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. 2013 Jun;162(2):1006-17.
doi: 10.1104/pp.113.218164. Epub 2013 Apr 30.

Negative Feedback Control of Jasmonate Signaling by an Alternative Splice Variant of JAZ10

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Free PMC article

Negative Feedback Control of Jasmonate Signaling by an Alternative Splice Variant of JAZ10

Javier E Moreno et al. Plant Physiol. .
Free PMC article

Abstract

The plant hormone jasmonate (JA) activates gene expression by promoting ubiquitin-dependent degradation of jasmonate ZIM domain (JAZ) transcriptional repressor proteins. A key feature of all JAZ proteins is the highly conserved Jas motif, which mediates both JAZ degradation and JAZ binding to the transcription factor MYC2. Rapid expression of JAZ genes in response to JA is thought to attenuate JA responses, but little is known about the mechanisms by which newly synthesized JAZ proteins exert repression in the presence of the hormone. Here, we show in Arabidopsis (Arabidopsis thaliana) that desensitization to JA is mediated by an alternative splice variant (JAZ10.4) of JAZ10 that lacks the Jas motif. Unbiased protein-protein interaction screens identified three related basic helix-loop-helix transcription factors (MYC2, MYC3, and MYC4) and the corepressor NINJA as JAZ10.4-binding partners. We show that the amino-terminal region of JAZ10.4 contains a cryptic MYC2-binding site that resembles the Jas motif and that the ZIM motif of JAZ10.4 functions as a transferable repressor domain whose activity is associated with the recruitment of NINJA. Functional studies showed that the expression of JAZ10.4 from the native JAZ10 promoter complemented the JA-hypersensitive phenotype of a jaz10 mutant. Moreover, treatment of these complemented lines with JA resulted in the rapid accumulation of JAZ10.4 protein. Our results provide an explanation for how the unique domain architecture of JAZ10.4 links transcription factors to a corepressor complex and suggest how JA-induced transcription and alternative splicing of JAZ10 premessenger RNA creates a regulatory circuit to attenuate JA responses.

Figures

Figure 1.
Figure 1.
The N-terminal region of JAZ10.4 has a cryptic MYC2-interacting domain. A, Y2H assays of JAZ10.4 deletion constructs (DNA-binding domain bait fusions) with MYC2 and JAZ10.4 (activation domain prey fusions). Yeast strains coexpressing the indicated bait and prey proteins were plated on medium containing 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside. Blue-color formation in streaked yeast cells is indicative of protein-protein interaction. Photographic images were taken after 30 h of incubation at 30°C. JAZ10.1 is included as a positive control. The ZIM (Z) and Jas domains are indicated. The solid black line denotes the C-terminal region that is specific for the JAZ10.4 isoform. B, C-terminal deletion constructs of JAZ10.4 were tested for interaction with MYC2 as described in A. C, Sequence similarity between the conserved Jas motif of JAZ10 and JAZ9 and the CMID near the N terminus of JAZ10. The underlined sequence shows the region of conservation, which includes a tribasic motif (boldface).
Figure 2.
Figure 2.
An RRR motif in the N-terminal region of JAZ10.4 is required for interaction with MYC2. A, Site-directed mutagenesis of the RRR motif within the CMID of JAZ10.4. The indicated R→A substitution mutants of JAZ10.4 were tested for interaction with MYC2 or JAZ10.4 as a positive control. Y2H assays were performed as described in Figure 1. B, In vitro pull-down assay of the JAZ10.4-MYC2 interaction. Assays were performed using the indicated JAZ10.4-His recombinant proteins and crude extracts from leaves of wild-type (WT) or 35S:cMyc-MYC2 transgenic (T) plants. Protein bound to JAZ-His was separated by SDS-PAGE and analyzed by immunoblotting (anti-cMyc antibody) for the presence of cMyc-MYC2. The Coomassie blue-stained gel shows total input protein as a loading control. C, The CMID of JAZ10.4 is sufficient for MYC2 binding. In vitro pull-down assays were performed as described in B using JAZ3, JAZ3ΔJas, or a chimeric JAZ (JAZ3ΔJas-J101-78) in which the CMID of JAZ10.4 was fused to the C terminus of JAZ3ΔJas. [See online article for color version of this figure.]
Figure 3.
Figure 3.
The RRR motif is required for the JAZ10.4-mediated repression of JA responses. A, The photograph shows silique development in the wild type (WT) and transgenic lines that overexpress JAZ10.4 or JAZ10.4RRR→AAA. B, The photograph shows seedlings of the indicated genotypes grown for 10 d on Murashige and Skoog medium containing 20 μm MeJA. C, Quantification of JA-induced root growth inhibition in the wild type, 35S:JAZ10.4 (10.4), and eight independent JAZ10.4RRR→AAA lines. Seedlings were grown for 10 d on Murashige and Skoog agar plates containing or not containing 20 µm MeJA. The root length ratio was calculated by dividing the average root length of MeJA-treated seedlings by the average root length of seedlings of the same genotype grown in the absence of MeJA. Data show means ± se (n = 12 seedlings per genotype). The asterisk denotes a significant difference (P < 0.05, Student’s t test) in comparison with the wild type. [See online article for color version of this figure.]
Figure 4.
Figure 4.
The ZIM domain of JAZ10 interacts with NINJA and is required for the repression of JA responses. A, Y2H assays depicting the interaction of JAZ10 or JAZ8 chimeric proteins (DNA binding-domain fusions [DB]) with MYC2, JAZ1, NINJA (NJA), and TPL (activation domain fusions [AD]). Empty vectors were included as negative controls. Sequence regions derived from JAZ10 and JAZ8 are shown in white and gray, respectively, together with various protein domains. E, EAR motif. Y2H assays were performed as described in Figure 1, except that the photographic image was taken after 48 h of incubation of yeast cells at 30°C. B, Overexpression of a JAZ8ΔEAR-ZIM10 chimeric protein confers insensitivity to JA in root growth assays. Seedlings were grown for 8 d on Murashige and Skoog agar plates containing or not containing 20 µm MeJA, and root length ratios were calculated as described in Figure 3C. Data show means ± se of at least 11 seedlings per genotype. Asterisks denote significant differences (P < 0.05, Student’s t test) in comparison with the wild type (WT).
Figure 5.
Figure 5.
Ile-107 in the TIFY motif is involved in JAZ10.4 interaction with both JAZ10 and NINJA. A, Y2H assay depicting the role of Thr-106 and Ile-107 (within the TIFY motif) in JAZ10.4 dimerization and JAZ10.4 interaction with NINJA. Y2H assays were performed as described in Figure 1, except that the photographic image was taken after 24 h of incubation of yeast cells at 30°C. B, In vitro pull-down assay of the JAZ10.4-JAZ10.4 interaction. Assays were performed using the indicated wild-type (JAZ10.4) and mutant (JAZ10.4I107A) recombinant proteins and crude extract from a yeast strain expressing a JAZ10.4-HA fusion protein. Protein bound to JAZ-His was separated by SDS-PAGE and analyzed by immunoblotting with an anti-HA antibody. A Coomassie blue-stained gel is shown as a loading control. C, In vitro pull-down assay of the JAZ10.4-NINJA interaction. Assays were performed as described in B except for the use of a crude extract from a yeast strain expressing a NINJA-HA fusion protein. [See online article for color version of this figure.]
Figure 6.
Figure 6.
JA-induced expression of JAZ10.4 complements the JA-hypersensitive phenotype of jaz10-1. A, Root growth inhibition assay of the wild type (WT), jaz10-1 (jaz10), and five independent lines in which the pJAZ10:HA-JAZ10.4 transgene was introduced into the jaz10-1 mutant background. Root length was measured in 8-d-old seedlings grown on Murashige and Skoog medium containing 0, 10, or 20 µm MeJA. The root length ratio was calculated by dividing the average root length of seedlings grown in the presence of either 10 µm (gray bars) or 20 µm (black bars) MeJA by the average root length of seedlings of the same genotype grown in the absence of MeJA. Data show means ± se of 12 to 14 seedlings per genotype. Asterisks indicate significant differences in root length (P < 0.05, Student’s t test) in comparison between wild-type and jaz10 seedlings grown at the same concentration of MeJA or between wild-type and pJAZ10:HA-JAZ10.4 seedlings grown at the same concentration of MeJA. B, Accumulation of HA-JAZ10.4 protein in response to JA treatment. Ten-day-old liquid-grown seedlings of the indicated pJAZ10:HA-JAZ10.4 line were treated with 50 µm MeJA and harvested at various times (h) thereafter. As controls, seedlings were harvested immediately prior to treatment (0) and 8 h after mock treatment (C). Total protein was subjected to immunoblot analysis with an anti-HA antibody to detect HA-JAZ10.4. A Coomassie blue-stained gel of protein extracts was used as a loading control (CB). [See online article for color version of this figure.]

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