A critical role for the TIFY motif in repression of jasmonate signaling by a stabilized splice variant of the JASMONATE ZIM-domain protein JAZ10 in Arabidopsis

Abstract

JASMONATE ZIM-domain (JAZ) proteins act as repressors of jasmonate (JA) signaling. Perception of bioactive JAs by the F-box protein CORONATINE INSENSITIVE1 (COI1) causes degradation of JAZs via the ubiquitin-proteasome pathway, which in turn activates the expression of genes involved in plant growth, development, and defense. JAZ proteins contain two highly conserved sequence regions: the Jas domain that interacts with COI1 to destabilize the repressor and the ZIM domain of unknown function. Here, we show that the conserved TIFY motif (TIFF/YXG) within the ZIM domain mediates homo- and heteromeric interactions between most Arabidopsis thaliana JAZs. We have also identified an alternatively spliced form (JAZ10.4) of JAZ10 that lacks the Jas domain and, as a consequence, is highly resistant to JA-induced degradation. Strong JA-insensitive phenotypes conferred by overexpression of JAZ10.4 were suppressed by mutations in the TIFY motif that block JAZ10.4-JAZ interactions. We conclude that JAZ10.4 functions to attenuate signal output in the presence of JA and further suggest that the dominant-negative action of this splice variant involves protein-protein interaction through the ZIM/TIFY domain. The ability of JAZ10.4 to interact with MYC2 is consistent with a model in which a JAZ10.4-containing protein complex directly represses the activity of transcription factors that promote expression of JA response genes.

Figure 1.

Homo- and Heteromeric Interaction of Arabidopsis JAZ Proteins. (A) Y2H assay for homomeric JAZ interactions. Yeast strains coexpressing AD and BD fusions to each of the 12 full-length JAZ proteins were plated on media containing X-gal. Based on the intensity of LacZ-mediated blue-color formation, the strength of each interaction was rated as strong (e.g., JAZ1), medium (e.g., JAZ5), weak (e.g., JAZ6), or undetectable (e.g., JAZ8). See Table 1 for ratings on all interactions. (B) Immunoblot analysis of JAZ proteins in yeast strains used for Y2H assays shown in (A). Each lane contains total protein extracted from cells expressing AD and BD fusions of the indicated JAZ protein (e.g., “1” corresponds to JAZ1). AD-JAZ and BD-JAZ fusion proteins were detected with anti-HA (left panel) and anti-LexA (right panel) antibodies, respectively. (C) BiFC assay of JAZ3-JAZ3 homomeric interaction in planta. YFP fluorescence was detected in N. tabacum leaves coinfiltrated with Agrobacterium strains expressing JAZ3-nYFP and cYFP-JAZ3. 4',6-Diamidino-2-phenylindole (DAPI) staining shows the location of nuclei. The merged image shows colocalization of DAPI and YFP fluorescence.

Figure 2.

Requirement for the TIFY Motif in Homo- and Heteromeric Interaction of JAZ Proteins. (A) Schematic diagram of JAZ3 deletion constructs analyzed in (B) and (C). The diagram shows the highly conserved Jas (blue) and ZIM (pink, with TIFY motif) domains as well as the weakly conserved sequence (orange, labeled “N”) at the N terminus (Thines et al., 2007). (B) Y2H assay to assess the interaction of each JAZ3 deletion construct (AD fusion) shown in (A) with full-length JAZ3 and JAZ10.1 (BD fusions). (C) Immunoblot analysis of JAZ3 deletion proteins in yeast strains tested in (B). AD-JAZ3 fusion proteins were detected with an anti-HA antibody. (D) Amino acid sequence alignment of the ZIM domain in 12 Arabidopsis JAZs. The highly conserved TIFY motif (TIFF/YXG) is boxed. The in-color alignment depicts amino acids with similar hydrophobicity and was generated with the software BioEdit v 7.0.9. (E) Ala-scanning mutagenesis of the JAZ3 TIFY motif. Wild-type and mutant forms (e.g., T180A) of JAZ3 (AD fusions) were tested in the Y2H system for interaction with wild-type JAZ3 and JAZ10.1 (BD fusions). (F) Ala-scanning mutagenesis of the JAZ10.1 TIFY motif. Wild-type and mutant forms (e.g., T106A) of JAZ10.1 (AD fusions) were tested in the Y2H system for interaction with wild-type JAZ10.1 and JAZ6 (BD fusions).

Figure 3.

Identification of Three JAZ10 Variants Produced by Alternative Splicing. (A) Schematic diagram of alternatively spliced transcripts At5g13220.1, At5g13220.3, and At5g13220.4 encoding JAZ10.1, JAZ10.3, and JAZ10.4, respectively. Thick lines (black, translated; gray, untranslated) represent exons (labeled A to E). Modified exons in At5g13220.3 and At5g13220.4 result from use of alternative splice sites marked D′ and C′, respectively. (B) Alternative splicing differentially affects the sequence of the C-terminal Jas domain in the three JAZ10 splice variants. Amino acid sequences N-terminal to Ser-147 are identical in the three isoforms and are not shown. Amino acids comprising the Jas domain are underlined, whereas amino acids encoded by exon D are in bold. A frame shift in exon D of At5g13220.4 (resulting from alternative splicing of exon C) removes the Jas domain and adds 20 unrelated amino acid residues C-terminal to Ser-147. Asterisks indicate the stop codon. (C) RT-PCR analysis of JAZ10 transcripts in RNA isolated from wounded leaves. The location of primers used for RT-PCR is indicated by arrows in (A). PCR products were separated by gel electrophoresis, and the resulting gel was stained with ethidium bromide. Arrows denote transcripts encoding the three JAZ10 splice variants.

Figure 4.

Protein–Protein Interaction Characteristics of JAZ10 Isoforms. (A) Coronatine-dependent interaction of three JAZ10 splice variants with COI1 in the Y2H system. Yeast strains cotransformed with AD-JAZ10 isoforms (JAZ10.1, JAZ10.3, or JAZ10.4) and BD-COI1 were plated on media containing the indicated concentration of coronatine (COR). (B) Quantification of β-galactosidase activity in yeast strains shown in (A). Each strain was grown in the absence of coronatine (“0”) or in the presence of 30 or 200 μM coronatine (COR). Data show the mean ± sd of triplicate technical replicates. (C) Immunoblot analysis of JAZ10 and COI1 proteins in yeast cells tested in (A). JAZ10 splice variants and COI1 were detected with anti-HA and anti-LexA antibodies, respectively. (D) Interaction of three JAZ10 splice variants with MYC2 in the Y2H system. Yeast strains cotransformed with AD-MYC2 and BD-JAZ10 variants (JAZ10.1, JAZ10.3, and JAZ10.4) were plated on media containing X-gal. A yeast strain cotransformed with AD-MYC2 and an empty BD vector (pGILDA) is shown as a control (empty + MYC2). (E) Homo- and heteromeric interaction of JAZ10 splice variants. Yeast strains cotransformed with one of the three JAZ10 isoforms (as an AD fusion) and other members (as a BD fusion) of the JAZ family were plated on media containing X-gal. (F) Protein gel blot analysis of JAZ10 isoforms and JAZ3 in yeast strains used for the Y2H experiment shown in (E). JAZ10 splice variants and JAZ3 were detected with anti-HA and anti-LexA antibodies, respectively.

Figure 5.

Subcellular Localization of JAZ10 Splice Variants. JAZ10-YFP fusion proteins were transiently expressed by Agrobacterium-mediated transformation of tobacco epidermal cells. YFP fluorescence was detected with a confocal laser scanning microscope. DAPI fluorescence was used as a marker for the nucleus. YFP alone (not fused to JAZ10) is located in both the nucleus and the cytosol.

Figure 6.

Overexpression of JAZ10 Splice Variants Differentially Affects JA Responses. (A) to (D) JAZ10.4-overexpressing plants are male sterile. (A) Wild-type (left) and 35S-JAZ10.4 (right) inflorescence. (B) to (D) Close-up view of a wild-type flower (B) and JAZ10.4 flowers ([C] and [D]) that have short filaments and nondehisced anthers. (E) Differential effect of JAZ10 isoforms on JA-mediated inhibition of root growth. Germinated seedlings of the indicated genotype were grown for 7 d on MS medium containing 50 μM MeJA. Bar = 5 mm. (F) Quantification of JA-induced root growth inhibition of seedlings shown in (E). Data show the mean ± sd (n = 15 seedlings per genotype). [See online article for color version of this figure.]

Figure 7.

Stability of JAZ10 Splice Variants in Vivo. (A) Differential stability of three JAZ10 splice variants in response to JA. Transgenic seedlings expressing JAZ10.1-YFP, JAZ10.3-YFP, or JAZ10.4-YFP fusion proteins were treated either with water (Mock) or with the indicated concentration of MeJA (JA). Two hours after treatment, YFP signal in root tissue was visualized by fluorescence microscopy. The exposure times for each image in (A) and (B) were identical. (B) Proteasome-dependent degradation of JAZ10.1-YFP. Seedlings expressing the 35S-JAZ10.1-YFP transgene were pretreated with water or the 26S proteasome–specific inhibitor MG132 (50 μM) for 2 h, at which time seedlings were treated with either MeJA (JA; 10 μM) or water (Mock) for 2 h. YFP signal in root tissue was visualized by fluorescence microscopy.

Figure 8.

The TIFY Motif Is Required for Repression of JA Responses by JAZ10.4. (A) Y2H analysis of the effect of I107A and G111A TIFY mutations on the interaction of JAZ10.4 with JAZ6, JAZ10.1, JAZ10.3, and JAZ10.4. Yeast strains cotransformed with AD fusions of JAZ10.4 (or JAZ10.4^I->A and JAZ10.4^G->A mutants) and BD-JAZ fusions (JAZ6, JAZ10.1, JAZ10.3, and JAZ10.4) were plated on X-gal–containing medium. (B) Immunoblot analysis of JAZ proteins in yeast cells cotransformed with AD fusions of JAZ10.4 (or JAZ10.4^I->A and JAZ10.4^G->A mutants) and BD-JAZ10.1, which were tested in (A). AD- and BD-JAZ fusions were detected with anti-HA and anti-LexA antibodies, respectively. (C) to (F) Mutations (I107A and G111A) in the TIFY motif differentially lack the male-sterile phenotype conferred by overexpression of wild-type JAZ10.4. Complete lack of sterility is shown by the inflorescence ([C], left) and floral (D) phenotype of 35S-JAZ10.4^I->A plants. Partial lack of JAZ10.4-mediated sterility is indicated by development of a few mature siliques on the inflorescence of 35S-JAZ10.4^G->A plants ([C] and [D], right). 35S-JAZ10.4^G->A plants produced a mixture of wild-type-like flowers (E) and flowers with shortened anther filaments (F). (G) Differential effect of I107A and G111A mutations on JAZ10.4-mediated changes in root growth in the presence of JA. Seedlings of the indicated genotype were grown for 6 d on MS medium containing 50 μM MeJA. Bar = 5 mm. (H) Measurement of primary root length of seedlings shown in (G). Data are the mean ± sd (n = 19 seedlings per genotype).

Figure 9.

The I107A Mutant Form of JAZ10.4 Is Expressed and Targeted to the Nucleus. (A) Histochemical GUS staining of 35S-JAZ10.4-GUS and 35S-JAZ10.4^I->A-GUS transgenic seedlings. Seedlings were grown on MS medium containing kanamycin (50 μg/mL) for 14 d prior to transferring to a 48-well microtiter plate for GUS staining. (B) The male-sterile phenotype conferred by overexpression of JAZ10.4-GUS (left) is not shown by plants overexpressing the I107A mutant form of JAZ10.4-GUS (right). (C) The JA-insensitive root growth phenotype conferred by overexpression of JAZ10.4-GUS is not shown by plants overexpressing the I107A mutant form of JAZ10.4-GUS. (D) Subcellular localization of JAZ10.4-YFP and JAZ10.4^I->A-YFP fusion proteins that were transiently expressed by Agrobacterium-mediated transformation of tobacco epidermal cells. YFP and DAPI fluorescence was detected by confocal laser scanning microscopy.