Cellular Complexity in MAPK Signaling in Plants: Questions and Emerging Tools to Answer Them

Mitogen activated protein kinase (MAPK) cascades play an important role in many aspects of plant growth, development, and environmental response. Because of their central role in many important processes, MAPKs have been extensively studied using biochemical and genetic approaches. This work has allowed for the identification of the MAPK genes and proteins involved in a number of different signaling pathways. Less well developed, however, is our understanding of how MAPK cascades and their corresponding signaling pathways are organized at subcellular levels. In this review, we will provide an overview of plant MAPK signaling, including a discussion of what is known about cellular mechanisms for achieving signaling specificity. Then we will explore what is currently known about the subcellular localization of MAPK proteins in resting conditions and after pathway activation. Finally, we will discuss a number of new experimental methods that have not been widely deployed in plants that have the potential to provide a deeper understanding of the spatial and temporal dynamics of MAPK signaling

Biochemical and Genetic Interactions of Phospholipase D Alpha 1 and Mitogen-Activated Protein Kinase 3 Affect Arabidopsis Stress Response

Phospholipase D alpha 1 (PLDα1, AT3G15730) and mitogen-activated protein kinases (MAPKs) participate on signaling-dependent events in plants. MAPKs are able to phosphorylate a wide range of substrates putatively including PLDs. Here we have focused on functional regulations of PLDα1 by interactions with MAPKs, their co-localization and impact on salt stress and abscisic acid (ABA) tolerance in Arabidopsis. Yeast two-hybrid and bimolecular fluorescent assays showed that PLDα1 interacts with MPK3. Immunoblotting analyses likewise confirmed connection between both these enzymes. Subcellularly we co-localized PLDα1 with MPK3 in the cortical cytoplasm close to the plasma membrane and in cytoplasmic strands. Moreover, genetic interaction studies revealed that pldα1mpk3 double mutant was resistant to a higher salinity and showed a higher tolerance to ABA during germination in comparison to single mutants and wild type. Thus, this study revealed importance of new biochemical and genetic interactions between PLDα1 and MPK3 for Arabidopsis stress (salt and ABA) response

Functional analysis of Arabidopsis immune-related MAPKs uncovers a role for MPK3 as negative regulator of inducible defences

Background : Mitogen-activated protein kinases (MAPKs) are key regulators of immune responses in animals and plants. In Arabidopsis, perception of microbe-associated molecular patterns (MAMPs) activates the MAPKs MPK3, MPK4 and MPK6. Increasing information depicts the molecular events activated by MAMPs in plants, but the specific and cooperative contributions of the MAPKs in these signalling events are largely unclear.[br/] Results: In this work, we analyse the behaviour of MPK3, MPK4 and MPK6 mutants in early and late immune responses triggered by the MAMP flg22 from bacterial flagellin. A genome-wide transcriptome analysis reveals that 36% of the flg22-upregulated genes and 68% of the flg22-downregulated genes are affected in at least one MAPK mutant. So far MPK4 was considered as a negative regulator of immunity, whereas MPK3 and MPK6 were believed to play partially redundant positive functions in defence.[br/] Our work reveals that MPK4 is required for the regulation of approximately 50% of flg22-induced genes and we identify a negative role for MPK3 in regulating defence gene expression, flg22-induced salicylic acid accumulation and disease resistance to Pseudomonas syringae. Among the MAPK-dependent genes, 27% of flg22-upregulated genes and 76% of flg22-downregulated genes require two or three MAPKs for their regulation. The flg22-induced MAPK activities are differentially regulated in MPK3 and MPK6 mutants, both in amplitude and duration, revealing a highly interdependent network.[br/] Conclusions : These data reveal a new set of distinct functions for MPK3, MPK4 and MPK6 and indicate that the plant immune signalling network is choreographed through the interplay of these three interwoven MAPK pathways

Assessing associations between the AURKAHMMR-TPX2-TUBG1 functional module and breast cancer risk in BRCA1/2 mutation carriers

While interplay between BRCA1 and AURKA-RHAMM-TPX2-TUBG1 regulates mammary epithelial polarization, common genetic variation in HMMR (gene product RHAMM) may be associated with risk of breast cancer in BRCA1 mutation carriers. Following on these observations, we further assessed the link between the AURKA-HMMR-TPX2-TUBG1 functional module and risk of breast cancer in BRCA1 or BRCA2 mutation carriers. Forty-one single nucleotide polymorphisms (SNPs) were genotyped in 15,252 BRCA1 and 8,211 BRCA2 mutation carriers and subsequently analyzed using a retrospective likelihood appr

Common breast cancer susceptibility alleles are associated with tumor subtypes in BRCA1 and BRCA2 mutation carriers : results from the Consortium of Investigators of Modifiers of BRCA1/2.

Abstract Introduction Previous studies have demonstrated that common breast cancer susceptibility alleles are differentially associated with breast cancer risk for BRCA1 and/or BRCA2 mutation carriers. It is currently unknown how these alleles are associated with different breast cancer subtypes in BRCA1 and BRCA2 mutation carriers defined by estrogen (ER) or progesterone receptor (PR) status of the tumour. Methods We used genotype data on up to 11,421 BRCA1 and 7,080 BRCA2 carriers, of whom 4,310 had been affected with breast cancer and had information on either ER or PR status of the tumour, to assess the associations of 12 loci with breast cancer tumour characteristics. Associations were evaluated using a retrospective cohort approach. Results The results suggested stronger associations with ER-positive breast cancer than ER-negative for 11 loci in both BRCA1 and BRCA2 carriers. Among BRCA1 carriers, single nucleotide polymorphism (SNP) rs2981582 (FGFR2) exhibited the biggest difference based on ER status (per-allele hazard ratio (HR) for ER-positive = 1.35, 95% CI: 1.17 to 1.56 vs HR = 0.91, 95% CI: 0.85 to 0.98 for ER-negative, P-heterogeneity = 6.5 × 10-6). In contrast, SNP rs2046210 at 6q25.1 near ESR1 was primarily associated with ER-negative breast cancer risk for both BRCA1 and BRCA2 carriers. In BRCA2 carriers, SNPs in FGFR2, TOX3, LSP1, SLC4A7/NEK10, 5p12, 2q35, and 1p11.2 were significantly associated with ER-positive but not ER-negative disease. Similar results were observed when differentiating breast cancer cases by PR status. Conclusions The associations of the 12 SNPs with risk for BRCA1 and BRCA2 carriers differ by ER-positive or ER-negative breast cancer status. The apparent differences in SNP associations between BRCA1 and BRCA2 carriers, and non-carriers, may be explicable by differences in the prevalence of tumour subtypes. As more risk modifying variants are identified, incorporating these associations into breast cancer subtype-specific risk models may improve clinical management for mutation carriers

Assessing Associations between the AURKA-HMMR-TPX2-TUBG1 Functional Module and Breast Cancer Risk in BRCA1/2 Mutation Carriers

While interplay between BRCA1 and AURKA-RHAMM-TPX2-TUBG1 regulates mammary epithelial polarization, common genetic variation in HMMR (gene product RHAMM) may be associated with risk of breast cancer in BRCA1 mutation carriers. Following on these observations, we further assessed the link between the AURKA-HMMR-TPX2-TUBG1 functional module and risk of breast cancer in BRCA1 or BRCA2 mutation carriers. Forty-one single nucleotide polymorphisms (SNPs) were genotyped in 15,252 BRCA1 and 8,211 BRCA2 mutation carriers and subsequently analyzed using a retrospective likelihood approach. The association of HMMR rs299290 with breast cancer risk in BRCA1 mutation carriers was confirmed: per-allele hazard ratio (HR) = 1.10, 95% confidence interval (CI) 1.04 - 1.15, p = 1.9 x 10(-4) (false discovery rate (FDR)-adjusted p = 0.043). Variation in CSTF1, located next to AURKA, was also found to be associated with breast cancer risk in BRCA2 mutation carriers: rs2426618 per-allele HR = 1.10, 95% CI 1.03 - 1.16, p = 0.005 (FDR-adjusted p = 0.045). Assessment of pairwise interactions provided suggestions (FDR-adjusted p(interaction) values > 0.05) for deviations from the multiplicative model for rs299290 and CSTF1 rs6064391, and rs299290 and TUBG1 rs11649877 in both BRCA1 and BRCA2 mutation carriers. Following these suggestions, the expression of HMMR and AURKA or TUBG1 in sporadic breast tumors was found to potentially interact, influencing patients' survival. Together, the results of this study support the hypothesis of a causative link between altered function of AURKA-HMMR-TPX2-TUBG1 and breast carcinogenesis in BRCA1/2 mutation carriers.Peer reviewe

LES CANAUX ANIONIQUES DE LA MEMBRANE PLASMIQUE CHEZ ARABIDOPSIS THALIANA: CARACTÉRISATION FONCTIONNELLE ET MISE EN PLACE D'OUTILS POUR L'IDENTIFICATION MOLÉCULAIRE

The function of plasma membrane anion channels in plant cells is well understood only in a few cases of specialized cells: osmotic regulation (for stomatal guard cells) and stress responses (for root epidermal cells). The laboratory where I prepared my thesis is involved in studies of Arabidopsis thaliana hypocotyl, an organ with less specialized cells. My PhD work is divided into two parts: first I was involved in the characterization of two anion channels at the plasma membrane of hypocotyl epidermal cells and I have looked for their involvement in a particular function, and second I have developed functional screens to identify anion channel genes. The first anion channel that I contributed to characterize is a channel with rapid kinetics, permeable to several anions. Among them, sulfate exerts an activating effect. Moreover, I have described the biophysical mechanism of the channel gating by voltage: the channel pore is blocked by a nucleotide molecule at very negative potentials. Extracellular anions are able to exert a repulsion on the nucleotide. This dual regulation modulates channel activity in the range of physiological voltages. I have also contributed to the characterisation of a second anion channel with slow kinetics, present on the same cells. This channel is weakly voltage-dependent, shows a high nitrate permeability, and is regulated by phosphorylation. To look for the physiological function of these channels, I have chosen to characterize anion effluxes from Arabidopsis suspension cells, which also posses these two types of channels. In resting conditions, no efflux was observed: channels are closed. Only few signals, hypoosmotic chock and bacterial and fungal elicitors, are able to trigger an efflux. Considering the pharmacological and selectivity profiles of these effluxes, we have not be able to clearly involve any of the rapid-type or the slow-type anion channels. On the basis of anion channel properties described using the patch-clamp technique, I have developed two functional screens using heterologous systems to identify channel cDNAs. The first one, based on channel selectivity for toxic anions, is developed in yeast. Positive controls did not allow us to define conditions for a positive screen but might provide conditions for a negative screen. The second one, based of the activating effect of sulfate on the rapid anion channel, is currently being investigated in Xenopus oocytes.La fonction des canaux anioniques présents sur la membrane plasmique des cellules végétales n'est comprise que dans un nombre restreint de cas correspondant à des activités particulières dans des cellules spécialisées: la régulation osmotique (cellules de garde des stomates) et la tolérance aux stress (cellules épidermiques de racine). Le laboratoire dans lequel j'ai effectué ma thèse s'intéresse plus particulièrement à l'hypocotyle d'Arabidopsis thaliana, organe ayant des types cellulaires moins clairement spécialisés. Mon travail de thèse comprend deux volets, d'une part j'ai contribué à la caractérisation de deux canaux anioniques de la membrane plasmique et j'ai recherché leur implication dans certaines réponses cellulaires, d'autre part j'ai développé des cribles fonctionnels afin d'identifier les gènes codant pour ces canaux. Le premier canal que j'ai contribué à caractériser dans les cellules épidermiques de l'hypocotyle, est un canal à cinétiques rapides perméant à différents anions. Parmi ceux-ci, le sulfate a un rôle activateur du canal. J'ai de plus décrit le mécanisme biophysique de la régulation par le voltage du canal rapide: le pore du canal est obstrué par une molécule de nucléotide aux potentiels hyperpolarisés. Les anions externes sont capables de chasser le nucléotide hors du pore par un phénomène répulsif. Cette double régulation module l'activité du canal dans une gamme de potentiels physiologiques. Enfin, j'ai participé à l'identification d'un second canal à cinétiques lentes sur les mêmes cellules. Ce canal est faiblement régulé par le potentiel de membrane et présente une perméabilité très en faveur du nitrate. Il est régulé par phosphorylation. Pour rechercher la fonction physiologique de ces deux canaux, j'ai choisi de caractériser les efflux d'anions dans le système des cellules d'Arabidopsis en suspension, qui sont aussi équipées de ces canaux. Au repos, aucun efflux d'anions n'est mesuré: les canaux sont fermés. Peu de signaux sont capables de les activer, parmi lesquels un choc hypoosmotique et certains éliciteurs de type fongique et bactérien. La caractérisation de ces efflux, grâce aux outils pharmacologiques et à la sélectivité ionique, n'a pas permis d'impliquer clairement un des canaux anioniques décrits en patch-clamp. Sur la base des propriétés des canaux décrites en patch-clamp, j'ai aussi développé deux cribles fonctionnels en système hétérologue pour tenter d'identifier les ADNc correspondants. Le premier, basé sur la sélectivité des canaux pour les anions toxiques, est réalisé en levure. Mes contrôles positifs n'ont pas permis de définir de conditions pour un crible positif mais ont ouvert des possibilités de cribles négatifs. Le second, fondé sur l'effet activateur du sulfate sur le canal rapide, est effectué en ovocyte de xénope et est en cours de validation

Sustained Incompatibility between MAPK Signaling and Pathogen Effectors

International audienceIn plants, Mitogen-Activated Protein Kinases (MAPKs) are important signaling components involved in developemental processes as well as in responses to biotic and abiotic stresses. In this review, we focus on the roles of MAPKs in Effector-Triggered Immunity (ETI), a specific layer of plant defense responses dependent on the recognition of pathogen effector proteins. Having inspected the literature, we synthesize the current state of knowledge concerning this topic. First, we describe how pathogen effectors can manipulate MAPK signaling to promote virulence, and how in parallel plants have developed mechanisms to protect themselves against these interferences. Then, we discuss the striking finding that the recognition of pathogen effectors can provoke a sustained activation of the MAPKs MPK3/6, extensively analyzing its implications in terms of regulation and functions. In line with this, we also address the question of how a durable activation of MAPKs might affect the scope of their substrates, and thereby mediate the emergence of possibly new ETI-specific responses. By highlighting the sometimes conflicting or missing data, our intention is to spur further research in order to both consolidate and expand our understanding of MAPK signaling in immunity

Les canaux anioniques de la membrane plasmique chez Arabidopsis thaliana (caractérisation fonctionnelle et mise en place d'outils pour l'identification moléculaire)

PARIS-BIUSJ-Thèses (751052125) / SudocCentre Technique Livre Ens. Sup. (774682301) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

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

International audienceMany 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