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

Abstract

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.

Cell wall‐derived mixed‐linked β‐1,3/1,4‐glucans trigger immune responses and disease resistance in plants

“…CERK1 does not seem to be involved in the perception of b-1,4-glucans (e.g. cellohexaose) in Arabidopsis as immune responses triggered by this DAMP were not impaired in cerk1 mutant, as predicted by in silico structural molecular dynamics simulations (del Hierro et al, 2020).…”

“…Specifically, a b-1,3-glucan hexasaccharide (laminarihexaose or Lam6) is an immuneactive structure whose recognition is dependent in Arabidopsis on the LysM-PRR CERK1 (Chitin Elicitor Receptor Kinase 1) (M elida et al, 2018). However, a direct binding of laminarihexaose to CERK1 extracellular ectodomain (ECD) has not been either observed in isothermal titration calorimetry binding assays performed with purified ECD-CERK1 nor predicted using recently developed in silico structural molecular dynamics simulations (del Hierro et al, 2020). These data suggest that CERK1 function as coreceptor rather than a receptor in b-1,3-glucan hexasaccharide perception.…”

“…Notably, cross-elicitation experiments in Col-0 AEQ seedlings demonstrated the absence of such a refractory period, further suggesting that the mechanisms of perception of these glycans and the PRRs involved in their perception are not identical (Figure 4a). In addition to CERK1, the LysM-PRRs LYK5 and LYK4 have been involved in chitohexaose perception as receptor and co-receptor, respectively (Cao et al, 2014;del Hierro et al, 2020;Liu et al, 2012). To characterize the molecular mechanisms of MLG43-mediated immunity further, phosphorylation of mitogen-activated protein kinase (MAPK) was tested by western blots in wild-type plants and cerk1 single and lyk4 lyk5 double mutants upon MLG43 treatment and we found that MAPK phosphorylation levels were weaker in these mutants than in wild-type plants, whereas they were fully impaired upon chitohexaose treatment, as reported previously (Cao et al, 2014;Figure 4b).…”

“…Geometries for MLG43-PRRs complexes were firstly obtained with docking calculations using the crystal structures of AtCERK1-ECD (PDB code: 4ebz), AtBAK1-ECD (PDB code: 4mn8) and AtSOBIR1-ECD (PDB code: 6rih). MLG43 ligand was built and optimized in vacuum as described (del Hierro et al, 2020). Energy terms contributing to protein-ligand interactions were computed using molecular mechanics following a previously established pipeline (del Hierro et al, 2020).…”

“…b-Glucans are well-known modulators of the immune system in mammals, but less is known about their roles in the plant counterpart (Fesel and Zuccaro, 2016;M elida et al, 2018;Rovenich et al, 2016;Sharp et al, 1984;Wanke et al, 2020). Interestingly, this field of b-glucan perception by plants has regained momentum due to recent discoveries demonstrating that glucans containing either b-1,3 or b-1,4 glycosidic linkages in their main backbones trigger PTI responses in plants (Claverie et al, 2018;del Hierro et al, 2020;Johnson et al, 2018;M elida et al, 2018;Souza et al, 2017;Wanke et al, 2020;Wawra et al, 2016). However, it remained elusive whether glucans containing both types of linkages (MLGs) can be perceived by plant cells as well (Aziz et al, 2007;Johnson et al, 2018;Klarzynski et al, 2000;Locci et al, 2019;M elida et al, 2018;M enard et al, 2004;Souza et al, 2017).…”