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

Crystal structures of two tandem malectin-like receptor kinases involved in plant reproduction.

Complex cell-to-cell communication between the male pollen tube and the female reproductive organs is required for plant fertilization. A family of Catharanthus roseus receptor kinase 1-like (CrRLK1L) membrane receptors has been genetically implicated in this process. Here, crystal structures of the CrRLK1Ls ANXUR1 and ANXUR2 are reported at 1.48 and 1.1 Å resolution, respectively. The structures reveal a novel arrangement of two malectin-like domains connected by a short β-hairpin linker and stabilized by calcium ions. The canonical carbohydrate-interaction surfaces of related animal and bacterial carbohydrate-binding modules are not conserved in plant CrRLK1Ls. In line with this, the binding of chemically diverse oligosaccharides to ANXUR1 and HERCULES1 could not be detected. Instead, CrRLK1Ls have evolved a protein-protein interface between their malectin domains which forms a deep cleft lined by highly conserved aromatic and polar residues. Analysis of the glycosylation patterns of different CrRLK1Ls and their oligomeric states suggests that this cleft could resemble a binding site for a ligand required for receptor activation of CrRLK1Ls

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

Dossier céréales. Cuisson des produits céréaliers

National audienc

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

Dryer modelling

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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