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

Pattern Recognition Receptors—Versatile Genetic Tools for Engineering Broad-Spectrum Disease Resistance in Crops

Infestations of crop plants with pathogens pose a major threat to global food supply. Exploiting plant defense mechanisms to produce disease-resistant crop varieties is an important strategy to control plant diseases in modern plant breeding and can greatly reduce the application of agrochemicals. The discovery of different types of immune receptors and a detailed understanding of their activation and regulation mechanisms in the last decades has paved the way for the deployment of these central plant immune components for genetic plant disease management. This review will focus on a particular class of immune sensors, termed pattern recognition receptors (PRRs), that activate a defense program termed pattern-triggered immunity (PTI) and outline their potential to provide broad-spectrum and potentially durable disease resistance in various crop species—simply by providing plants with enhanced capacities to detect invaders and to rapidly launch their natural defense program

Prominent examples of functions of the different LPS domains in mammalian and plant immunity^*.

<p>Prominent examples of functions of the different LPS domains in mammalian and plant immunity^{<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005596#t001fn001" target="_blank">*</a>}.</p

Immune Sensing of Lipopolysaccharide in Plants and Animals: Same but Different - Fig 1

<p><b>Scheme of LPS structures of gram-negative bacteria (A) and the currently known LPS sensing systems in humans and cruciferous plants (B). A.</b> LPS structure showing a representative core region and LA from <i>Escherichia coli</i> K12 and <i>Pseudomonas aeruginosa</i>. The typical enterobacterial LA consists of a di-phosphorylated di-glucosamine with four primary and two secondary FAs (all C12/14) attached in an asymmetric fashion [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005596#ppat.1005596.ref003" target="_blank">3</a>]. <i>Pseudomonas</i> spp. mostly produce penta-acylated and symmetrically hexa-acylated LA with shorter fatty acids (C10/12) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005596#ppat.1005596.ref036" target="_blank">36</a>]. The FAs are embedded in the OM, and the di-glucosamine is linked to the core oligosaccharide composed of about ten monosaccharides, which is conceptually subdivided into the rather variable outer core and the more conserved inner core region that usually contains heptose and the LPS-specific monosaccharide “Kdo” [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005596#ppat.1005596.ref003" target="_blank">3</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005596#ppat.1005596.ref036" target="_blank">36</a>]. Often, an OPS consisting of repetitive units composed of several monosaccharides is attached to the core region. Dotted/grey lines indicate non-stoichiometric substitutions. Structures according to [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005596#ppat.1005596.ref036" target="_blank">36</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005596#ppat.1005596.ref037" target="_blank">37</a>]. Abbreviations: Ara4N, 4-amino-4-deoxy-L-arabinose; Cm, carbamoyl; Etn, ethanolamine; Gal, galactose; GalN, galactosamine; Glc, glucose; GlcN, glucosamine; Hep, L-<i>glycero</i>-D-<i>manno</i>-heptose; Kdo, 3-deoxy-D-<i>manno</i>-oct-2-ulosonic acid; <i>P</i>, phosphate; Rha, L-rhamnose. <b>B.</b> In humans (left panel), LPS is sensed by different immune cells through different extra- and intracellular receptors [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005596#ppat.1005596.ref014" target="_blank">14</a>]. LPS is disaggregated from the bacterial membrane by the serum protein LBP and transferred to CD14, which occurs as a soluble (sCD14) and membrane-linked (mCD14) version. Dependent on the cell type, CD14 can trigger LPS signaling itself, such as calcium signaling and activation of NFAT transcription factors in dendritic cells, or further transfers LPS to the membrane-resident TLR4/MD-2 receptor complex. LA binding to a preformed TLR4/MD-2 hetero-dimer leads to association with another TLR4/MD-2-dimer and initiates intracellular signaling. Depending on the cellular localization (at the plasma membrane or in endosomes upon CD14-dependent endocytosis), TLR4/MD-2/LPS complexes activate production of either interferons or cytokines through distinct signaling adapters (TIRAP/MyD88 or TRIF/TRAM) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005596#ppat.1005596.ref014" target="_blank">14</a>]. Intracellular LPS leads to oligomerization of caspase-4, activation of the non-canonical inflammasome and pyroptotic cell death [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005596#ppat.1005596.ref019" target="_blank">19</a>]. In plants (right panel), the bulb-type lectin S-domain-1 RLK LORE (LipoOligosaccharide-specific Reduced Elicitation) was identified as the first LPS receptor component in plants and mediates sensitive perception of <i>Pseudomonas</i> LA [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005596#ppat.1005596.ref030" target="_blank">30</a>]. If and how LPS is processed in the apoplast to make the membrane-embedded LA accessible for receptor binding, if LA directly binds to LORE or to an LPS-binding co-receptor and how the receptor complex and downstream signaling is activated, is yet unknown. In analogy to other SD-RLKs, LORE presumably forms dimers and is activated through mutual phosphorylation by the cytosolic kinase domain [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005596#ppat.1005596.ref038" target="_blank">38</a>]. Taken together, both mammals and plants sense LA as pathogen-associated molecular pattern (PAMP) but with distinct epitope specificities and through different types of receptors.</p

Microbe-associated molecular pattern-induced calcium signaling requires the receptor-like cytoplasmic kinases, PBL1 and BIK1

BACKGROUND: Plant perception of conserved microbe-derived or damage-derived molecules (so-called microbe- or damage-associated molecular patterns, MAMPs or DAMPs, respectively) triggers cellular signaling cascades to initiate counteracting defence responses. Using MAMP-induced rise in cellular calcium levels as one of the earliest biochemical readouts, we initiated a genetic screen for components involved in early MAMP signaling in Arabidopsis thaliana. RESULTS: We characterized here the “changed calcium elevation 5” (cce5) mutant, where five allelic cce5 mutants were isolated. They all show reduced calcium levels after elicitation with peptides representing bacteria-derived MAMPs (flg22 and elf18) and endogenous DAMP (AtPep1), but a normal response to chitin octamers. Mapping, sequencing of the mutated locus and complementation studies revealed CCE5 to encode the receptor-like cytoplasmic kinase (RLCK), avrPphB sensitive 1-like 1 (PBL1). Kinase activities of PBL1 derived from three of the cce5 alleles are abrogated in vivo. Validation with T-DNA mutants revealed that, besides PBL1, another RLCK, Botrytis-induced kinase 1 (BIK1), is also required for MAMP/DAMP-induced calcium elevations. CONCLUSIONS: Hence, PBL1 and BIK1 (but not two related RLCKs, PBS1 and PBL2) are required for MAMP/DAMP-induced calcium signaling. It remains to be investigated if the many other RLCKs encoded in the Arabidopsis genome affect early calcium signal transduction – perhaps in dependence on the type of MAMP/DAMP ligands. A future challenge would be to identify the substrates of these various RLCKs, in order to elucidate their signaling role between the receptor complexes at the plasma membrane and downstream cellular signaling components

The cell wall-localized atypical β-1,3 glucanase ZERZAUST controls tissue morphogenesis in Arabidopsis thaliana

Orchestration of cellular behavior in plant organogenesis requires integration of intercellular communication and cell wall dynamics. The underlying signaling mechanisms are poorly understood. Tissue morphogenesis in Arabidopsis depends on the receptor-like kinase STRUBBELIG. Mutations in ZERZAUST were previously shown to result in a strubbelig-like mutant phenotype. Here, we report on the molecular identification and functional characterization of ZERZAUST. We show that ZERZAUST encodes a putative GPIanchored β-1,3 glucanase suggested to degrade the cell wall polymer callose. However, a combination of in vitro, cell biological and genetic experiments indicate that ZERZAUST is not involved in the regulation of callose accumulation. Nonetheless, Fourier-transformed infraredspectroscopy revealed that zerzaust mutants show defects in cell wall composition. Furthermore, the results indicate that ZERZAUST represents a mobile apoplastic protein, and that its carbohydrate-binding module family 43 domain is required for proper subcellular localization and function whereas its GPI anchor is dispensable. Our collective data reveal that the atypical β-1,3 glucanase ZERZAUST acts in a non-cell-autonomous manner and is required for cell wall organization during tissue morphogenesis.</p

The cell wall-localized atypical β-1,3 glucanase ZERZAUST controls tissue morphogenesis in Arabidopsis thaliana

<p>Orchestration of cellular behavior in plant organogenesis requires integration of intercellular communication and cell wall dynamics. The underlying signaling mechanisms are poorly understood. Tissue morphogenesis in Arabidopsis depends on the receptor-like kinase STRUBBELIG. Mutations in ZERZAUST were previously shown to result in a strubbelig-like mutant phenotype. Here, we report on the molecular identification and functional characterization of ZERZAUST. We show that ZERZAUST encodes a putative GPIanchored β-1,3 glucanase suggested to degrade the cell wall polymer callose. However, a combination of in vitro, cell biological and genetic experiments indicate that ZERZAUST is not involved in the regulation of callose accumulation. Nonetheless, Fourier-transformed infraredspectroscopy revealed that zerzaust mutants show defects in cell wall composition. Furthermore, the results indicate that ZERZAUST represents a mobile apoplastic protein, and that its carbohydrate-binding module family 43 domain is required for proper subcellular localization and function whereas its GPI anchor is dispensable. Our collective data reveal that the atypical β-1,3 glucanase ZERZAUST acts in a non-cell-autonomous manner and is required for cell wall organization during tissue morphogenesis.</p

Remodeling of Lipid A in <i>Pseudomonas syringae</i> pv. <i>phaseolicola</i> In Vitro

Pseudomonas species infect a variety of organisms, including mammals and plants. Mammalian pathogens of the Pseudomonas family modify their lipid A during host entry to evade immune responses and to create an effective barrier against different environments, for example by removal of primary acyl chains, addition of phosphoethanolamine (P-EtN) to primary phosphates, and hydroxylation of secondary acyl chains. For Pseudomonas syringae pv. phaseolicola (Pph) 1448A, an economically important pathogen of beans, we observed similar lipid A modifications by mass spectrometric analysis. Therefore, we investigated predicted proteomes of various plant-associated Pseudomonas spp. for putative lipid A-modifying proteins using the well-studied mammalian pathogen Pseudomonas aeruginosa as a reference. We generated isogenic mutant strains of candidate genes and analyzed their lipid A. We show that the function of PagL, LpxO, and EptA is generally conserved in Pph 1448A. PagL-mediated de-acylation occurs at the distal glucosamine, whereas LpxO hydroxylates the secondary acyl chain on the distal glucosamine. The addition of P-EtN catalyzed by EptA occurs at both phosphates of lipid A. Our study characterizes lipid A modifications in vitro and provides a useful set of mutant strains relevant for further functional studies on lipid A modifications in Pph 1448A