Novel cell wall-derived oligosaccharides trigger immune responses and disease resistance in plants

Resumo

Plant cell walls are dynamic and highly controlled structures mainly made up of carbohydrate polymers (glycans), lignin and proteins that are the first point of contact during a plant-microbe interaction. Pathogens have coevolved with plants developing a wide set of tools to breakthrough that barrier, releasing cell wall-derived damage-associated molecular patterns (DAMPs) that are able to trigger pattern-triggered immunity (PTI) upon recognition by pattern recognition receptors (PRRs) in a similar way to microbe-associated molecular patterns (MAMPs). In this Thesis, to better understand plants mechanisms involved in the perception of carbohydrate based-structures recognised as DAMPs/MAMPs, we have studied the ability of mixed-linked β-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 obtained and tested for their capability to induce PTI hallmarks, such as cytoplasmic Ca2+ elevations, reactive oxygen species (ROS) production, phosphorylation of mitogen-activated protein kinases (MAPKs), and gene transcriptional reprogramming. These analyses revealed that MLG oligosaccharides are perceived by Arabidopsis thaliana and identified a trisaccharide, β-D-cellobiosyl-1,3-β-D-glucose (MLG43), as the smallest MLG structure triggering strong PTI responses. MLG43 is also perceived by crops such as tomato, canola, soybean, maize, pepper and wheat and confers enhanced disease resistance in Arabidopsis to the oomycete Hyaloperonospora arabidopsidis and in tomato, pepper and wheat to different bacterial and fungal pathogens. MLG43 perception mechanism in Arabidopsis was explored. The MLG43-mediated PTI responses are partially dependent on LysM PRRs CERK1, LYK4 and LYK5, since they were weaker in cerk1 and lyk4 lyk5 mutants than in wild-type plants. Cross-elicitation experiments between MLG43 and the carbohydrate MAMP chitohexaose [β-1,4-D-(GlcNAc)6], that is also perceived by these LysM PRRs, indicated that the mechanism of MLG43 recognition could differ from that of chitohexaose, that is fully impaired in cerk1 and lyk4 lyk5 plants. Moreover, after a screening of knockout mutant Arabidopsis lines impaired in genes encoding different putative carbohydrate-binding ectodomains from Wall-associated kinases (WAKs) and Malectin PRR subclasses, several Malectin PRRs resulted as candidates to be involved in the perception of MLGs, which should be confirmed in further studies. Identification and characterization of cell wall-derived DAMPs/MAMPs implies their extraction by using hazardous chemicals and time-consuming methodologies. Subcritical water extraction (SWE) has been previously shown to be an environmentally sustainable alternative method for the generation of glycan-enriched fractions from cell walls since it only involves the use of water. We have explored two different SWE sequential extractions to obtain glycans from Equisetum arvense cell walls, that contain PTI-active MLGs, and we have compared the SWE methodology with standard wall fractionations based in the use of alkali solutions. We found that SWE fractions were able to trigger PTI hallmarks such as Ca2+ influxes, ROS production, MAPKs phosphorylation and the overexpression of immune-related genes. Enzymatic digestion of SWE fractions with lichenase enriched the availability of active MLGs in the fractions and enhanced PTI responses. Notably, application of SWE fractions to pepper prior to pathogen inoculation triggered disease resistance against the fungal pathogen Sclerotinia sclerotiorum in comparison with mock-treated plants. These data support the potential of SWE technology in extracting PTI-active fractions from different plant materials in a more sustainable way. The data generated in the frame of this Thesis support the classification of MLGs as a novel group of carbohydrate-based molecular patterns, present both in microbes and plants, that are perceived by plants and trigger immune responses and disease resistance. This data, together with modern and sustainable cell wall extraction technologies represent an opportunity to enhance crop production by the application in the field of molecules obtained from leftovers of different industries, thus strengthening the circular bioeconomy.