Project description:We studied the phosphorylation of Arabidopsis LAZY4 proteins during gravistimulation, and searched for its interaction proteins using Immunoprecipitation-Mass Spectrometry (IP-MS). For the identified kinases MPK3 and MKK5, we carried out in vitro kinase assays using recombinant LAZY4 as the substrate.
Project description:In Arabidopsis, mitogen-activated protein kinases MPK3, MPK4 and MPK6 constitute essential relays for a variety of functions including cell division, development and innate immunity. While some substrates of MPK3, MPK4 and MPK6 have been identified, the picture is still far from complete. To identify substrates of these MAPKs in cell division, growth and development we compared the phosphoproteomes of wild-type and mpk3, mpk4 and mpk6. To study the function of these MAPKs in innate immunity, we analyzed their phosphoproteomes following activation by a microbe-associated molecular pattern (MAMP). Partially overlapping substrates were retrieved for all three MAPKs, showing target specificity to one, two or all three MAPKs in different biological processes. More precisely, our results illustrate the fact that the entity to be defined as a specific or a shared substrate for MAPKs is not a phosphoprotein but a particular (S/T)P phosphorylation site in a given protein. As a whole, 152 peptides were identified to be differentially phosphorylated in response to MAMP treatment and/or when compared between genotypes and 70 of them could be classified as putative MAPK targets. Biochemical analysis of a number of putative MAPK substrates by phosphorylation and interaction assays confirmed the global phosphoproteome approach. Our study finally expands the set of MAPK substrates to involve other protein kinases, including calcium-dependent (CDPK) and sugar non-fermenting (SnRK) protein kinases.
Project description:MicroRNA microarray profiling was performed in Arabidopsis seedlings - Col-0, atmpk3 and atmpk6 mutants to uncover the roles of MPK3 and MPK6 in microRNA biogenesis pathway.
Project description:Agrobacterium tumefaciens is a special plant pathogen causing crown gall disease. This pathogen is well known for the technology Agrobacterium-mediated transformation. As a pathogen, Agrobacterium triggers plant immunity, and this affects transformation. But the signaling components and pathways in plant immunity to Agrobacterium remain elusive. We demonstrate two Arabidopsis MAPKKs MKK4/MKK5 and their downstream MAPKs MPK3/MPK6 play a major role in both Agrobacterium-triggered immunity and Agrobacterium-mediated transformation. Agrobacteria induce MPK3/MPK6 activity and plant defense responsive genes expression in a very early stage. This process is dependent on MKK4/MKK5 function. Loss of function of MKK4 and MKK5 or their downstream MPK3 and MPK6 abolishes plant immunity to agrobacteria, and increases the transformation frequency, while activation of MKK4 and MKK5 enhances the plant immunity and represses the transformation. Global transcriptome indicates agrobacteria induce various plant defense pathways, including ROS production, ethylene and SA-mediated defense responses, and MKK4/MKK5 is essential for these pathways induction. Activation of MKK4 and MKK5 promotes ROS production and cell death in agrobacteria infection process. Ethylene and SA act bypass of MKK4/MKK5 signaling to regulate transformation. Based on these results, we propose MKK4/5-MPK3/6 cascade is an essential signaling pathway to regulate Agrobacterium-mediated transformation by modulating Agrobacterium-triggered plant immunity.
Project description:inra12-05_mut_flg_ii-FLG-MPKs ra12-05_mut_flg_ii - inra12-05_mut_flg_ii - Analysis of flg22-induced transcriptional changes in mpk3, mpk4 and mpk6 mutants. - 2 weeks old seedlings compared for transcriptome after a 30min mock or 1µM flg22 treatment.
Project description:Mitogen-activated protein kinases constitute essential signaling mechanisms linking external signal perception to gene expression in all eukaryotes. In Arabidopsis, MAPKs are found in the cytoplasmic and nuclear compartments, but no nuclear phosphoproteomic studies of mapk mutants were performed so far. In this work, we report a panel of nuclear and chromatin-associated targets by performing phosphoproteome analyses of wild-type and mpk3, mpk4, and mpk6 mutant plants. Since the three MAPKs play a role in cell division, development and innate immunity, we also analyzed the phosphoproteomes following microbe-associated molecular pattern (MAMP) treatment. A total of 165 proteins were identified to be differentially phosphorylated when compared between genotypes and in response to MAMP treatment. Using different criteria, including different biochemical and interaction assays, a number of the putative MAPK substrates were validated as true substrates for different MAPKs. Signaling networks for the three MAPKs revealed their common role in RNA transcription, RNA processing and chromatin organization, but also specific roles in development and organelle organization. Overall, this study unravels a set of chromatin-associated targets for these three MAPKs and promises novel insights into yet undiscovered domains linking MAPK signaling to a number of chromatin-related events.
Project description:Mitogen-activated protein kinases (MAPKs) target a variety of protein substrates to regulate cellular signaling processes in eukaryotes. In plants, the number of identified MAPK substrates that control plant defense responses is still limited. Here, we generated transgenic Arabidopsis thaliana plants with an inducible system to simulate in vivo activation of two stress-activated MAPKs, MPK3 and MPK6. Metabolome analysis revealed that this artificial MPK3/6 activation (without any exposure to pathogens or other stresses) is sufficient to drive the production of major defense-related metabolites, including various camalexin, indole glucosinolate and agmatine derivatives. An accompanying (phospho)proteome analysis led to detection of hundreds of potential phosphoproteins downstream of MPK3/6 activation. Besides known MAPK substrates, many candidates on this list possess typical MAPK-targeted phosphosites and in many cases, the corresponding phosphopeptides were detected by mass spectrometry. Notably, several of these putative phosphoproteins have been reported to be associated with the biosynthesis of antimicrobial defense substances (e.g. WRKY transcription factors and proteins encoded by the genes from the “PEN” pathway required for penetration resistance to filamentous pathogens). Thus, this work provides an inventory of candidate phosphoproteins, including putative direct MAPK substrates, for future analysis of MAPK-mediated defense control.
Project description:inra12-05_mut_flg_ii-FLG-MPKs ra12-05_mut_flg_ii - inra12-05_mut_flg_ii - Analysis of flg22-induced transcriptional changes in mpk3, mpk4 and mpk6 mutants. - 2 weeks old seedlings compared for transcriptome after a 30min mock or 1µM flg22 treatment. 21 dye-swap - gene knock out,normal vs mutant comparison,treated vs untreated comparison