Improvement in 5'-position-selective glucosylation of pyridoxine by Verticillium dahliae TPU 4900.
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Optimization of culture and reaction conditions for 5'-position-selective transglucosylation to pyridoxine by Verticillium dahliae TPU 4900 was investigated. V. dahliae TPU 4900 had high transglucosylation activity when grown with soluble starch as a carbon source and organic nitrogens such as Esusan meat as a nitrogen source at 15-20 degrees C. Both the yield of pyridoxine 5'-alpha-D-glucoside (PN-5'-alpha-G) and the 5'-position-selectivity reached a maximum when an intact-cell reaction was done at 50-60 degrees C and pH 7 with additions of dextrin. The transglucosylation activity in culture broth was 71 times with the optimization of culture conditions that under the conditions used for screening. The productivity of PN-5'-alpha-G synthesis was 6.9 times that under the initial conditions when the reaction conditions of intact cells were optimized. From 1000 mM (206 g/L) pyridoxine hydrochloride, PN-5'-alpha-G was synthesized to the concentration of 300 mM (98.4 g/L as PN-5'-alpha-G) with 5'-selectivity of 85% in 53 h by intact cells of V. dahliae TPU 4900. (+info)
New diketopiperazines from the entomopathogenic fungus Verticillium hemipterigenum BCC 1449.
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Two new diketopiperazines, bisdethiodi(methylthio)-1-demethylhyalodendrin and 1-demethylhyalodendrin tetrasulfide, together with two known cyclodepsipeptides, enniatins B and B4, and two known pyrones, pyrenocines A and B, were isolated from a culture broth of the entomopathogenic fungus Verticillium hemipterigenum BCC 1449. These structures were elucidated using spectroscopic methods and X-ray crystallography. Antimalarial and cytotoxic activities of these compounds were evaluated. (+info)
Use of borate to control the 5'-position-selective microbial glucosylation of pyridoxine.
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Nearly 100% 5'-position selectivity of transglucosylation from maltodextrin to pyridoxine (PN) by cells of Verticillium dahliae TPU 4900 was observed when the reaction was carried out with borate. The same effect of borate was observed not only during synthesis of pyridoxine 5'-alpha-D-glucoside by partially purified enzyme of this strain but also during synthesis of this compound by other microorganisms and with other enzymes (alpha-glucosidase and cyclomaltodextrin glucanotransferase). The effect was thought to be caused by the formation of a borate complex with 3- and 4'-position hydroxyl groups of PN. A decrease in the formation of pyridoxine 5'-alpha-D-glucoside was observed in the reaction with borate, but this decrease was overcome by optimizing the pH and increasing the amount of cells in the reaction mixture. (+info)
Antimicrobial activity of a chelatable poly(arginyl-histidine) produced by the ergot fungus Verticillium kibiense.
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We have recently developed a convenient method of screening a broad range of microorganisms that produce epsilon-poly-L-lysine (M. Nishikawa and K. Ogawa, Appl. Environ. Microbiol. 68:3575-3581, 2002). Using this method, we found an ergot fungus that secretes a charged polypeptide other than epsilon-poly-L-lysine. It was identified as a new species on the basis of its 28S rRNA sequence and was named Verticillium kibiense (formerly Epichloe kibiensis). Peptide sequencing and mass spectrometry revealed that the polypeptide is a linear peptide composed of repeated units of arginyl-histidine. The numbers of repeated units were in most cases five and in some cases four or six. This peptide showed activity against a broad range of bacteria and fungi but lost its activity under conditions of high ionic strength. Zinc and copper ions specifically changed the circular dichroism spectra of the peptide and restored the antimicrobial activity from abrogation under high ionic conditions, although these ions had no reinforcing effect on antimicrobial activity when they were added to solutions at a low ionic strength. The peptide labeled with fluorescein was able to permeate the cell membranes of target microbes, but its ability to permeate cell membranes decreased under conditions of high ionic strength. This decreased ability was partially recovered specifically by the addition of zinc and copper ions. These results indicate that poly(arginyl-histidine) is a cationic polypeptide characterized by specific metal binding and resistance to salts. (+info)
A novel ascochlorin glycoside from the insect pathogenic fungus Verticillium hemipterigenum BCC 2370.
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Vertihemipterin A, the ascochlorin glycoside, and its aglycone, 4',5'-dihydro-4'-hydroxyascochlorin, were isolated from the insect pathogenic fungus Verticillium hemipterigenum BCC 2370. A new analog, 8'-hydroxyascochlorin, and five known compounds, ascochlorin, LL-Z1272delta, 8',9'-dehydroascochlorin, ascofuranone and ascofuranol, were also isolated from the same fermentation broth. Structures of these compounds were elucidated by spectroscopic methods. Stereochemistry of the known compound, 4',5'-dihydro-4'-hydroxyascochlorin, was addressed by NMR spectral analyses and the modified Mosher's method. Antiviral (HSV-1) and cytotoxic activities of these ascochlorin analogs were evaluated. (+info)
Characterization of GaWRKY1, a cotton transcription factor that regulates the sesquiterpene synthase gene (+)-delta-cadinene synthase-A.
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The cotton (+)-delta-cadinene synthase (CAD1), a sesquiterpene cyclase, catalyzes a branch-point step leading to biosynthesis of sesquiterpene phytoalexins, including gossypol. CAD1-A is a member of CAD1 gene family, and its promoter contains a W-box palindrome with two reversely oriented TGAC repeats, which are the proposed binding sites of WRKY transcription factors. We isolated several WRKY cDNAs from Gossypium arboreum. One of them, GaWRKY1, encodes a protein containing a single WRKY domain and a putative N-terminal Leu zipper. Similar to genes encoding enzymes of cotton sesquiterpene pathway, GaWRKY1 was down-regulated in a glandless cotton cultivar that contained much less gossypol. GaWRKY1 showed a temporal and spatial pattern of expression comparable to that of CAD1-A in various aerial organs examined, including sepal, stigma, anther, and developing seeds. In suspension cells, expression of both GaWRKY1 and CAD1-A genes and biosynthesis of sesquiterpene aldehydes were strongly induced by a fungal elicitor preparation and methyl jasmonate. GaWRKY1 interacted with the 3x W-box derived from CAD1-A promoter in yeast (Saccharomyces cerevisiae) one-hybrid system and in vitro. Furthermore, in transgenic Arabidopsis plants, overexpression of GaWRKY1 highly activated the CAD1-A promoter, and transient assay in tobacco (Nicotiana tabacum) leaves demonstrated that W-box was required for this activation. These results suggest that GaWRKY1 participates in regulation of sesquiterpene biosynthesis in cotton, and CAD1-A is a target gene of this transcription factor. (+info)
The role of the jasmonate response in plant susceptibility to diverse pathogens with a range of lifestyles.
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Plants defend themselves against attack from insects and pathogens with various resistance strategies. The jasmonate and salicylate signaling pathways are two induced responses that protect plants against these attackers. Knowledge of the range of organisms that are affected by each response is important for understanding how plants coordinate their defenses against multiple attackers and the generality of effect of different resistance mechanisms. The jasmonate response is known to protect plants against a wide range of insect herbivores; in this study, we examined the role of the jasmonate response in susceptibility to eight pathogens with diverse lifestyles in the laboratory and field. Recent biochemical models suggest that the lifestyle of the pathogen (necrotroph versus biotroph) should predict whether the jasmonate response will be involved in resistance. We tested this by examining the susceptibility of wild-type (cv Castlemart with no known genes for resistance to the pathogens used) and jasmonate-deficient mutant tomato (Lycopersicon esculentum) plants (def1) and by employing rescue treatments of the mutant. Plant susceptibility to five of the eight pathogens we examined was reduced by the jasmonate response, including two bacteria (Pseudomonas syringae and Xanthomonas campestris), two fungi (Verticillium dahliae and Fusarium oxysporum f. sp. lycopersici), and an oomycete (Phytophthora infestans). Susceptibility to three fungi was unaffected (Cladosporium fulvum, Oidium neolycopersici, and Septoria lycopersici). Our results indicate that the jasmonate response reduces damage by a wide range of pathogens from different lifestyles, a result that contrasts with the emerging picture of diseases on Arabidopsis. Thus, the generality of jasmonate-based resistance of tomato challenges the view that ecologically distinct plant parasites are resisted via different mechanisms. (+info)
VdNEP, an elicitor from Verticillium dahliae, induces cotton plant wilting.
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Verticillium wilt is a vascular disease of cotton. The causal fungus, Verticillium dahliae, secretes elicitors in culture. We have generated approximately 1,000 5'-terminal expressed sequence tags (ESTs) from a cultured mycelium of V. dahliae. A number of ESTs were found to encode proteins harboring putative signal peptides for secretion, and their cDNAs were isolated. Heterologous expression led to the identification of a protein with elicitor activities. This protein, named V. dahliae necrosis- and ethylene-inducing protein (VdNEP), is composed of 233 amino acids and has high sequence identities with fungal necrosis- and ethylene-inducing proteins. Infiltration of the bacterially expressed His-VdNEP into Nicotiana benthamiana leaves resulted in necrotic lesion formation. In Arabidopsis thaliana, the fusion protein also triggered production of reactive oxygen species and induced the expression of PR genes. When added into suspension cultured cells of cotton (Gossypium arboreum), the fusion protein elicited the biosynthesis of gossypol and related sesquiterpene phytoalexins at low concentrations, and it induced cell death at higher concentrations. On cotton cotyledons and leaves, His-VdNEP induced dehydration and wilting, similar to symptoms caused by a crude preparation of V. dahliae elicitors. Northern blotting showed a low level of VdNEP expression in the mycelium during culture. These data suggest that VdNEP is a wilt-inducing factor and that it participates in cotton-V. dahliae interactions. (+info)