Bacteria may induce the secretion of mucin-like proteins by the diatom Phaeodactylum tricornutum.

Benthic diatoms live in photoautotrophic/heterotrophic biofilm communities embedded in a matrix of secreted extracellular polymeric substances. Closely associated bacteria influence their growth, aggregation, and secretion of exopolymers. We have studied a diatom/bacteria model community, in which a marine Roseobacter strain is able to grow with secreted diatom exopolymers as a sole source of carbon. The strain influences the aggregation of Phaeodactylum tricornutum by inducing a morphotypic transition from planktonic, fusiform cells to benthic, oval cells. Analysis of the extracellular soluble proteome of P. tricornutum in the presence and absence of bacteria revealed constitutively expressed newly identified proteins with mucin-like domains that appear to be typical for extracellular diatom proteins. In contrast to mucins, the proline-, serine-, threonine-rich (PST) domains in these proteins were also found in combination with protease-, glucosidase- and leucine-rich repeat-domains. Bioinformatic functional predictions indicate that several of these newly identified diatom-specific proteins may be involved in algal defense, intercellular signaling, and aggregation.

Comprehensive computational analysis of leucine-rich repeat (LRR) proteins encoded in the genome of the diatom Phaeodactylum tricornutum.

We have screened the genome of the marine diatom Phaeodactylum tricornutum for gene models encoding proteins exhibiting leucine-rich repeat (LRR) structures. In order to reveal the functionality of these proteins, their amino acid sequences were scanned for known domains and for homologies to other proteins. Additionally, proteins were categorized into different LRR-families according to the variable sequence part of their LRR. This approach enabled us to group proteins with potentially similar functionality and to classify also LRR proteins where no characterized homologues in other organisms exist. Most interestingly, we were able to indentify several transmembrane LRR-proteins, which are likely to function as receptor-like molecules. However, none of them carry additional domains that are typical for mammalian or plant-like receptors. Thus, the respective signal recognition pathways seem to be substantially different in diatoms. Moreover, P. tricornutum encodes a family of secreted LRR proteins likely to function as adhesion or binding proteins as part of the extracellular matrix. Additionally, intracellular LRR-only proteins were divided into proteins similar to RasGTPase activators, regulators of nuclear transport, and mitotic regulation. Our approach allowed us to draw a detailed picture of the conservation and diversification of LRR proteins in the marine diatom P. tricornutum.

The anticipation of danger: microbe-associated molecular pattern perception enhances AtPep-triggered oxidative burst.

The endogenous Arabidopsis (Arabidopsis thaliana) peptides, AtPeps, elicit an innate immune response reminiscent of pattern-triggered immunity. Detection of various danger signals, including microbe-associated molecular patterns (MAMPs), leads to elevated transcription of PROPEPs, the AtPep precursors, and PEPRs, the AtPep receptors. It has been hypothesized that AtPeps are involved in enhancing pattern-triggered immunity. Following this idea, we analyzed the relationship between MAMP- and AtPep-elicited signaling. We found that the perception of MAMPs enhanced a subsequent AtPep-triggered production of reactive oxygen species (ROS). Intriguingly, other components of AtPep-triggered immunity like Ca(2+) influx, mitogen-activated protein kinase phosphorylation, ethylene production, and expression of early defense genes, as well as ROS-activated genes, remained unchanged. By contrast, treatment with methyl jasmonate promoted an increase of all analyzed AtPep-triggered responses. We positively correlated the intensities of generic AtPep-triggered responses with the abundance of the two AtPep receptors by generating constitutively expressing PEPR1 and PEPR2 transgenic lines and by analyzing pepr1 and pepr2 mutants. Further, we show that enhanced, as well as basal, ROS production triggered by AtPeps is absent in the double mutant of the respiratory burst oxidase homologs D and F (rbohD rbohF). We present evidence that the enhancement of AtPep-triggered ROS is not based on changes in the ROS detoxification machinery and is independent of mitogen-activated protein kinase and Ca(2+) signaling pathways. Taken together, these results indicate an additional level of regulation besides receptor abundance for the RbohD/RbohF-dependent production of AtPep-elicited ROS, which is specifically operated by MAMP-triggered pathways.

The lectin receptor kinase-VI.2 is required for priming and positively regulates Arabidopsis pattern-triggered immunity.

Plant cells can be sensitized toward a subsequent pathogen attack by avirulent pathogens or by chemicals such as ß-aminobutyric acid (BABA). This process is called priming. Using a reverse genetic approach in Arabidopsis thaliana, we demonstrate that the BABA-responsive L-type lectin receptor kinase-VI.2 (LecRK-VI.2) contributes to disease resistance against the hemibiotrophic Pseudomonas syringae and the necrotrophic Pectobacterium carotovorum bacteria. Accordingly, LecRK-VI.2 mRNA levels increased after bacterial inoculation or treatments with microbe-associated molecular patterns (MAMPs). We also show that LecRK-VI.2 is required for full activation of pattern-triggered immunity (PTI); notably, lecrk-VI.2-1 mutants show reduced upregulation of PTI marker genes, impaired callose deposition, and defective stomatal closure. Overexpression studies combined with genome-wide microarray analyses indicate that LecRK-VI.2 positively regulates the PTI response. LecRK-VI.2 is demonstrated to act upstream of mitogen-activated protein kinase signaling, but independently of reactive oxygen production and Botrytis-induced kinase1 phosphorylation. In addition, complex formation between the MAMP receptor flagellin sensing2 and its signaling partner brassinosteroid insensitive1-associated kinase1 is observed in flg22-treated lecrk-VI.2-1 mutants. LecRK-VI.2 is also required for full BABA-induced resistance and priming of PTI. Our work identifies LecRK-VI.2 as a novel mediator of the Arabidopsis PTI response and provides insight into molecular mechanisms governing priming.

Rapid heteromerization and phosphorylation of ligand-activated plant transmembrane receptors and their associated kinase BAK1.

In plants leucine-rich repeat receptor kinases (LRR-RKs) located at the plasma membrane play a pivotal role in the perception of extracellular signals. For two of these LRR-RKs, the brassinosteroid receptor BRI1 and the flagellin receptor FLS2, interaction with the LRR receptor-like kinase BAK1 (BRI1-associated receptor kinase 1) was shown to be required for signal transduction. Here we report that FLS2.BAK1 heteromerization occurs almost instantaneously after perception of the ligand, the flagellin-derived peptide flg22. Flg22 can induce formation of a stable FLS2.BAK1 complex in microsomal membrane preparations in vitro, and the kinase inhibitor K-252a does not prevent complex formation. A kinase dead version of BAK1 associates with FLS2 in a flg22-dependent manner but does not restore responsiveness to flg22 in cells of bak1 plants, demonstrating that kinase activity of BAK1 is essential for FLS2 signaling. Furthermore, using in vivo phospholabeling, we are able to detect de novo phosphorylation of both FLS2 and BAK1 within 15 s of stimulation with flg22. Similarly, brassinolide induces BAK1 phosphorylation within seconds. Other triggers of plant defense, such as bacterial EF-Tu and the endogenous AtPep1 likewise induce rapid formation of heterocomplexes consisting of de novo phosphorylated BAK1 and proteins representing the ligand-specific binding receptors EF-Tu receptor and Pep1 receptor 1, respectively. Thus, we propose that several LRR-RKs form tight complexes with BAK1 almost instantaneously after ligand binding and that the subsequent phosphorylation events are key initial steps in signal transduction.

Capsaicin: tailored chemical defence against unwanted "frugivores".

Why are chilli peppers hot? The vanillyl amide, capsaicin, has long been known as the pungent principle of peppers, but only in their recent work have Tewksbury et al. addressed its ecological roles: to distract unsuitable seed dispensers and to protect the seeds against fungal infection by Fusarium.

Herbivore-induced terpenoid emission in Medicago truncatula: concerted action of jasmonate, ethylene and calcium signaling.

Plant volatiles emitted by Medicago truncatula in response to feeding larvae of Spodoptera exigua are composed of a complex blend of terpenoids. The cDNAs of three terpene synthases (TPSs), which contribute to the blend of terpenoids, were cloned from M. truncatula. Their functional characterization proved MtTPS1 to be a beta-caryophyllene synthase and MtTPS5 to be a multi-product sesquiterpene synthase. MtTPS3 encodes a bifunctional enzyme producing (E)-nerolidol and geranyllinalool (precursors of C11 and C16 homoterpenes) from different prenyl diphosphates serving as substrates. The addition of jasmonic acid (JA) induced expression of the TPS genes, but terpenoid emission was higher from plants treated with JA and the ethylene precursor 1-amino-cyclopropyl-1-carboxylic acid. Compared to infested wild-type M. truncatula plants, lower amounts of various sesquiterpenes and a C11-homoterpene were released from an ethylene-insensitive mutant skl. This difference coincided with lower transcript levels of MtTPS5 and of 1-deoxy-D: -xylulose-5-phosphate synthase (MtDXS2) in the damaged skl leaves. Moreover, ethephon, an ethylene-releasing compound, modified the extent and mode of the herbivore-stimulated Ca2+ variations in the cytoplasm that is necessary for both JA and terpene biosynthesis. Thus, ethylene contributes to the herbivory-induced terpenoid biosynthesis at least twice: by modulating both early signaling events such as cytoplasmic Ca2+-influx and the downstream JA-dependent biosynthesis of terpenoids.

Rapid enzymatic isomerization of 12-oxophytodienoic acid in the gut of lepidopteran larvae.

In response to feeding larvae of the Mediterranean climbing cutworm (Spodoptera littoralis), leaves of the lima bean (Phaseolus lunatus) produce fatty acid-derived signaling compounds (oxylipins). The major products are the phytohormones jasmonic acid and its biosynthetic precursor 12-oxophytodienoic acid (OPDA), along with 13-hydroxy-12-oxooctadeca-9,15-dienoic acid, 9-hydroxy-12-oxooctadeca-10,15-dienoic acid (alpha- and gamma-ketol), as well as unsaturated aldehydes. Oxylipin production is highest at the feeding zone of the insect and decreases with distance from the damaged area. Accordingly, the feeding insect experiences high local concentrations of oxylipins, which are taken up into the alimentary canal and are finally excreted with the feces. In contrast to most other oxylipins, OPDA was not detectable in the insect's gut; instead the structurally related tetrahydrodicranenone B (iso-OPDA) was identified. Feeding experiments with deuterium-labeled OPDA proved that the isomerization is catalyzed by an enzyme from the insect's gut tissue. The phenomenon appears to be widespread among Lepidopteran larvae.

Profiling of structurally labile oxylipins in plants by in situ derivatization with pentafluorobenzyl hydroxylamine.

A GC-MS-based method for the simultaneous quantification of common oxylipins along with labile and highly reactive compounds based on in situ derivatization with pentafluorobenzyl hydroxylamine to the corresponding O-2,3,4,5,6-pentafluorobenzyl oximes (PFB oximes) is presented. The approach covers oxo derivatives such as jasmonic acid (JA), 12-oxophytodienoic acid (OPDA), certain phytoprostanes, unsaturated oxo-acids, oxo-hydroxy acids, and aldehyde fragments from the polar head of fatty acids. In the positive electron impact-MS mode, the PFB oximes display characteristic fragment ions that greatly facilitate the identification of oxylipins in complex matrices. In addition, the fluorinated derivatives allow a highly selective and low-background analysis by negative chemical ionization. Besides showing the general value of the method for the identification of a broad range of oxylipins (18 examples), we also demonstrate sensitivity, linearity, and reproducibility for the quantification of JA, OPDA, 11-oxo-9-undecenoic acid, and 13-oxo-9,11-tridecadienoic acid. The efficiency of the method is demonstrated by differential profiling of these four oxylipins in lima bean leaves after mechanical wounding and feeding by the herbivore Spodoptera littoralis. Caterpillar feeding induced several oxylipins, whereas after wounding only the level of JA increased. The rapid in situ derivatization prevents the isomerization of cis-JA to trans-JA. The resting level of JA in lima beans showed an isomer ratio of 80:20 for trans/cis-JA. After wounding, de novo synthesis of JA alters the ratio to 20:80 in favor of the cis isomer.

Biotic and heavy metal stress response in plants: evidence for common signals.

In higher plants, biotic stress (e.g., herbivore or pathogen attack) as well as abiotic stress (in particular heavy metals) often induce the synthesis and accumulation of the same defense-related secondary metabolites. This well-known finding still awaits an explanation regarding the common features of both stress types. In this study, a mechanism is proposed that links reactive oxygen species (ROS) generation with lipid oxidation processes, ultimately resulting in the formation of similar, highly active signalling compounds. The generation of ROS is a common event in both heavy metal treatment and biotic stress although it can depend on quite different, enzymatic and non-enzymatic reactions. Regardless, ROS are involved in the oxidation of unsaturated fatty acids which initiate the formation of oxylipins, a highly variable class of lipid-derived compounds in plants. Oxylipins represent new endogenous signals involved in biotic- and abiotic-induced stress responses.

Study of 30 patients with unexplained developmental delay and dysmorphic features or congenital abnormalities using conventional cytogenetics and multiplex FISH telomere (M-TEL) integrity assay.

Cryptic subtelomeric chromosome rearrangements are a major cause of mild to severe mental retardation pointing out the necessity of sensitive screening techniques to detect such aberrations among affected patients. In this prospective study a group of 30 patients with unexplained developmental retardation and dysmorphic features or congenital abnormalities were analysed using the recently published multiplex FISH telomere (M-TEL) integrity assay in combination with conventional G-banding analysis. The patients were selected by one or more of the following criteria defined by de Vries et al.: (a) family history with two or more affected individuals, (b) prenatal onset growth retardation, (c) postnatal growth abnormalities, (d) facial dysmorphic features, (e) non-facial dysmorphism and congenital abnormalities. In addition, we included two patients who met these criteria and revealed questionable chromosome regions requiring further clarification. In four patients (13.3%) cryptic chromosome aberrations were successfully determined by the M-TEL integrity assay and in two patients with abnormal chromosome regions intrachromosomal aberrations were characterized by targetted FISH experiments. Our results accentuate the requirement of strict selection criteria prior to patient testing with the M-TEL integrity assay. Another essential precondition is high-quality banding analysis to identify structural abnormal chromosomes. The detection of familial balanced translocation carriers in 50% of the cases emphasizes the significance of such an integrated approach for genetic counselling and prenatal diagnosis.