Ghent University Faculty of Sciences – Department of Plant Biotechnology and Bioinformatics Identification and characterization of E3 ubiquitin ligases involved in jasmonate signalling Astrid Nagels Durand Thesis submitted in partial fulfilment of requirements for the degree of Doctor (PhD) in Sciences: Biotechnology July 2015 Promotors: Prof. Dr. Alain Goossens Faculty of Sciences, Department Plant Biotechnology and Bioinformatics, Ghent University, Belgium VIB Department of Plant Systems Biology, Secondary Metabolism group, B-9052 Ghent, Belgium Dr. Laurens Pauwels Faculty of Sciences, Department Plant Biotechnology and Bioinformatics, Ghent University, Belgium VIB Department of Plant Systems Biology, Secondary Metabolism group, B-9052 Ghent, Belgium This research was performed in the Department of Plant Systems Biology of the Flanders Institute for Biotechnology This work was supported by The Research Foundation-Flanders (FWO) through the projects GA13111N and G005212N. ii EXAM COMMISSION Chairman Prof. Dr. Lieven De Veylder Department of Plant Biotechnology and Bioinformatics Faculty of Sciences Ghent University Promotors Prof. Dr. Alain Goossens Department of Plant Biotechnology and Bioinformatics (Secretary) Faculty of Sciences Ghent University Dr. Laurens Pauwels Department of Plant Biotechnology and Bioinformatics (Secretary) Faculty of Sciences Ghent University Promotion commission Prof. Dr. Rudi Beyaert Department of Biomedical Molecular Biology Faculty of Sciences Ghent University Dr. Steven Spoel * Institute of Molecular Plant Sciences School of Biological Sciences University of Edinburgh (UK) Prof. Dr. Steven Maere Department of Plant Biotechnology and Bioinformatics Faculty of Sciences Ghent University Prof. Dr. Tina Kyndt * Department of Molecular Biotechnology Faculty of Bioscience Engineering Ghent University Dr. Barbara De Coninck * Centre of Microbial and Plant Genetics Katholieke Universiteit Leuven Dr. Jefri Heyman * Department of Plant Biotechnology and Bioinformatics Faculty of Sciences Ghent University *Members of the reading committee iii iv Summary Summary Ubiquitination, the covalent attachment of ubiquitin (Ub) to a target protein, is a post- translational modification that is involved in most, if not all, biological processes in eukaryotic organisms. Ubiquitination of a target protein requires the consecutive action of three enzymes (E1, E2 and E3). The E3 Ub ligase is of special interest because it is responsible for recruiting the target protein, determining the specificity of this modification. The Ub system contributes to the regulation of the production, perception and signal transduction of plant hormones. These hormones are critical for correct plant development and adaptation to the variable environmental conditions plants are exposed to during their life cycle. Jasmonic acid and its derivatives, known as jasmonates (JAs), act as signalling compounds regulating plant development and plant responses to various biotic and abiotic stress conditions. Even though more than 1500 E3 ligases are encoded by the genome of the model plant Arabidopsis thaliana , very few have been associated with JA biosynthesis or signal transduction. Best characterized is the SCF COI1 E3 ligase, a component of the JA-receptor. Making use of transcriptomic data and a bio-informatics approach, we have identified a set of 22 E3 ligases with a putative role in JA-signalling (see Chapter 3), further extending the connection between the Ub system and JA-signalling. Despite the large number of E3s present in plants, very few ubiquitination targets have been associated with a specific E3 ligase. Here, we used a customized Tandem Affinity Purification (TAP) platform to identify E3-interacting proteins that constitute potential ubiquitination substrates. Customization consisted of inactivation of the Ub-ligase activity of the E3s by introduction of specific mutations in their RING domain, preventing target ubiquitination and resulting in stabilization of the E3-target interaction. Mutated E3s where then used as baits in purification experiments. This approach led to the identification of potential targets of several E3 ligases with a predicted function in JA-signalling, including AE31, ATL23, BRIZ1/2, KEG and RGLG3/4 (see Chapter 3 and 5). To cope with the restraints that in vitro ubiquitination assays impose on the ability to demonstrate ubiquitination of a protein by a specific E3 ligase, we attempted to develop an alternative in vivo assay. The yeast S. cerevisiae was chosen as a heterologous host to maintain modification of the potential target protein by related or unrelated E3s to a minimum. The principle of this assay is based on the assembly of plant E3 ligases with the yeast ubiquitination machinery. Upon expression of the suspected target-E3 couple, the target would be ubiquitinated, and this should be dependent on co-expression of the (functional) E3. Because yeast is relatively resistant to proteasome inhibition, and target ubiquitination in most cases leads to proteasome mediated degradation of the latter, we first engineered a strain that had increased sensitivity to proteasome inhibition and determined which treatment was most efficient to attain this inhibition (see Chapter 3). We also generated a set of MultiSite Gateway compatible vectors for the expression of genes in S. cerevisiae , as this cloning technology is vi Summary widely used by plant researchers (see Chapter 4). The assay was tested using the heterodimeric E3 ligase BRIZ1/2 and its candidate target LARP6b. Although we could not detect ubiquitination of the LARP6b protein after purification of the protein under denaturing conditions, we did detect a modified form of the protein and this was dependent on co- expression of both BRIZ1 and BRIZ2 (see Chapter 3). Further work focussed on the redundant E3 ligases RGLG3/4 that were reported to be involved in JA-signalling. We identified PKC-θ INTERACTING PROTEIN COUSIN OF THIOREDOXIN 1 (PICOT1/GRXS17) and HEMOGLOBIN 3 (GLB3) as potential targets of RGLG3/4 using the customized TAP approach described above, followed by confirmation of direct interaction using yeast two-hybrid (Y2H). We further demonstrated RGLG3/4 mediate PICOT1 poly-ubiquitination and subsequent proteasomal degradation. PICOT1 is an iron-sulfur (Fe-S) cluster binding glutathioredoxin that localizes to the nucleus and the cytoplasm in Arabidopsis. The use of PICOT1 as a bait in TAP experiments further showed that PICOT1 forms a protein complex with nearly all components of the cytosolic Fe-S assembly pathway as well as with some other known Fe-S proteins. In contrast to the human and yeast orthologs of PICOT1, which function in Fe-homeostasis and Fe-S cluster assembly, the activity of the PICOT1- interacting Fe-S protein XANTHINE DEHYDROGENASE 1 was only marginally affected in picot1 seedlings. In addition to Fe-S proteins, PICOT1 interacted with several proteins involved in tRNA thiolation. Accordingly, plants carrying mutations in genes involved in tRNA modification were found to resemble picot1 plants phenotypically (see Chapter 5). Our most remarkable finding, however, was that PICOT1 potentially regulates the JA- receptor F-box component COI1. The thioredoxin domain of PICOT1 mediates direct interaction with COI1 in the nucleus. In addition, the short-root phenotype of picot1 seedlings was partially rescued in coi1-16picot1 lines. Despite their physical and genetic interaction however, picot1 seedlings did not show alterations in their response to JA. Transcriptome analysis of picot1, coi1-16 and coi1-16picot1 seedlings revealed a set of genes are upregulated in coi1-16 in a PICOT1-dependent manner. These genes were not related to JA-signalling, and included a set of co-regulated genes that are expressed in response to infection with Hyaloperonospera arabidopsidis (previously known as H. parasitica ). We therefore propose PICOT1 regulates the JA-independent function of COI1 (see Chapter 6). In conclusion, a bio-informatics approach combined with customized protein-protein interaction mining was effective in extending our knowledge on the role of the Ub system in JA-signalling. Additionally, we have identified the E3 ligases RGLG3/4 and their target PICOT1 as molecular players in COI1-dependent but JA-independent signalling. vii Summary viii Samenvatting Samenvatting Ubiquitinatie, de covalente binding van ubiquitine (Ub) aan een substraateiwit, is een post-translationele modificatie die betrokken is in bijna alle biologische processen in eukaryote organismen. Ubiquitinatie van een substraat vereist de opeenvolgende werking van drie enzymen (E1, E2 en E3). Het E3 ligase is van uitzonderlijk belang omdat het verantwoordelijk is voor de rekrutering van het substraateiwit en op die manier de specificiteit van de modificatie bepaalt. Het Ub systeem draagt bij aan de regulatie van de aanmaak, de perceptie en de signaaltransductie van plantenhormonen. Deze hormonen zijn van cruciaal belang voor de correcte ontwikkeling en voor aanpassing van planten aan de wisselende omstandigheden waaraan deze gedurende hun levenscyclus worden blootgesteld. Jasmijnzuur en derivaten, gekend als jasmonaten (JAs), werken als signaalmoleculen die plantenontwikkeling en verdedigingsresponsen tegen verscheidene biotische en abiotische stresscondities reguleren. Hoewel er meer dan 1500 E3 ligasen gecodeerd zijn in het genoom van de modelplant Arabidopsis thaliana (zandraket), zijn er slechts enkelen geassocieerd met JA-biosynthese of signaaltransductie.