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

Reconstructing the TIR side of the Myddosome: a paradigm for TIR-TIR interactions?

Members of the Toll-like receptor and interleukin-1 (IL-1) receptor families all signal via Toll/IL-1R (TIR) domain-driven assemblies with adaptors such as MyD88. We here combine the mammalian two-hybrid system MAPPIT and saturation mutagenesis to complement and extend crystallographic and nuclear magnetic resonance data, and reveal how TIR domains interact. We fully delineate the interaction sites on the MyD88 TIR domain for homo-oligomerization and for interaction with Mal and TLR4. Interactions between three sites drive MyD88 homo-oligomerization. The BB-loop interacts with the alpha E-helix, explaining how BB-loop mimetics inhibit MyD88 signaling. The alpha C'-helix interacts symmetrically. The MyD88 TIR domains thus assemble into a left-handed helix, compatible with the Myddosome death domain crystal structure. This assembly explains activation of MyD88 by Mal and by an oncogenic mutation, and regulation by phosphorylation. These findings provide a paradigm for the interaction of mammalian TIR domains

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

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

A dual sgRNA approach for functional genomics in Arabidopsis thaliana

Reverse genetics uses loss-of-function alleles to interrogate gene function. The advent of CRISPR/Cas9-based gene editing now allows the generation of knock-out alleles for any gene and entire gene families. Even in the model plant Arabidopsis thaliana, gene editing is welcomed as T-DNA insertion lines do not always generate null alleles. Here, we show efficient generation of heritable mutations in Arabidopsis using CRISPR/Cas9 with a workload similar to generating overexpression lines. We obtain for several different genes Cas9 null-segregants with bi-allelic mutations in the T2 generation. While somatic mutations were predominantly generated by the canonical non-homologous end joining (cNHEJ) pathway, we observed inherited mutations that were the result of synthesis-dependent microhomology-mediated end joining (SD-MMEJ), a repair pathway linked to polymerase theta (PolQ). We also demonstrate that our workflow is compatible with a dual sgRNA approach in which a gene is targeted by two sgRNAs simultaneously. This paired nuclease method results in more reliable loss-of-function alleles that lack a large essential part of the gene. The ease of the CRISPR/Cas9 workflow should help in the eventual generation of true null alleles of every gene in the Arabidopsis genome, which will advance both basic and applied plant research

The molecular basis of JAZ-MYC coupling, a protein-protein interface essential for plant response to stressors

The jasmonic acid (JA) signaling pathway is one of the primary mechanisms that allow plants to respond to a variety of biotic and abiotic stressors. Within this pathway, the JAZ repressor proteins and the basic helix-loop-helix (bHLH) transcription factor MYC3 play a critical role. JA is a volatile organic compound with an essential role in plant immunity. The increase in the concentration of JA leads to the decoupling of the JAZ repressor proteins and the bHLH transcription factor MYC3 causing the induction of genes of interest. The primary goal of this study was to identify the molecular basis of JAZ-MYC coupling. For this purpose, we modeled and validated 12 JAZ-MYC3 3D in silico structures and developed a molecular dynamics/machine learning pipeline to obtain two outcomes. First, we calculated the average free binding energy of JAZ-MYC3 complexes, which was predicted to be -10.94 +/-2.67 kJ/mol. Second, we predicted which ones should be the interface residues that make the predominant contribution to the free energy of binding (molecular hotspots). The predicted protein hotspots matched a conserved linear motif SL••FL•••R, which may have a crucial role during MYC3 recognition of JAZ proteins. As a proof of concept, we tested, both in silico and in vitro, the importance of this motif on PEAPOD (PPD) proteins, which also belong to the TIFY protein family, like the JAZ proteins, but cannot bind to MYC3. By mutating these proteins to match the SL••FL•••R motif, we could force PPDs to bind the MYC3 transcription factor. Taken together, modeling protein-protein interactions and using machine learning will help to find essential motifs and molecular mechanisms in the JA pathway

A network of stress-related genes regulates hypocotyl elongation downstream of selective auxin perception

The plant hormone auxin, a master coordinator of development, regulates hypocotyl elongation during seedling growth. We previously identified the synthetic molecule RubNeddin 1 (RN1), which induces degradation of the AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) transcriptional repressors INDOLE-3-ACETIC ACID-INDUCIBLE3 (IAA3) and IAA7 in planta and strongly promotes hypocotyl elongation. In the present study, we show that despite the structural similarity of RN1 to the synthetic auxin 2,4-dichlorophenoxyacetic-acid (2,4-D), direct treatments with these compounds in Arabidopsis (Arabidopsis thaliana) result in distinct effects, possibly due to enhanced uptake of RN1 and low-level, chronic release of 2,4-D from RN1 in planta. We confirm RN1-induced hypocotyl elongation occurs via specific TRANSPORT INHIBITOR RESISTANT1 (TIR1)/AUXIN SIGNALING F-BOX (AFB) receptor-mediated auxin signaling involving TIR1, AFB2, and AFB5. Using a transcriptome profiling strategy and candidate gene approach, we identify the genes ZINC FINGER OF ARABIDOPSIS THALIANA10 (ZAT10), ARABIDOPSIS TOXICOS EN LEVADURA31 (ATL31), and WRKY DNA-BINDING PROTEIN33 (WRKY33) as being rapidly upregulated by RN1, despite being downregulated by 2,4-D treatment. RN1-induced expression of these genes also occurs via TIR1/AFB-mediated auxin signaling. Our results suggest both hypocotyl elongation and transcription of these genes are induced by RN1 via the promoted degradation of the AUX/IAA transcriptional repressor IAA7. Moreover, these three genes, which are known to be stress-related, act in an inter-dependent transcriptional regulatory network controlling hypocotyl elongation. Together, our results suggest ZAT10, ATL31, and WRKY33 take part in a common gene network regulating hypocotyl elongation in Arabidopsis downstream of a selective auxin perception module likely involving TIR1, AFB2, and AFB5 and inducing the degradation of IAA7