URGV - Plant Genomics Research
Unité de Recherche en Génomique Végétale

The Dark Side of the Salad: Salmonella typhimurium Overcomes the Innate Immune Response of Arabidopsis thaliana and Shows an Endopathogenic Lifestyle

Schikora A, Carreri A, Charpentier E, Hirt H.
PLoS One. 2008 May 28;3(5):e2279. PMID: 18509467

Trojan Horse Strategy in Agrobacterium Transformation: Abusing MAPK Defense Signaling

Djamei A, Pitzschke A, Nakagami H, Rajh I, Hirt H.
Science. 2007 Oct 19;318(5849):453-6. PMID: 17947581

The MKK2 pathway mediates cold and salt stress signaling in Arabdiopsis.

Teige M, Scheikl E, Eulgem T, Dóczi R, Ichimura K, Shinozaki K, Dangl JL, Hirt H.
Mol Cell. 2004 Jul 2;15(1):141-52. PMID: 15225555

Convergence and divergence of stress-induced MAPK signaling pathways at the level of two distinct MAP kinase kinases.

Cardinale F, Meskiene I, Ouaked F, Hirt H.
Plant Cell. 2002 Mar;14(3):703-11. PMID: 11910015

Receptor-mediated activation of a MAP kinase in pathogen defense of plants.

Science 276, 2054-2057. PMID: 9197271
Ligterink, T. Kroj, U. zur Nieden, H.. Hirt, and D. Scheel (1997)

Plants as alternative hosts for Salmonella.

Trends Plant Sci. 2012 Apr 16. PMID: 22513107
Schikora A, Garcia AV, Hirt H.
Institute for Plant Pathology and Applied Zoology, Research Centre for BioSystems, Land Use and Nutrition, JL University Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany.

Recent findings show that many human pathogenic bacteria can use multiple host organisms. For example, Salmonella Typhimurium can use plants as alternative hosts to humans and other animals. These bacteria are able to adhere to plant surfaces and actively infect the interior of plants. Similarly to the infection of animal cells, S. Typhimurium suppresses plant defense responses by a type III secretion mechanism, indicating that these bacteria possess a dedicated multi-kingdom infection strategy, raising the question of host specificity. In addition, evidence is accumulating that the interaction of Salmonella with plants is an active process with different levels of specificity, because different Salmonella serovars show variations in pathogenicity, and different plant species reveal various levels of resistance towards these bacteria.

Improvement of stress tolerance in plants by genetic manipulation of mitogenactivated protein kinases

Biotechnology Advances 2011, Dec 06, PMID: 22198202
Samajova O,Plihal O,Yousif MA,Hirt H,Samaj J

Plant stress tolerance depends on many factors among which signaling by mitogen-activated protein-kinase (MAPK) modules plays a crucial role. Reversible phosphorylation of MAPKs, their upstream activators and downstream targets such as transcription factors can trigger a myriad of transcriptomic, cellular and physiological responses. Genetic manipulation of abundance and/or activity of some of these modular MAPK components can lead to better stress tolerance in Arabidopsis and crop plant species such as tobacco and cereals. The main focus of this review is devoted to the MAPK-related signaling components which show the most promising biotechnological potential. Additionally, recent studies identified MAPK components to be involved both in plant development as well as in stress responses, suggesting that these processes are tightly linked in plants.

Conservation of Salmonella Infection Mechanisms in Plants and Animals

PLoS One September 2011 Volume 6 Issue 9, e24112
Adam Schikora, Isabelle Virlogeux-Payant, Eduardo Bueso, Ana V. Garcia, Theodora Nilau, Amélie Charrier, Sandra Pelletier, Pierrette Menanteau, Manuela Baccarini, Philippe Velge, Heribert Hirt

Salmonella virulence in animals depends on effectors injected by Type III Secretion Systems (T3SSs). In this report we demonstrate that Salmonella mutants that are unable to deliver effectors are also compromised in infection of Arabidopsis thaliana plants. Transcriptome analysis revealed that in contrast to wild type bacteria, T3SS mutants of Salmonella are compromised in suppressing highly conserved Arabidopsis genes that play a prominent role during Salmonella infection of animals. We also found that Salmonella originating from infected plants are equally virulent for human cells and mice. These results indicate a high degree of conservation in the defense and infection mechanism of animal and plant hosts during Salmonella infection.

Press Release Sept 09, 2011:
Salmonella uses similar mechanism to infect plants and humans
INRA - CNRS - Université d'Evry

AGC kinases in plant development and defense

Plant Signaling & Behavior 6:7, 1-4; July 2011; © 2011 Landes Bioscience
Heribert Hirt, Ana V. Garcia, Ralf Oelmüller

More than 100,000 publications demonstrate that AGC kinases are important regulators of growth, metabolism, proliferation, cell divison, survival and apoptosis in mammalian systems.1 Mutation and/or dysregulation of these kinases contribute to the pathogenesis of many human diseases, including cancer and diabetes. Although AGC kinases are also present in plants, little is known about their functions. We demonstrated that the AGC kinase OXIDATIVE SIGNAL-INDUCIBLE1 (OXI1/AGC2-1) regulate important developmental processes and defense responses in plants. The summary of recent progress also demonstrates that we are only beginning to understand the role of this kinase pathway in plants.

The OXI1 Kinase Pathway Mediates Piriformospora indica-Induced Growth Promotion in Arabidopsis

PLOS Pathogens May 2011 doi:10.1371/journal.ppat.1002051
Iris Camehl, Corinna Drzewiecki, Jyothilakshmi Vadassery, Bationa Shahollari, Irena Sherameti, Celine Forzani, Teun Munnik, Heribert Hirt, Ralf Oelmüller

Piriformospora indica is an endophytic fungus that colonizes roots of many plant species and promotes growth and resistance to certain plant pathogens. Despite its potential use in agriculture, little is known on the molecular basis of this beneficial plant-fungal interaction. In a genetic screen for plants, which do not show a P. indica- induced growth response, we isolated an Arabidopsis mutant in the OXI1 (Oxidative Signal Inducible1) gene. OXI1 has been characterized as a protein kinase which plays a role in pathogen response and is regulated by H2O2 and PDK1 (3-PHOSPHOINOSITIDE-DEPENDENT PROTEIN KINASE1). A genetic analysis showed that double mutants of the two closely related PDK1.1 and PDK1.2 genes are defective in the growth response to P. indica. While OXI1 and PDK1 gene expression is upregulated in P. indica-colonized roots, defense genes are downregulated, indicating that the fungus suppresses plant defense reactions. PDK1 is activated by phosphatidic acid (PA) and P. indica triggers PA synthesis in Arabidopsis plants. Under beneficial co-cultivation conditions, H2O2 formation is even reduced by the fungus. Importantly, phospholipase D (PLD)a1 or PLDd mutants, which are impaired in PA synthesis do not show growth promotion in response to fungal infection. These data establish that the P. indica-stimulated growth response is mediated by a pathway consisting of the PLD-PDK1-OXI1 cascade.

Isolation and characterization of plant protein complexes by mass spectrometry.

Proteomics. 2010 Nov 2. doi: 10.1002/pmic.201000635. PMID: 21472857
Pflieger D, Bigeard J, Hirt H.

Abstract
The components that enable cells and organisms to fulfill a plethora of chemical and physical reactions, including their ability to metabolize, replicate, repair and communicate with their environment are mostly based on the functioning of highly complex cellular machines which are to a large extent composed of proteins. With the development of MS techniques compatible with the analysis of minute amounts of biological material, it has become more and more feasible to dissect the composition and modification of these protein machineries. Indeed, new purification methods of protein complexes followed by MS analysis together with the genomic sequencing of various organisms - and in particular of crop species - now provide unforeseen insight to understand biological processes at a molecular level. We here review the current state of the art of in vivo protein complex isolation and their MS-based analytical characterization, emphasizing on the tandem affinity purification approach.

Linking the proteins-Elucidation of proteome-scale networks using mass spectrometry.

Mass Spectrom Rev. 2011 Mar;30(2):268-97. doi: 10.1002/mas.20278. Epub 2010 May 24. PMID: 21337599
Pflieger D, Gonnet F, de la Fuente van Bentem S, Hirt H, de la Fuente A.
Laboratoire Analyse et Modélisation pour la Biologie et l'Environnement, Université d'Evry Val d'Essonne, CNRS UMR 8587, Evry, France

Proteomes are intricate. Typically, thousands of proteins interact through physical association and post-translational modifications (PTMs) to give rise to the emergent functions of cells. Understanding these functions requires one to study proteomes as "systems" rather than collections of individual protein molecules. The abstraction of the interacting proteome to "protein networks" has recently gained much attention, as networks are effective representations, that lose specific molecular details, but provide the ability to see the proteome as a whole. Mostly two aspects of the proteome have been represented by network models: proteome-wide physical protein-protein-binding interactions organized into Protein Interaction Networks (PINs), and proteome-wide PTM relations organized into Protein Signaling Networks (PSNs). Mass spectrometry (MS) techniques have been shown to be essential to reveal both of these aspects on a proteome-wide scale. Techniques such as affinity purification followed by MS have been used to elucidate protein-protein interactions, and MS-based quantitative phosphoproteomics is critical to understand the structure and dynamics of signaling through the proteome. We here review the current state-of-the-art MS-based analytical pipelines for the purpose to characterize proteome-scale networks. © 2010 Wiley Periodicals, Inc., Mass Spec Rev 30:268-297, 2011.

The Arabidopsis protein kinase PTI1-4 is a common target of the oxidative signal-inducible1 (OXI1) and MAP kinases.

FEBS J. 2011 Jan 29. doi: 10.1111/j.1742-4658.2011.08033.x. PMID: 21276203
Forzani C, Carreri A, de la Fuente van Bentem S, Lecourieux D, Lecourieux F, Hirt H.
Max Perutz Laboratories, Dr. Bohrgasse 9, A-1030 Vienna, Austria URGV Plant Genomics, INRA-CNRS-University of Evry, 2 rue Gaston Cremieux, F-91057

In Arabidopsis thaliana the serine/threonine protein kinase oxidative signal-inducible1 (OXI1), mediates oxidative stress signalling. Its activity is required for the full activation of the mitogen-activated protein kinases (MAPKs), MPK3 and MPK6 in response to oxidative stress. In addition, the serine/threonine protein kinase PTI1-2 has been positioned downstream from OXI1 but whether PTI1-2 signals through MAPK cascades is unclear. Using a yeast two-hybrid screen we show that OXI1 also interacts with PTI1-4. OXI1 and PTI1-4 are stress-responsive genes and are expressed in the same tissues. Therefore, studies were undertaken to determine whether PTI1-4 is positioned in the OXI1/MAPK signalling pathway. The interaction between OXI1 and PTI1-4 was confirmed by using in vivo co-immunoprecipitation experiments. OXI1 as well as PTI1-4 were substrates of MPK3 and MPK6 in vitro. Whereas no direct interaction was detected between MPK3, MPK6 and OXI1, in vitro binding studies showed an interaction between MPK3, MPK6 and PTI1-4. In addition, PTI1-4 and MPK6 were found in vivo in the same protein complex. These results demonstrate that PTI1-4 signals via OXI1 and MPK6 signalling cascades.
Journal compilation © 2011 Federation of European Biochemical Societies.

The MAP Kinase MPK4 Is Required for Cytokinesis in Arabidopsis thaliana

Plant Cell Advance Online Publication Published on November 23, 2010; 10.1105/tpc.110.077164 PMID: 21098735
Ken Kosetsu (a), Sachihiro Matsunaga (b), Hirofumi Nakagami (c), Jean Colcombet (d), Michiko Sasabe (a), Takashi Soyano (a), Yuji Takahashi (a), Heribert Hirt (c, d) and Yasunori Machida (a, 3)
(a) Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan
(b) Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan
(c) Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, A-1030 Vienna, Austria
(d) Unité de Recherche en Génomique Végétale Plant Genomics, Institut National de la Recherche Agronomique, Centre National de la Recherche Scientifique, Université d’Evry, 91057 Evry, France
(3) Address correspondence to yas@bio.nagoya-u.ac.jp

Cytokinesis in plants is achieved by the formation of the cell plate. A pathway that includes mitogen-activated protein (MAP) kinase kinase kinase and MAP kinase kinase (MAPKK) plays a key role in the control of plant cytokinesis. We show here that a MAP kinase, MPK4, is required for the formation of the cell plate in Arabidopsis thaliana. Single mutations in MPK4 caused dwarfism and characteristic defects in cytokinesis, such as immature cell plates, which became much more prominent upon introduction of a mutation in MKK6/ANQ, the MAPKK for cytokinesis, into mpk4. MKK6/ANQ strongly activated MPK4 in protoplasts, and kinase activity of MPK4 was detected in wild-type tissues that contained dividing cells but not in mkk6/anq mutants. Fluorescent protein–fused MPK4 localized to the expanding cell plates in cells of root tips. Expansion of the cell plates in mpk4 root tips appeared to be retarded. The level of MPK11 transcripts was markedly elevated in mpk4 plants, and defects in the mpk4 mpk11 double mutant with respect to growth and cytokinesis were more severe than in the corresponding single mutants. These results indicate that MPK4 is the downstream target of MKK6/ANQ in the regulation of cytokinesis in Arabidopsis and that MPK11 is also involved in cytokinesis.

New insights into an old story: Agrobacterium-induced tumour formation in plants by plant transformation.

EMBO J. 2010 Feb 11. PMID: 20150897
Andrea Pitzschke (1) and Heribert Hirt (2,3,*)
(1) Department of Applied Genetics and Cell Biology, University of Natural Resources and Applied Life Sciences, Muthgasse 18, Vienna, Austria,
(2) Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, Dr-Bohr-Gasse 9, Vienna, Austria and
(3) URGV Plant Genomics, INRA-University of Evry, 2 Rue Gaston Cremieux, Evry, France

Agrobacterium tumefaciens causes tumour formation in plants. Plant signals induce in the bacteria the expression of a range of virulence (Vir) proteins and the formation of a type IV secretion system (T4SS). On attachment to plant cells, a transfer DNA (T-DNA) and Vir proteins are imported into the host cells through the bacterial T4SS. Through interaction with a number of host proteins, the Vir proteins suppress the host innate immune system and support the transfer, nuclear targeting, and integration of T-DNA into host cell chromosomes. Owing to extensive genetic analyses, the bacterial side of the plant-Agrobacterium interaction is well understood. However, progress on the plant side has only been achieved recently, revealing a highly complex molecular choreography under the direction of the Vir proteins that impinge on multiple processes including transport, transcription, and chromosome status of their host cells.

Mechanism of MAPK-targeted gene expression unraveled in plants.

Cell Cycle. 2010 Jan 1;9(1):18-9. Epub 2010 Jan 14. PMID: 20016264
Andrea Pitschke (1, +), Heribert Hirt (1, 2)
(1) Department of Plant Molecular Biology; Max F. Perutz Laboratories; University of Vienna; Vienna, Austria;
(2) URGV Plant Genomics Laboratory; Evry, France
(+) Present address: Department of Applied Genetics and Cell Biology; University of Natural Resources and Applied Life Sciences; Vienna, Austria

Mitogen-activated protein kinase (MAPK) cascades—phosphorelay modules minimally composed of a MAPK kinase kinase, a MAPK kinase and a MAPK—are key players in eukaryotic cell signaling, linking developmental or environmental stimulus perception to alteration/adaptation of gene expression. Their prominent role in mammalian cancer development, but also in regulating plant development and stress adaptation are well-documented (reviewed in refs. 1–4). Through their kinase activity, MAPK cascades translate incoming environmental signals into post-translational modification of target proteins, e.g., transcription factors, to ultimately reorganize gene expression and stress adaptation.

Bioinformatic and systems biology tools to generate testable models of signalling pathways and their targets

Plant Physiology Preview. Published on November 13, 2009, as DOI:10.1104/pp.109.149583, PMID: 19915012
Andrea Pitzschke (a) and Heribert Hirt (b,c)
(a) Department of Applied Genetics and Cell Biology; University of Natural Resources and Applied Life Sciences, Muthgasse 18, 1190 Vienna, Austria
(b) Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, Dr.-Bohr-Gasse 9, 1030 Vienna, Austria
(c) URGV Plant Genomics Laboratory, 2 Rue Gaston Cremieux, 91057 Evry, France

Over the last years a number of bioinformatic software programs have been developed in the area of molecular biology. The application of these bioinformatic tools to the wealth of existing transcriptomic and proteomic data can be used to predict the structure and hierarchy of signalling pathways and gene networks. In genetically tractable model organisms such as Arabidopsis thaliana, these hyphotheses can be validated experimentally and modified in reiterative cycles, giving hypothesis-driven research high feasibility. These predictive systems biology approaches significantly reduce the scale, time and manpower usually required in classical approaches. Here, we provide an overview on the use of currently available tools in deciphering signalling pathways in Arabidopsis research.

VIP1 response elements mediate mitogen-activated protein kinase 3-induced stress gene expression.

Proc Natl Acad Sci U S A. 2009 Oct 9. PMID: 19820165
Pitzschke A, Djamei A, Teige M, Hirt H.
Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, Dr.-Bohr-Gasse 9, 1030 Vienna, Austria.
URGV Plant Genomics Laboratory, 2 Rue Gaston Crémieux, 91057 Evry, France

The plant pathogen Agrobacterium tumefaciens transforms plant cells by delivering its T-DNA into the plant cell nucleus where it integrates into the plant genome and causes tumor formation. A key role of VirE2-interacting protein 1 (VIP1) in the nuclear import of T-DNA during Agrobacterium-mediated plant transformation has been unravelled and VIP1 was shown to undergo nuclear localization upon phosphorylation by the mitogen-activated protein kinase MPK3. Here, we provide evidence that VIP1 encodes a functional bZIP transcription factor that stimulates stress-dependent gene expression by binding to VIP1 response elements (VREs), a DNA hexamer motif. VREs are overrepresented in promoters responding to activation of the MPK3 pathway such as Trxh8 and MYB44. Accordingly, plants overexpressing VIP1 accumulate high levels of Trxh8 and MYB44 transcripts, whereas stress-induced expression of these genes is impaired in mpk3 mutants. Trxh8 and MYB44 promoters are activated by VIP1 in a VRE-dependent manner. VIP1 strongly enhances expression from a synthetic promoter harboring multiple VRE copies and directly interacts with VREs in vitro and in vivo. Chromatin immunoprecipitation assays of the MYB44 promoter confirm that VIP1 binding to VREs is enhanced under conditions of MPK3 pathway stimulation. These results provide molecular insight into the cellular mechanism of target gene regulation by the MPK3 pathway.

MAP KINASE PHOSPHATASE1 and PROTEIN TYROSINE PHOSPHATASE1 Are Repressors of Salicylic Acid Synthesis and SNC1-Mediated Responses in Arabidopsis

Plant Cell Advance Online Publication
Published on September 29, 2009; 10.1105/tpc.109.067678
Sebastian Bartels (1), Jeffrey C. Anderson (2), Marina A. González Besteiro (3), Alessandro Carreri (4), Heribert Hirt (5), Antony Buchala (6), Jean-Pierre Métraux (6), Scott C. Peck (2), and Roman Ulm (7*)
(1) Faculty of Biology, Institute of Biology II, University of Freiburg, D-79104 Freiburg, Germany
(2) Department of Biochemistry, University of Missouri, Columbia, Missouri 65211
(3) Faculty of Biology, Institute of Biology II, University of Freiburg, D-79104 Freiburg, Germany; Spemann Graduate School of Biology and Medicine, University of Freiburg, D-79104 Freiburg, Germany
(4) Max F. Perutz Laboratories, University of Vienna, A-1030 Vienna, Austria
(5) Max F. Perutz Laboratories, University of Vienna, A-1030 Vienna, Austria; URGV - Plant Genomics, INRA, CNRS, University Evry, F-91057 Evry Cedex, France
(6) Department of Biology, University of Fribourg, CH-1700 Fribourg, Switzerland
(7) Faculty of Biology, Institute of Biology II, University of Freiburg, D-79104 Freiburg, Germany; Centre for Biological Signaling Studies (bioss), University of Freiburg, D-79104 Freiburg, Germany
(*) To whom correspondence should be addressed: roman.ulm@biologie.uni-freiburg.de

Mitogen-activated protein (MAP) kinase phosphatases are important negative regulators of the levels and kinetics of MAP kinase activation that modulate cellular responses. The dual-specificity phosphatase MAP KINASE PHOSPHATASE1 (MKP1) was previously shown to regulate MAP KINASE6 (MPK6) activation levels and abiotic stress responses in Arabidopsis thaliana. Here, we report that the mkp1 null mutation in the Columbia (Col) accession results in growth defects and constitutive biotic defense responses, including elevated levels of salicylic acid, camalexin, PR gene expression, and resistance to the bacterial pathogen Pseudomonas syringae. PROTEIN TYROSINE PHOSPHATASE1 (PTP1) also interacts with MPK6, but the ptp1 null mutant shows no aberrant growth phenotype. However, the pronounced constitutive defense response of the mkp1 ptp1 double mutant reveals that MKP1 and PTP1 repress defense responses in a coordinated fashion. Moreover, mutations in MPK3 and MPK6 distinctly suppress mkp1 and mkp1 ptp1 phenotypes, indicating that MKP1 and PTP1 act as repressors of inappropriate MPK3/MPK6-dependent stress signaling. Finally, we provide evidence that the natural modifier of mkp1 in Col is largely the disease resistance gene homolog SUPPRESSOR OF npr1-1, CONSTITUTIVE 1 (SNC1) that is absent in the Wassilewskija accession. Our data thus indicate a major role of MKP1 and PTP1 in repressing salicylic acid biosynthesis in the autoimmune-like response caused by SNC1.

MAPK cascade signalling networks in plant defence.

Curr Opin Plant Biol. 2009 Jul 14. PMID: 19608449
Pitzschke A, Schikora A, Hirt H.
Max F. Perutz Laboratories, University of Vienna, Dr. Bohrgasse 9, 1030 Vienna, Austria.
URGV Plant Genomics, INRA-CNRS-University of Evry, 2 rue Gaston Cremieux, 91057 Evry, France

The sensing of stress signals and their transduction into appropriate responses is crucial for the adaptation and survival of plants. Kinase cascades of the mitogen-activated protein kinase (MAPK) class play a remarkably important role in plant signalling of a variety of abiotic and biotic stresses. MAPK cascade-mediated signalling is an essential step in the establishment of resistance to pathogens. Here, we describe the most recent insights into MAPK-mediated pathogen defence response regulation with a particular focus on the cascades involving MPK3, MPK4 and MPK6. We also discuss the strategies developed by plant pathogens to circumvent, inactivate or even 'hijack' MAPK-mediated defence responses.

Transgenerational stress memory is not a general response in Arabidopsis.

PLoS ONE. 2009;4(4):e5202. Epub 2009 Apr 21. PMID: 19381297
Pecinka A (1), Rosa M (1), Schikora A (2), Berlinger M (3), Hirt H (2), Luschnig C (3), Mittelsten Scheid O (1)
(1) Gregor Mendel Institute of Molecular Plant Biology (GMI), Austrian Academy of Sciences, Vienna, Austria,
(2) INRA – URGV, Plant Genomics Research Unit, Evry, France
(3) University of Natural Resources and Applied Life Sciences (BOKU), Vienna, Austria

Adverse conditions can trigger DNA damage as well as DNA repair responses in plants. A variety of stress factors are known to stimulate homologous recombination, the most accurate repair pathway, by increasing the concentration of necessary enzymatic components and the frequency of events. This effect has been reported to last into subsequent generations not exposed to the stress. To establish a basis for a genetic analysis of this transgenerational stress memory, a broad range of treatments was tested for quantitative effects on homologous recombination in the progeny. Several Arabidopsis lines, transgenic for well-established recombination traps, were exposed to 10 different physical and chemical stress treatments, and scored for the number of somatic homologous recombination (SHR) events in the treated generation as well as in the two subsequent generations that were not treated. These numbers were related to the expression level of genes involved in homologous recombination and repair. SHR was enhanced after the majority of treatments, confirming previous data and adding new effective stress types, especially interference with chromatin. Compounds that directly modify DNA stimulated SHR to values exceeding previously described induction rates, concomitant with an induction of genes involved in SHR. In spite of the significant stimulation in the stressed generations, the two subsequent non-treated generations only showed a low and stochastic increase in SHR that did not correlate with the degree of stimulation in the parental plants. Transcripts coding for SHR enzymes generally returned to pre-treatment levels in the progeny. Thus, transgenerational effects on SHR frequency are not a general response to abiotic stress in Arabidopsis and may require special conditions.

Disentangling the complexity of mitogen-activated protein kinases and reactive oxygen species signaling.

Plant Physiol. 2009 Feb;149(2):606-15. PMID: 19201916
Andrea Pitzschke (1) and Heribert Hirt (1, 2)
(1) Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, 1030 Vienna, Austria (A.P., H.H.)
(2) URGV Plant Genomics, INRA-CNRS-Université d'Evry, 91057 Evry, France (H.H.)

For about 2 million years, molecular oxygen arising from photosynthetic processes has become pivotal to almost all organisms. Reactive oxygen species (ROS), the partially reduced or activated derivatives of oxygen (hydrogen peroxide [H₂O₂], HO·, ¹O₂, O₂–), are the highly reactive by-products of aerobic metabolism. They arise from various chemical reactions and can lead to oxidative damage of cells.
Plants possess a sophisticated ROS network, comprising antioxidative enzymes, antioxidants, and ROS-producing enzymes, which allow them to keep ROS levels under tight control. Moreover, as research of the past few years has shown, plants have developed efficient strategies for targeted production of ROS. For instance, ROS play a role in programmed cell death (PCD), development, and stress response. Mitogen-activated protein kinase (MAPK) cascades are key players in ROS signaling. Several studies have shown that MAPK signaling pathways are not only induced by ROS but can also regulate ROS production. MAPK cascades are signaling modules that minimally consist of a MAPK kinase kinase (MAPKKK/MEKK), a MAPK kinase (MAPKK/MKK), and MAPK. Upon a stimulus-triggered activation of a MAPKKK, the signal is transduced via phosphorylation-mediated activation of a corresponding downstream MAPKK, which in turn phosphorylates and thereby activates a specific MAPK. The Arabidopsis (Arabidopsis thaliana) genome contains more than 60 MAPKKKs, 20 MAPKs, and 10 MAPKs, which can, depending on the environmental stimulus or developmental stage, engage in different MAPK modules. With the characterization of mutants affected in pathogen response as well as the development and dynamics of stomata, the network of MAPK cascade activation and ROS is being disentangled. Here, we discuss the most recent insights into ROS production and perception involving MAPK-mediated signaling.

Protein tyrosine phosphorylation in plants: more abundant than expected?

Trends Plant Sci. 2009 Jan 20. PMID: 19162527
de la Fuente van Bentem S (1), Hirt H. (1,2)
(1) Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, Dr. Bohr-Gasse 9, 1030 Vienna, Austria
(2) Unité de Recherche en Génomique Végétale, INRA-CNRS-UEVE, 2 rue Gaston Crémieux, 91057 Evry Cédex, France

Protein phosphorylation in eukaryotes predominantly occurs on serine (Ser) and threonine (Thr) residues, whereas phosphorylation on tyrosine (Tyr) residues is less abundant. Plants lack classic Tyr kinases, such as the epidermal growth factor receptor, that govern Tyr phosphorylation in animals. A long-standing debate questions whether plants have any Tyr-specific kinases and, although several protein kinases with both Ser/Thr and Tyr specificities exist, data supporting the existence of other such kinases are scarce. As we discuss here, mass-spectrometry-based analyses now indicate that Tyr phosphorylation is as extensive in plants as it is in animals. However, careful inspection of available data indicates that these promising mass spectrometry studies have to be interpreted with caution before current ideas on Tyr phosphorylation in plants are revised.

A Major Role of the MEKK1–MKK1/2–MPK4 Pathway in ROS Signalling

Molecular Plant Advance Access published January 6, 2009 | Molecular Plant • Pages 1–18, 2008
Andrea Pitzschke (a), Armin Djamei (a,b,) Frédérique Bitton (c) and Heribert Hirt (a,c,1)
(a) Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, Dr.-Bohr-Gasse 9, 1030 Vienna, Austria
(b) Present address: Max-Planck-Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse, 35043 Marburg, Germany
(c) URGV Plant Genomics Laboratory, 2 Rue Gaston Cre´ mieux, 91057 Evry, France
(1) To whom correspondence should be addressed. E-mail hirt@evry.inra.fr

ABSTRACT Over the last few years, it has become evident that reactive oxygen species (ROS) signalling plays an important role in various physiological responses, including pathogen defense and stomatal opening/closure. On the other hand, ROS overproduction is detrimental for proper plant growth and development, indicating that the regulation of an appropriate redox balance is essential for plants. ROS homeostasis in plants involves the mitogen-activated protein kinase (MAPK) pathway consisting of the MAPK kinase kinase MEKK1 and the MAPK MPK4. Phenotypic and molecular analysis revealed that the MAPK kinases MKK1 and MKK2 are part of a cascade, regulating ROS and salicylic acid (SA) accumulation. Gene expression analysis shows that of 32 transcription factors reported to be highly responsive to multiple ROS-inducing conditions, 20 are regulated by the MEKK1, predominantly via the MEKK1–MKK1/2–MPK4 pathway. However, MEKK1 also functions on other as yet unknown pathways and part of the MEKK1-dependent MPK4 responses are regulated independently of MKK1 and MKK2. Overall, this analysis emphasizes the central role of this MAPK cascade in oxidative stress signalling, but also indicates the high level of complexity revealed by this signalling network.

Towards functional phosphoproteomics by mapping differential phosphorylation events in signaling networks.

Proteomics. 2008 Oct 29;8(21):4453-4465. PMID: 18972525
de la Fuente van Bentem S, Mentzen WI, de la Fuente A, Hirt H.
Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, Vienna, Austria.

Protein phosphorylation plays a central role in many signal transduction pathways that mediate biological processes. Novel quantitative mass spectrometry-based methods have recently revealed phosphorylation dynamics in animals, yeast, and plants. These methods are important for our understanding of how differential phosphorylation participates in translating distinct signals into proper physiological responses, and shifted research towards screening for potential cancer therapies and in-depth analysis of phosphoproteomes. In this review, we aim to describe current progress in quantitative phosphoproteomics. This emerging field has changed numerous static pathways into dynamic signaling networks, and revealed protein kinase networks that underlie adaptation to environmental stimuli. Mass spectrometry enables high-throughput and high-quality analysis of differential phosphorylation at a site-specific level. Although determination of differential phosphorylation between treatments is analogous to detecting differential gene expression, the large body of statistical techniques that has been developed for analysis of differential gene expression is not generally applied for detecting differential phosphorylation. We suggest possible improvements for analysis of quantitative phosphorylation by increasing the number of biological replicates and adapting statistical tests used for gene expression profiling and widely implemented in freely available software tools.

Possible involvement of MAP kinase pathways in acquired metal-tolerance induced by heat in plants.

Planta. 2008 Aug;228(3):499-509. Epub 2008 May 28. doi 10.1007/s00425-008-0753-x PMID: 18506480
Po-Yu Chen (1), Kuo-Ting Lee (1), Wen-Chang Chi (1), Heribert Hirt (2),
Ching-Chun Chang (3) and Hao-Jen Huang (1)
(1) Department of Life Sciences, National Cheng Kung University, Tainan, 701, Taiwan, ROC
(2) Department of Plant Molecular Biology, Max F. Perutz Laboratories,
University of Vienna, Dr. Bohr-Gasse 9, 1030 Vienna, Austria
(3) Institute of Biotechnology, National Cheng Kung University, Tainan, 701, Taiwan, ROC

Cross tolerance is a phenomenon that occurs when a plant, in resisting one form of stress, develops a tolerance to another form. Pretreatment with nonlethal heat shock has been known to protect cells from metal stress. In this study, we found that the treatment of rice roots with more than 25 muM of Cu(2+) caused cell death. However, heat shock pretreatment attenuated Cu(2+)-induced cell death. The mechanisms of the cross tolerance phenomenon between heat shock and Cu(2+) stress were investigated by pretreated rice roots with the protein synthesis inhibitor cycloheximide (CHX). CHX effectively block heat shock protection, suggesting that protection of Cu(2+)-induced cell death by heat shock was dependent on de novo protein synthesis. In addition, heat pretreatment downregulated ROS production and mitogen-activated protein kinase (MAPK) activities, both of which can be greatly elicited by Cu(2+) stress in rice roots. Moreover, the addition of purified recombinant GST-OsHSP70 fusion proteins inhibited Cu(2+)-enhanced MAPK activities in an in vitro kinase assay. Furthermore, loss of heat shock protection was observed in Arabidopsis mkk2 and mpk6 but not in mpk3 mutants under Cu(2+) stress. Taken together, these results suggest that the interaction of OsHSP70 with MAPKs may contribute to the cellular protection in rice roots from excessive Cu(2+) toxicity.

Arabidopsis MAPKs: a complex signalling network involved in multiple biological processes.

Biochem J. 2008 Jul 15;413(2):217-26. PMID: 18570633
Colcombet J, Hirt H.
URGV INRA-CNRS-UEVE,

Many changes in environmental conditions and hormones are mediated by MAPK (mitogen-activated protein kinase) cascades in all eukaryotes, including plants. Studies of MAPK pathways in genetic model organisms are especially informative in revealing the molecular mechanisms by means of which MAPK cascades are controlled and modulate cellular processes. The present review highlights recent insights into MAPK-based signalling in Arabidopsis thaliana (thale cress), revealing the complexity and future challenges to understanding signal-transduction networks on a global scale.

The Dark Side of the Salad: Salmonella typhimurium Overcomes the Innate Immune Response of Arabidopsis thaliana and Shows an Endopathogenic Lifestyle

PLoS One May 28, 2008 3(5): e2279. doi:10.1371/journal.pone.0002279 PMID: 18509467
Adam Schikora¹, Alessandro Carreri², Emmanuelle Charpentier³, Heribert Hirt¹,²*
¹ URGV Unité de Recherche en Génomique Végétale, INRA Institut National de la Recherche Agronomique/CNRS Centre National de la Recherche Scientifique/University of Evry Val d'Essonne, Evry, France
² Department of Plant Molecular Biology, Max F. Perutz Laboratories, Vienna, Austria
³ Department of Microbiology and Immunobiology, Max F. Perutz Laboratories, Vienna, Austria

Salmonella enterica serovar typhimurium contaminated vegetables and fruits are considerable sources of human infections. Bacteria present in raw plant-derived nutrients cause salmonellosis, the world wide most spread food poisoning. This facultative endopathogen enters and replicates in host cells and actively suppresses host immune responses. Although Salmonella survives on plants, the underlying bacterial infection mechanisms are only poorly understood. In this report we investigated the possibility to use Arabidopsis thaliana as a genetically tractable host system to study Salmonella-plant interactions. Using green fluorescent protein (GFP) marked bacteria, we show here that Salmonella can infect various Arabidopsis tissues and proliferate in intracelullar cellular compartments. Salmonella infection of Arabidopsis cells can occur via intact shoot or root tissues resulting in wilting, chlorosis and eventually death of the infected organs. Arabidopsis reacts to Salmonella by inducing the activation of mitogen-activated protein kinase (MAPK) cascades and enhanced expression of pathogenesis related (PR) genes. The induction of defense responses fails in plants that are compromised in ethylene or jasmonic acid signaling or in the MKK3-MPK6 MAPK pathway. These findings demonstrate that Arabidopsis represents a true host system for Salmonella, offering unique possibilities to study the interaction of this human pathogen with plants at the molecular level for developing novel drug targets and addressing current safety issues in human nutrition.
FWF Austrian Science Fund Press Release
INRA La bactérie Salmonella typhimurium peut infecter les plantes
La bactérie Agrobacterium tumefaciens utilise le système de défense des plantes pour les transformer
INRA 2007 Oct 19
CNRS 2007 Oct 19
Objectif Sciences Comprendre le stress des plantes pour attenuer les effets du rechauffement terrestre

Site-Specific Phosphorylation Profiling of Arabidopsis Proteins by Mass Spectrometry and Peptide Chip Analysis.

J Proteome Res. 2008 Apr 24 [Epub ahead of print] PMID: 18433157
de la Fuente van Bentem S ¹, Anrather D, Dohnal I, Roitinger E, Csaszar E, Joore J, Buijnink J, Carreri A ¹, Forzani C ¹, Lorkovic ZJ, Barta A, Lecourieux D ¹, Verhounig A, Jonak C, Hirt H. ¹*
¹ Department of Plant Molecular Biology, and Department of Biochemistry, Max F. Perutz Laboratories, University of Vienna, Dr. Bohr-Gasse 9, 1030 Vienna, Austria
* Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, 1030 Vienna, Austria
* Pepscan Presto, Zuidersluisweg 2, 8243 RC Lelystad, The Netherlands
* Department of Medical Biochemistry, Max F. Perutz Laboratories, Medical University of Vienna, Dr. Bohr-Gasse 9, 1030 Vienna, Austria
* Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Vienna Biocenter, Dr. Bohrgasse 3, 1030 Vienna, Austria
* URGV Plant Genomics, 2 rue Gaston Cremieux, 91057 Evry, France

An estimated one-third of all proteins in higher eukaryotes are regulated by phosphorylation by protein kinases (PKs). Although plant genomes encode more than 1000 PKs, the substrates of only a small fraction of these kinases are known. By mass spectrometry of peptides from cytoplasmic- and nuclear-enriched fractions, we determined 303 in vivo phosphorylation sites in Arabidopsis proteins. Among 21 different PKs, 12 were phosphorylated in their activation loops, suggesting that they were in their active state. Immunoblotting and mutational analysis confirmed a tyrosine phosphorylation site in the activation loop of a GSK3/shaggy-like kinase. Analysis of phosphorylation motifs in the substrates suggested links between several of these PKs and many target sites. To perform quantitative phosphorylation analysis, peptide arrays were generated with peptides corresponding to in vivo phosphorylation sites. These peptide chips were used for kinome profiling of subcellular fractions as well as H₂O₂-treated Arabidopsis cells. Different peptide phosphorylation profiles indicated the presence of overlapping but distinct PK activities in cytosolic and nuclear compartments. Among different H₂O₂-induced PK targets, a peptide of the serine/arginine-rich (SR) splicing factor SCL30 was most strongly affected. SRPK4 (SR protein-specific kinase 4) and MAPKs (mitogen-activated PKs) were found to phosphorylate this peptide, as well as full-length SCL30. However, whereas SRPK4 was constitutively active, MAPKs were activated by H₂O₂. These results suggest that SCL30 is targeted by different PKs. Together, our data demonstrate that a combination of mass spectrometry with peptide chip phosphorylation profiling has a great potential to unravel phosphoproteome dynamics and to identify PK substrates.

Protein networking: insights into global functional organization of proteomes

Proteomics 2008, 8, 799–816 DOI 10.1002/pmic.200700767
Enrico Pieroni¹, Sergio de la Fuente van Bentem², Gianmaria Mancosu¹, Enrico Capobianco¹, Heribert Hirt², ³ and Alberto de la Fuente¹
¹ CRS4 Bioinformatica, c/o Parco Tecnologico POLARIS, Pula, Italy
² Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, Vienna, Austria
³ Plant Genomics Research Unit, Unité de Recherche en Génomique Végétale (URGV), INRA/CNRS, Evry, France

The formulation of network models from global protein studies is essential to understand the functioning of organisms. Network models of the proteome enable the application of Complex Network Analysis, a quantitative framework to investigate large complex networks using techniques from graph theory, statistical physics, dynamical systems and other fields. This approach has provided many insights into the functional organization of the proteome so far and will likely continue to do so. Currently, several network concepts have emerged in the field of proteomics. It is important to highlight the differences between these concepts, since different representations allow different insights into functional organization. One such concept is the protein interaction network, which contains proteins as nodes and undirected edges representing the occurrence of binding in large-scale protein-protein interaction studies. A second concept is the protein-signaling network, in which the nodes correspond to levels of post-translationally modified forms of proteins and directed edges to causal effects through post-translational modification, such as phosphorylation. Several other network concepts were introduced for proteomics. Although all formulated as networks, the concepts represent widely different physical systems. Therefore caution should be taken when applying relevant topological analysis. We review recent literature formulating and analyzing such networks.

Trojan Horse Strategy in Agrobacterium Transformation: Abusing MAPK Defense Signaling

Science 19 October 2007: Vol. 318. no. 5849, pp. 453 - 456 DOI: 10.1126/science.1148110
Armin Djamei,¹* Andrea Pitzschke,¹ Hirofumi Nakagami,¹ Iva Rajh,¹ Heribert Hirt¹²
¹ Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, Dr.-Bohr-Gasse 9, 1030 Vienna, Austria.
² URGV

Nuclear import of transfer DNA (T-DNA) is a central event in Agrobacterium transformation of plant cells and is thought to occur by the hijacking of certain host cell proteins. The T-DNA–associated virulence protein VirE2 mediates this process by binding to the nuclear import machinery via the host cell factor VIP1, whose role in plants has been so far unknown. Here we show that VIP1 is a transcription factor that is a direct target of the Agrobacterium-induced mitogen-activated protein kinase (MAPK) MPK3. Upon phosphorylation by MPK3, VIP1 relocalizes from the cytoplasm to the nucleus and regulates the expression of the PR1 pathogenesis-related gene. MAPK-dependent phosphorylation of VIP1 is necessary for VIP1-mediated Agrobacterium T-DNA transfer, indicating that Agrobacterium abuses the MAPK-targeted VIP1 defense signaling pathway for nuclear delivery of the T-DNA complex as a Trojan horse.
Materials and Methods, Figs. S1 to S4, References

The Arabidopsis Mitogen-Activated Protein Kinase Kinase MKK3 Is Upstream of Group C Mitogen-Activated Protein Kinases and Participates in Pathogen Signaling

The Plant Cell, Vol. 19: 3266–3279, October 2007, www.plantcell.org © 2007 American Society of Plant Biologists PMID: 17933903
Róbert Dóczi (1), Günter Brader (2), Aladár Pettkó-Szandtner (1), Iva Rajh (1), Armin Djamei (1), Andrea Pitzschke (1), Markus Teige (1), and Heribert Hirt (1, 3, *)
(1) Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, A-1030 Vienna, Austria
(2) Viikki Biocenter, Department of Biological and Environmental Sciences, Division of Genetics, Faculty of Biosciences, University of Helsinki, FIN-00014 Helsinki, Finland
(3) Unité de Recherche en Génomique Végétale, Plant Genomics Research Unit, F-91057 Evry, France
(*) To whom correspondence should be addressed: heribert.hirt@univie.ac.at

Although the Arabidopsis thaliana genome contains genes encoding 20 mitogen-activated protein kinases (MAPKs) and 10 MAPK kinases (MAPKKs), most of them are still functionally uncharacterized. In this work, we analyzed the function of the group B MAPK kinase, MKK3. Transgenic ProMKK3:GUS lines showed basal expression in vascular tissues that was strongly induced by Pseudomonas syringae pv tomato strain DC3000 (Pst DC3000) infection but not by abiotic stresses. The growth of virulent Pst DC3000 was increased in mkk3 knockout plants and decreased in MKK3-overexpressing plants. Moreover, MKK3 overexpression lines showed increased expression of several PR genes. By yeast two-hybrid analysis, coimmunoprecipitation, and protein kinase assays, MKK3 was revealed to be an upstream activator of the group C MAPKs MPK1, MPK2, MPK7, and MPK14. Flagellin-derived flg22 peptide strongly activated MPK6 but resulted in poor activation of MPK7. By contrast, MPK6 and MPK7 were both activated by H2O2, but only MPK7 activation was enhanced by MKK3. In agreement with the notion that MKK3 regulates the expression of PR genes, ProPR1:GUS expression was strongly enhanced by coexpression of MKK3-MPK7. Our results reveal that the MKK3 pathway plays a role in pathogen defense and further underscore the importance and complexity of MAPK signaling in plant stress responses.

The PP2C-Type Phosphatase AP2C1 Negatively Regulates MPK4 and MPK6 and Modulates Innate Immunity and Jasmonic Acid and Ethylene Levels in Arabidopsis

The Plant Cell , published July 13, 2007
Alois Schweighofer^a, Vaiva Kazanaviciute^a, Elisabeth Scheikl^a, Markus Teige^a, Róbert Dóczi^a, Heribert Hirt^a, Manfred Schwanninger^b, Merijn Kant^c, Robert Schuurink^c, Felix Mauch^d, Antony Buchala^d, Francesca Cardinale^e, and Irute Meskiene^a,
^aMax F. Perutz Laboratories of the University of Vienna, 1030 Vienna, Austria
^bDepartment of Chemistry, University of Natural Resources and Applied Life Sciences, 1190 Vienna, Austria
^cDepartment of Plant Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, 1098 SM Amsterdam, The Netherlands
^dDépartement de Biologie, Université de Fribourg, CH-1700 Fribourg, Switzerland
^eDipartimento di Valorizzazione e Protezione delle Risorse Agroforestali, Plant Pathology, University of Turin, I-10095 Grugliasco, Italy

Summery
Wound signaling pathways in plants are mediated by mitogen-activated protein kinases (MAPKs) and stress hormones, such as ethylene and jasmonates. In Arabidopsis thaliana, the transmission of wound signals by MAPKs has been the subject of detailed investigations; however, the involvement of specific phosphatases in wound signaling is not known. Here, we show that AP2C1, an Arabidopsis Ser/Thr phosphatase of type 2C, is a novel stress signal regulator that inactivates the stress-responsive MAPKs MPK4 and MPK6. Mutant ap2c1 plants produce significantly higher amounts of jasmonate upon wounding and are more resistant to phytophagous mites (Tetranychus urticae). Plants with increased AP2C1 levels display lower wound activation of MAPKs, reduced ethylene production, and compromised innate immunity against the necrotrophic pathogen Botrytis cinerea. Our results demonstrate a key role for the AP2C1 phosphatase in regulating stress hormone levels, defense responses, and MAPK activities in Arabidopsis and provide evidence that the activity of AP2C1 might control the plant’s response to B. cinerea.

The BRI1-Associated Kinase 1, BAK1, Has a Brassinolide-Independent Role in Plant Cell-Death Control

Current Biology 17, 1116–1122, July 3, 2007 ©2007 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2007.05.046

Birgit Kemmerling¹, Anne Schwedt¹, Patricia Rodriguez¹, Sara Mazzotta¹, Markus Frank², Synan Abu Qamar³, Tesfaye Mengiste³, Shigeyuki Betsuyaku⁴, Jane E. Parker⁴, Carsten Müssig⁵, Bart P.H.J. Thomma⁶, Catherine Albrecht⁷, Sacco C. de Vries⁷, Heribert Hirt⁸, and Thorsten Nürnberger^1*
1Department of Plant Biochemistry, Center for Plant Molecular Biology, Eberhard-Karls-University Tübingen, 72076 Tübingen, Germany
²BASF Plant Science GmbH, BPS-LI444, 67117 Limburgerhof, Germany
³Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907-2054, USA
⁴Department of Plant-Microbe Interactions, Max-Planck-Institute for Plant Breeding Research, 50829 Köln, Germany
⁵Department of Genetics, University of Potsdam, 14476 Potsdam-Golm, Germany
⁶Laboratory of Phytopathology, Wageningen University, Wageningen 6709 PD, The Netherlands
⁷Laboratory of Biochemistry, Wageningen University, Wageningen 6703 HA, The Netherlands
⁸Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, 1030 Vienna, Austria

Summary: Programmed cell death (PCD) is a common host response to microbial infection [1–3]. In plants, PCD is associated with immunity to biotrophic pathogens, but it can also promote disease upon infection by necrotrophic pathogens [4]. Therefore, plant cell-suicide programs must be strictly controlled. Here we demonstrate that the Arabidopsis thaliana Brassinosteroid Insensitive 1 (BRI1)-associated receptor Kinase 1 (BAK1), which operates as a coreceptor of BRI1 in brassinolide (BL)-dependent plant development, also regulates the containment of microbial infectioninduced cell death. BAK1-deficient plants develop spreading necrosis upon infection. This is accompanied by production of reactive oxygen intermediates and results in enhanced susceptibility to necrotrophic fungal pathogens. The exogenous application of BL rescues growth defects of bak1 mutants but fails to restore immunity to fungal infection. Moreover, BL -insensitive and -deficient mutants do not exhibit spreading necrosis or enhanced susceptibility to fungal infections. Together, these findings suggest that plant steroid-hormone signaling is dispensable for the containment of infection-induced PCD. We propose a novel, BL-independent function of BAK1 in plant celldeath control that is distinct from its BL -dependent role in plant development.

The MAP Kinase Kinase MKK2 Affects Disease Resistance in Arabidopsis

PMID: 17506336 MPMI Vol. 20, No. 5, 2007, pp. 589–596. doi:10.1094/MPMI -20-5-0589. © 2007 The American Phytopathological Society
Günter Brader ¹, Armin Djamei ², Markus Teige ², E. Tapio Palva ¹, and Heribert Hirt², ³

¹ Viikki Biocenter, Faculty of Biosciences, Department of Biological and Environmental Sciences, Division of Genetics, P.O. Box 56, FIN-00014 University of Helsinki, Finland
² Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, Dr. Bohrgasse 9, A-1030 Vienna, Austria
³ Unité de Recherche en Génomique Végétale, 2 rue Gaston Crémieux, 91057 Evry cedex, France

Summary: The Arabidopsis mitogen-activated protein kinase (MAPK) kinase 2 (MKK2) was shown to mediate cold and salt stress responses through activation of the two MAP kinases MPK4 and MPK6. Transcriptome analysis of plants expressing constitutively active MKK2 (MKK2-EE plants) showed altered expression of genes induced by abiotic stresses but also a significant number of genes involved in defense responses. Both MPK4 and MPK6 became rapidly activated upon Pseudomonas syringae pv. tomato DC3000 infection and MKK2-EE plants showed enhanced levels of MPK4 activation. Although MKK2-EE plants shared enhanced expression of genes encoding enzymes of ethylene (ET) and jasmonic acid (JA) synthesis, ET, JA, and salicylic acid (SA) levels did not differ dramatically from those of wild-type or mkk2-null plants under ambient growth conditions. Upon P. syringae pv. tomato DC3000 infection, however, MKK2-EE plants showed reduced increases of JA and SA levels. These results indicate that MKK2 is involved in regulating hormone levels in response to pathogens. MKK2-EE plants were more resistant to infection by P. syringae pv. tomato DC3000 and Erwinia carotovora subsp. carotovora, but showed enhanced sensitivity to the fungal necrotroph Alternaria brassicicola. Our data indicate that MKK2 plays a role in abiotic stress tolerance and plant disease resistance.

A plastid-localized glycogen synthase kinase 3 modulatesstress tolerance and carbohydrate metabolism

The Plant Journal (2007) 49, 1076–1090 doi: 10.1111/j.1365-313X.2006.03025.x
Stefan Kempa¹, Wilfried Rozhon¹, Jozef Šamaj^2,3, Alexander Erban⁴, Frantiŝek Baluŝka³, Thomas Becker⁵, Joachim Haselmayer⁶, Enrico Schleiff⁵, Joachim Kopka⁴, Heribert Hirt⁶ and Claudia Jonak^1
1Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Vienna Biocenter, Dr Bohrgasse 3, A-1030 Vienna, Austria
²Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademická 2, PO Box 39A, SK-950 07 Nitra, Slovak Republic
³Institute of Cellular and Molecular Botany, University of Bonn, Kirschallee 1, D-53115 Bonn, Germany
⁴Max Plank Institute of Molecular Plant Biology, Am Mühlenberg 1, D-14467 Golm, Germany
⁵Department of Biology I, Ludwig-Maximilians-University Munich, Menzinger Straße 67, D-80638 Munich, Germany
⁶Max F. Perutz Laboratories, University of Vienna, Vienna Biocenter, Dr Bohrgasse 9, A-1030 Vienna, Austria

Summary: Glycogen synthase kinase 3 (GSK-3) was originally identified as a regulator of glycogen synthesis in mammals. Like starch in plants, glycogen is a polymer of glucose, and serves as an energy and carbon store. Starch is the main carbohydrate store in plants. Regulation of starch metabolism, in particular in response to environmental cues, is of primary importance for carbon and energy flow in plants but is still obscure. Here, we provide evidence that MsK4, a novel Medicago sativa GSK-3-like kinase, connects stress signalling with carbon metabolism. MsK4 was found to be a plastid-localized protein kinase that is associated with starch granules. High-salt stress rapidly induced the in vivo kinase activity of MsK4. Metabolic profiling of MsK4 over-expressor lines revealed changes in sugar metabolism, including increased amounts of maltose, the main degradation product of starch in leaves. Plants over-expressing MsK4 showed improved tolerance to salt stress. Moreover, under high-salinity conditions, MsK4-over-expressing plants accumulated significantly more starch and showed modified carbohydrate content compared with wild-type plants. Overall, these data indicate that MsK4 is an important regulator that adjusts carbohydrate metabolism to environmental stress.

Using phosphoproteomics to reveal signalling dynamics in plants

Trends Plant Sci. 2007 Sep;12(9):404-11. Epub 2007 Aug 31. PMID: 17765599
Sergio de la Fuente van Bentem ¹ and Heribert Hirt ¹, ²
¹ Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, Dr. Bohr-Gasse 9, A-1030 Vienna, Austria
² URGV Plant Genomics, 2 rue Gaston Cremieux, F-91057 Evry, France

To ensure appropriate responses to stimuli, organisms have evolved signalling networks that rely on post-translational modifications of their components. Among these, protein phosphorylation has a prominent role and much research in plants has focused on protein kinases and phosphatases, which, respectively, catalyse phosphorylation and dephosphorylation of specific substrates. Technical limitations, however, have hampered the identification of these substrates. As reviewed here, novel mass spectrometry-based techniques have enabled the large-scale mapping of *in vivo* phosphorylation sites. Alternatively, methods based on peptide and protein microarrays have revealed protein kinase activities in cell extracts, in addition to kinase substrates. A combined phosphoproteomic approach of mass spectrometry and microarray technology could enhance the construction of dynamic plant signalling networks that underlie plant biology.