3 research outputs found
Novel cell wall-derived oligosaccharides trigger immune responses and disease resistance in plants
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. ----------RESUMEN---------- Las paredes celulares de las plantas son estructuras dinámicas y altamente controladas, compuestas principalmente por carbohidratos (glicanos), lignina y proteínas, que suponen el primer punto de contacto durante las interacciones planta-microorganismo. Los patógenos de las plantas han coevolucionado con sus huéspedes desarrollando un amplio conjunto de herramientas moleculares para romper esta barrera física. Dicha degradación de las paredes celulares de las plantas provoca la liberación patrones moleculares asociados a daño (“damage-associated molecular patterns”; DAMPs), que son capaces de inducir el proceso conocido como inmunidad mediada por patrones (“pattern-triggered immunity”; PTI) al ser reconocidos por receptores de reconocimiento de patrones (“pattern recognition receptors”; PRRs) de forma similar a los patrones moleculares asociados a microorganismos (“microbe-associated molecular patterns”; MAMPs). Con el objetivo de comprender mejor los mecanismos de las plantas que intervienen en la percepción de DAMPs/MAMPs de naturaleza glicídica, en esta Tesis se estudió la capacidad de los β-1,3/1,4-glucanos mixtos (mixed-linked glucans; MLGs), presentes en las paredes celulares de algunas especies de plantas y microorganismos, de inducir respuestas inmunes y resistencia a las enfermedades en plantas. Se obtuvieron y analizaron una serie de estructuras de MLGs, que se usaron para determinar su capacidad de inducir eventos marcadores de PTI, como flujos intracelulares de Ca2+, producción de especies reactivas de oxígeno (ROS), fosforilación de proteínas quinasas activadas por mitógeno (MAPKs) y la reprogramación transcripcional génica. Estos análisis revelaron que los oligosacáridos de MLG son percibidos por la especie modelo Arabidopsis thaliana y se identificó un trisacárido, β-D-celobiosil-1,3-β-D-glucosa (MLG43), como la estructura mínima de glucano mixto que desencadena fuertes respuestas inmunes tempranas. Además, se demostró que MLG43 también es percibido por plantas de cultivo como el tomate, la canola, la soja, el maíz, el pimiento y el trigo y confiere una mayor resistencia a las plantas de Arabidopsis frente al oomiceto Hyaloperonospora arabidopsidis y en tomate, pimiento y trigo frente a diferentes patógenos bacterianos y fúngicos. A continuación, se exploró el mecanismo de percepción de MLG43 en Arabidopsis. Se observó, que las respuestas de tipo PTI inducidas por MLG43 resultaron parcialmente dependientes de los PRRs de tipo LysM CERK1, LYK4 y LYK5, ya que fueron más tenues en los mutantes cerk1 y lyk4 lyk5 que en las plantas silvestres. Sin embargo, los experimentos de elicitación cruzada entre MLG43 y el MAMP oligosacarídico quitohexaosa [β-1,4-D-(GlcNAc)6], que también es percibido por estos PRRs de tipo LysM, indicaron que el mecanismo de reconocimiento de MLG43 difiere del de quitohexaosa, ya que en el caso de este último es totalmente dependiente de CERK1, LYK4 y LYK5. Por otro lado, a través un cribado masivo de líneas de Arabidopsis con mutaciones en los genes que codifican para diferentes ectodominios con potencial unión a carbohidratos pertenecientes a las subclases de PRRs de quinasas asociadas a pared (wall-associated kinase; WAKs) y de malectinas, se determinó que varios PRRs de tipo malectina son candidatos a estar implicados en la percepción de MLGs. Estos resultados abren la puerta a investigaciones futuras acerca del papel de las malectinas en la percepción de oligosacáridos de MLG. La identificación y caracterización de DAMPs/MAMPs derivados de pared celular implica generalmente su extracción mediante el uso de productos químicos peligrosos y metodologías complejas. La extracción con agua en condiciones subcríticas (“subcritical water extraction”; SWE) ha sido mostrada previamente como un método alternativo sostenible para la generación de fracciones enriquecidas en glicanos a partir de paredes celulares, ya que sólo implica el uso de agua. En esta Tesis, se ha explorado el posible uso de la tecnología SWE para obtener glicanos a partir de paredes celulares de Equisetum arvense, que contienen MLGs inmunoactivos en plantas, y se ha comparado la metodología de SWE con las extracciones clásicas de paredes celulares basadas en el uso de soluciones alcalinas. Esta comparación demostró que las fracciones SWE indujeron marcadores de PTI similares a los anteriormente indicados para MLG43. Además, la digestión enzimática de las fracciones de SWE con liquenasa, enzima que hidroliza específicamente glucanos mixtos poliméricos liberando sus oligosacáridos, mejoró la capacidad de estas fracciones para inducir respuestas tipo PTI. Finalmente, la aplicación de fracciones SWE a plantas de pimiento antes de la inoculación con patógeno indujo una mayor resistencia contra el patógeno fúngico Sclerotinia sclerotiorum en comparación con las plantas control. Estos datos apoyan el potencial de la tecnología SWE en la extracción de fracciones inmunoactivas de forma sostenible. Los datos generados en esta Tesis demuestran que los oligosacáridos de glucano mixto constituyen un nuevo grupo de patrones moleculares, presentes tanto en microorganismos como en plantas (MAMPs y DAMPs), que son percibidos por las plantas y desencadenan respuestas inmunes y resistencia a enfermedades causadas por fitopatógenos. Estos datos, junto con el uso de una tecnología de extracción de componentes de pared celular más eficiente que los métodos clásicos, representan una oportunidad para mejorar la producción de cultivos mediante la aplicación de moléculas obtenidas a partir de residuos de otras actividades industriales en el marco de economía circular
Arabinoxylan-Oligosaccharides Act as Damage Associated Molecular Patterns in Plants Regulating 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 β-1,4-xylooligosaccharides of specific degrees of polymerization and carrying arabinose decorations are sensed as DAMPs by plants. Moreover, the pentasaccharide 33-α-L-arabinofuranosyl-xylotetraose (XA3XX) was found as a highly active DAMP structure triggering strong immune responses in Arabidopsis thaliana and enhancing crop disease resistance
Cell wall-derived mixed-linked β-1,3/1,4-glucans trigger immune responses and disease resistance in plants
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 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 tested for their capacity to induce PTI hallmarks, such as cytoplasmic Ca2+ 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, β-d-cellobiosyl-(1,3)-β-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 [β-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