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.

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.

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Plant cell walls are complex structures mainly made up of carbohydrate and phenolic polymers. In addition to their structural roles, cell walls function as external barriers against pathogens and are also reservoirs of glycan structures that can be perceived by plant receptors, activating Pattern-Triggered Immunity (PTI). Since these PTI-active glycans are usually released upon plant cell wall degradation, they are classified as Damage Associated Molecular Patterns (DAMPs). Identification of DAMPs imply their extraction from plant cell walls by using multistep methodologies and hazardous chemicals. Subcritical water extraction (SWE) has been shown to be an environmentally sustainable alternative and a simplified methodology for the generation of glycan-enriched fractions from different cell wall sources, since it only involves the use of water. Starting from Equisetum arvense cell walls, we have explored two different SWE sequential extractions (isothermal at 160 ºC and using a ramp of temperature from 100 to 160 ºC) to obtain glycans-enriched fractions, and we have compared them with those generated with a standard chemical-based wall extraction. We obtained SWE fractions enriched in pectins that triggered PTI hallmarks in Arabidopsis thaliana such as calcium influxes, reactive oxygen species production, phosphorylation of mitogen activated protein kinases and overexpression of immune-related genes. Notably, application of selected SWE fractions to pepper plants enhanced their disease resistance against the fungal pathogen Sclerotinia sclerotiorum. These data support the potential of SWE technology in extracting PTI-active fractions from plant cell wall biomass containing DAMPs and the use of SWE fractions in sustainable crop production.

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.