Computational prediction method to decipher receptor–glycoligand interactions in plant immunity

[EN] Microbial and plant cell walls have been selected by the plant immune system as a source of microbe- and plant damage-associated molecular patterns (MAMPs/DAMPs) that are perceived by extracellular ectodomains (ECDs) of plant pattern recognition receptors (PRRs) triggering immune responses. From the vast number of ligands that PRRs can bind, those composed of carbohydrate moieties are poorly studied, and only a handful of PRR/glycan pairs have been determined. Here we present a computational screening method, based on the first step of molecular dynamics simulation, that is able to predict putative ECD-PRR/ glycan interactions. This method has been developed and optimized with Arabidopsis LysM-PRR members CERK1 and LYK4, which are involved in the perception of fungal MAMPs, chitohexaose (1,4-b-D-(GlcNAc)6) and laminarihexaose (1,3-b-D-(Glc)6). Our in silico results predicted CERK1 interactions with 1,4-b-D-(GlcNAc)6 whilst discarding its direct binding by LYK4. In contrast, no direct interaction between CERK1/laminarihexaose was predicted by the model despite CERK1 being required for laminarihexaose immune activation, suggesting that CERK1 may act as a co-receptor for its recognition. These in silico results were validated by isothermal titration calorimetry binding assays between these MAMPs and recombinant ECDs-LysM-PRRs. The robustness of the developed computational screening method was further validated by predicting that CERK1 does not bind the DAMP 1,4-b-D-(Glc)6 (cellohexaose), and then probing that immune responses triggered by this DAMP were not impaired in the Arabidopsis cerk1 mutant. The computational predictive glycan/ PRR binding method developed here might accelerate the discovery of protein–glycan interactions and provide information on immune responses activated by glycoligands.SIThis work was also financially supported by the ‘Severo Ochoa Programme for Centers of Excellence in R&D(2017–2021) from the Agencia Estatal de Investigaci on of Spain (grant SEV-2016-0672 to CBGP). In the frame of this program HM was supported with a postdoctoral fellow supported by SEV-2016-0672. IdH was the recipient of a PhD FPU fellow (FPU16/07118) from the Spanish Ministry of Education and from an EMBO Short-Term Fellowship (7985). Research in JS’s lab was financially supported by the European Research Council (ERC) grant agreement no. 716358, the Swiss National Science Foundation grants no. 31003A_173101 and the Programme Fondation Philanthropique Famille Sandoz

Plant cell wall patterning and expansion mediated by protein-peptide-polysaccharide interaction

Assembly of cell wall polysaccharides into specific patterns is required for plant growth. A complex of RAPID ALKALINIZATION FACTOR 4 (RALF4) and its cell wall-anchored LEUCINE-RICH REPEAT EXTENSIN 8 (LRX8)-interacting protein is crucial for cell wall integrity during pollen tube growth, but its molecular connection with the cell wall is unknown. Here, we show that LRX8-RALF4 complexes adopt a heterotetrametric configuration in vivo, displaying a dendritic distribution. The LRX8-RALF4 complex specifically interacts with demethylesterified pectins in a charge-dependent manner through RALF4's polycationic surface. The LRX8-RALF4-pectin interaction exerts a condensing effect, patterning the cell wall's polymers into a reticulated network essential for wall integrity and expansion. Our work uncovers a dual structural and signaling role for RALF4 in pollen tube growth and in the assembly of complex extracellular polymers

A mechanistic model of heat and mass transfer used as a tool to bring insight into chemical reactivity during baking of sponge-cake products

National audienceDuring cooking of bakery products, heat and mass transfer phenomena occur as well as many chemical reactions including Maillard reaction. All these physico-chemical phenomena are strongly inter-related and a modeling approach can help to bring insight into these interactions. This paper presents a mechanistic heat and mass transfer model able to predict the nature and level of the transfer phenomena occurring within the product and between the product and its environment during baking of a sponge cake. The developed model takes into account internal moisture evaporation and vapour migration within the open porosity of the product during heating as well as apparent liquid moisture migration described by pseudo-Fick’s law. Internal heat transfer phenomena were described using Fourier’s law and apparent thermal heat conductivity. External heat and mass transfer phenomena taken into account were convective drying, convective and radiative heat transfer for both heated product and baking tray. Measurements of product core and surface temperatures variations as well as product moisture content and air hygrometry were acquired using an instrumented baking oven specifically designed for the project. Baking trials were performed in order to identify the unknown product properties of the model and to validate the assumptions made. The state variables predicted by the model such as local temperature and moisture content could become the input variables of a stoechio-kinetic model of the Maillard reaction, allowing a better understanding of the interactions between transfer and reaction phenomena as well as a better control of the physico-chemical processes involved during baking

Structural basis for recognition of RALF peptides by LRX proteins during pollen tube growth

Plant reproduction relies on the highly regulated growth of the pollen tube for sperm delivery. This process is controlled by secreted RALF signaling peptides, which have previously been shown to be perceived by Catharanthus roseus RLK1-like (CrRLK1Ls) membrane receptor-kinases/LORELEI-like GLYCOLPHOSPHATIDYLINOSITOL (GPI)-ANCHORED PROTEINS (LLG) complexes, or by leucine-rich repeat (LRR) extensin proteins (LRXs). Here, we demonstrate that RALF peptides fold into bioactive, disulfide bond-stabilized proteins that bind the LRR domain of LRX proteins with low nanomolar affinity. Crystal structures of LRX2-RALF4 and LRX8-RALF4 complexes at 3.2- and 3.9-Å resolution, respectively, reveal a dimeric arrangement of LRX proteins, with each monomer binding one folded RALF peptide. Structure-based mutations targeting the LRX-RALF4 complex interface, or the RALF4 fold, reduce RALF4 binding to LRX8 in vitro and RALF4 function in growing pollen tubes. Mutants targeting the disulfide-bond stabilized LRX dimer interface fail to rescue lrx infertility phenotypes. Quantitative biochemical assays reveal that RALF4 binds LLGs and LRX cell-wall modules with drastically different binding affinities, and with distinct and mutually exclusive binding modes. Our biochemical, structural, and genetic analyses reveal a complex signaling network by which RALF ligands instruct different signaling proteins using distinct targeting mechanisms

Microscale Thermophoresis (MST) to Study Rapid Alkalinization Factor (RALF)-Receptor Interactions

Abstract: Microscale thermophoresis (MST) is a simple but powerful tool to study the in vitro interaction among biomolecules, and to quantify binding affinities. MST curves describe the change in the fluorescence level of a fluorescent target as a result of an IR-laser-induced temperature change. The degree and nature of the change in fluorescence signal depends on the size, charge, and solvation shell of the molecules, properties that change in function of the binding of a ligand to the fluorescent target. We used MST to describe the interaction between components of a regulatory module involved in plant cell wall integrity control. This module comprises the secreted peptide Rapid Alkalinization Factor 23 (RALF23) and its receptor complex consisting of the GPI-anchored receptor Lorelei-Like Glycoprotein 1 (LLG1) and a receptor kinase of the CrRLK1L family, FERONIA. Here we show how MST can also be used to study three-partner interactions

Microscale Thermophoresis (MST) to Study Rapid Alkalinization Factor (RALF)-Receptor Interactions

Microscale thermophoresis (MST) is a simple but powerful tool to study the in vitro interaction among biomolecules, and to quantify binding affinities. MST curves describe the change in the fluorescence level of a fluorescent target as a result of an IR-laser-induced temperature change. The degree and nature of the change in fluorescence signal depends on the size, charge, and solvation shell of the molecules, properties that change in function of the binding of a ligand to the fluorescent target.We used MST to describe the interaction between components of a regulatory module involved in plant cell wall integrity control. This module comprises the secreted peptide Rapid Alkalinization Factor 23 (RALF23) and its receptor complex consisting of the GPI-anchored receptor Lorelei-Like Glycoprotein 1 (LLG1) and a receptor kinase of the CrRLK1L family, FERONIA. Here we show how MST can also be used to study three-partner interactions

Coupling between heat and mass transfer and stoechio-kinetic models to bring insight into Maillard reaction kinetics during baking of sponge-cake products

Penicaud11couplingThe objective of this work is to present the methodology used to bring insight into interactions between heat and mass transfer phenomena (within the heating product and between the product and its environment) and the extent of Maillard reaction (influenced by the variations of local values of product moisture content, temperature and reactants concentration) during baking of a sponge-cake type bakery product. Therefore, a heat and mass transfer model coupled with a stoechio-kinetic model has been developed. This model aimed at mechanistically inter-relate the level of operating conditions and the initial composition of the bakery products (in terms of reducing sugars and amino-acid contents) with the nature and extent of Maillard reactions occurring during baking. The developed heat and mass transfer model takes into account internal moisture evaporation and vapour migration within the open porosity of the product during heating as well as apparent liquid moisture migration described by Fick's law. Internal heat transfer phenomena were described using Fourier's law and apparent thermal heat conductivity. External heat and mass transfer phenomena taken into account were convective drying, convective and radiative heat transfer for both heating product and baking tray. Concerning the stoechio-kinetic model developed in the present work, attention was paid to the nature of simplifying assumptions made to deal with phenomena of concern. This point is particularly important considering the complex reaction scheme of Maillard reaction which was necessarily reduced to a simplified observable scheme taking into account bibliographical references about the subject and available experimental data about reactivity acquired during the study. The level of physical realism of heat and mass transfer model previously discussed has hence been adapted to the level of complexity of the stoechio-kinetic model

Coupling between heat and mass transfer and stoechio-kinetic models to bring insight into Maillard reaction kinetics during baking of sponge-cake products

Penicaud11couplingThe objective of this work is to present the methodology used to bring insight into interactions between heat and mass transfer phenomena (within the heating product and between the product and its environment) and the extent of Maillard reaction (influenced by the variations of local values of product moisture content, temperature and reactants concentration) during baking of a sponge-cake type bakery product. Therefore, a heat and mass transfer model coupled with a stoechio-kinetic model has been developed. This model aimed at mechanistically inter-relate the level of operating conditions and the initial composition of the bakery products (in terms of reducing sugars and amino-acid contents) with the nature and extent of Maillard reactions occurring during baking. The developed heat and mass transfer model takes into account internal moisture evaporation and vapour migration within the open porosity of the product during heating as well as apparent liquid moisture migration described by Fick's law. Internal heat transfer phenomena were described using Fourier's law and apparent thermal heat conductivity. External heat and mass transfer phenomena taken into account were convective drying, convective and radiative heat transfer for both heating product and baking tray. Concerning the stoechio-kinetic model developed in the present work, attention was paid to the nature of simplifying assumptions made to deal with phenomena of concern. This point is particularly important considering the complex reaction scheme of Maillard reaction which was necessarily reduced to a simplified observable scheme taking into account bibliographical references about the subject and available experimental data about reactivity acquired during the study. The level of physical realism of heat and mass transfer model previously discussed has hence been adapted to the level of complexity of the stoechio-kinetic model

Structural basis for recognition of RALF peptides by LRX proteins during pollen tube growth

Plant reproduction relies on the highly regulated growth of the pollen tube for proper sperm delivery. This process is controlled by secreted RALF signaling peptides, which have been previously shown to be perceived by CrRLK1Ls membrane receptor-kinases and leucine-rich (LRR) extensin proteins (LRXs). Here we demonstrate that RALF peptides are active as folded, disulfide bond-stabilized proteins, which can bind to the LRR domain of LRX proteins with nanomolar affinity. Crystal structures of the LRX-RALF signaling complexes reveal LRX proteins as constitutive dimers. The N-terminal LRR domain containing the RALF binding site is tightly linked to the extensin domain via a cysteine-rich tail. Our biochemical and structural work reveals a complex signaling network by which RALF ligands may instruct different signaling proteins – here CrRLK1Ls and LRXs – through structurally different binding modes to orchestrate cell wall remodeling in rapidly growing pollen tubes.SignificancePlant reproduction relies on proper pollen tube growth to reach the female tissue and release the sperm cells. This process is highly regulated by a family of secreted signaling peptides that are recognized by cell-wall monitoring proteins to enable plant fertilization. Here, we report the crystal structure of the LRX-RALF cell-wall complex and we demonstrate that RALF peptides are active as folded proteins. RALFs are autocrine signaling proteins able to instruct LRX cell-wall modules and CrRKL1L receptors, through structurally different binding modes to coordinate pollen tube integrity.</jats:sec