Contains a functional EAR-repression motifIncreased JA-sensitivity and JA-mediated gene expression in jaz7-1D suggests a part for JAZ7 in activation of JA responses. Having said that, JAZ proteins repress JA-responses via direct (EAR repressor motif) or indirect (NINJA-mediated) recruitment of co-repressor(s) plus the EAR motif associated with gene repression is present in JAZ7 (Kazan, 2006; Kagale et al., 2010). JAZ5, JAZ6, JAZ8 and JAZ13 also include EAR motifs needed for interactions using the co-repressor protein TOPLESS (TPL) (Kagale et al., 2010; Causier et al., 2012; Shyu et al. 2012; Thireault et al., 2015) along with the transcriptional repression functionActivation-tagged jaz7-1D mutant confers susceptibility to Fusarium oxysporum |Fig. 8. jaz7-1D shows improved JA-responsive gene expression below Fusarium infection. Gene expression was monitored in leaf tissue of untreated or F. oxysporum-challenged wild-type (WT), jaz7-1D and jaz7-1 plants at 2 and four d post-infection (dpi). Values are averages E of 3 biological replicates consisting of pools of five plants. Gene expression levels are relative towards the internal manage -actin genes.of this motif in JAZ8 has been confirmed (Shyu et al., 2012). The functionality on the predicted EAR motif found in JAZ7 has, having said that, not been experimentally tested. To decide no matter whether the JAZ7 EAR motif acts as a functional repressor, we fused an extended version of this motif to the carboxy terminus on the CUC1 (CUP-SHAPED COTYLEDON1) H-D-Asn-OH Autophagy transcription element which really should convert this transcriptional activator to an active repressor and generate plants with a cup-shaped cotyledon phenotype (Hiratsu et al., 2003). As shown in Fig. 11A, B, Arabidopsis plants transformed with 35S:CUC1-JAZ7EAR showed a common cup-shaped cotyledon formation, indicating that the JAZ7EAR motif can act as a repression domain. In the 13 selected T1 seedlings, severity of fused cotyledons varied, with eight displaying the cup-shaped cotyledon phenotype.JAZ7 interacts using the co-repressor TPLThe getting that ectopic overexpression of the JAZ7 EAR motif has repressor activity prompted us to determinewhether JAZ7 could interact with the co-repressor TPL, an interaction which had not but been demonstrated. Using JAZ5-TPL and JAZ8-TPL interactions as constructive controls (Causier et al., 2012; Shyu et al., 2012), an interaction in between JAZ7 and TPL in Y2H studies was observed (Fig. 11C). To decide if the interaction is mediated by way of the EAR motif of JAZ7, we generated a JAZ7 construct containing a mutated version of the EAR motif (JAZ7mEAR). No interaction involving JAZ7mEAR and TPL was observed (Fig. 11C). In stick to up in vivo studies, we conducted co-immunoprecipitation experiments amongst TPL and JAZ7, JAZ7mEAR, JAZ8 and JAZ5 through transient expression in Nicotiana benthamiana. Correlating using the Y2H final results, TPL interactions with JAZ7, JAZ5 and JAZ8 had been observed, but when the EAR motif of JAZ7 was mutated, the TPL and JAZ7 interaction was strongly diminished (Fig. 12). These results strongly assistance an in planta interaction involving TPL and JAZ7 mediated by means of its EAR-motif.2378 | Thatcher et al.Fig. 9. JAZ transcripts are up-regulated in jaz7-1D. JAZ expression was monitored in leaf tissue of (A) untreated or (B) F. oxysporum-challenged wildtype (WT), jaz7-1D and jaz7-1 plants at 4 d post-infection (dpi). Values are expressed relative to WT values at that time-point. Values are averages E of 3 biological replicates.
