RESUMEN
Phytocytokines regulate plant immunity by cooperating with cell-surface proteins. Populus trichocarpa RUST INDUCED SECRETED PEPTIDE 1 (PtRISP1) exhibits an elicitor activity in poplar, as well as a direct antimicrobial activity against rust fungi. PtRISP1 gene directly clusters with a gene encoding a leucine-rich repeat receptor protein (LRR-RP), that we termed RISP-ASSOCIATED LRR-RP (PtRALR). In this study, we used phylogenomics to characterize the RISP and RALR gene families, and molecular physiology assays to functionally characterize RISP/RALR pairs. Both RISP and RALR gene families specifically evolved in Salicaceae species (poplar and willow), and systematically cluster in the genomes. Despite a low sequence identity, Salix purpurea RISP1 (SpRISP1) shows properties and activities similar to PtRISP1. Both PtRISP1 and SpRISP1 induced a reactive oxygen species (ROS) burst and mitogen-activated protein kinases (MAPKs) phosphorylation in Nicotiana benthamiana leaves expressing the respective clustered RALR. PtRISP1 also triggers a rapid stomatal closure in poplar. Altogether, these results suggest that plants evolved phytocytokines with direct antimicrobial activities, and that the genes coding these phytocytokines co-evolved and physically cluster with genes coding LRR-RPs required to initiate immune signaling.
RESUMEN
The eukaryotic translation elongation factor 1Bγ (eEF1Bγ) is an atypical member of the glutathione transferase (GST) superfamily. Contrary to more classical GSTs having a role in toxic compound detoxification, eEF1Bγ is suggested to act as a scaffold protein, anchoring the elongation factor complex EF1B to the endoplasmic reticulum. In this study, we show that eEF1Bγ from the basidiomycete Phanerochaete chrysosporium is fully active as a glutathione transferase in vitro and undergoes conformational changes upon binding of oxidized glutathione. Using real-time analyses of biomolecular interactions, we show that GSSG allows eEF1Bγ to physically interact with other GSTs from the Ure2p class, opening new perspectives for a better understanding of the role of eEF1Bγ in cellular oxidative stress response.
Asunto(s)
Glutatión Peroxidasa/genética , Estrés Oxidativo/genética , Factor 1 de Elongación Peptídica/ultraestructura , Phanerochaete/genética , Priones/genética , Proteínas de Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos/genética , Animales , Cristalografía por Rayos X , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/ultraestructura , Glutatión/genética , Disulfuro de Glutatión/genética , Glutatión Peroxidasa/ultraestructura , Glutatión Transferasa/genética , Humanos , Ratones , Factor 1 de Elongación Peptídica/genética , Phanerochaete/ultraestructura , Priones/ultraestructura , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestructura , Factores de Transcripción de Dominio TEA , Factores de Transcripción/genética , Factores de Transcripción/ultraestructuraRESUMEN
BACKGROUND: Glutaredoxins (GRXs) are oxidoreductases involved in diverse cellular processes through their capacity to reduce glutathionylated proteins and/or to coordinate ironsulfur (Fe-S) clusters. Among class II GRXs, the plant-specific GRXS16 is a bimodular protein formed by an N-terminal endonuclease domain fused to a GRX domain containing a 158CGFS signature. METHODS: The biochemical properties (redox activity, sensitivity to oxidation, pKa of cysteine residues, midpoint redox potential) of Arabidopsis thaliana GRXS16 were investigated by coupling oxidative treatments to alkylation shift assays, activity measurements and mass spectrometry analyses. RESULTS: Activity measurements using redox-sensitive GFP2 (roGFP2) as target protein did not reveal any significant glutathione-dependent reductase activity of A. thaliana GRXS16 whereas it was able to catalyze the oxidation of roGFP2 in the presence of glutathione disulfide. Accordingly, Arabidopsis GRXS16 reacted efficiently with oxidized forms of glutathione, leading to the formation of an intramolecular disulfide between Cys158 and the semi-conserved Cys215, which has a midpoint redox potential of - 298â¯mV at pHâ¯7.0 and is reduced by plastidial thioredoxins (TRXs) but not GSH. By promoting the formation of this disulfide, Cys215 modulates GRXS16 oxidoreductase activity. CONCLUSION: The reduction of AtGRXS16, which is mandatory for its oxidoreductase activity and the binding of Fe-S clusters, depends on light through the plastidial FTR/TRX system. Hence, disulfide formation may constitute a redox switch mechanism controlling GRXS16 function in response to day/night transition or oxidizing conditions. GENERAL SIGNIFICANCE: From the in vitro data obtained with roGFP2, one can postulate that GRXS16 would mediate protein glutathionylation/oxidation in plastids but not their deglutathionylation.