Genetically-clustered antifungal phytocytokines and receptor protein family members cooperate to trigger plant immune signaling.

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.

Oxidized glutathione promotes association between eukaryotic translation elongation factor 1Bγ and Ure2p glutathione transferase from Phanerochaete chrysosporium.

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.

The thioredoxin-mediated recycling of Arabidopsis thaliana GRXS16 relies on a conserved C-terminal cysteine.

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 - 298 mV at pH 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.

Transcriptomic responses of Phanerochaete chrysosporium to oak acetonic extracts: focus on a new glutathione transferase.

The first steps of wood degradation by fungi lead to the release of toxic compounds known as extractives. To better understand how lignolytic fungi cope with the toxicity of these molecules, a transcriptomic analysis of Phanerochaete chrysosporium genes was performed in the presence of oak acetonic extracts. It reveals that in complement to the extracellular machinery of degradation, intracellular antioxidant and detoxification systems contribute to the lignolytic capabilities of fungi, presumably by preventing cellular damages and maintaining fungal health. Focusing on these systems, a glutathione transferase (P. chrysosporium GTT2.1 [PcGTT2.1]) has been selected for functional characterization. This enzyme, not characterized so far in basidiomycetes, has been classified first as a GTT2 compared to the Saccharomyces cerevisiae isoform. However, a deeper analysis shows that the GTT2.1 isoform has evolved functionally to reduce lipid peroxidation by recognizing high-molecular-weight peroxides as substrates. Moreover, the GTT2.1 gene has been lost in some non-wood-decay fungi. This example suggests that the intracellular detoxification system evolved concomitantly with the extracellular ligninolytic machinery in relation to the capacity of fungi to degrade wood.

Retinoic acid biosynthesis catalyzed by retinal dehydrogenases relies on a rate-limiting conformational transition associated with substrate recognition.

Retinoic acid (RA), a metabolite of vitamin A, exerts pleiotropic effects throughout life in vertebrate organisms. Thus, RA action must be tightly regulated through the coordinated action of biosynthetic and degrading enzymes. The last step of retinoic acid biosynthesis is irreversibly catalyzed by the NAD-dependent retinal dehydrogenases (RALDH), which are members of the aldehyde dehydrogenase (ALDH) superfamily. Low intracellular retinal concentrations imply efficient substrate molecular recognition to ensure high affinity and specificity of RALDHs for retinal. This study addresses the molecular basis of retinal recognition in human ALDH1A1 (or RALDH1) and rat ALDH1A2 (or RALDH2), through the comparison of the catalytic behavior of retinal analogs and use of the fluorescence properties of retinol. We show that, in contrast to long chain unsaturated substrates, the rate-limiting step of retinal oxidation by RALDHs is associated with acylation. Use of the fluorescence resonance energy transfer upon retinol interaction with RALDHs provides evidence that retinal recognition occurs in two steps: binding into the substrate access channel, and a slower structural reorganization with a rate constant of the same magnitude as the kcat for retinal oxidation: 0.18 vs. 0.07 and 0.25 vs. 0.1 s(-1) for ALDH1A1 and ALDH1A2, respectively. This suggests that the conformational transition of the RALDH-retinal complex significantly contributes to the rate-limiting step that controls the kinetics of retinal oxidation, as a prerequisite for the formation of a catalytically competent Michaelis complex. This conclusion is consistent with the general notion that structural flexibility within the active site of ALDH enzymes has been shown to be an integral component of catalysis.

Adenine binding mode is a key factor in triggering the early release of NADH in coenzyme A-dependent methylmalonate semialdehyde dehydrogenase.

Structural dynamics associated with cofactor binding have been shown to play key roles in the catalytic mechanism of hydrolytic NAD(P)-dependent aldehyde dehydrogenases (ALDH). By contrast, no information is available for their CoA-dependent counterparts. We present here the first crystal structure of a CoA-dependent ALDH. The structure of the methylmalonate semialdehyde dehydrogenase (MSDH) from Bacillus subtilis in binary complex with NAD(+) shows that, in contrast to what is observed for hydrolytic ALDHs, the nicotinamide ring is well defined in the electron density due to direct and H(2)O-mediated hydrogen bonds with the carboxamide. The structure also reveals that a conformational isomerization of the NMNH is possible in MSDH, as shown for hydrolytic ALDHs. Finally, the adenine ring is substantially more solvent-exposed, a result that could be explained by the presence of a Val residue at position 229 in helix α(F) that reduces the depth of the binding pocket and the absence of Gly-225 at the N-terminal end of helix α(F). Substitution of glycine for Val-229 and/or insertion of a glycine residue at position 225 resulted in a significant decrease of the rate constant associated with the dissociation of NADH from the NADH/thioacylenzyme complex, thus demonstrating that the weaker stabilization of the adenine ring is a key factor in triggering the early NADH release in the MSDH-catalyzed reaction. This study provides for the first time structural insights into the mechanism whereby the cofactor binding mode is responsible at least in part for the different kinetic behaviors of the hydrolytic and CoA-dependent ALDHs.