Functional characterization of non-JAZ TIFY proteins in Arabidopsis thaliana

In this thesis, we initiated the characterization of the non-JAZ TIFY proteins TIFY8 and PPD proteins. Given their similarity to the JAZ proteins, we decided to study their putative role in JA signalling by analysis of JA-responsiveness. Moreover, and since we previously demonstrated that both PPD and TIFY8 proteins are able to interact with NINJA, we also focused on the study of their interacting partners by means of Tandem affinity purification (TAP) and yeast two-hybrid (Y2H), with the aim to unravel the formation of specific protein complexes and, ultimately, the function these non-JAZ TIFY proteins

Із зали засідань Президії НАН України

20 червня 2012 року відбулося виїзне засідання Президії Національної академії наук України на запрошення президента — генерального конструктора Державного підприємства «АНТОНОВ» академіка НАН України Д.С. Ківи

Jasmonate signalling: a copycat of auxin signalling?

Plant hormones regulate almost all aspects of plant growth and development. The past decade has provided breakthrough discoveries in phytohormone sensing and signal transduction, and highlighted the striking mechanistic similarities between the auxin and jasmonate (JA) signalling pathways. Perception of auxin and JA involves the formation of co-receptor complexes in which hormone-specific E3-ubiquitin ligases of the SKP1-Cullin-F-box protein (SCF) type interact with specific repressor proteins. Across the plant kingdom, the Aux/IAA and the JASMONATE-ZIM DOMAIN (JAZ) proteins correspond to the auxin- and JA-specific repressors, respectively. In the absence of the hormones, these repressors form a complex with transcription factors (TFs) specific for both pathways. They also recruit several proteins, among which the general co-repressor TOPLESS, and thereby prevent the TFs from activating gene expression. The hormone-mediated interaction between the SCF and the repressors targets the latter for 26S proteasome-mediated degradation, which, in turn, releases the TFs to allow modulating hormone-dependent gene expression. In this review, we describe the similarities and differences in the auxin and JA signalling cascades with respect to the protein families and the protein domains involved in the formation of the pathway-specific complexes

Yeast two-hybrid analysis of jasmonate signaling proteins

Protein-protein interaction studies are crucial to unravel how jasmonate (JA) signals are transduced. Among the different techniques available, yeast two-hybrid (Y2H) is commonly used within the JA research community to identify proteins belonging to the core JA signaling module. The technique is based on the reconstitution of a transcriptional activator that drives the reporter gene expression upon protein-protein interactions. The method is sensitive and straightforward and can be adapted for different approaches. In this chapter, we provide a detailed protocol to perform targeted Y2H assays to test known proteins and/or protein domains for direct interaction in a pairwise manner and present the possibility to study ternary protein complexes through Y3H

El dictado

Tras definir qué es el dictado y las posibilidades que ofrece, se recopilan diferentes métodos para el dictado y su corrección, explicando variaciones de los mismos, realizando sugerencias e indicando el objetivo que se persigue con cada uno de ellos. Finalmente se recopilan actividades que tienen como eje central el dictado, señalando el nivel educativo al que van dirigidos, el tema que tratan, el método recomendado para llevarlo a cabo y el estilo del texto.ExtremaduraCPR de Navalmoral de la Mata (Cáceres); Avda. San Isidro, 10; Apdo. 221; 10300 Navalmoral de la Mata (Cáceres); +34927016720; +34927016721; [email protected]

The non-JAZ TIFY protein TIFY8 from Arabidopsis thaliana is a transcriptional repressor

Jasmonate (JA) signalling is mediated by the JASMONATE-ZIM DOMAIN (JAZ) repressor proteins, which are degraded upon JA perception to release downstream responses. The ZIM protein domain is characteristic of the larger TIFY protein family. It is currently unknown if the atypical member TIFY8 is involved in JA signalling. Here we show that the TIFY8 ZIM domain is functional and mediated interaction with PEAPOD proteins and NINJA. TIFY8 interacted with TOPLESS through NINJA and accordingly acted as a transcriptional repressor. TIFY8 expression was inversely correlated with JAZ expression during development and after infection with Pseudomonas syringae. Nevertheless, transgenic lines with altered TIFY8 expression did not show changes in JA sensitivity. Despite the functional ZIM domain, no interaction with JAZ proteins could be found. In contrast, TIFY8 was found in protein complexes involved in regulation of dephosphorylation, deubiquitination and O-linked N-acetylglucosamine modification suggesting an important role in nuclear signal transduction

The non-JAZ TIFY protein TIFY8 from Arabidopsis is a transcriptional repressor

Jasmonate (JA) signalling is mediated by the JASMONATE-ZIM DOMAIN (JAZ) repressor proteins, which are degraded upon JA perception to release downstream responses. The ZIM protein domain is characteristic of the larger TIFY protein family. It is currently unknown if the atypical member TIFY8 is involved in JA signalling. Here we show that the TIFY8 ZIM domain is functional and mediated interaction with PEAPOD proteins and NINJA. TIFY8 interacted with TOPLESS through NINJA and accordingly acted as a transcriptional repressor. TIFY8 expression was inversely correlated with JAZ expression during development and after infection with Pseudomonas syringae. Nevertheless, transgenic lines with altered TIFY8 expression did not show changes in JA sensitivity. Despite the functional ZIM domain, no interaction with JAZ proteins could be found. In contrast, TIFY8 was found in protein complexes involved in regulation of dephosphorylation, deubiquitination and O-linked N-acetylglucosamine modification suggesting an important role in nuclear signal transduction

The ZIM domain of TIFY8 is functional.

<p><b>A</b>, Y2H analysis of TIFY8 interaction with class II TIFY proteins. NINJA was included as a positive control for JAZ interaction. <b>B, C</b>, Analysis of TIFY8 truncations to map the interaction domain with NINJA (<b>B</b>) and PPD2 (<b>C</b>). Co-transformation of the PJ69-4A yeast strain with TIFY8 or NINJA and all TIFY family members in Gateway-compatible pGADT7 and pGBKT7 vectors, respectively. Transformed yeasts were spotted on control medium lacking Leu and Trp (-2) or selective medium additionally lacking His (-3). AD: activation domain; BD: DNA-binding domain. Controls for autoactivation are provided by transformation with the corresponding empty vector. <b>D</b>, Immunoblot analysis of 7 day-old Arabidopsis seedlings overexpressing the TIFY8- or JAZ1-GS fusions after 1 h treatment with either 50 µM JA or ethanol (mock). Immunoblot using the Peroxidase Anti-Peroxidase (PAP) (top) and anti-cdc2 (bottom) antibodies.</p

The TIFY protein family in Arabidopsis.

<p>Phylogenetic tree of the Arabidopsis TIFY family members based on the ZIM domain (Z) protein sequence. AT4G27110 and AT3G20580 were chosen as the outgroup. AT4G27110 contains a TIFY motif but is not conserved in the domain outside this motif. Consequently, it is not considered to be a real TIFY protein. The second protein, AT3G20580, is its closest homologue within the parsed region. The numbers above the branches are bootstrap values from 100 replicates and assess the robustness of the tree. Additional protein domains are shown. C: CONSTANS, CO-like, and TOC1 (CCT) domain; G: C2C2-GATA Zn-finger; P: PEAPOD domain; J: Jas domain; J* Jas-like domain; E: EAR domain. Figure adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084891#pone.0084891-Vanholme1" target="_blank">[20]</a>.</p

Overview of prey proteins identified through TAP using TIFY8 as bait.

<p>Proteins were identified using peptide-based homology analysis of MS data. Background proteins identified in control experiments were withdrawn. Number indicates the times the prey was identified in 2 experiments with each bait protein. Abbreviations: AGI, Arabidopsis Genome Identifier; PPD2, PEAPOD2; NINJA, NOVEL INTERACTOR OF JAZ; TPL, TOPLESS; SPY, SPINDLY; TTA2, TITANIA2; PP2A, PROTEIN PHOSPHATASE2A; UBP12, UBIQUITIN-SPECIFIC PROTEASE. Detailed MS data can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084891#pone.0084891.s003" target="_blank">Table S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084891#pone.0084891.s005" target="_blank">Dataset S1</a>.</p