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1.
The response of 13 European cauliflower cultivars to Verticillium longisporum was evaluated using two greenhouse tests and one in vitro inoculation test. The greenhouse tests involved dipping roots of 3‐week‐old seedlings in a conidial suspension or inoculating the soil of 3‐week‐old seedlings with Verticillium microsclerotia. The in vitro test involved the inoculation of 9‐day‐old seedlings with Verticillium conidia. Useful disease parameters were the area under disease progress curve and plant growth reduction for the greenhouse tests and fresh weight reduction for the in vitro test. Significant correlations were found among the three inoculation methods. Irrespective of the inoculation method used, cultivar ‘Sernio’ was most resistant to V. longisporum, while ‘Minaret’ was the most susceptible cultivar. The pathogen could be re‐isolated from the hypocotyls and from the stem of ‘Minaret’ 4 and 49 days after inoculation respectively, whereas V. longisporum could never be re‐isolated from ‘Sernio’. These results suggest that the more resistant cauliflower cultivar ‘Sernio’ can suppress the ascent and the proliferation of V. longisporum into the plant.  相似文献   

2.
The devastating soilborne fungal pathogen Verticillium longisporum is host specific to members of the family Brassicaceae, including oilseed rape (Brassica napus) as the economically most important crop. The fungus infects through the roots and causes stunting and early senescence of susceptible host plants and a marked decrease in crop yield. We show here that V. longisporum reacts to the presence of B. napus xylem sap with the production of six distinct upregulated and eight downregulated proteins visualized by two-dimensional gel electrophoresis. Identification of 10 proteins by mass spectrometry revealed that all upregulated proteins are involved in oxidative stress response. The V. longisporum catalase peroxidase (VlCPEA) was the most upregulated protein and is encoded by two isogenes, VlcpeA-1 and VlcpeA-2. Both genes are 98% identical, corroborating the diploid or "amphihaploid" status of the fungus. Knock downs of both VlcpeA genes reduced protein expression by 80% and resulted in sensitivity against reactive oxygen species. Whereas saprophytic growth and the initial phase of the plant infection were phenotypically unaffected, the mutants were not able to perform the late phases of disease. We propose that the catalase peroxidase plays a role in protecting the fungus from the oxidative stress generated by the host plant at an advanced phase of the disease.  相似文献   

3.
4.
The resurgence of drug-resistant apicomplexa, in particular Plasmodium falciparum, the most fatal human malarial parasite, has focused attention on the recent discovery of the shikimate pathway in these organisms, as it may provide the urgently required, novel drug targets resulting from the absence of this pathway in mammals. The direction of a parasiticidal drug design programme obviously requires knowledge of the subcellular localization and indeed full characterization of the possible enzyme targets. Here, we report the cloning and characterization of chorismate synthase from P. falciparum and present the first biochemical and immunological studies of an enzyme of the shikimate pathway from an apicomplexan parasite. We show that this chorismate synthase does not possess an intrinsic flavin reductase activity and is therefore monofunctional like the plant and bacterial chorismate synthases. Highest immunological cross-reactivity was found with a plant chorismate synthase. However, in contrast to the plant enzyme, which is located to the plastid, P. falciparum chorismate synthase is found in the parasite cytosol, akin to the fungal enzymes that possess an intrinsic flavin reductase activity (i.e. are bifunctional). Thus, P. falciparum chorismate synthase has a combination of properties that distinguishes it from other described chorismate synthases.  相似文献   

5.
6.
Verticillium wilt of oilseed rape is caused by the host-adapted pathogen Verticillium longisporum comb. nov. With one set of nuclear SSU-rRNA gene primers, a PCR amplification product of ca. 2.5 kb was generated from all isolates of V. longisporum tested (36 from Europe, Japan, and USA), with the exception of two recombinant isolates. On the contrary, all the other phytopathogenic and non-phytopathogenic species of Verticillium tested (18 species, 46 isolates), with the exception of one isolate of V. lecanii and two of Cordyceps sp., generated a product of ca. 1.65 kb. Sequence analysis of the SSU-rRNA gene of two typical isolates of V. longisporum (wild radish, Japan, and oilseed rape, Germany) revealed that this dimorphism was due to the presence of an identical 839-bp intron located in a highly conserved insertion position (nt 1165 of Saccharomyces cerevisiae). The intron sequence was classified as group-I intron on the basis of conserved sequence and secondary structural elements. Primers designed from the 839-bp intron sequence amplified only the V. longisporum. Phylogenetic analysis based on SSU-rDNA sequences showed that V. longisporum was closely related to the genera of other filamentous Ascomycetes with fruiting bodies. Received: 24 August 2000 / Accepted: 25 September 2000  相似文献   

7.
Chorismate synthase is the last enzyme of the common shikimate pathway, which catalyzes the anti-1,4-elimination of the 3-phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate (EPSP) to generate chorismate, a precursor for the biosynthesis of aromatic compounds. Enzyme activity relies on reduced FMN, which is thought to donate an electron transiently to the substrate, facilitating C(3)-O bond breakage. The crystal structure of the enzyme with bound EPSP and the flavin cofactor highlighted two invariant serine residues interacting with a bound water molecule that is close to the C(3)-O of EPSP. In this article we present the results of a mutagenesis study where we replaced the two invariant serine residues at positions 16 and 127 of the Neurospora crassa chorismate synthase with alanine, producing two single-mutant proteins (Ser16Ala and Ser127Ala) and a double-mutant protein (Ser16AlaSer127Ala). The residual activity of the Ser127Ala and Ser16Ala single-mutant proteins was found to be six-fold and 70-fold lower, respectively, than that of the wild-type protein. No residual activity was detected for the Ser16AlaSer127Ala double-mutant protein, and formation of the typical transient intermediate, characteristic for the chorismate synthase-catalysed reaction, was not observed, in contrast to the single-mutant proteins. On the basis of the structure of the enzyme, we propose that Ser16 and Ser127 form part of a proton relay system among the isoalloxazine ring of FMN, histidine 106 and the phosphate group of EPSP that is essential for the formation of the transient intermediate and for substrate turnover.  相似文献   

8.
9.
The shikimate pathway is essential for the biosynthesis of aromatic compounds. The seventh and last step is catalysed by chorismate synthase, which has an absolute requirement for reduced FMN in its active site. There are two classes of this enzyme, which are distinguished according to the origin of the reduced cofactor. Monofunctional chorismate synthases sequester it from the cellular environment whereas bifunctional enzymes can generate reduced FMN at the expense of NADPH. These bifunctional enzymes are found in fungi and the ciliated protozoan Euglena gracilis while all bacterial and plant enzymes are monofunctional. In this study, we introduce an in vivo screen, which is based on a chorismate synthase-deficient Saccharomyces cerevisiae strain, allowing the classification of hitherto uncharacterized chorismate synthases. This analysis revealed that bifunctionality is present in the enzymes of protozoan species. In contrast, all bacterial and plant enzymes tested are monofunctional. In addition, we demonstrate that a monofunctional chorismate synthase confers prototrophy in conjunction with a NADPH : FMN oxidoreductase indicating that bifunctionality is required due to the lack of free reduced FMN in fungal and possibly protozoan species. Interestingly, the distribution of bifunctional chorismate synthase concurs with the presence of a pentafunctional enzyme complex.  相似文献   

10.
Chorismate synthase catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate. The strict requirement for a reduced FMN cofactor and a trans-1,4-elimination are unusual. (6R)-6-Fluoro-EPSP was shown to be converted to chorismate stoichiometrically with enzyme-active sites in the presence of dithionite. This conversion was associated with the oxidation of FMN to give a stable flavin semiquinone. The IC(50) of the fluorinated substrate analogue was 0.5 and 250 microm with the Escherichia coli enzyme, depending on whether it was preincubated with the enzyme or not. The lack of dissociation of the flavin semiquinone and chorismate from the enzyme appears to be the basis of the essentially irreversible inhibition by this analogue. A dithionite-dependent transient formation of flavin semiquinone during turnover of (6S)-6-fluoro-EPSP has been observed. These reactions are best rationalized by radical chemistry that is strongly supportive of a radical mechanism occurring during normal turnover. The lack of activity with 5-deaza-FMN provides additional evidence for the role of flavin in catalysis by the E. coli enzyme.  相似文献   

11.
Chorismate synthase catalyzes the last step in the common shikimate pathway leading to aromatic compounds such as the aromatic amino acids. The reaction consists of the 1,4-anti-elimination of the 3-phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate to yield chorismate. Although this reaction does not involve a net redox change, the enzyme has an absolute requirement for reduced flavin mononucleotide, which is not consumed during the reaction. Two invariant histidine residues are found in the active site of the enzyme: His(17) and His(106). Using site-directed mutagenesis, both histidines were replaced by alanine, reducing the activity 10- and 20-fold in the H106A and H17A mutant protein, respectively. Based on the characterization of the two single mutant proteins, it is proposed that His(106) serves to protonate the monoanionic reduced FMN, whereas His(17) protonates the leaving phosphate group of the substrate. An enzymatic reaction mechanism in keeping with the experimental results is presented.  相似文献   

12.
《Biological Control》2013,66(3):293-301
The yeast Meyerozyma caribbica was evaluated for their effectiveness against Colletotrichum gloeosporioides in the mango (Mangifera indica L.) cv. “Ataulfo” and to identify the possible mechanisms of action involved in the inhibition. M. caribbica showed a high antagonistic potential in vivo, with significant inhibition of 86.7% of anthracnose. M. caribbica competed for the nutrients sucrose and fructose (p < 0.05). Electron microscopy showed that the yeast produces a biofilm adhering to the fruit and to C. gloeosporioides hyphae. M. caribbica showed competition for space and parasitism to the phytopathogen, furthermore it produces hydrolytic enzymes such as chitinase, N-acetyl-β-d-glucosaminidase and β-1, 3-glucanase. These enzymes caused notched and non-lethal deformations on the fungal hyphae through this specific action mechanism. According to the results obtained here, the combination of the different action mechanisms of the yeast increases their ability to control C. gloeosporioides. The use of biological agents to control C. gloeosporioides may contribute to the integrated management of disease caused by this pathogen.  相似文献   

13.
14.
Context: Integrin-linked kinase (ILK), a multidomain focal adhesion protein serine/threonine kinase, plays an essential role in ovarian carcinoma. There are reports that the expression and activity of ILK are increased in ovarian cancer.

Objective: To test the hypothesis that ILK pathway mediates the apoptosis of ovarian carcinoma SKOV3 cell influencing the cell survival, we performed these studies.

Materials and methods: We applied lentivirus transfection, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT), apoptotic proteins expressions assay, and Hoechst to study our hypothesis.

Results: We found that silencing of the ILK increases the cell cytotoxic, growth inhibition, and apoptosis. Moreover, after blocking the activation of ILK with ILK shRNA, up-regulation of pro-apoptotic bax expression and down-regulation of the anti-apoptotic bcl-2 expression were found in ovarian cancer SKOV3 cell line. These were associated with an increasing cleaved caspase-3 activity and chromatin condensation of cell nuclear. Furthermore, the expressions of fas and fas ligand (fasL), belonging to the tumor necrosis factor family and controlling the cell apoptosis, were also enhanced.

Conclusions: Thus, these findings indicate that both the intrinsic pathway and the extrinsic death receptor pathway are involved in the process that silencing of the ILK gene induces the apoptosis in ovarian carcinoma SKOV3 cell.  相似文献   

15.
The initial interaction of a pathogenic fungus with its host is complex and involves numerous metabolic pathways and regulatory proteins. Considerable attention has been devoted to proteins that play a crucial role in these interactions, with an emphasis on so‐called effector molecules that are secreted by the invading microbe to establish the symbiosis. However, the contribution of other types of molecules, such as glycans, is less well appreciated. Here, we present a random genetic screen that enabled us to identify 58 novel candidate genes that are involved in the pathogenic potential of the fungal pathogen Verticillium dahliae, which causes vascular wilt diseases in over 200 dicotyledonous plant species, including economically important crops. One of the candidate genes that was identified concerns a putative biosynthetic gene involved in nucleotide sugar precursor formation, as it encodes a putative nucleotide‐rhamnose synthase/epimerase‐reductase (NRS/ER). This enzyme has homology to bacterial enzymes involved in the biosynthesis of the nucleotide sugar deoxy‐thymidine diphosphate (dTDP)‐rhamnose, a precursor of L‐rhamnose, which has been shown to be required for virulence in several human pathogenic bacteria. Rhamnose is known to be a minor cell wall glycan in fungi and has therefore not been suspected as a crucial molecule in fungal–host interactions. Nevertheless, our study shows that deletion of the VdNRS/ER gene from the V. dahliae genome results in complete loss of pathogenicity on tomato and Nicotiana benthamiana plants, whereas vegetative growth and sporulation are not affected. We demonstrate that VdNRS/ER is a functional enzyme in the biosynthesis of uridine diphosphate (UDP)‐rhamnose, and further analysis has revealed that VdNRS/ER deletion strains are impaired in the colonization of tomato roots. Collectively, our results demonstrate that rhamnose, although only a minor cell wall component, is essential for the pathogenicity of V. dahliae.  相似文献   

16.
PevD1, a novel protein elicitor from the pathogenic cotton verticillium wilt fungus, Verticillium dahliae, induced a hypersensitive response in tobacco plants. In this paper, the elicitor was purified and analyzed using de novo sequencing. The protein-encoding pevD1 gene consists of a 468-bp open reading frame that produces a polypeptide of 155 amino acids, with a theoretical molecular weight of 16.23 kDa. The sequence of elicitor protein PevD1 was matched to the genomic sequence (GenBank accession no. ABJE 01000445.1) of a putative protein from V. dahliae strain vdls.17, but a function had not yet been reported. The pevD1 gene was expressed in Escherichia coli, and the recombinant protein was characterized for its ability to confer systemic acquired resistance to tobacco mosaic virus (TMV). Recombinant PevD1-treated plants exhibited enhanced systemic resistance compared to control, including a significant reduction in the number and size of TMV lesions on tobacco leaves. The elicitor protein-induced hydrogen peroxide production, extracellular-medium alkalization, callose deposition, phenolics metabolism, and lignin synthesis in tobacco. Our results demonstrate that elicitor-PevD1 triggers defense responses in intact tobacco plants.  相似文献   

17.
Chorismate synthase catalyzes the last common step in the biosynthesis of the three aromatic amino acids in microorganisms and plants. We have cloned a cDNA for this enzyme from the higher plant Corydalis sempervirens. This is the first chorismate synthase cDNA from a eukaryotic organism. The nucleotide sequence was determined and the identity of the cDNA was confirmed by the amino acid sequence of tryptic peptides obtained from purified chorismate synthase. The homology to the two known bacterial sequences is about 48%. The cDNA contains an open reading frame of 1341 base pairs, encoding a protein of 447 amino acids. This protein with a molecular mass of 48,100 daltons resembles a chorismate synthase precursor targeted for chloroplast import. Multiple sites of polyadenylation were observed in chorismate synthase mRNAs.  相似文献   

18.
NADPH-dependent flavin reductase (required for the activation of chorismate synthase) was purified to homogeneity from cell-free extracts of Bacillus subtilis. The enzyme has a molecular weight of 13,000 as determined by sodium dodecyl sulfate-gel electrophoresis, is specific for NADPH, and requires a divalent metal ion and either FMN or FAD for maximal rates of NADPH oxidation. The enzyme is able to reduce 2,6-dichlorophenolindophenol (DCIP) in the presence of NADPH and a divalent metal ion. Both catalytic activities were completely inhibited by EDTA. The Km for FMN is 1.25 X 10(-5) M and for NADPH 7.8 X 10(-5) M with oxygen as the final electron acceptor, and 3.85 X 10(-4) M with DCIP as the final electron acceptor. The enzyme was also isolated in association with chorismate synthase and dehydroquinate synthase. The enzyme associated with the complex has the same catalytic properties as the dissociated enzyme except that it requires both a divalent metal ion and FMN for DCIP reduction. Maximal enzyme activity was observed when the enzyme was preincubated with FMN and the divalent metal ion. The enzyme complex is easily dissociable and the dissociation of the enzyme complex resulted in the failure of NADPH-dependent flavin reductase to adsorb to phosphocellulose.  相似文献   

19.
Chorismate synthase, the seventh enzyme in the shikimate pathway, catalyzes the transformation of 5-enolpyruvylshikimate 3-phosphate to chorismate which is the last common precursor in the biosynthesis of numerous aromatic compounds in bacteria, fungi and plants. The enzyme has an absolute requirement for reduced FMN as a cofactor, although the 1,4-anti elimination of phosphate and the C(6proR)-hydrogen does not involve a net redox change. The role of the reduced FMN in catalysis has long been elusive. However, recent detailed kinetic and bioorganic approaches have fundamentally advanced our understanding of the mechanism of action, suggesting an initial electron transfer from tightly bound reduced flavin to the substrate, a process which results in C—O bond cleavage. Studies on chorismate synthases from bacteria, fungi and plants revealed that in these organisms the reduced FMN cofactor is made available in different ways to chorismate synthase: chorismate synthases in fungi – in contrast to those in bacteria and plants – carry a second enzymatic activity which enables them to reduce FMN at the expense of NADPH. Yet, as shown by the analysis of the corresponding genes, all chorismate synthases are derived from a common ancestor. However, several issues revolving around the origin of reduced FMN, as well as the possible regulation of the enzyme activity by means of the availability of reduced FMN, remain poorly understood. This review summarizes recent developments in the biochemical and genetic arena and identifies future aims in this field. Received: 22 June 1998 / Accepted: 7 August 1998  相似文献   

20.
Chorismate mutase catalyzes a key step in the shikimate biosynthetic pathway towards phenylalanine and tyrosine. Curiously, the intracellular chorismate mutase of Mycobacterium tuberculosis (MtCM; Rv0948c) has poor activity and lacks prominent active‐site residues. However, its catalytic efficiency increases >100‐fold on addition of DAHP synthase (MtDS; Rv2178c), another shikimate‐pathway enzyme. The 2.35 Å crystal structure of the MtCM–MtDS complex bound to a transition‐state analogue shows a central core formed by four MtDS subunits sandwiched between two MtCM dimers. Structural comparisons imply catalytic activation to be a consequence of the repositioning of MtCM active‐site residues on binding to MtDS. The mutagenesis of the C‐terminal extrusion of MtCM establishes conserved residues as part of the activation machinery. The chorismate‐mutase activity of the complex, but not of MtCM alone, is inhibited synergistically by phenylalanine and tyrosine. The complex formation thus endows the shikimate pathway of M. tuberculosis with an important regulatory feature. Experimental evidence suggests that such non‐covalent enzyme complexes comprising an AroQδ subclass chorismate mutase like MtCM are abundant in the bacterial order Actinomycetales.  相似文献   

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