Phytohormones are endogenous signalling molecules that regulate plant development, adaptation to the environment, and survival. Upon internal or external stimuli, hormones are quickly accumulated and ...perceived, which in turn activates specific signalling cascades regulating the appropriate physiological responses. In the last decade, great advances in understanding plant hormone perception mechanisms have been achieved. Among different methodological approaches, yeast-two hybrid (Y2H) assays played a pivotal role in the identification and analysis of plant hormone perception complexes. The Y2H assay is a rapid and straightforward technique that can be easily employed to identify small molecules directly modulating plant hormone perception complexes in a high-throughput manner. However, an Y2H chemical screen tends to isolate false positive molecules, and therefore a secondary in planta screen is required to confirm the genuine bioactivity of putative positive hits. This two-step screening approach can substantially save time and manual labor. This chapter focuses on the prospects of Y2H-based chemical genomic high-throughput screens applied to plant hormone perception complexes. Specifically, the method employed to carry out a chemical genomic screen to identify agonist and antagonist molecules of the phytohormone jasmonic acid in its conjugated form jasmonic acid-isoleucine (JA-Ile) is described. An easy in planta confirmation assay is also illustrated. However, this methodology can be easily extended to detect novel chemical compounds perturbing additional plant hormone receptor complexes. Finally, the high-throughput approach described here can also be implemented for the identification of molecules interfering with protein-protein interaction of plant complexes other than hormone receptors.
The TIFY proteins constitute a plant-specific super-family and they are involved in regulating many plant processes, such as development, defences and stress responses. The Jasmonate-ZIM-Domain (JAZ) ...proteins, the best-characterized sub-group of the TIFY family are key regulator of the jasmonic acid (JA) signalling pathway. Jasmonates regulate several aspects of plant development, and play a primary role in defence mechanisms as well as in plant responses to abiotic stresses. The TIFY family is well studied in dicots but poorly investigated in monocots. The present study reports an extensive genomic identification of TIFY proteins from Triticum aestivum. We identified 49 TIFY genes, which were annotated according to three sub-genomes (AABBDD) of T. aestivum. Following their clustering with Oryza sativa and Brachypodium distachyon, the 49 genes were grouped in 18 different TIFY homeologous subsets. Expression analyses of 6 representative TIFY genes on Tunisian durum wheat seedlings revealed their differential regulation by various stress treatment, including JA, ABA and salt stress. TIFY11a was specifically induced after salt treatment. Transgenic lines over-expressing TdTIFY11a showed higher germination and growth rates under high salinity conditions, compared to wild type plants. In summary, our results outline a relevant role of wheat TIFY proteins in promoting germination under salt stress.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Plant phenotypic plasticity determines plant adaptation to changing environments and agricultural productivity. Phytohormones are essential plant signalling molecules regulating this plasticity ...through complex signalling networks. Jasmonates (JAs) are key phytohormones regulating many aspects of growth, development and defence responses. An important role of JAs in tolerance to abiotic stresses is also emerging. The expression of JAZ (JASMONATE-ZIM-DOMAIN PROTEIN) genes, encoding for the key repressors in the JA-pathway, is regulated by multiple abiotic stresses, suggesting a role for the JAZ proteins in response to these stresses. The JAZ proteins belong to the TIFY family, well described in many plant species. However, only the role of few tomato JAZ proteins in response to microbial infection has been analysed so far. Here, we identify the members of the tomato TIFY family, and characterize them phylogenetically. In addition, we analyse the transcriptional regulation of several SlJAZ in response to abiotic stresses and hormone treatments both in root and leaves to assess their specific expression in response to stresses. Most SlJAZ are JA-induced and responsive to one or more abiotic stresses, providing clues for functional analysis of JAZ genes in abiotic responses in tomato.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Living organisms are part of a highly interconnected web of interactions, characterised by species nurturing, competing, parasitizing and preying on one another. Plants have evolved cooperative as ...well as defensive strategies to interact with neighbour organisms. Among these, the plant-fungus associations are very diverse, ranging from pathogenic to mutualistic. Our current knowledge of plant-fungus interactions suggests a sophisticated coevolution to ensure dynamic plant responses to evolving fungal mutualistic/pathogenic strategies. The plant-fungus communication relies on a rich chemical language. To manipulate the plant defence mechanisms, fungi produce and secrete several classes of biomolecules, whose modeof- action is largely unknown. Upon perception of the fungi, plants produce phytohormones and a battery of secondary metabolites that serve as defence mechanism against invaders or to promote mutualistic associations. These mutualistic chemical signals can be co-opted by pathogenic fungi for their own benefit. Among the plant molecules regulating plant-fungus interaction, phytohormones play a critical role since they modulate various aspects of plant development, defences and stress responses. Intriguingly, fungi can also produce phytohormones, although the actual role of fungalproduced phytohormones in plant-fungus interactions is poorly understood. Here, we discuss the recent advances in fungal production of phytohormone, their putative role as endogenous fungal signals and how fungi manipulate plant hormone balance to their benefits.
Coronatine (COR) facilitates entry of bacteria into the plant apoplast by stimulating stomata opening. COR-induced signaling events at stomata remain unclear. We found that the COR and jasmonate ...isoleucine (JA-Ile) co-receptor JAZ2 is constitutively expressed in guard cells and modulates stomatal dynamics during bacterial invasion
We analyzed tissue expression patterns of AtJAZ genes and measured stomata opening and pathogen resistance in loss- and gain-of-function mutants.
Arabidopsis jaz2 mutants are partially impaired in pathogen-induced stomatal closing and more susceptible to Pseudomonas. Gain-of-function mutations in JAZ2 prevent stomatal reopening by COR and are highly resistant to bacterial penetration. The JAZ2 targets MYC2, MYC3 and MYC4 directly regulate the expression of ANAC19, ANAC55 and ANAC72 to modulate stomata aperture. Due to the antagonistic interactions between the salicylic acid (SA) and JA defense pathways, efforts to increase resistance to biotrophs result in enhanced susceptibility to necrotrophs, and vice versa. Remarkably, dominant jaz2Djas mutants are resistant to Pseudomonas syringae but retain unaltered resistance against necrotrophs.
Our results demonstrate the existence of a COI1-JAZ2-MYC2,3,4-ANAC19,55,72 module responsible for the regulation of stomatal aperture that is hijacked by bacterial COR to promote infection. They also provide novel strategies for crop protection against biotrophs without compromising resistance to necrotrophs.
The phytohormone jasmonoyl-L-isoleucine (JA-Ile) regulates many stress responses and developmental processes in plants. A co-receptor complex formed by the F-box protein Coronatine Insensitive 1 ...(COI1) and a Jasmonate (JA) ZIM-domain (JAZ) repressor perceives the hormone. JA-Ile antagonists are invaluable tools for exploring the role of JA-Ile in specific tissues and developmental stages, and for identifying regulatory processes of the signaling pathway. Using two complementary chemical screens, we identified three compounds that exhibit a robust inhibitory effect on both the hormone-mediated COI-JAZ interaction and degradation of JAZ1 and JAZ9 in vivo. One molecule, J4, also restrains specific JA-induced physiological responses in different angiosperm plants, including JA-mediated gene expression, growth inhibition, chlorophyll degradation, and anthocyanin accumulation. Interaction experiments with purified proteins indicate that J4 directly interferes with the formation of the Arabidopsis (Arabidopsis thaliana) COI1-JAZ complex otherwise induced by JA. The antagonistic effect of J4 on COI1-JAZ also occurs in the liverwort Marchantia polymorpha, suggesting the mode of action is conserved in land plants. Besides JA signaling, J4 works as an antagonist of the closely related auxin signaling pathway, preventing Transport Inhibitor Response1/Aux-indole-3-acetic acid interaction and auxin responses in planta, including hormone-mediated degradation of an auxin repressor, gene expression, and gravitropic response. However, J4 does not affect other hormonal pathways. Altogether, our results show that this dual antagonist competes with JA-Ile and auxin, preventing the formation of phylogenetically related receptor complexes. J4 may be a useful tool to dissect both the JA-Ile and auxin pathways in particular tissues and developmental stages since it reversibly inhibits these pathways. One-sentence summary: A chemical screen identified a molecule that antagonizes jasmonate perception by directly interfering with receptor complex formation in phylogenetically distant vascular and nonvascular plants.
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 SCFCOI¹, 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.
The phytohormone 7-iso-(+)-jasmonoyl-L-isoleucine (JA-Ile) mediates plant defense responses against herbivore and pathogen attack, and thus increases plant resistance against foreign invaders. ...However, JA-Ile also causes growth inhibition; and therefore JA-Ile is not a practical chemical regulator of plant defense responses. Here, we describe the rational design and synthesis of a small molecule agonist that can upregulate defense-related gene expression and promote pathogen resistance at concentrations that do not cause growth inhibition in Arabidopsis. By stabilizing interactions between COI1 and JAZ9 and JAZ10 but no other JAZ isoforms, the agonist leads to formation of JA-Ile co-receptors that selectively activate the JAZ9-EIN3/EIL1-ORA59 signaling pathway. The design of a JA-Ile agonist with high selectivity for specific protein subtypes may help promote the development of chemical regulators that do not cause a tradeoff between growth and defense.
Plant immunity relies on a complex network of hormone signaling pathways in which jasmonic acid (JA) plays a central role. Successful microbial pathogens or symbionts have developed strategies to ...manipulate plant hormone signaling pathways to cause hormonal imbalances for their own benefit. These strategies include the production of plant hormones, phytohormone mimics, or effector proteins that target host components to disrupt hormonal signaling pathways and enhance virulence. Here, we describe the molecular details of the most recent and best-characterized examples of specific JA hormonal manipulation by microbes, which exemplify the ingenious ways by which pathogens can take control over the plant's hormone signaling network to suppress host immunity.
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