Identification of ε-Poly-L-lysine as an Antimicrobial Product from an Epichloë Endophyte and Isolation of Fungal ε-PL Synthetase Gene.

The endophytic fungus Epichloë festucae is known to produce bioactive metabolites, which consequently protect the host plants from biotic and abiotic stresses. We previously found that the overexpression of vibA (a gene for transcription factor) in E. festucae strain E437 resulted in the secretion of an unknown fungicide. In the present study, the active substance was purified and chemically identified as ε-poly-L-lysine (ε-PL), which consisted of 28-34 lysine units. The productivity was 3.7-fold compared with that of the wild type strain E437. The isolated ε-PL showed inhibitory activity against the spore germination of the plant pathogens Drechslera erythrospila, Botrytis cinerea, and Phytophthora infestans at 1-10 µg/mL. We also isolated the fungal gene "epls" encoding ε-PL synthetase Epls. Overexpression of epls in the wild type strain E437 resulted in the enhanced production of ε-PL by 6.7-fold. Interestingly, overexpression of epls in the different strain E. festucae Fl1 resulted in the production of shorter ε-PL with 8-20 lysine, which exhibited a comparable antifungal activity to the longer one. The results demonstrate the first example of ε-PL synthetase gene from the eukaryotic genomes and suggest the potential of enhanced expression of vibA or/and epls genes in the Epichloë endophyte for constructing pest-tolerant plants.

Nitrate analogs as attractants for soybean cyst nematode.

Soybean cyst nematode (SCN) Heterodera glycines Ichinohe, a plant parasite, is one of the most serious pests of soybean. In this paper, we report that SCN is attracted to nitrate and its analogs. We performed attraction assays to screen for novel attractants for SCN and found that nitrates were attractants for SCN and SCN recognized nitrate gradients. However, attraction of SCN to nitrates was not observed on agar containing nitrate. To further elucidate the attraction mechanism in SCN, we performed attraction assays using nitrate analogs ([Formula: see text], [Formula: see text], [Formula: see text]). SCN was attracted to all nitrate analogs; however, attraction of SCN to nitrate analogs was not observed on agar containing nitrate. In contrast, SCN was attracted to azuki root, irrespective of presence or absence of nitrate in agar media. Our results suggest that the attraction mechanisms differ between plant-derived attractant and nitrate.

1,10-Phenanthroline and its derivatives are novel hatching stimulants for soybean cyst nematodes.

Soybean cyst nematode (SCN), Heterodera glycines Ichinohe, is a plant-parasitic nematode and one of the most serious soybean pests. Herein, we present the heterocyclic compound 1,10-phenanthroline (Phen) and its derivatives as novel hatching stimulants for SCN. Phen treatment promoted hatching of second-stage juveniles of SCNs in a concentration-dependent manner. In addition, the hatching of SCNs following treatment with Phen occurred more rapidly than that following treatment with the known hatching stimulant, glycinoeclepin A (GEA). Furthermore, the co-application of Phen and GEA enhanced SCN hatching rate compared with that of Phen or GEA alone. A structure-activity relationship study for Phen derivatives suggested that 2,2'-bipyridine is the essential structure of the SCN-hatching stimulants. These results suggest that Phen and its derivatives activate different hatching pathways of SCNs from GEA.

Toll-like receptors as a target of food-derived anti-inflammatory compounds.

Toll-like receptors (TLRs) play a key role in linking pathogen recognition with the induction of innate immunity. They have been implicated in the pathogenesis of chronic inflammatory diseases, representing potential targets for prevention/treatment. Vegetable-rich diets are associated with the reduced risk of several inflammatory disorders. In the present study, based on an extensive screening of vegetable extracts for TLR-inhibiting activity in HEK293 cells co-expressing TLR with the NF-κB reporter gene, we found cabbage and onion extracts to be the richest sources of a TLR signaling inhibitor. To identify the active substances, we performed activity-guiding separation of the principal inhibitors and identified 3-methylsulfinylpropyl isothiocyanate (iberin) from the cabbage and quercetin and quercetin 4'-O-ß-glucoside from the onion, among which iberin showed the most potent inhibitory effect. It was revealed that iberin specifically acted on the dimerization step of TLRs in the TLR signaling pathway. To gain insight into the inhibitory mechanism of TLR dimerization, we developed a novel probe combining an isothiocyanate-reactive group and an alkyne functionality for click chemistry and detected the probe bound to the TLRs in living cells, suggesting that iberin disrupts dimerization of the TLRs via covalent binding. Furthermore, we designed a variety of iberin analogues and found that the inhibition potency was influenced by the oxidation state of the sulfur. Modeling studies of the iberin analogues showed that the oxidation state of sulfur might influence the global shape of the isothiocyanates. These findings establish the TLR dimerization step as a target of food-derived anti-inflammatory compounds.

Differential effects of the peptides Stomagen, EPF1 and EPF2 on activation of MAP kinase MPK6 and the SPCH protein level.

The positioning and density of leaf stomata are regulated by three secretory peptides, EPIDERMAL PATTERNING FACTOR 1 (EPF1), EPF2 and stomagen. Several lines of published evidence have suggested a regulatory pathway as follows. EPF1 and EPF2 are perceived by receptor complexes consisting of a receptor-like protein, TOO MANY MOUTHS (TMM), and receptor kinases, ERECTA (ER), ERECTA-LIKE (ERL) 1 and ERL2. These receptors activate a mitogen-activated protein (MAP) kinase module. MAP kinases phosphorylate and destabilize the transcription factor SPEECHLESS (SPCH), resulting in a decrease in the number of stomatal lineage cells. Stomagen acts antagonistically to EPF1 and EPF2. However, there is no direct evidence that EPF1 and EPF2 activate or that stomagen inactivates the MAP kinase cascade, through which they might regulate the SPCH level. Experimental modulation of these peptides in Arabidopsis thaliana would change the number of stomatal lineage cells in developing leaves, which in turn would change the expression of SPCH, making the interpretation difficult. Here we reconstructed this signaling pathway in differentiated leaf cells of Nicotiana benthamiana to examine signaling without the confounding effect of cell type change. We show that EPF1 and EPF2 are able to activate the MAP kinase MPK6, and that both EPF1 and EPF2 are able to decrease the SPCH level, whereas stomagen is able to increase it. Our data also suggest that EPF1 can be recognized by TMM together with any ER family receptor kinase, whereas EPF2 can be recognized by TMM together with ERL1 or ERL2, but not by TMM together with ER.

EPIDERMAL PATTERNING FACTOR LIKE5 peptide represses stomatal development by inhibiting meristemoid maintenance in Arabidopsis thaliana.

Stomatal development in Arabidopsis epidermis is both positively and negatively regulated by a family of Cys-rich peptides, EPIDERMAL PATTERNING FACTOR LIKEs (EPFLs). We synthesized biologically active synthetic EPFL5 (sEPFL5) peptide, which reduced the number of stoma in leaves and cotyledons. The sEPFL5 possesses three disulfide bonds at positions identical to those of a positive development factor, stomagen. Application of sEPFL5 had little inhibitory effect on protodermal cells entering the stomatal lineage, but did inhibit the maintenance of meristemoid activity, resulting in the differentiation of arrested meristemoids into pavement cells. This phenotype was enhanced in the too many mouths (tmm) mutant background. RNA analysis revealed that sEPFL5 application halved SPEECHLESS expression and abolished MUTE expression in tmm mutants, explaining the phenotype observed. The action of sEPFL5 was mediated by ERECTA family receptors. We propose that EPFL5 functions to establish the differentiation of stomatal lineage cells to pavement cells.

Root-knot nematode modulates plant CLE3-CLV1 signaling as a long-distance signal for successful infection.

Plants use many long-distance and systemic signals to modulate growth and development, as well as respond to biotic and abiotic stresses. Parasitic nematodes infect host plant roots and cause severe damage to crop plants. However, the molecular mechanisms that regulate parasitic nematode infections are still unknown. Here, we show that plant parasitic root-knot nematodes (RKNs), Meloidogyne incognita, modulate the host CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION (CLE)-CLV1 signaling module to promote the infection progression. Plants deficient in the CLE signaling pathway show enhanced RKN resistance, whereas CLE overexpression leads to increased susceptibility toward RKN. Grafting analysis shows that CLV1 expression in the shoot alone is sufficient to positively regulate RKN infection. Together with results from the split-root culture system, infection assays, and CLE3-CLV1 binding assays, we conclude that mobile root-derived CLE signals are perceived by CLV1 in the shoot, which subsequently produce systemic signals to promote gall formation and RKN reproduction.

Analogs of the CLV3 peptide: synthesis and structure-activity relationships focused on proline residues.

CLAVATA3 (CLV3) is a plant peptide hormone in which the proline residues are post-translationally hydroxylated and glycosylated. CLV3 plays a key role in controlling the stem cell mass in the shoot meristem of Arabidopsis thaliana. In a previous report, we identified a dodecapeptide (MCLV3) from CLV3-overexpressing Arabidopsis calli; MCLV3 was the smallest functional peptide derived from the CLV3 precursor. Here, we designed a series of MCLV3 analogs in which proline residues were substituted with proline derivatives or N-substituted glycines (peptoids). Peptoid substitution at Pro9 decreased bioactivity without affecting specific binding to the CLV1-related protein in cauliflower membrane. These findings suggest that peptoid-substituted peptides would be lead compounds for developing potential agonists and antagonists of CLV3.

Stomatal density is controlled by a mesophyll-derived signaling molecule.

Stomata are composed of a pair of guard cells and a pore between them, and their density and positions are regulated by developmental and environmental signals. In a screen in which we overexpressed many genes coding for putative secretory proteins one by one in Arabidopsis, we identified a gene named STOMAGEN, which increases stomatal density when overexpressed. The STOMAGEN gene encodes a small peptide with a putative secretory signal sequence at its N-terminus and is expressed preferentially in mesophyll cells. This peptide belongs to the EPIDERMAL PATTERNING FACTOR (EPF) family of the cysteine-rich peptides superfamily. The mature form was a 45-amino-acid peptide (stomagen) with three intramolecular disulfide bonds. Stomagen treatment at very low concentrations, as low as 10 nM, increased the stomatal density of wild-type Arabidopsis plants. We propose that stomagen is a mesophyll-to-epidermis signaling molecule that positively regulates stomatal density. We also suggest that stomagen increases stomatal density by competing with negative regulators EPF1 and EPF2 for the receptor-like protein TOO MANY MOUTHS.

RNAi of the sesquiterpene cyclase gene for phytoalexin production impairs pre- and post-invasive resistance to potato blight pathogens.

Potato antimicrobial sesquiterpenoid phytoalexins lubimin and rishitin have been implicated in resistance to the late blight pathogen, Phytophthora infestans and early blight pathogen, Alternaria solani. We generated transgenic potato plants in which sesquiterpene cyclase, a key enzyme for production of lubimin and rishitin, is compromised by RNAi to investigate the role of phytoalexins in potato defence. The transgenic tubers were deficient in phytoalexins and exhibited reduced post-invasive resistance to an avirulent isolate of P. infestans, resulting in successful infection of the first attacked cells without induction of cell death. However, cell death was observed in the subsequently penetrated cells. Although we failed to detect phytoalexins and antifungal activity in the extract from wild-type leaves, post-invasive resistance to avirulent P. infestans was reduced in transgenic leaves. On the other hand, A. solani frequently penetrated epidermal cells of transgenic leaves and caused severe disease symptoms presumably from a deficiency in unidentified antifungal compounds. The contribution of antimicrobial components to resistance to penetration and later colonization may vary depending on the pathogen species, suggesting that sesquiterpene cyclase-mediated compounds participate in pre-invasive resistance to necrotrophic pathogen A. solani and post-invasive resistance to hemibiotrophic pathogen P. infestans.

Dual assay for MCLV3 activity reveals structure-activity relationship of CLE peptides.

The dodecapeptide MCLV3 is a functional peptide, derived from the CLV3 precursor protein, which is a candidate ligand of the CLV1/CLV2 receptor complex that restricts the stem cell population in the shoot apical meristem (SAM). MCLV3 can induce shoot and root meristem consumption, the typical phenotype of transgenic plants overexpressing CLV3. We investigated the bioactivities of a series of alanine-substituted MCLV3 and related peptides on the root growth of Arabidopsis. The structure-activity relationship (SAR) of MCLV3 had high similarity with that of tracheary element differentiation inhibitory factor (TDIF). We also evaluated the binding activities of the peptides by a competitive receptor binding assay using tritiated MCLV3 and the membrane fraction of a tobacco BY-2 cell line overexpressing the MCLV3 ectodomain. This dual assay, combining a biological and receptor binding assay for evaluating the activities of MCLV3-related peptides, uncovered the SAR of MCLV3, and indicated that the terminal residues play critical roles in exerting its activity and are important for specific binding to the receptor, CLV1.

Articulospora sp. produces Art1, an inhibitor of bacterial histidine kinase.

A two-component system (TCS) comprising a histidine kinase (HK) sensor and a response regulator (RR) plays important roles in regulating the virulence of many pathogenic bacteria. We used a new screening method to isolate novel inhibitor Art1 against bacterial sensory HK from an acetone extract of solid cultures of Articulospora sp., an aquatic hypomycete. Art1 inhibited the ATP-dependent autophosphorylation of recombinant glutathione S-transferase-fusion protein SasA, a cyanobacterial HK, with an IC50 value of 9.5 microg/ml.

Identification of lactate dehydrogenase as a mammalian pyrroloquinoline quinone (PQQ)-binding protein.

Pyrroloquinoline quinone (PQQ), a redox-active o-quinone, is an important nutrient involved in numerous physiological and biochemical processes in mammals. Despite such beneficial functions, the underlying molecular mechanisms remain to be established. In the present study, using PQQ-immobilized Sepharose beads as a probe, we examined the presence of protein(s) that are capable of binding PQQ in mouse NIH/3T3 fibroblasts and identified five cellular proteins, including l-lactate dehydrogenase (LDH) A chain, as potential mammalian PQQ-binding proteins. In vitro studies using a purified rabbit muscle LDH show that PQQ inhibits the formation of lactate from pyruvate in the presence of NADH (forward reaction), whereas it enhances the conversion of lactate to pyruvate in the presence of NAD(+) (reverse reaction). The molecular mechanism underlying PQQ-mediated regulation of LDH activity is attributed to the oxidation of NADH to NAD(+) by PQQ. Indeed, the PQQ-bound LDH oxidizes NADH, generating NAD(+), and significantly catalyzes the conversion of lactate to pyruvate. Furthermore, PQQ attenuates cellular lactate release and increases intracellular ATP levels in the NIH/3T3 fibroblasts. Our results suggest that PQQ, modulating LDH activity to facilitate pyruvate formation through its redox-cycling activity, may be involved in the enhanced energy production via mitochondrial TCA cycle and oxidative phosphorylation.

Oxidative Deamination of Serum Albumins by (-)-Epigallocatechin-3-O-Gallate: A Potential Mechanism for the Formation of Innate Antigens by Antioxidants.

(-)-Epigallocatechin-3-O-gallate (EGCG), the most abundant polyphenol in green tea, mediates the oxidative modification of proteins, generating protein carbonyls. However, the underlying molecular mechanism remains unclear. Here we analyzed the EGCG-derived intermediates generated upon incubation with the human serum albumin (HSA) and established that EGCG selectively oxidized the lysine residues via its oxidative deamination activity. In addition, we characterized the EGCG-oxidized proteins and discovered that the EGCG could be an endogenous source of the electrically-transformed proteins that could be recognized by the natural antibodies. When HSA was incubated with EGCG in the phosphate-buffered saline (pH 7.4) at 37°C, the protein carbonylation was associated with the formation of EGCG-derived products, such as the protein-bound EGCG, oxidized EGCG, and aminated EGCG. The aminated EGCG was also detected in the sera from the mice treated with EGCG in vivo. EGCG selectively oxidized lysine residues at the EGCG-binding domains in HSA to generate an oxidatively deaminated product, aminoadipic semialdehyde. In addition, EGCG treatment results in the increased negative charge of the protein due to the oxidative deamination of the lysine residues. More strikingly, the formation of protein carbonyls by EGCG markedly increased its cross-reactivity with the natural IgM antibodies. These findings suggest that many of the beneficial effects of EGCG may be partly attributed to its oxidative deamination activity, generating the oxidized proteins as a target of natural antibodies.

O-linked N-acetylglucosamine is present on the extracellular domain of notch receptors.

Rare types of glycosylation often occur in a domain-specific manner and are involved in specific biological processes. In particular, O-fucose glycans are reported to regulate the functions of EGF domain-containing proteins such as Notch receptors. In the course of mass spectrometric analysis of O-glycans displayed on Drosophila Notch receptors expressed in S2 cells, we found an unusual O-linked N-acetylhexosamine (HexNAc) modification which occurs at a site distinct from those of O-fucose and O-glucose glycosylations. Modification site mapping by mass spectrometry and amino acid substitution studies revealed that O-HexNAc modification occurs on a serine or threonine located between the fifth and sixth cysteines within the EGF domain. This modification occurs simultaneously along with other closely positioned O-glycosylations. This modification was determined to be O-beta-GlcNAc by galactosyltransferase labeling and beta-N-acetyl-hexosaminidase digestion experiments and by immunoblotting with a specific antibody. O-GlcNAc modification occurs at multiple sites on Notch epidermal growth factor repeats. O-GlcNAc modification was also found on the extracellular domain of Delta, a ligand for Notch receptors. Although the O-GlcNAc modification is known to regulate a wide range of cellular processes, the list of known modified proteins has previously been limited to intracellular proteins in animals. Thus, the finding of O-GlcNAc modification in extracellular environments predicts a distinct glycosylation process that might be associated with a novel regulatory mechanism for Notch receptor activity.

Dioctatin A is a strong inhibitor of aflatoxin production by Aspergillus parasiticus.

Dioctatin A (DotA), a metabolite of Streptomyces, is known to be an inhibitor of human dipeptidyl aminopeptidase II. Here, it was found that DotA strongly inhibited aflatoxin production by Aspergillus parasiticus, with an IC50 value of 4.0 microM. The mycelial growth of the fungus was not affected by the addition of DotA at a concentration of 50 microM, but inhibition of conidiation was observed at the same concentration. DotA inhibited production of norsolorinic acid, an early biosynthetic intermediate of aflatoxin, and it strongly reduced the mRNA levels of genes encoding aflatoxin biosynthetic enzymes, and significantly decreased the mRNA level of aflR, which encodes a key regulatory protein for aflatoxin biosynthesis. In addition to these genes, the mRNA level of brlA, which encodes a conidiation-specific transcription factor, was also reduced by the addition of DotA. It was also found that DotA dramatically enhanced kojic acid production by the fungus. Furthermore, DotA inhibited production of sterigmatocystin, which is a toxic aflatoxin biosynthetic intermediate, and it also inhibited conidiation in Aspergillus nidulans. These results indicate that DotA has pleiotropic effects on regulatory mechanisms of fungal secondary metabolite production and differentiation, leading to inhibition of aflatoxin production.

A plant peptide encoded by CLV3 identified by in situ MALDI-TOF MS analysis.

The Arabidopsis CLAVATA3 (CLV3) gene encodes a stem cell-specific protein presumed to be a precursor of a secreted peptide hormone. Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) applied to in situ Arabidopsis tissues determined the structure of a modified 12-amino acid peptide (MCLV3), which was derived from a conserved motif in the CLV3 sequence. Synthetic MCLV3 induced shoot and root meristem consumption as cells differentiated into other organs, displaying the typical phenotype of transgenic plants overexpressing CLV3. These results suggest that the functional peptide of CLV3 is MCLV3.