay. Moreover, of great interest is our finding that ERF15 but not bZIP53 or ORA59 serves as a scaffold for the JAV1/JUL1 system, indicating that there is narrow selectivity of the transcriptional reprogramming by the JAV1/JUL1 system.

Keywords: Arabidopsis thaliana; ERF15; JAV1; JAV1-associated ubiquitin ligase1 (JUL1); Spodoptera litura; abscisic acid; jasmonate.

Figure 1

Transcriptional activity of JiTFs. Transient activation of a firefly luciferase (Fluc) reporter gene under the control of PDF1.2 promoter (PDF1.2P) or GEA6 promoter (GEA6P) following expression with or without (−) JiTFs in Arabidopsis thaliana protoplasts. Data represent the mean and standard error (n = 5). Data marked with asterisks are significantly different from those obtained without JiTFs, based on an ANOVA with Holm’s sequential Bonferroni post hoc test (**, p < 0.01).

Figure 2

Anti-herbivore traits of JiTF-deficient mutant plants. (A) Relative transcript levels of PDF1.2 and GEA6 in leaves of wild-type (WT) Arabidopsis thaliana plants and T-DNA insertion mutant plants, corresponding to the respective JiTFs (JiTF3 [erf15], JiTF5 [bzip53], and JiTF7 [ora59]), were exposed to methyl jasmonate (MeJA) for 12 h, abscisic acid (ABA) for 6 h, or Spodoptera litura larvae for 24 h. Plants with application of aqueous solution (0.1% ethanol) alone for 12 h and for 6 h and uninfested healthy plants served as control for MeJA, ABA and S. litura-exposed plants, respectively. (B) Foliage damage areas of mutant plants, relative to those of WT plants, were assessed after incubation with S. litura larvae for 24 h. Data represent the mean and standard error (n = 6 and 20 for (A) and (B), respectively). Data marked with an asterisk(s) are significantly different from those in WT among each data set, based on an ANOVA with Holm’s sequential Bonferroni post hoc test (**, p < 0.01; *, 0.01 ≤ p < 0.05). ns, not significant.

Figure 3

Modulation of JiTF1-mediated PDF1.2 promoter (PDF1.2P) and GEA6 promoter (GEA6P) activities via JAV1/JUL1. Transient activation of a firefly luciferase (Fluc) reporter gene under the control of PDF1.2P and GEA6P following expression with (+) or without (−) JiTFs (JiTF3 [ERF15], JiTF5 [bZIP5] and JiTF7 [ORA59]), JAV1 or JUL1 in Arabidopsis thaliana protoplasts. Data represent the mean and standard error (n = 5). The means indicated by different small letters are significantly different among data of each day, based on an ANOVA with post hoc Tukey’s HSD (p < 0.05). ns, not significant.

Figure 4

In vitro JUL1 ubiquitination activity on JAV1 and JiTFs (JiTF3 [ERF15], JiTF5 [bZIP5] and JiTF7 [ORA59]) as substrates. (A) Biotinylated JiTFs and JAV1 proteins were incubated with FLAG-tagged ubiquitin (FLAG-Ub), UBCH5b (E2) and AGIA-tagged JUL1. (B) Likewise, biotinylated JAV1 proteins were incubated with FLAG-Ub, UBCH5b, and AGIA-tagged JUL1 in the presence of AGIA-tagged GFP or ERF15. The incubated reaction mixtures were immunoprecipitated using streptavidin-linked magnetic beads, subjected to SDS-PAGE, and probed with an anti-FLAG-HRP (FLAG-Ub) antibody. The smeared signals indicate possible ubiquitinated substrate proteins.

Figure 5

Bimolecular fluorescence complementation analysis of in planta interaction of JUL1, JAV1 or JAZ8 fused to the N-terminal fragment of Venus with ERF15 fused to the C-terminal fragment of Venus in Nicotiana benthamiana leaf cells. The photographs with reconstructed Venus signal, DAPI (4′,6-diamidino-2-phenylindole) fluorescence, and the merged image with bright field are shown.

Figure 6

A possible model of the two ways of JUL1-catalyzed JAV1 ubiquitination when ERF15 or WRKY is captured by JAV1 in Arabidopsis thaliana. In brief, in the steady state of the plant, the JAV1/ERF15 complex has different constituent molecules from the JAV1/WRKY51 complex which harbors JAZ8. In response to herbivory, JUL1 starts ubiquitination of JAV1 with a scaffold on ERF15, resulting in degradation of JAV1 via the ubiquitin-26S proteasome system. On the other hand, the JAV1/WRKY51/JAZ8 complex is decomposed with degradation of JAV1 and JAZ8 via JUL1 and SCF(COI1) ubiquitin-ligase complex, respectively. The consequent releases of ERF15 and WRKY51 act at the respective specific cis-elements, leading to gene regulation.