The ubiquitin system and jasmonate signaling

The ubiquitin (Ub) system is involved in most, if not all, biological processes in eukaryotes. The major specificity determinants of this system are the E3 ligases, which bind and ubiquitinate specific sets of proteins and are thereby responsible for target recruitment to the proteasome or other cellular processing machineries. The Ub system contributes to the regulation of the production, perception and signal transduction of plant hormones. Jasmonic acid (JA) and its derivatives, known as jasmonates (JAs), act as signaling compounds regulating plant development and plant responses to various biotic and abiotic stress conditions. We provide here an overview of the current understanding of the Ub system involved in JA signaling

The JAZ proteins: a crucial interface in the jasmonate signaling cascade

Jasmonates are phytohormones that regulate many aspects of plant growth, development, and defense. Within the signaling cascades that are triggered by jasmonates, the JASMONATE-ZIM DOMAIN (JAZ) repressor proteins play a central role. The endogenous bioactive JA-Ile conjugate mediates the binding of JAZ proteins to the F-box protein CORONATINE INSENSITIVE1 (COI1), part of the Skp1/Cullin/F-box SCF(COI1) ubiquitin E3 ligase complex. Upon the subsequent destruction of the JAZ proteins by the 26S proteasome, multiple transcription factors are relieved from JAZ-mediated repression, allowing them to activate their respective downstream responses. However, many questions remain regarding the targets, specificity, function, and regulation of the different JAZ proteins. Here, we review recent studies on the model plant Arabidopsis thaliana that provided essential and novel insights. JAZ proteins have been demonstrated to interact with a broad array of transcription factors that each control specific downstream processes. Recruitment of the corepressor TOPLESS unveiled a mechanism for JAZ-mediated gene repression. Finally, the presence of JAZ proteins was also found to be regulated by alternative splicing and interactions with proteins from other hormonal signaling pathways. Overall, these contemporary findings underscore the value of protein-protein interaction studies to acquire fundamental insight into molecular signaling pathways

A multiSite gateway™ vector set for the functional analysis of genes in the model Saccharomyces cerevisiae

<p>Abstract</p> <p>Background</p> <p>Recombinatorial cloning using the Gateway^TM technology has been the method of choice for high-throughput omics projects, resulting in the availability of entire ORFeomes in Gateway^TM compatible vectors. The MultiSite Gateway^TM system allows combining multiple genetic fragments such as promoter, ORF and epitope tag in one single reaction. To date, this technology has not been accessible in the yeast <it>Saccharomyces cerevisiae</it>, one of the most widely used experimental systems in molecular biology, due to the lack of appropriate destination vectors.</p> <p>Results</p> <p>Here, we present a set of three-fragment MultiSite Gateway^TM destination vectors that have been developed for gene expression in <it>S. cerevisiae</it> and that allow the assembly of any promoter, open reading frame, epitope tag arrangement in combination with any of four auxotrophic markers and three distinct replication mechanisms. As an example of its applicability, we used yeast three-hybrid to provide evidence for the assembly of a ternary complex of plant proteins involved in jasmonate signalling and consisting of the JAZ, NINJA and TOPLESS proteins.</p> <p>Conclusion</p> <p>Our vectors make MultiSite Gateway^TM cloning accessible in <it>S. cerevisiae</it> and implement a fast and versatile cloning method for the high-throughput functional analysis of (heterologous) proteins in one of the most widely used model organisms for molecular biology research.</p

Jasmonate and auxin perception : how plants keep F-boxes in check

Phytohormones regulate the plasticity of plant growth and development, and responses to biotic and abiotic stresses. Many hormone signal transduction cascades involve ubiquitination and subsequent degradation of proteins by the 26S proteasome. The conjugation of ubiquitin to a substrate is facilitated by the E1 activating, E2 conjugating, and the substrate-specifying E3 ligating enzymes. The most prevalent type of E3 ligase in plants is the Cullin-RING ligase (CRL)-type, with F-box proteins (FBPs) as the substrate recognition component. The activity of these SKP-Cullin-F-box (SCF) complexes needs to be tightly regulated in time and place. Here, we review the regulation of SCF function in plants on multiple levels, with a focus on the auxin and jasmonate SCF-type receptor complexes. We discuss in particular the relevance of protein-protein interactions and post-translational modifications as mechanisms to keep SCF functioning under control. Additionally, we highlight the unique property of SCFTIR1/AFB and SCFCOI1 to recognize substrates by forming co-receptor complexes. Finally, we explore how engineered selective agonists can be used to study and uncouple the outcomes of the complex auxin and jasmonate signaling networks that are governed by these FBPs

Assessment of the susceptibility to pests and diseases of 36 apple cultivars in four low-input organic orchards in France

One of the keystones of the organic orchard is the cultivar choice as one element of pest and disease control. However, few exhaustive data sets concerning the cultivar susceptibility to pests and diseases are available for growers. In order to identify cultivars adapted to organic production methods, the susceptibility to scab, aphids and powdery mildew and the agronomic properties of 36 cultivars were assessed in four French sites under different pedo-climatic conditions. Different levels of susceptibility to scab were observed for 23 scab Vf-resistant and 13 other cultivars. In the North of France where Vf resistance is overcome, the Vf cultivars displayed different levels of scab severity. A high variability in the susceptibility to aphids was observed whereas susceptibility to powdery mildew varied less. The analysis of susceptibility properties, yield and fruit quality, fruit storability and tree behaviour permitted to identify a set of interesting cultivars according to the site

A user-friendly platform for yeast two-hybrid library screening using next generation sequencing

Yeast two-hybrid (Y2H) is a well-established genetics-based system that uses yeast to selectively display binary protein-protein interactions (PPIs). To meet the current need to unravel complex PPI networks, several adaptations have been made to establish mediumto high-throughput Y2H screening platforms, with several having successfully incorporated the use of the next-generation sequencing (NGS) technology to increase the scale and sensitivity of the method. However, these have been to date mainly restricted to the use of fully annotated custom-made open reading frame (ORF) libraries and subject to complex downstream data processing. Here, a streamlined Y2H library screening strategy, based on integration of Y2H with NGS, called Y2H-seq, was developed, which allows efficient and reliable screening of Y2H cDNA libraries. To generate proof of concept, the method was applied to screen for interaction partners of two key components of the jasmonate signaling machinery in the model plant Arabidopsis thaliana, resulting in the identification of several previously reported as well as hitherto unknown interactors. Our Y2H-seq method offers a user-friendly, specific and sensitive screening method that allows identification of PPIs without prior knowledge of the organism's ORFs, thereby extending the method to organisms of which the genome has not entirely been annotated yet. The quantitative NGS readout allows to increase genome coverage, thereby overcoming some of the bottlenecks of current Y2H technologies, which will further strengthen the value of the Y2H technology as a discovery platform

Central Effects of Beta-Blockers May Be Due to Nitric Oxide and Hydrogen Peroxide Release Independently of Their Ability to Cross the Blood-Brain Barrier

Propranolol is the first-line treatment for infants suffering from infantile hemangioma. Recently, some authors raised the question of potential neurologic side effects of propranolol due to its lipophilic nature and thus its ability to passively cross the blood-brain barrier (BBB) and accumulate into the brain. Hydrophilic beta-blockers, such as atenolol and nadolol, where therefore introduced in clinical practice. In addition to their classical mode of action in the brain, circulating factors may modulate the release of reactive oxygen/nitrogen species (ROS/RNS) from endothelial cells that compose the BBB without entering the brain. Due to their high capacity to diffuse across membranes, ROS/RNS can reach neurons and modify their activity. The aim of this study was to investigate other mechanisms of actions in which these molecules may display a central effect without actually crossing the BBB. We first performed an oral treatment in mice to measure the accumulation of propranolol, atenolol and nadolol in different brain regions in vivo. We then evaluated the ability of these molecules to induce the release of nitric oxide (NO) and hydrogen peroxide (H2O2) ex vivo in the hypothalamus. As expected, propranolol is able to cross the BBB and is found in brain tissue in higher amounts than atenolol and nadolol. However, all of these beta-blockers are able to induce the secretion of signaling molecules (i.e., NO and/or H2O2) in the hypothalamus, independently of their ability to cross the BBB, deciphering a new potential deleterious impact of hydrophilic beta-blockers in the brain