Jasmonates (JAs) trigger an important transcriptional reprogramming of plant cells to modulate both basal development and stress responses. In spite of the importance of transcriptional regulation, only one transcription factor (TF), the Arabidopsis thaliana basic helix-loop-helix MYC2, has been described so far as a direct target of JAZ repressors. By means of yeast two-hybrid screening and tandem affinity purification strategies, we identified two previously unknown targets of JAZ repressors, the TFs MYC3 and MYC4, phylogenetically closely related to MYC2. We show that MYC3 and MYC4 interact in vitro and in vivo with JAZ repressors and also form homo- and heterodimers with MYC2 and among themselves. They both are nuclear proteins that bind DNA with sequence specificity similar to that of MYC2. Loss-of-function mutations in any of these two TFs impair full responsiveness to JA and enhance the JA insensitivity of myc2 mutants. Moreover, the triple mutant myc2 myc3 myc4 is as impaired as coi1-1 in the activation of several, but not all, JA-mediated responses such as the defense against bacterial pathogens and insect herbivory. Our results show that MYC3 and MYC4 are activators of JA-regulated programs that act additively with MYC2 to regulate specifically different subsets of the JA-dependent transcriptional response.

Summary: Discovery of the jasmonate ZIM‐domain (JAZ) repressors defined the core jasmonate (JA) signalling module as COI1–JAZ–MYC2, and allowed a full view of the JA signalling pathway from hormone perception to transcriptional reprogramming. JAZ proteins are repressors of MYC2 and targets of SCF^COI1, which is the likely jasmonate receptor. Upon hormone perception, JAZ repressors are degraded by the proteasome releasing MYC2 and allowing the activation of JA responses. All members of the JAZ family share two conserved domains, the Jas motif, required for JAZ interactions with MYC2 and COI1, and the ZIM domain, the function of which is so far unknown. Here, we show that the ZIM domain acts as a protein–protein interaction domain mediating homo‐ and heteromeric interactions between JAZ proteins. These JAZ–JAZ interactions are independent of the presence of the hormone. The observation that only a few members of the JAZ family form homo‐ and heteromers may suggest the relevance of these proteins in the regulation of JA signalling. Interestingly, the JAZ3ΔJas protein interacts with several JAZ proteins, providing new clues to understanding the dominant JA insensitivity promoted by truncated JAZΔJas proteins. We also provide evidence that the Jas motif mediates the hormone‐dependent interaction between Arabidopsis JAZ3 and COI1, and further confirm that the Jas motif is required and sufficient for Arabidopsis JAZ3–MYC2 interaction. Finally, we show that interaction with MYC2 is a common feature of the JAZ family, as most JAZ proteins can bind MYC2 in pull‐down and yeast two‐hybrid assays.

Pathogenicity of Pseudomonas syringae is dependent on a type III secretion system, which secretes a suite of virulence effector proteins into the host cytoplasm, and the production of a number of toxins such as coronatine (COR), which is a mimic of the plant hormone jasmonate-isoleuce (JA-Ile). Inside the plant cell, effectors target host molecules to subvert the host cell physiology and disrupt defenses. However, despite the fact that elucidating effector action is essential to understanding bacterial pathogenesis, the molecular function and host targets of the vast majority of effectors remain largely unknown. Here, we found that effector HopX1 from Pseudomonas syringae pv. tabaci (Pta) 11528, a strain that does not produce COR, interacts with and promotes the degradation of JAZ proteins, a key family of JA-repressors. We show that hopX1 encodes a cysteine protease, activity that is required for degradation of JAZs by HopX1. HopX1 associates with JAZ proteins through its central ZIM domain and degradation occurs in a COI1-independent manner. Moreover, ectopic expression of HopX1 in Arabidopsis induces the expression of JA-dependent genes, represses salicylic acid (SA)-induced markers, and complements the growth of a COR-deficient P. syringae pv. tomato (Pto) DC3000 strain during natural bacterial infections. Furthermore, HopX1 promoted susceptibility when delivered by the natural type III secretion system, to a similar extent as the addition of COR, and this effect was dependent on its catalytic activity. Altogether, our results indicate that JAZ proteins are direct targets of bacterial effectors to promote activation of JA-induced defenses and susceptibility in Arabidopsis. HopX1 illustrates a paradigm of an alternative evolutionary solution to COR with similar physiological outcome. Bacterial plant pathogens secrete toxins and inject effector proteins into the host cells to promote infection, and the identification of the individual functions of these molecules is essential to understand the infective process. Remarkably, some Pseudomonas strains have evolved a sophisticated strategy for manipulating hormonal balance by producing the toxin coronatine (COR), which mimics the plant hormone jasmonate-isoleucine (JA-Ile). The JA-Ile pathway plays a key role in plant immunity by activating defenses against fungal pathogens, while promoting bacterial growth by inhibiting the salicylic acid (SA)-dependent defenses required for Pseudomonas resistance. Here, we report that the effector HopX1 from a Pseudomonas syringae strain that does not produce COR exploits an alternative evolutionary strategy to activate the JA-Ile pathway. We show that HopX1 encodes a cysteine protease that interacts with and promotes the degradation of key JA pathway repressors, the JAZ proteins. Correspondingly, ectopically expressing HopX1 in the model plant Arabidopsis induces the expression of JA-dependent genes, and natural infection with Pseudomonas producing HopX1 promotes bacterial growth in a similar fashion to COR. Our results highlight a novel example by which a bacterial effector directly manipulates core regulators of hormone signaling to facilitate infection.