Laboratoire de Glycobiologie et Matrice Extracellulaire Végétale

















Topic I. Glycomolecules in root defense

Principal Investigators: M-L. Follet-Gueye, Associate Professor and M. Vicré-Gibouin, Associate Professor.
Participants: A. Driouich, Professor; M. Bardor, Professor; I. Boulogne, Associate ProfessorJ-C. Mollet, Professor; B. Gugi, Associate Professor; M-C. Kiefer-Meyer, Research Engineer; C. Burel, Technician; C. Plasson, Technician. PhD students: A. Planchon (2015-present), M. Ropitaux (2015-present), M. Chambard (2017-present).

The root system is important for plant development and survival. Nevertheless, the role of root cells in defense and the impact of cell wall components in root-microbe interaction are still poorly understood (Attard et al., 2010; Millet et al., 2010). The root tip is known to release border cells and border-like cells that influence the rhizosphere functional properties (Hawes et al., 2000; Vicré et al., 2005; Driouich et al., 2007). Recent findings have shown that these cells secrete abundant mucilage composed of a mixture of polysaccharides, proteoglycans, secondary metabolites, anti-microbial proteins and extracellular DNA (Wen et al., 2007; Durand et al., 2009; Wen et al., 2009; Koroney et al., 2016). This molecular network is believed to function to attract and entrap pathogens, thereby retarding and/or preventing root infection (Hawes et al., 2011; Driouich et al., 2013). We have hypothesized that mucilage together with root border cells form a defensive web-like structure- termed  “Root Extracellular Trap (RET)” - similar to  the “Neutrophil Extracellular Trap (NET)” found in mammalian cells that is able to destroy pathogens (Brinkmann et al., 2004; Driouich et al. 2013). Common features to both extracellular traps are the presence of defensive components and extracellular DNA (Wen et al., 2009; Hawes 2011; Driouich et al., 2013). Unlike the NET, the formation, composition and role of plant RET are not fully characterized. Here, we propose to investigate i) the functional characterization of the RET in root defense, ii) the role of cell wall extensin in root-microbe interaction, and iii) the specific response of root cells to novel elicitors/biological agents.

The Root Extracellular Trap, composition and function in root protection

This part of the project aims to gain a better understanding of the composition of the RET and to investigate the function of the RET in root protection against soil-borne pathogens. The pathosystems selected for this study are Arabidopsis thaliana / Phytophtora parasitica and pea (Pisum sativum) / Aphanomyces euteiches. Pea is the plant model used to study the role of border cells in root-microbe interactions and the function of the RET in root defense. A. thaliana is known to produce atypical border cells and only low amounts of mucilage. For this reason, it is not clear whether A. thaliana possesses a RET and further investigations are required to determine its composition and contribution to root defense.
The RET characterization will be performed using cell imaging (confocal microscopy, transmission electron microscopy, immunocytochemistry), biochemical characterization of glycomolecules including AGP, pectins… (by gas chromatography, mass spectrometry, electrophoresis) and metabolomics (in collaboration with Dr A. Lanoux at the University of Tours). The occurrence of polysaccharide-modifying enzymes (e.g., PME, PG, peroxidase …) will be also assessed in RET (collaboration with Topic II). Furthermore we will determine whether elicitors/pathogens alter RET composition. Finally, the involvement of the oxidative burst (ROS production) in RET formation and function will be also investigated using pharmacological approaches. The biosynthesis of the glycomolecules (AGP, pectins, enzymes, PR proteins …) within the endomembrane system of border cells and their secretion into the mucilage will be followed using cryo-transmission electron microscopy coupled to immunocytochemistry (developed in the transverse Project 1). Whether these different molecules are localized within distinct sites of the RET network will be determined. In addition, the impact of elicitors /pathogens on the secretion activity of border cells, cell wall structure and mucilage production will be investigated.
Finally, we will investigate the synergy between root border cells, glycomolecules, and ex-DNA in preventing root infection by pathogens. In mammals, ex-DNA of the NET is described as a thick scaffold embedding anti-microbial molecules able to combat infection by trapping and killing pathogens (Brinkmann et al., 2004). A pharmacological approach will be used to investigate the interconnectivity between different compounds of the RET (AGP, pectins, ex-DNA, enzymes…). Disorganization of the RET will be performed using various enzymes (DNAse, pectinases, proteases …) or chemical dyes and drugs such as β-glucosyl Yariv reagent and DHP (3,4-dehydroproline).

Role of extensin in root protection

Our recent data have highlighted the importance of cell wall HRGPs (AGPs and extensins) of root border cells and in mucilage in root-microbe interactions (Cannesan et al., 2012 ; Plancot et al., 2013). However, the role of extensin in root protection against soil-borne pathogens is not well established. This project aims to decipher the role of extensin in root resistance by using the pathosystem A. thaliana / Phytophtora parasitica. We will take advantage of available mutants affected in extensin biosynthesis and/or cross-linking (e.g Lrx1, Rra1 …) to investigate root colonization/infection by the pathogen using microscopy. The selected mutants will be investigated further in terms of resistance to the pathogen, extensin content and localization using biochemical and immunocytochemical techniques. Pathogen quantification within root tissues and regulation of defense gene expression in response to pathogen will be investigated in the mutants and WT plants (in collaboration with Dr A. Attard at the INRA Nice Sofia Antipolis).

Identification of novel elicitors and impact on root protection

We will investigate the impact of novel natural products on root growth and protection against soil-borne pathogens of economically important crops. Linum / Fusarium oxysporum, soybean / Phytophtora sojae and A. thaliana / Phytophtora parasitica are the pathosystems investigated here.
Several natural-occurring compounds including glyco-extracts from microalgae (in collaboration with the Topic III) and biological agents such as plant-growth promoting rhizobacteria, will be tested for their elicitor activities. The originality of this project is to focus on the impact of biostimulants specifically on root response (root mucilage, glycomolecules and border cells production, defense-related genes) and protection against pathogens in order to provide new strategies for plant protection against soil-borne diseases. This project will be conducted in tight collaboration with industrial partners (CMI, Groupe Rouiller International, Societé Agricole Terre de Lin, SIPRE).

References: Attard et al., (2010) New Phytol. 187,449-60 ; Brinkmann et al. (2004) Science 303, 1532–1535; Driouich et al. (2007) Trends Plant Sci. 12, 14–19 ; Durand et al (2009) Plant Physiol. 150, 1411–1421 ; Hawes et al (2000) Trends in Plant Science 5, 128-133 ; Hawes et al. (2011) Plant Science 180: 741–745 ; Koroney et al. (2016) Annals of Botany 118(4): 797–808. Millet et al. (2010) Plant Cell 22, 973-990; Wen et al. (2007) Plant Physiol. 143, 773–783; Wen et al. (2009) Plant Physiol. 151, 820–829. Vicré et al. (2005) Plant Physiol. 138, 998–1008.