Voir la page en français

Stress signaling

Team STRESS / Jean Colcombet

 

The team offers Master and PhD opportunities

  • M2 subject: “Post-translational regulation of Arabidopsis thaliana MAPKs”

Supervisors: Jean Bigeard (jean.bigeard @ u-psud.fr) et Benedicte Sturbois (benedicte.sturbois @ inra.fr)

Fichier PDF

 

Arabidopsis genome codes for more than 1000 protein kinases and the majority of plant proteins are expected to be phosphorylated. The Stress Signalling group focuses its work on two particular kinase-related modules, which have been shown to be activated by stresses: the Mitogen-Activated Protein Kinases (MAPKs) and the Calcium-Dependent Protein Kinases (CDPKs or CPKs). MAPK modules are constituted of three kinases -a MAP3K, a MAP2K and a MAPK- activating each other by phosphorylation in a serial manner. In plants, they are encoded by rather large multi-genic families and theoretically may define many functional signaling modules. Three MAPKs, namely MPK3, MPK4 and MPK6 and their cognate MAP2Ks and MAP3Ks have been the focus of the majority of reports. They were shown to be activated very rapidly by both abiotic and biotic stresses and regulate various aspects of plant stress responses ranging from hormone and phytoalexin synthesis to transcriptional reprogramming. Beside stress responses, the same kinases are also involved in various aspects of plant development such as stomatal patterning, cytokinesis and abscission. Comparatively, data about other MAPK family members are rather scarce. CDPKs constitute a 34-member family able to translate cytosolic and nuclear calcium elevations into phosphorylation signals. Our group is mainly focusing on CPK5 and CPK6, two key regulators of both biotic and abiotic stress responses. They redundantly trigger disease resistance through NADPH-dependent oxidative burst and transcriptional reprogramming. Yet, only few substrates have been identified and the molecular mechanisms underlying CDPK functions in stress responses remain elusive.

The Stress Signalling group  tries to develop original approaches to better understand signalling mechanisms in MAPK and CDPK modules in the context of stress responses. Notably, we developed original genetic material (project 1 ) and proteomic-based studies (projects 2, 3 and 5). We also aim to characterize the function of atypical MAPK modules (project 4).

 

Project 1

Use of mutations conferring constitutive activity to MAPKs allows deciphering their function(Julien Lang (julien.lang @ ips2.universite-paris-saclay.fr) )

Loss-of-function T-DNA mutants have been successfully used to analyze MAPK functions during stresses. Nevertheless, in some cases, MAPK mutants show either no phenotype or very pleiotropic ones.

To bypass these problems, we proposed 10 years ago to develop plants expressing constitutively active (CA) forms of MAPKs. To do so, we developed an original functional screen to identify mutations triggering MAP2K-independent MAPK constitutive activity (Hudik et al. 2014 Meth Mol Bio). Our proof of concept focused on MPK4, which is an atypical stress-activated MAPK, and allowed clarifying some of its roles in immunity (Berriri et al. 2012 Plant Cell, Colcombet et al. 2013 PSB).

We more recently created lines expressing CA MPK3. These plants are dwarf (figure) and exhibit constitutive stress responses leading to a higher resistance to pathogens (Genot et al. 2017 Plant Physiol, Lang et al. 2017 PSB). In the future we will try to transfer the CA strategy to other poorly studied MAPKs. We also developed epistatic studies which should help us better understand the gene network involving MPK3 in response to stress.

 

Publications

  • Lang J, Genot B, Hirt H, Colcombet J. Constitutive activity of the Arabidopsis MAP Kinase 3 confers resistance to Pseudomonas syringae and drives robust immune responses. Plant Signal Behav. 2017 Aug 3;12(8):e1356533
  • Genot B, Lang J, Berriri S, Garmier M, Gilard F, Pateyron S, Haustraete K, Van Der Straeten D, Hirt H, Colcombet J. Constitutively Active Arabidopsis MAP Kinase 3 Triggers Defense Responses Involving Salicylic Acid and SUMM2 Resistance Protein. Plant Physiol. 2017 Jun;174(2):1238-1249
  • Hudik E, Berriri S, Hirt H, Colcombet J.Identification of constitutively active AtMPK6 mutants using a functional screen in Saccharomyces cerevisiae. Methods Mol Biol. 2014;1171:67-77.
  • Colcombet J, Berriri S, Hirt H. Constitutively active MPK4 helps to clarify its role in plant immunity. Plant Signal Behav. 2013 Feb;8(2):e22991
  • Berriri S, Garcia AV, Frei dit Frey N, Rozhon W, Pateyron S, Leonhardt N, Montillet JL, Leung J, Hirt H, Colcombet J. Constitutively active mitogen-activated protein kinase versions reveal functions of Arabidopsis MPK4 in pathogen defense signaling. Plant Cell. 2012 Oct;24(10):4281-93.

 

Collaboration

  • Heribert Hirt. Desert Agriculture Initiative, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia

 

 

Project 2

Identification of MAPK signaling network during defense (Jean Bigeard (jean.bigeard @ u-psud.fr)), in collaboration with Delphine Pflieger and Heribert Hirt)

In Arabidopsis, 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. The aim of this project is to identify the MAPK signaling network during defense through first the identification of MAPK substrates, then their functional characterization. For this, we use comparative quantitative phosphoproteomic approaches, following or not MAMP treatment, of wild type and mpk3, mpk4 and mpk6 mutant plants (figure). We recently compared the cytoplasmic phosphoproteomes of these different genotypes (Rayapuram et al. 2018 MCP). Partially overlapping substrate networks were retrieved for all three MAPKs, showing target specificity to one, two or all three MAPKs in different biological processes. The functional characterization of some putative MAPK substrates identified through these approaches is currently in progress.

 

Publications

  • Bigeard J and Hirt H. Nuclear Signaling of Plant MAPKs. Frontiers in Plant Science (2018) 9:469
  • Rayapuram N*, Bigeard J*, Alhoraibi H*, Bonhomme L, Hesse AM, Vinh J, Hirt H, Pflieger D. Quantitative Phosphoproteomic Analysis Reveals Shared and Specific Targets of Arabidopsis Mitogen-Activated Protein Kinases (MAPKs) MPK3, MPK4, and MPK6. Molecular & Cellular Proteomics 2018 Jan;17(1):61-80
  • Bigeard J, Colcombet J, Hirt H. Signaling Mechanisms in Pattern-Triggered Immunity (PTI). Molecular Plant 2015 Jan 9. pii: S1674-2052(15)00087-8
  • Bigeard J, Rayapuram N, Bonhomme L, Hirt H and Pflieger D. Proteomic and phosphoproteomic analyses of chromatin-associated proteins from Arabidopsis thaliana. Proteomics 2014 Oct;14(19):2141-55
  • Bigeard J, Rayapuram N, Pflieger D and Hirt H. Phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins. Proteomics 2014 Oct;14(19):2127-40
  • Rayapuram N*, Bonhomme L*, Bigeard J, Haddadou K, Przybylski C, Hirt H and Pflieger D. Paired MSA and ETcaD fragmentation spectra improve phosphopeptide identification of PAMP-triggered phosphorylation/dephosphorylation in Arabidopsis thaliana. Journal of Proteome Research 2014, Apr 4;13(4):2137-51
  • Vandenbogaert M, Hourdel V, Jardin-Mathé O, Bigeard J, Bonhomme L, Legros V, Hirt H, Schwikowski B, Pflieger D. Automated phosphopeptide identification using multiple MS/MS fragmentation modes. Journal of Proteome Research 2012, Dec 7;11(12):5695-703

 

Collaborations

  • Delphine Pflieger. Université Grenoble Alpes, CEA, Inserm, BIG-BGE, Grenoble, France. CNRS, BIG-BGE FR3425, Grenoble, France
  • Heribert Hirt. Desert Agriculture Initiative, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia

 

 

Project 3 

Post-translational regulation of Arabidopsis MAPK modules (Jean Bigeard (jean.bigeard @ u-psud.fr) and Bénédicte Sturbois (benedicte.sturbois @ inra.fr))

While biological functions have been attributed to some plant MAPK modules, there is very little information about their own post-translational regulation. Protein-protein interactions and protein post-translational modifications (PTMs) represent the most important mechanisms of protein post-translational regulation. PTMs notably may affect important properties, such as protein stability, enzyme activity or subcellular localization. MAPKs are known to be activated by the dual phosphorylation of their activation loop, as MAPKKs, but besides this regulatory mechanism almost nothing is known. The aim of this project is to better understand the post-translational regulation of Arabidopsis MAPK modules. For this, we developed a tandem affinity purification (TAP) approach coupled to mass spectrometry (MS) analyses to identify PTMs and interactors of our proteins of interest (figure). The functional study of several regulatory candidates is currently in progress.

 

Publications

  • Bigeard J, Pflieger D, Colcombet J, Gérard L, Mireau H, Hirt H. Protein Complexes Characterization in Arabidopsis thaliana by Tandem Affinity Purification Coupled to Mass Spectrometry Analysis. Methods in Molecular Biology 2014;1171:237-50
  • Pflieger D*, Bigeard J* and Hirt H. Isolation and characterization of plant protein complexes by mass spectrometry. Proteomics 2011, 11, 1–10

 

Collaborations

  • Delphine Pflieger. Université Grenoble Alpes, CEA, Inserm, BIG-BGE, Grenoble, France. CNRS, BIG-BGE FR3425, Grenoble, France
  • Heribert Hirt. Desert Agriculture Initiative, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia

 

Project 4 

Identification of new MAPK modules activated atypically late by stresses (Jean Colcombet (jean.colcombet @ u-psud.fr))

Despite a large number of genes encoding protein kinases potentially involved in MAPK cascades, only few complete modules have been published so far. In 2015, we reported the identification of a new complete module, constituted of MAP3K17/18-MKK3-MPK1/2/7/14, and showed its activation by ABA and its function in plant adaptation to drought (Danquah et al. 2015 Plant J, de Zelicourt et al. 2016 TIPS). We also showed that, whereas MAPKs are usually activated by stresses within minutes, MPK1/2/7/14 are activated by ABA after several hours. 

This activation relies on the de novo synthesis of MAP3K17/18, whose transcripts and proteins are not detectable in resting conditions (Danquah et al. 2015 Plant J, Boudsocq et al.2015 PSB). To our knowledge, this is the first time that such MAPK regulation is described in plants and animals. The consequence of such protein synthesis-dependent activation of the module is that it does not probably regulate early drought-dependent responses but is rather involved in the modulation of long-term responses.

Interestingly, MPK1 and MPK2 were shown to be activated by other stresses such as wounding, H2O2 and JA, suggesting that they could regulate a set of responses shared by distinct stresses. Integrating spare data from the literature as well as lab results, we hypothesized that MKK3-MPK1/2/7/14 module could be activated unspecifically by any of the eight MAP3Ks of the sub-MEKK-like clade III through de novo protein synthesis. Coherently, these genes are strongly transcriptionally regulated (Colcombet et al. 2016 Frontiers in Plant Science). Currently, we are testing this hypothesis by studying this MAPK module in other contexts (figure).

 

 

 

 

 

 

 

 

Publications

  • Colcombet J, Sözen C, Hirt H. Convergence of Multiple MAP3Ks on MKK3 Identifies a Set of Novel Stress MAPK Modules (2017) Front Plant Sci. 7:1941
  • Boudsocq M, Danquah A, de Zélicourt A, Hirt H, Colcombet J. Plant MAPK cascade: just rapid signaling modules? (2015) Plant Signal behav. 10:e1062197
  • Danquah A+, de Zélicourt A+, Boudsocq M+, Neubauer J, Frei Dit Frey N, Leonhardt N, Pateyron S, Gwinner F, Tamby JP, Ortiz-Masia D, Marcote MJ, Hirt H*, Colcombet J. Identification and characterization of an ABA-activated MAP kinase cascade in Arabidopsis thaliana (2015) Plant J. 82:232

 

Collaborations

  • Axel Mithöfer, Max Planck Institute for Chemical Ecology, Jena, Germany
  • Anne Krapp, Annie Marion Poll, Loic Rajjou, IJPB, Versailles, France
  • Heribert Hirt. Desert Agriculture Initiative, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia

 

Project 5

Deciphering CPK5/6 functions in stress responses through the identification of substrates (Marie Boudsocq (marie.boudsocq @ u-psud.fr))

CPK5/6 are 2 key players of calcium-mediated stress responses, either in flg22 signaling (Boudsocq et al. 2010) or in response to salt stress. To uncover the underlying molecular mechanisms, we undertook 2 complementary approaches to identify CPK substrates. On one hand, we performed a high throughput yeast-2-hybrid screen (collaboration with the InterAtome platform, IPS2) and retrieved 8 putative substrates. On the other hand, we took advantage of the constitutively active forms of CPKs (CA CPKs) by deleting the auto-inhibitory domain and the C-terminal calcium-binding domain to identify biologically relevant substrates of CPK5/6 in vivo.

We developed a phosphoproteomic approach using transgenic Arabidopsis lines that express CA-CPK5 or CA-CPK6 under the control of a dexamethasone (DEX)-inducible promoter, either in the WT form or mutated in the active site of the kinase (CPK-dead) to kill the activity (figure). We first validated the lines by showing that upon DEX treatment, only the CA-CPK but not the CPK-dead could trigger the CPK-dependent physiological responses such as gene expression and cell death. By comparing the phosphoproteome of CA-CPK5 and CPK5-dead lines upon DEX treatment, we could identify 25 proteins whose phosphorylation specifically increased in the CA-CPK5 lines. The validation of the candidates is in progress. In particular, we could already confirm the in vitro phosphorylation of most of them. Importantly, the CPK5 targeted phosphorylation site has been identified and validated for 2 selected substrates whose functional characterization is under way.

 

Collaborations

  • Michel Zivy, PAPPSO, INRA, Gif-sur-Yvette, France
  • Tina Romeis, University of Berlin, Germany
  • Heribert Hirt. Desert Agriculture Initiative, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia