A mutation in Asparagine-Linked Glycosylation 12 (ALG12) leads to receptor misglycosylation and attenuated responses to multiple microbial elicitors.

Changes in cellular calcium levels are one of the earliest signalling events in plants exposed to pathogens or other exogenous factors. In a genetic screen, we identified an Arabidopsis thaliana 'changed calcium elevation 1' (cce1) mutant with attenuated calcium response to the bacterial flagellin flg22 peptide and several other elicitors. Whole-genome resequencing revealed a mutation in asparagine-linked glycosylation 12 that encodes the mannosyltransferase responsible for adding the eighth mannose residue in an α-1,6 linkage to the dolichol-PP-oligosaccharide N-glycosylation glycan tree precursors. While properly targeted to the plasma membrane, misglycosylation of several receptors in the cce1 background suggests that N-glycosylation is required for proper functioning of client proteins.

pH effects on plant calcium fluxes: lessons from acidification-mediated calcium elevation induced by the γ-glutamyl-leucine dipeptide identified from Phytophthora infestans.

Cytosolic Ca2+ ([Ca2+]cyt) elevation is an early signaling response upon exposure to pathogen-derived molecules (so-called microbe-associated molecular patterns, MAMPs) and has been successfully used as a quantitative read-out in genetic screens to identify MAMP receptors or their associated components. Here, we isolated and identified by mass spectrometry the dipeptide γ-Glu-Leu as a component of a Phytophthora infestans mycelium extract that induces [Ca2+]cyt elevation. Treatment of Arabidopsis seedlings with synthetic γ-Glu-Leu revealed stimulatory effects on defense signaling, including a weak enhancement of the expression of some MAMP-inducible genes or affecting the refractory period to a second MAMP elicitation. However, γ-Glu-Leu is not a classical MAMP since pH adjustment abolished these activities and importantly, the observed effects of γ-Glu-Leu could be recapitulated by mimicking extracellular acidification. Thus, although γ-Glu-Leu can act as a direct agonist of calcium sensing receptors in animal systems, the Ca2+-mobilizing activity in plants reported here is due to acidification. Low pH also shapes the Ca2+ signature of well-studied MAMPs (e.g. flg22) or excitatory amino acids such as glutamate. Overall, this work serves as a cautionary reminder that in defense signaling studies where Ca2+ flux measurements are concerned, it is important to monitor and consider the effects of pH.

Plant-to-Plant Variability in Root Metabolite Profiles of 19 Arabidopsis thaliana Accessions Is Substance-Class-Dependent.

Natural variation of secondary metabolism between different accessions of Arabidopsis thaliana (A. thaliana) has been studied extensively. In this study, we extended the natural variation approach by including biological variability (plant-to-plant variability) and analysed root metabolic patterns as well as their variability between plants and naturally occurring accessions. To screen 19 accessions of A. thaliana, comprehensive non-targeted metabolite profiling of single plant root extracts was performed using ultra performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC/ESI-QTOF-MS) and gas chromatography/electron ionization quadrupole mass spectrometry (GC/EI-QMS). Linear mixed models were applied to dissect the total observed variance. All metabolic profiles pointed towards a larger plant-to-plant variability than natural variation between accessions and variance of experimental batches. Ratios of plant-to-plant to total variability were high and distinct for certain secondary metabolites. None of the investigated accessions displayed a specifically high or low biological variability for these substance classes. This study provides recommendations for future natural variation analyses of glucosinolates, flavonoids, and phenylpropanoids and also reference data for additional substance classes.

Natural variation of root exudates in Arabidopsis thaliana-linking metabolomic and genomic data.

Many metabolomics studies focus on aboveground parts of the plant, while metabolism within roots and the chemical composition of the rhizosphere, as influenced by exudation, are not deeply investigated. In this study, we analysed exudate metabolic patterns of Arabidopsis thaliana and their variation in genetically diverse accessions. For this project, we used the 19 parental accessions of the Arabidopsis MAGIC collection. Plants were grown in a hydroponic system, their exudates were harvested before bolting and subjected to UPLC/ESI-QTOF-MS analysis. Metabolite profiles were analysed together with the genome sequence information. Our study uncovered distinct metabolite profiles for root exudates of the 19 accessions. Hierarchical clustering revealed similarities in the exudate metabolite profiles, which were partly reflected by the genetic distances. An association of metabolite absence with nonsense mutations was detected for the biosynthetic pathways of an indolic glucosinolate hydrolysis product, a hydroxycinnamic acid amine and a flavonoid triglycoside. Consequently, a direct link between metabolic phenotype and genotype was detected without using segregating populations. Moreover, genomics can help to identify biosynthetic enzymes in metabolomics experiments. Our study elucidates the chemical composition of the rhizosphere and its natural variation in A. thaliana, which is important for the attraction and shaping of microbial communities.

Altered glycosylation of exported proteins, including surface immune receptors, compromises calcium and downstream signaling responses to microbe-associated molecular patterns in Arabidopsis thaliana.

BACKGROUND: Calcium, as a second messenger, transduces extracellular signals into cellular reactions. A rise in cytosolic calcium concentration is one of the first plant responses after exposure to microbe-associated molecular patterns (MAMPs). We reported previously the isolation of Arabidopsis thaliana mutants with a "changed calcium elevation" (cce) response to flg22, a 22-amino-acid MAMP derived from bacterial flagellin. RESULTS: Here, we characterized the cce2 mutant and its weaker allelic mutant, cce3. Besides flg22, the mutants respond with a reduced calcium elevation to several other MAMPs and a plant endogenous peptide that is proteolytically processed from pre-pro-proteins during wounding. Downstream defense-related events such flg22-induced mitogen-activated protein kinase activation, accumulation of reactive oxygen species and growth arrest are also attenuated in cce2/cce3. By genetic mapping, next-generation sequencing and allelism assay, CCE2/CCE3 was identified to be ALG3 (Asparagine-linked glycosylation 3). This encodes the α-1,3-mannosyltransferase responsible for the first step of core oligosaccharide Glc3Man9GlcNAc2 glycan assembly on the endoplasmic reticulum (ER) luminal side. Complementation assays and glycan analysis in yeast alg3 mutant confirmed the reduced enzymatic function of the proteins encoded by the cce2/cce3 alleles - leading to accumulation of M5(ER), the immature five mannose-containing oligosaccharide structure found in the ER. Proper protein glycosylation is required for ER/Golgi processing and trafficking of membrane proteins to the plasma membrane. Endoglycosidase H-insensitivity of flg22 receptor, FLS2, in the cce2/cce3 mutants suggests altered glycan structures in the receptor. CONCLUSION: Proper glycosylation of MAMP receptors (or other exported proteins) is required for optimal responses to MAMPs and is important for immune signaling of host plants.

A lectin S-domain receptor kinase mediates lipopolysaccharide sensing in Arabidopsis thaliana.

The sensing of microbe-associated molecular patterns (MAMPs) triggers innate immunity in animals and plants. Lipopolysaccharide (LPS) from Gram-negative bacteria is a potent MAMP for mammals, with the lipid A moiety activating proinflammatory responses via Toll-like receptor 4 (TLR4). Here we found that the plant Arabidopsis thaliana specifically sensed LPS of Pseudomonas and Xanthomonas. We isolated LPS-insensitive mutants defective in the bulb-type lectin S-domain-1 receptor-like kinase LORE (SD1-29), which were hypersusceptible to infection with Pseudomonas syringae. Targeted chemical degradation of LPS from Pseudomonas species suggested that LORE detected mainly the lipid A moiety of LPS. LORE conferred sensitivity to LPS onto tobacco after transient expression, which demonstrated a key function in LPS sensing and indicated the possibility of engineering resistance to bacteria in crop species.

Expression of Caenorhabditis elegans†PCS in the AtPCS1-deficient Arabidopsis thaliana†cad1-3 mutant separates the metal tolerance and non-host resistance functions of phytochelatin synthases.

Phytochelatin synthases (PCS) play key roles in plant metal tolerance. They synthesize small metal-binding peptides, phytochelatins, under conditions of metal excess. Respective mutants are strongly cadmium and arsenic hypersensitive. However, their ubiquitous presence and constitutive expression had long suggested a more general function of PCS besides metal detoxification. Indeed, phytochelatin synthase1 from Arabidopsis thaliana (AtPCS1) was later implicated in non-host resistance. The two different physiological functions may be attributable to the two distinct catalytic activities demonstrated for AtPCS1, that is the dipeptidyl transfer onto an acceptor molecule in phytochelatin synthesis, and the proteolytic deglycylation of glutathione conjugates. In order to test this hypothesis and to possibly separate the two biological roles, we expressed a phylogenetically distant PCS from Caenorhabditis elegans in an AtPCS1 mutant. We confirmed the involvement of AtPCS1 in non-host resistance by showing that plants lacking the functional gene develop a strong cell death phenotype when inoculated with the potato pathogen Phytophthora infestans. Furthermore, we found that the C. elegans gene rescues phytochelatin synthesis and cadmium tolerance, but not the defect in non-host resistance. This strongly suggests that the second enzymatic function of AtPCS1, which remains to be defined in detail, is underlying the plant immunity function.

Mutations in the EDR1 Gene Alter the Response of Arabidopsis thaliana to Phytophthora infestans and the Bacterial PAMPs flg22 and elf18.

Mechanistically, nonhost resistance of Arabidopsis thaliana against the oomycete Phytophthora infestans is not well understood. Besides PEN2 and PEN3, which contribute to penetration resistance, no further components have been identified so far. In an ethylmethane sulphonate-mutant screen, we mutagenized pen2-1 and screened for mutants with an altered response to infection by P. infestans. One of the mutants obtained, enhanced response to Phytophthora infestans6 (erp6), was analyzed. Whole-genome sequencing of erp6 revealed a single nucleotide polymorphism in the coding region of the kinase domain of At1g08720, which encodes the putative MAPKKK ENHANCED DISEASE RESISTANCE1 (EDR1). We demonstrate that three independent lines with knock-out alleles of edr1 mount an enhanced response to P. infestans inoculation, mediated by increased salicylic acid signaling and callose deposition. Moreover, we show that the single amino acid substitution in erp6 causes the loss of in vitro autophosphorylation activity of EDR1. Furthermore, growth inhibition experiments suggest a so-far-unknown involvement of EDR1 in the response to the pathogen-associated molecular patterns flg22 and elf18. We conclude that EDR1 contributes to the defense response of A. thaliana against P. infestans. Our data position EDR1 as a negative regulator in postinvasive nonhost resistance.

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.

Growing and processing conditions lead to changes in the carotenoid profile of spinach.

This study aimed to evaluate the influence of different light regimens during spinach cultivation on the isomeric composition of ß-carotene. Irradiation with a halogen lamp, which has a wavelength spectrum close to that of daylight, was used to mimic field-grown conditions. The additional use of optical filters was established as a model system for greenhouse cultivation. Field-grown model systems led to a preferential increase of 9-cis-ß-carotene, whereas 13-cis-ß-carotene was just formed at the beginning of irradiation. Additionally 9,13-di-cis-ß-carotene decreased significantly in the presence of energy-rich light. Isomerization of ß-carotene was strongly suppressed during irradiation in greenhouse-grown model systems and led to significant differences. These results were verified in biological samples. Authentic field-grown spinach (Spinacia oleracea L.) showed among changes of other isomers a significantly higher level of 9-cis-isomers (7.52 ± 0.14%) and a significantly lower level of 9,13-di-cis-isomers (0.25 ± 0.03%) compared to authentic greenhouse-grown spinach (6.49 ± 0.11 and 0.76 ± 0.05%). Almost all analyzed commercial spinach samples (fresh and frozen) were identified as common field-grown cultivation. Further investigations resulted in a clear differentiation of frozen commercial samples from fresh spinach, caused by significantly higher levels of 13-cis- and 15-cis-ß-carotene as a result of industrial blanching processes.

Analyses of wrky18 wrky40 plants reveal critical roles of SA/EDS1 signaling and indole-glucosinolate biosynthesis for Golovinomyces orontii resistance and a loss-of resistance towards Pseudomonas syringae pv. tomato AvrRPS4.

Simultaneous mutation of two WRKY-type transcription factors, WRKY18 and WRKY40, renders otherwise susceptible wild-type Arabidopsis plants resistant towards the biotrophic powdery mildew fungus Golovinomyces orontii. Resistance in wrky18 wrky40 double mutant plants is accompanied by massive transcriptional reprogramming, imbalance in salicylic acid (SA) and jasmonic acid (JA) signaling, altered ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) expression, and accumulation of the phytoalexin camalexin. Genetic analyses identified SA biosynthesis and EDS1 signaling as well as biosynthesis of the indole-glucosinolate 4MI3G as essential components required for loss-of-WRKY18 WRKY40-mediated resistance towards G. orontii. The analysis of wrky18 wrky40 pad3 mutant plants impaired in camalexin biosynthesis revealed an uncoupling of pre- from postinvasive resistance against G. orontii. Comprehensive infection studies demonstrated the specificity of wrky18 wrky40-mediated G. orontii resistance. Interestingly, WRKY18 and WRKY40 act as positive regulators in effector-triggered immunity, as the wrky18 wrky40 double mutant was found to be strongly susceptible towards the bacterial pathogen Pseudomonas syringae DC3000 expressing the effector AvrRPS4 but not against other tested Pseudomonas strains. We hypothesize that G. orontii depends on the function of WRKY18 and WRKY40 to successfully infect Arabidopsis wild-type plants while, in the interaction with P. syringae AvrRPS4, they are required to mediate effector-triggered immunity.

The exocyst subunit Exo70B1 is involved in the immune response of Arabidopsis thaliana to different pathogens and cell death.

Components of the vesicle trafficking machinery are central to the immune response in plants. The role of vesicle trafficking during pre-invasive penetration resistance has been well documented. However, emerging evidence also implicates vesicle trafficking in early immune signaling. Here we report that Exo70B1, a subunit of the exocyst complex which mediates early tethering during exocytosis is involved in resistance. We show that exo70B1 mutants display pathogen-specific immuno-compromised phenotypes. We also show that exo70B1 mutants display lesion-mimic cell death, which in combination with the reduced responsiveness to pathogen-associated molecular patterns (PAMPs) results in complex immunity-related phenotypes.

Impaired sterol ester synthesis alters the response of Arabidopsis thaliana to Phytophthora infestans.

Non-host resistance of Arabidopsis thaliana against Phytophthora infestans, the causal agent of late blight disease of potato, depends on efficient extracellular pre- and post-invasive resistance responses. Pre-invasive resistance against P. infestans requires the myrosinase PEN2. To identify additional genes involved in non-host resistance to P. infestans, a genetic screen was performed by re-mutagenesis of pen2 plants. Fourteen independent mutants were isolated that displayed an enhanced response to Phytophthora (erp) phenotype. Upon inoculation with P. infestans, two mutants, pen2-1 erp1-3 and pen2-1 erp1-4, showed an enhanced rate of mesophyll cell death and produced excessive callose deposits in the mesophyll cell layer. ERP1 encodes a phospholipid:sterol acyltransferase (PSAT1) that catalyzes the formation of sterol esters. Consistent with this, the tested T-DNA insertion lines of PSAT1 are phenocopies of erp1 plants. Sterol ester levels are highly reduced in all erp1/psat1 mutants, whereas sterol glycoside levels are increased twofold. Excessive callose deposition occurred independently of PMR4/GSL5 activity, a known pathogen-inducible callose synthase. A similar formation of aberrant callose deposits was triggered by the inoculation of erp1 psat1 plants with powdery mildew. These results suggest a role for sterol conjugates in cell non-autonomous defense responses against invasive filamentous pathogens.

The multifunctional enzyme CYP71B15 (PHYTOALEXIN DEFICIENT3) converts cysteine-indole-3-acetonitrile to camalexin in the indole-3-acetonitrile metabolic network of Arabidopsis thaliana.

Accumulation of camalexin, the characteristic phytoalexin of Arabidopsis thaliana, is induced by a great variety of plant pathogens. It is derived from Trp, which is converted to indole-3-acetonitrile (IAN) by successive action of the cytochrome P450 enzymes CYP79B2/B3 and CYP71A13. Extracts from wild-type plants and camalexin biosynthetic mutants, treated with silver nitrate or inoculated with Phytophthora infestans, were comprehensively analyzed by ultra-performance liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry. This metabolomics approach was combined with precursor feeding experiments to characterize the IAN metabolic network and to identify novel biosynthetic intermediates and metabolites of camalexin. Indole-3-carbaldehyde and indole-3-carboxylic acid derivatives were shown to originate from IAN. IAN conjugates with glutathione, gamma-glutamylcysteine, and cysteine [Cys(IAN)] accumulated in challenged phytoalexin deficient3 (pad3) mutants. Cys(IAN) rescued the camalexin-deficient phenotype of cyp79b2 cyp79b3 and was itself converted to dihydrocamalexic acid (DHCA), the known substrate of CYP71B15 (PAD3), by microsomes isolated from silver nitrate-treated Arabidopsis leaves. Surprisingly, yeast-expressed CYP71B15 also catalyzed thiazoline ring closure, DHCA formation, and cyanide release with Cys(IAN) as substrate. In conclusion, in the camalexin biosynthetic pathway, IAN is derivatized to the intermediate Cys(IAN), which serves as substrate of the multifunctional cytochrome P450 enzyme CYP71B15.

A cysteine-rich receptor-like kinase NCRK and a pathogen-induced protein kinase RBK1 are Rop GTPase interactors.

In plants, Rop/Rac GTPases have emerged as central regulators of diverse signalling pathways in plant growth and pathogen defence. When active, they interact with a wide range of downstream effectors. Using yeast two-hybrid screening we have found three previously uncharacterized receptor-like protein kinases to be Rop GTPase-interacting molecules: a cysteine-rich receptor kinase, named NCRK, and two receptor-like cytosolic kinases from the Arabidopsis RLCK-VIb family, named RBK1 and RBK2. Uniquely for Rho-family small GTPases, plant Rop GTPases were found to interact directly with the protein kinase domains. Rop4 bound NCRK preferentially in the GTP-bound conformation as determined by flow cytometric fluorescence resonance energy transfer measurements in insect cells. The kinase RBK1 did not phosphorylate Rop4 in vitro, suggesting that the protein kinases are targets for Rop signalling. Bimolecular fluorescence complementation assays demonstrated that Rop4 interacted in vivo with NCRK and RBK1 at the plant plasma membrane. In Arabidopsis protoplasts, NCRK was hyperphosphorylated and partially co-localized with the small GTPase RabF2a in endosomes. Gene expression analysis indicated that the single-copy NCRK gene was relatively upregulated in vasculature, especially in developing tracheary elements. The seven Arabidopsis RLCK-VIb genes are ubiquitously expressed in plant development, and highly so in pollen, as in case of RBK2. We show that the developmental context of RBK1 gene expression is predominantly associated with vasculature and is also locally upregulated in leaves exposed to Phytophthora infestans and Botrytis cinerea pathogens. Our data indicate the existence of cross-talk between Rop GTPases and specific receptor-like kinases through direct molecular interaction.

Conserved requirement for a plant host cell protein in powdery mildew pathogenesis.

In the fungal phylum Ascomycota, the ability to cause disease in plants and animals has been gained and lost repeatedly during phylogenesis. In monocotyledonous barley, loss-of-function mlo alleles result in effective immunity against the Ascomycete Blumeria graminis f. sp. hordei, the causal agent of powdery mildew disease. However, mlo-based disease resistance has been considered a barley-specific phenomenon to date. Here, we demonstrate a conserved requirement for MLO proteins in powdery mildew pathogenesis in the dicotyledonous plant species Arabidopsis thaliana. Epistasis analysis showed that mlo resistance in A. thaliana does not involve the signaling molecules ethylene, jasmonic acid or salicylic acid, but requires a syntaxin, glycosyl hydrolase and ABC transporter. These findings imply that a common host cell entry mechanism of powdery mildew fungi evolved once and at least 200 million years ago, suggesting that within the Erysiphales (powdery mildews) the ability to cause disease has been a stable trait throughout phylogenesis.