Molecular identification and characterization of the tomato flagellin receptor LeFLS2, an orthologue of Arabidopsis FLS2 exhibiting characteristically different perception specificities

Bacterial flagellin is known to stimulate host immune responses in mammals and plants. In Arabidopsis thaliana, the receptor kinase FLS2 mediates flagellin perception through physical interaction with a highly conserved epitope in the N-terminus of flagellin, represented by the peptide flg22 derived from Pseudomonas syringae. The peptide flg22 is highly active as an elicitor in many plant species. In contrast, a shortened version of the same epitope derived from Escherichia coli, flg15E coli, is highly active as an elicitor in tomato but not in A. thaliana or Nicotiana benthamiana. Here, we make use of these species-specific differences in flagellin perception abilities to identify LeFLS2 as the flagellin receptor in tomato. LeFLS2 is most closely related to AtFLS2, indicating that it may represent the flagellin receptor of tomato. Expression of the LeFLS2 gene in Arabidopsis did not result in accumulation of its corresponding gene product, as indicated by experiments with LeFLS2-GFP fusions. In contrast, expression of LeFLS2-GFP fusions in N. benthamiana, a species that, like tomato, belongs to the Solanaceae, was obviously functional. N. benthamiana plants transiently expressing a LeFLS2-GFP fusion acquired responsiveness to flg15E coli to which they are normally unresponsive. Thus, LeFLS2 encodes a functional, specific flagellin receptor, the first to be identified in a plant family other than the Brassicacea

Le rôle des ankyrine protéines kinases dans le développement végétal

Les protéines kinases constituent des régulateurs essentiels des voies de signalisation. Chez Medicago spp., le gène Msapk1 " Medicago saliva ankyrin protéine kinase " a été identifié comme exprimé dans les nodosités spontanées de luzerne. Il code pour un nouveau type de kinase, composée d'un domaine aminoterminal à motif ankyrine fusionné à un domaine catalytique. Cette structure est retrouvée au sein de protéines "Integrin-Linked Kinases" animales, impliquées dans l'adhésion cellulaire. Mon travail de thèse a porté sur la caractérisation des gènes APK chez Medicago spp., et de leurs homologues chez Arabidopsis thaliana, afin de comprendre leur fonction dans le développement végétal. Chez Medicago spp., l'expression du gène Msapkl est induite dans les racines en réponse au choc osmotique. Par ailleurs, une protéine de fusion MsAPK1-GFP localiserait sur le cytosquelette des cellules d'oignon, en réponse à ce choc. Nous avons également montré que la protéine MsAPK1 purifiée chez E. coli phosphoryle la tubuline in vitro. Ces résultats suggèrent que MsAPK1 pourrait être un régulateur des modifications du cytosquelette observées en réponse au choc osmotique. Chez Arabidopsis, les gènes AtAPK, homologues à Msapkl, s'expriment différentiellement dans plusieurs organes. Malgré la forte conservation observée entre les gènes d'Arabidopsis et de Medicago truncatula, les AtAPK ne seraient pas impliqués dans la réponse osmotique. Plusieurs approches de génétique inverse, de type " perte de fonction " et " gain de fonction ", réalisées sur les APK suggèrent leur redondance fonctionnelle chez Arabidopsis. Parallèlement à cette étude, nous avons caractérisé l'induction de l'expression du gène MsCPK3, codant pour une CDPK, au cours du développement de la nodosité de la luzerne. Cette activation a lieu simultanément à une augmentation d'activité CDPK, durant les étapes précoces de la nodulation. Cette CDPK constitue une cible potentielle de l'action du calcium chez les Fabacées. Ces résultats contribuent à la caractérisation de nouvelles protéines kinases dans la réponse de Medicago spp. au choc osmotique et au cours du développement de la nodosité. En particulier, la caractérisation des APK ouvre de nouvelles perspectives sur la participation de ces kinases dans les modifications de l'adhésion cellulaire végétale.Protein kinases are key components of signalling pathways. The Msapk1 gene (for Medicago saliva ankyrin protein kinase), coding for a novel protein kinase, was isolated as expressed in spontaneous nodules in alfalfa. This kinase has an unique aminoterminal domain containing three ankyrin repeats. This structure resembles that from animal Integrin Linked Kinases which are involved in cell adhesion in mammalian cells. The major objective of this work was to characterize the APK gene in Medicago spp. and their homologues in Arabidopsis thaliana, in order to understand their function in plant development. Msapkl expression was round to be induced upon hyperosmotic stress in Medicago roots. Moreover, a MsAPK1-GFP fusion protein localized to a cytoskeletal network after an osmotic shock in onion cells. By heterologous expression in E. coli, we also showed that MsAPK1 phosphorylates tubulin in vitro. These results suggest that MsAPK1 may regulate cytoskeleton modifications occurring during cellular responses to osmotic stress in plants. In Arabidopsis, the AtAPK genes, homologues of Msapk1, were round to be differentially expressed in several organs. Despite the high conservation between Arabidopsis and Medicago truncatula genes, the AtAPKs seem not to be involved in osmotic responses in this species. Reverse genetics approaches on APK, developed to create "loss of function" and "gain of function" effects, suggest that APK genes are functionally redundant in Arabidopsis. In parallel, we characterized the induction of a CDPK gene expression, called MsCPK3, during nodule development in alfalfa. This activation takes place concomitantly with the induction of CDPK activity in alfalfa roots, during the early steps of nodulation. This CDPK is a potential target for calcium action in Fabaceae. These results add to the characterization of new protein kinases in Medicago spp. involved in osmotic stress responses and nodule organogenesis. Moreover, the studies on APK may open new perspectives on their participation in plant cell adhesion processes.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Ligand-induced endocytosis of the pattern recognition receptor FLS2 in Arabidopsis

Pattern-recognition receptors (PRRs) trigger innate immune responses in animals and plants. One such PRR is the flagellin receptor FLS2 in Arabidopsis. Here, we demonstrate that a functional fusion of FLS2 to the green fluorescent protein (GFP) resides in cell membranes of most tissues. Stimulation with the flagellin epitope flg22 induces its transfer into intracellular mobile vesicles, followed by degradation. FLS2 internalization depends on cytoskeleton and proteasome functions, and receptor activation. A variant FLS2 mutated in Thr 867, a potential phosphorylation site, binds flg22 normally, but is impaired in flg22 responses and FLS2 endocytosis. We propose that plant cells regulate pathogen-associated molecular pattern (PAMP)-mediated PRR activities by subcellular compartmentalization

The Arabidopsis Receptor Kinase FLS2 Binds flg22 and Determines the Specificity of Flagellin Perception

Flagellin, the main building block of the bacterial flagellum, acts as a pathogen-associated molecular pattern triggering the innate immune response in animals and plants. In Arabidopsis thaliana, the Leu-rich repeat transmembrane receptor kinase FLAGELLIN SENSITIVE2 (FLS2) is essential for flagellin perception. Here, we demonstrate the specific interaction of the elicitor-active epitope flg22 with the FLS2 protein by chemical cross-linking and immunoprecipitation. The functionality of this receptor was further tested by heterologous expression of the Arabidopsis FLS2 gene in tomato (Lycopersicon esculentum) cells. The perception of flg22 in tomato differs characteristically from that in Arabidopsis. Expression of Arabidopsis FLS2 conferred an additional flg22-perception system on the cells of tomato, which showed all of the properties characteristic of the perception of this elicitor in Arabidopsis. In summary, these results show that FLS2 constitutes the pattern-recognition receptor that determines the specificity of flagellin perception

A two-hybrid-receptor assay demonstrates heteromer formation as switch-on for plant immune receptors

Receptor kinases sense extracellular signals and trigger intracellular signaling and physiological responses. However, how does signal binding to the extracellular domain activate the cytoplasmic kinase domain? Activation of the plant immunoreceptor Flagellin sensing2 (FLS2) by its bacterial ligand flagellin or the peptide-epitope flg22 coincides with rapid complex formation with a second receptor kinase termed brassinosteroid receptor1 associated kinase1 (BAK1). Here, we show that the receptor pair of FLS2 and BAK1 is also functional when the roles of the complex partners are reversed by swapping their cytosolic domains. This reciprocal constellation prevents interference by redundant partners that can partially substitute for BAK1 and demonstrates that formation of the heteromeric complex is the molecular switch for transmembrane signaling. A similar approach with swaps between the Elongation factor-Tu receptor and BAK1 also resulted in a functional receptor/coreceptor pair, suggesting that a "two-hybrid-receptor assay" is of more general use for studying heteromeric receptor complexes