Identification and characterization of the Non-race specific Disease Resistance 1 (NDR1) orthologous protein in coffee

<p>Abstract</p> <p>Background</p> <p>Leaf rust, which is caused by the fungus <it>Hemileia vastatrix </it>(Pucciniales), is a devastating disease that affects coffee plants (<it>Coffea arabica </it>L.). Disadvantages that are associated with currently developed phytoprotection approaches have recently led to the search for alternative strategies. These include genetic manipulations that constitutively activate disease resistance signaling pathways. However, molecular actors of such pathways still remain unknown in <it>C. arabica</it>. In this study, we have isolated and characterized the coffee <it>NDR1 </it>gene, whose <it>Arabidopsis </it>ortholog is a well-known master regulator of the hypersensitive response that is dependent on coiled-coil type R-proteins.</p> <p>Results</p> <p>Two highly homologous cDNAs coding for putative NDR1 proteins were identified and cloned from leaves of coffee plants. One of the candidate coding sequences was then expressed in the <it>Arabidopsis </it>knock-out null mutant <it>ndr1-1</it>. Upon a challenge with a specific strain of the bacterium <it>Pseudomonas syringae </it>(DC3000::<it>AvrRpt2</it>), analysis of both macroscopic symptoms and <it>in planta </it>microbial growth showed that the coffee cDNA was able to restore the resistance phenotype in the mutant genetic background. Thus, the cDNA was dubbed <it>CaNDR1a </it>(standing for <it>Coffea arabica Non-race specific Disease Resistance 1a</it>). Finally, biochemical and microscopy data were obtained that strongly suggest the mechanistic conservation of the <it>NDR1</it>-driven function within coffee and <it>Arabidopsis </it>plants. Using a transient expression system, it was indeed shown that the CaNDR1a protein, like its <it>Arabidopsis </it>counterpart, is localized to the plasma membrane, where it is possibly tethered by means of a GPI anchor.</p> <p>Conclusions</p> <p>Our data provide molecular and genetic evidence for the identification of a novel functional <it>NDR1 </it>homolog in plants. As a key regulator initiating hypersensitive signalling pathways, <it>CaNDR1 </it>gene(s) might be target(s) of choice for manipulating the coffee innate immune system and achieving broad spectrum resistance to pathogens. Given the potential conservation of <it>NDR1</it>-dependent defense mechanisms between <it>Arabidopsis </it>and coffee plants, our work also suggests new ways to isolate the as-yet-unidentified <it>R</it>-gene(s) responsible for resistance to <it>H. vastatrix</it>.</p

A global LC-MS2 -based methodology to identify and quantify anionic phospholipids in plant samples

peer reviewedAnionic phospholipids (PS, PA, PI, PIPs) are low-abundant phospholipids with impactful functions in cell signaling, membrane trafficking and cell differentiation processes. They can be quickly metabolized and can transiently accumulate at defined spots within the cell or an organ to respond to physiological or environmental stimuli. As even a small change in their composition profile will produce a significant effect on biological processes, it is crucial to develop a sensitive and optimized analytical method to accurately detect and quantify them. While thin-layer chromatography (TLC) separation coupled with gas chromatography (GC) detection methods already exist, they do not allow for precise, sensitive, and accurate quantification of all anionic phospholipid species. Here we developed a method based on high-performance liquid chromatography (HPLC) combined with two-dimensional mass spectrometry (MS 2) by MRM mode to detect and quantify all molecular species and classes of anionic phospholipids in one shot. This method is based on a derivatization step by methylation that greatly enhances the ionization, the separation of each peak, the peak resolution as well as the limit of detection and quantification for each individual molecular species, and more particularly for PA and PS. Our method universally works in various plant samples. Remarkably, we identified that PS is enriched with very long chain fatty acids in the roots but not in aerial organs of Arabidopsis thaliana. Our work thus paves the way for new studies on how the composition of anionic lipids is finely tuned during plant development and environmental responses

Identification and characterization of the Non- race specific Disease Resistance 1 (NDR1) orthologous protein in coffee

Abstract Background: Leaf rust, which is caused by the fungus Hemileia vastatrix (Pucciniales), is a devastating disease that affects coffee plants (Coffea arabica L.). Disadvantages that are associated with currently developed phytoprotection approaches have recently led to the search for alternative strategies. These include genetic manipulations that constitutively activate disease resistance signaling pathways. However, molecular actors of such pathways still remain unknown in C. arabica. In this study, we have isolated and characterized the coffee NDR1 gene, whose Arabidopsis ortholog is a well-known master regulator of the hypersensitive response that is dependent on coiledcoil type R-proteins

REM1.3's phospho-status defines its plasma membrane nanodomain organization and activity in restricting PVX cell-to-cell movement

Plants respond to pathogens through dynamic regulation of plasma membrane-bound signaling pathways. To date, how the plant plasma membrane is involved in responses to viruses is mostly unknown. Here, we show that plant cells sense the Potato virus X (PVX) COAT PROTEIN and TRIPLE GENE BLOCK 1 proteins and subsequently trigger the activation of a membrane-bound calcium-dependent kinase. We show that the Arabidopsis thaliana CALCIUM-DEPENDENT PROTEIN KINASE 3-interacts with group 1 REMORINs in vivo, phosphorylates the intrinsically disordered N-terminal domain of the Group 1 REMORIN REM1.3, and restricts PVX cell-to-cell movement. REM1.3’s phospho-status defines its plasma membrane nanodomain organization and is crucial for REM1.3-dependent restriction of PVX cell-to-cell movement by regulation of callose deposition at plasmodesmata. This study unveils plasma membrane nanodomain-associated molecular events underlying the plant immune response to viruses

Mechanosensing and Sphingolipid-Docking Mediate Lipopeptide-Induced Immunity in Arabidopsis

Bacteria-derived lipopeptides are immunogenic triggers of host defenses in metazoans and plants. Root-associated rhizobacteria produce cyclic lipopeptides that activate systemically induced resistance (IR) against microbial infection in various plants. How these molecules are perceived by plant cells remains elusive. Here, we reveal that immunity activation inArabidopsis thalianaby the lipopeptide elicitor surfactin is mediated by docking into specific sphingolipid-enriched domains and relies on host membrane deformation and subsequent activation of mechanosensitive ion channels. This mechanism leads to host defense potentiation and resistance to the necrotrophB. cinereabut is distinct from host pattern recognition receptor-mediated immune activation and reminiscent of damage-induced plant immunity

Biophysical analysis of the plant-specific GIPC sphingolipids reveals multiple modes of membrane regulation

The plant plasma membrane (PM) is an essential barrier between the cell and the external environment, controlling signal perception and transmission. It consists of an asymmetrical lipid bilayer made up of three different lipid classes: sphingolipids, sterols, and phospholipids. The glycosyl inositol phosphoryl ceramides (GIPCs), representing up to 40% of total sphingolipids, are assumed to be almost exclusively in the outer leaflet of the PM. However, their biological role and properties are poorly defined. In this study, we investigated the role of GIPCs in membrane organization. Because GIPCs are not commercially available, we developed a protocol to extract and isolate GIPC-enriched fractions from eudicots (cauliflower and tobacco) and monocots (leek and rice). Lipidomic analysis confirmed the presence of trihydroxylated long chain bases and 2-hydroxylated very long-chain fatty acids up to 26 carbon atoms. The glycan head groups of the GIPCs from monocots and dicots were analyzed by gas chromatograph–mass spectrometry, revealing different sugar moieties. Multiple biophysics tools, namely Langmuir monolayer, ζ-Potential, light scattering, neutron reflectivity, solid state 2H-NMR, and molecular modeling, were used to investigate the physical properties of the GIPCs, as well as their interaction with free and conjugated phytosterols. We showed that GIPCs increase the thickness and electronegativity of model membranes, interact differentially with the different phytosterols species, and regulate the gel-to-fluid phase transition during temperature variations. These results unveil the multiple roles played by GIPCs in the plant PM.Vers un modèle intégratif de la bicouche lipidique de la membrane plasmique végétaleDéveloppement d’une infrastructure française distribuée pour la métabolomique dédiée à l’innovatio