Pattern-triggered immunity (PTI) is activated in plants upon recognition by pattern recognition receptors (PRRs) of damage-and microbe-associated molecular patterns (DAMPs and MAMPs) derived from plants or microorganisms, respectively. To understand better the plant mechanisms involved in the perception of carbohydrate-based structures recognized as DAMPs/MAMPs, we have studied the ability of mixed-linked b-1,3/1,4-glucans (MLGs), present in some plant and microbial cell walls, to trigger immune responses and disease resistance in plants. A range of MLG structures were tested for their capacity to induce PTI hallmarks, such as cytoplasmic Ca 2+ elevations, reactive oxygen species production, phosphorylation of mitogen-activated protein kinases and gene transcriptional reprogramming. These analyses revealed that MLG oligosaccharides are perceived by Arabidopsis thaliana and identified a trisaccharide, b-D-cellobiosyl-(1,3)-b-D-glucose (MLG43), as the smallest MLG structure triggering strong PTI responses. These MLG43-mediated PTI responses are partially dependent on LysM PRRs CERK1, LYK4 and LYK5, as they were weaker in cerk1 and lyk4 lyk5 mutants than in wild-type plants. Cross-elicitation experiments between MLG43 and the carbohydrate MAMP chitohexaose [b-1,4-D-(GlcNAc) 6 ], which is also perceived by these LysM PRRs, indicated that the mechanism of MLG43 recognition could differ from that of chitohexaose, which is fully impaired in cerk1 and lyk4 lyk5 plants. MLG43 treatment confers enhanced disease resistance in A. thaliana to the oomycete Hyaloperonospora arabidopsidis and in tomato and pepper to different bacterial and fungal pathogens. Our data support the classification of MLGs as a group of carbohydrate-based molecular patterns that are perceived by plants and trigger immune responses and disease resistance.

Immune responses in plants can be triggered by damage/microbe-associated molecular patterns (DAMPs/MAMPs) upon recognition by plant pattern recognition receptors (PRRs). DAMPs are signaling molecules synthesized by plants or released from host cellular structures (e.g., plant cell walls) upon pathogen infection or wounding. Despite the hypothesized important role of plant cell wall-derived DAMPs in plant-pathogen interactions, a very limited number of these DAMPs are well characterized. Recent work demonstrated that pectin-enriched cell wall fractions extracted from the cell wall mutant impaired in Arabidopsis Response Regulator 6 (arr6), that showed altered disease resistance to several pathogens, triggered more intense immune responses than those activated by similar cell wall fractions from wild-type plants. It was hypothesized that arr6 cell wall fractions could be differentially enriched in DAMPs. In this work, we describe the characterization of the previous immune-active fractions of arr6 showing the highest triggering capacities upon further fractionation by chromatographic means. These analyses pointed to a role of pentose-based oligosaccharides triggering plant immune responses. The characterization of several pentose-based oligosaccharide structures revealed that b-1,4-xylooligosaccharides of specific degrees of polymerization and carrying arabinose decorations are sensed as DAMPs by plants. Moreover, the pentasaccharide 3 3-a-L-arabinofuranosyl-xylotetraose (XA3XX) was found as a highly active DAMP structure triggering strong immune responses in Arabidopsis thaliana and enhancing crop disease resistance.

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.

SUMMARY The plant immune system perceives a diversity of carbohydrate ligands from plant and microbial cell walls through the extracellular ectodomains (ECDs) of pattern recognition receptors (PRRs), which activate pattern‐triggered immunity (PTI). Among these ligands are oligosaccharides derived from mixed‐linked β‐1,3/β‐1,4‐glucans (MLGs; e.g. β‐1,4‐D‐(Glc)2‐β‐1,3‐D‐Glc, MLG43) and cellulose (e.g. β‐1,4‐D‐(Glc)3, CEL3). The mechanisms behind carbohydrate perception in plants are poorly characterized except for fungal chitin oligosaccharides (e.g. β‐1,4‐d‐(GlcNAc)6, CHI6), which involve several receptor kinase proteins (RKs) with LysM‐ECDs. Here, we describe the isolation and characterization of Arabidopsis thaliana mutants impaired in glycan perception (igp) that are defective in PTI activation mediated by MLG43 and CEL3, but not by CHI6. igp1–igp4 are altered in three RKs – AT1G56145 (IGP1), AT1G56130 (IGP2/IGP3) and AT1G56140 (IGP4) – with leucine‐rich‐repeat (LRR) and malectin (MAL) domains in their ECDs. igp1 harbors point mutation E906K and igp2 and igp3 harbor point mutation G773E in their kinase domains, whereas igp4 is a T‐DNA insertional loss‐of‐function mutant. Notably, isothermal titration calorimetry (ITC) assays with purified ECD‐RKs of IGP1 and IGP3 showed that IGP1 binds with high affinity to CEL3 (with dissociation constant KD = 1.19 ± 0.03 μm) and cellopentaose (KD = 1.40 ± 0.01 μM), but not to MLG43, supporting its function as a plant PRR for cellulose‐derived oligosaccharides. Our data suggest that these LRR‐MAL RKs are components of a recognition mechanism for both cellulose‐ and MLG‐derived oligosaccharide perception and downstream PTI activation in Arabidopsis.

The fungal genus Plectosphaerella comprises species and strains with different lifestyles on plants, such as P. cucumerina, which has served as model for the characterization of Arabidopsis thaliana basal and non-host resistance to necrotrophic fungi. We have sequenced, annotated and compared the genomes and transcriptomes of three Plectosphaerella strains with different lifestyles on A. thaliana: PcBMM, a natural pathogen of wild-type plants (Col-0); Pc2127, a non-pathogenic strain on Col-0 but pathogenic on the immunocompromised cyp79B2 cyp79B3 mutant; and P0831 strain, that was isolated from a natural population of A. thaliana and is shown here to be non-pathogenic and to grow epiphytically on Col-0 and cyp79B2 cyp79B3 plants. The genomes of these Plectosphaerella strains are very similar and do not differ in the number of genes with pathogenesis-related functions, with the exception of secreted Carbohydrate-Active Enzymes (CAZymes) that are up to five times more abundant in the pathogenic strain PcBMM. Analysis of the fungal transcriptomes in inoculated Col-0 and cyp79B2 cyp79B3 plants at initial colonization stages confirm the key role of secreted CAZymes in the necrotrophic interaction, since PcBMM expresses more genes encoding secreted CAZymes than Pc2127 and P0831. We also show that P0831 epiphytic growth on A. thaliana involves the transcription of specific repertoires of fungal genes, that might be necessary for epiphytic growth adaptation. Overall these results suggest that in planta expression of specific sets of fungal genes at early stages of colonization determine the diverse lifestyles and pathogenicity of Plectosphaerella strains.

Plant immune system perceives through the extracellular ectodomains (ECDs) of Pattern Recognition Receptors (PRRs) a diversity of carbohydrate ligands from plant and microbial cell walls, which activate Pattern-Triggered Immunity (PTI). Among these ligands are oligosaccharides derived from mixed-linked β-1,3/β-1,4-glucans (MLGs, e.g., β-1,4-D-(Glc)2-β-1,3-D-Glc, MLG43) and cellulose (e.g., β-1,4-D-(Glc)3, CEL3). The mechanisms of perception of carbohydrates by plants are poorly characterized, with the exception of that determining recognition of fungal chitin oligosaccharides (e.g., β-1,4-D(GlcNAc)6, CHI6) that involves several PRRs with LysM-ECDs that function as receptor or co-receptors. Here, we describe the isolation and characterization of Arabidopsis thaliana mutants impaired in glycan perception (igp), which are defective in PTI activation mediated by MLG43 and CEL3, but not CHI6. igp1-igp4 are altered in receptor-like kinases [RLKs: AT1G56145 (IGP1), AT1G56130 (IGP2/3), and AT1G56140 (IGP4)] with Leucine-Rich-Repeat (LRR) and Malectin (MAL) domains in their ECDs. igp4 is a T-DNA insertional, loss of function mutant whereas igp1 and the allelic igp2/igp3 harbour point mutations (E906K and G773E, respectively) in their kinase domains, which impact their structure and surface electrostatic potential as revealed by in silico structural analyses. Notably, Isothermal Titration Calorimetry assays with purified ECD-RLKs showed that AT1G56145 binds with high affinity CEL3 (Kd = 1.19 ± 0.03 μM) and cellopentaose (Kd = 1.40 ± 0.01 μM), but not MLG43, supporting AT1G56145 function as a plant PRR for cellulose oligosaccharides. Our data suggest that these LRR-MAL RLKs are receptor/co-receptors of a novel mechanism of perception of cellulose and MLG-derived oligosaccharides and PTI activation in Arabidopsis thaliana.