D-lysergic acid-activating enzyme from the ergot fungus Claviceps purpurea.
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ABSTRACT:
SUBMITTER: Keller U
PROVIDER: S-EPMC1153415 | biostudies-other | 1984 Mar
REPOSITORIES: biostudies-other
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Project description:The ergot fungus Claviceps purpurea produces both ergopeptines and simple d-lysergic acid alkylamides. In the ergopeptines, such as ergotamine, d-lysergic acid is linked to a bicyclic tripeptide in amide-like fashion, whereas in the d-lysergylalkanolamides it is linked to an amino alcohol derived from alanine. We show here that these compound classes are synthesized by a set of three non-ribosomal lysergyl peptide synthetases (LPSs), which interact in a combinatorial fashion for synthesis of the relevant product. The trimodular LPS1 assembles with LPS2, the d-lysergic acid recruiting module, to synthesize the d-lysergyltripeptide precursors of ergopeptines from d-lysergic acid and the three amino acids of the peptide chain. Alternatively, LPS2 can assemble with a distinct monomodular non-ribosomal peptide synthetase (NRPS) subunit (ergometrine synthetase) to synthesize the d-lysergic acid alkanolamide ergometrine from d-lysergic acid and alanine. The synthesis proceeds via covalently bound d-lysergyl alanine and release of dipeptide as alcohol with consumption of NADPH. Enzymatic and immunochemical analyses showed that ergometrine synthetase is most probably the enzyme LPS3 whose gene had been identified previously as part of the ergot alkaloid biosynthesis gene cluster in C. purpurea. Inspections of all LPS sequences showed no recognizable peptide linkers for their protein-protein interactions as in NRPS subunits of bacteria. Instead, they all carry conserved N-terminal domains (C0-domains) with similarity to the C-terminal halves of NRPS condensation domains pointing to an alternative mechanism of subunit-subunit interactions in fungal NRPS systems. Phylogenetic analysis of LPS modules and the C0-domains suggests that these enzyme systems most probably evolved by module duplications and rearrangements from a bimodular ancestor.
| S-EPMC2652330 | biostudies-literature
Project description:To investigate its susceptibility to ergot infection, we inoculated Brachypodium distachyon with Claviceps purpurea and compared the infection symptoms with those on rye (Secale cereale). We showed that, after inoculation of Brachypodium with Claviceps, the same disease symptoms occurred in comparable temporal and spatial patterns to those on rye. The infection rate of Claviceps on this host was reduced compared with rye, but the disease could be surveyed by fungal genomic DNA quantification. Mutants of Claviceps which were virulence attenuated on rye were also affected on Brachypodium. We were able to show that pathogenesis-related gene expression changed in a typical manner for biotrophic pathogen attack. Our results indicated that the Claviceps-Brachypodium interaction was dependent on salicylic acid, cytokinin and auxin. We consider Brachypodium to be a suitable and useful alternative host; the increased sensitivity compared with rye will be valuable for the identification of infection mechanisms. Future progess in understanding the Claviceps-plant interaction will be facilitated by the use of a well-characterized model host system.
| S-EPMC6638158 | biostudies-literature
Project description:BACKGROUND:Claviceps purpurea is a phytopathogenic fungus infecting a broad range of grasses including economically important cereal crop plants. The infection cycle ends with the formation of the typical purple-black pigmented sclerotia containing the toxic ergot alkaloids. Besides these ergot alkaloids little is known about the secondary metabolism of the fungus. Red anthraquinone derivatives and yellow xanthone dimers (ergochromes) have been isolated from sclerotia and described as ergot pigments, but the corresponding gene cluster has remained unknown. Fungal pigments gain increasing interest for example as environmentally friendly alternatives to existing dyes. Furthermore, several pigments show biological activities and may have some pharmaceutical value. RESULTS:This study identified the gene cluster responsible for the synthesis of the ergot pigments. Overexpression of the cluster-specific transcription factor led to activation of the gene cluster and to the production of several known ergot pigments. Knock out of the cluster key enzyme, a nonreducing polyketide synthase, clearly showed that this cluster is responsible for the production of red anthraquinones as well as yellow ergochromes. Furthermore, a tentative biosynthetic pathway for the ergot pigments is proposed. By changing the culture conditions, pigment production was activated in axenic culture so that high concentration of phosphate and low concentration of sucrose induced pigment syntheses. CONCLUSIONS:This is the first functional analysis of a secondary metabolite gene cluster in the ergot fungus besides that for the classical ergot alkaloids. We demonstrated that this gene cluster is responsible for the typical purple-black color of the ergot sclerotia and showed that the red and yellow ergot pigments are products of the same biosynthetic pathway. Activation of the gene cluster in axenic culture opened up new possibilities for biotechnological applications like the dye production or the development of new pharmaceuticals.
| S-EPMC5611617 | biostudies-literature
Project description:The role of reactive oxygen species (ROS) in interactions between phytopathogenic fungi and their hosts is well established. An oxidative burst mainly caused by superoxide formation by membrane-associated NADPH oxidases is an essential element of plant defence reactions. Apart from primary effects, ROS play a major role as a second messenger in host response. Recently, NADPH oxidase (nox)-encoding genes have been identified in filamentous fungi. Functional analyses have shown that these fungal enzymes are involved in sexual differentiation, and there is growing evidence that they also affect developmental programmes involved in fungus-plant interactions. Here we show that in the biotrophic plant pathogen Claviceps purpurea deletion of the cpnox1 gene, probably encoding an NADPH oxidase, has impact on germination of conidia and pathogenicity: Deltacpnox1 mutants can penetrate the host epidermis, but they are impaired in colonization of the plant ovarian tissue. In the few cases where macroscopic signs of infection (honeydew) appear, they are extremely delayed and fully developed sclerotia have never been observed. C. purpurea Nox1 is important for the interaction with its host, probably by directly affecting pathogenic differentiation of the fungus.
| S-EPMC6640299 | biostudies-literature
Project description:Evidence suggests that sucrose is the main carbon source for growth of Claviceps spp. in the parasitic condition. The sucrose acts as substrate for an active beta-fructofuranosidase, produced by the fungus, which in the first instance converts the disaccharide into glucose and an oligofructoside. In this way, 50% of the glucose, supplied as sucrose, is made available to the parasite for assimilation. Subsequent action of the enzyme on both sucrose and the oligofructoside leads to the release of more glucose and the formation of additional oligosaccharides. The structures of the main oligosaccharides formed have been elucidated and the interactions of each compound studied. In experiments with purified enzyme in vitro the interaction of the oligosaccharides is rapid but in culture they are assimilated only slowly; in each case some free fructose is liberated. Free fructose is not assimilated in the presence of glucose and, further, inhibits growth at concentrations which might be expected to occur in the parasitic condition. A dual role has been suggested for the enzyme, with sucrose as substrate, in which glucose is made available to the growing parasite, while at the same time transfer of the fructose to form oligosaccharides prevents it from accumulating at inhibitory concentrations. Ultimately, when glucose becomes limiting, the fungus will adapt to fructose assimilation.
| S-EPMC1174076 | biostudies-other
Project description:We studied genetic variability of 100 isolates of Claviceps purpurea by using randomly amplified polymorphic DNA (RAPD), an EcoRI restriction site polymorphism in the 5.8S ribosomal DNA (rDNA), the alkaloids produced, and conidial morphology. We identified three groups: (i) group G1 from fields and open meadows (57 isolates), (ii) group G2 from shady or wet habitats (41 isolates), and (iii) group G3 from Spartina anglica from salt marshes (2 isolates). The sclerotia of G1 isolates contained ergotamines and ergotoxines; G2 isolates produced ergosine and ergocristine along with small amounts of ergocryptine; and G3 isolates produced ergocristine and ergocryptine. The conidia of G1 isolates were 5 to 8 microm long, the conidia of G2 isolates were 7 to 10 microm long, and the conidia of G3 isolates were 10 to 12 microm long. Sclerotia of the G2 and G3 isolates floated on water. In the 5.8S rDNA analysis, an EcoRI site was found in G1 and G3 isolates but not in G2 isolates. The host preferences of the groups were not absolute, and there were host genera that were common to both G1 and G2; the presence of members of different groups in the same locality was rare. Without the use of RAPD or rDNA polymorphism, it was not possible to distinguish the three groups solely on the basis of phenotype, host, or habitat. In general, populations of C. purpurea are not host specialized, as previously assumed, but they are habitat specialized, and collecting strategies and toxin risk assessments should be changed to reflect this paradigm shift.
| S-EPMC92477 | biostudies-literature
Project description:SUMMARY The ascomycete Claviceps purpurea (ergot) is a biotrophic flower pathogen of rye and other grasses. The deleterious toxic effects of infected rye seeds on humans and grazing animals have been known since the Middle Ages. To gain further insight into the molecular basis of this disease, we generated about 10 000 expressed sequence tags (ESTs)-about 25% originating from axenic fungal culture and about 75% from tissues collected 6-20 days after infection of rye spikes. The pattern of axenic vs. in planta gene expression was compared. About 200 putative plant genes were identified within the in planta library. A high percentage of these were predicted to function in plant defence against the ergot fungus and other pathogens, for example pathogenesis-related proteins. Potential fungal pathogenicity and virulence genes were found via comparison with the pathogen-host interaction database (PHI-base; http://www.phi-base.org) and with genes known to be highly expressed in the haustoria of the bean rust fungus. Comparative analysis of Claviceps and two other fungal flower pathogens (necrotrophic Fusarium graminearum and biotrophic Ustilago maydis) highlighted similarities and differences in their lifestyles, for example all three fungi have signalling components and cell wall-degrading enzymes in their arsenal. In summary, the analysis of axenic and in planta ESTs yielded a collection of candidate genes to be evaluated for functional roles in this plant-microbe interaction.
| S-EPMC6640482 | biostudies-literature
Project description:The ergot diseases of agricultural and nonagricultural grasses are caused by the infection of Claviceps spp. (Hypocreales, Ascomycota) on florets, producing dark spur-like sclerotia on spikes that are toxic to humans and animals, leading to detrimental impacts on agriculture and economy due to the downgrading of cereal grains, import-export barriers, reduced yield, and ecological concerns. At least seven phylogenetic lineages (phylogenetic species) were identified within the premolecular concept of C. purpurea s.l. (sensu lato) in agricultural areas and vicinities in Canada and the Western United States. Claviceps purpurea s.s (sensu stricto) remained as the most prevalent species with a wide host range, including cereal crops, native, invasive, and weedy grasses. The knowledge on genetic diversity and distribution of C. purpurea s.s. in North America is lacking. The objective of the present study was to shed light on genetic differentiation and evolution of the natural populations of C. purpurea s.s. Multilocus DNA sequences of samples from Canada and the Western USA were analyzed using a phylogenetic network approach, and population demographic parameters were investigated. Results showed that three distinct genetically subdivided populations exist, and the subdivision is not correlated with geographic or host differentiations. Potential intrinsic mechanisms that might play roles in leading to the cessation of gene flows among the subpopulations, that is, mating and/or vegetative incompatibility, genomic adaptation, were discussed. The neutrality of two house-keeping genes that are widely used for DNA barcoding, that is, translation elongation factor 1-α (TEF1-α) and RNA polymerase II second largest subunit (RPB2), was challenged and discussed.
| S-EPMC7790621 | biostudies-literature
Project description:High-yielding strains of Claviceps purpurea (Fr.) Tul, grown on a defined medium, have been used for a study of the biosynthesis of the peptide ergot alkaloid, ergotamine. l-[U-(14)C]tryptophan, dl-[2-(14)C]mevalonic acid lactone, sodium [2-(14)C]acetate, sodium [(14)C]formate and the methyl group of l-[methyl-(14)C]methionine were efficiently incorporated into the peptide alkaloids and specifically labelled the ergoline moiety of ergotamine. These results are the same as previously found for the biosynthesis of other ergot alkaloids. Time-course incubation experiments demonstrated that l-[U-(14)C]phenylalanine, l-[U-(14)C]proline and l-[U-(14)C]alanine were incorporated into the peptide ergot alkaloids. Chemical degradation of the radioactive alkaloid derived from additional precursor incubation experiments showed that phenylalanine and proline function as the most efficient precursors, and specifically label the constitutive side-chain phenylalanyl and prolyl moieties of the alkaloid. The evidence obtained from l-[U-(14)C]alanine-incorporation experiments was inconclusive. However, degradation of ergotamine isolated after incubation with dl-[1-(14)C]alanine, showed that the carboxyl group of the labelled amino acid was specifically incorporated into the alpha-hydroxy-alpha-amino acid residue of the alkaloid. This, in conjunction with the l-[U-(14)C]alanine-incorporation results, showed conclusively that all three carbon atoms of alanine were incorporated as a biosynthetic unit into the alpha-hydroxy-alpha-amino acid moiety of ergotamine.
| S-EPMC1177782 | biostudies-other
Project description:Claviceps purpurea is an important food contaminant and well known for the production of the toxic ergot alkaloids. Apart from that, little is known about its secondary metabolism and not all toxic substances going along with the food contamination with Claviceps are known yet. We explored the metabolite profile of a gene cluster in C. purpurea with a high homology to gene clusters, which are responsible for the formation of epipolythiodiketopiperazine (ETP) toxins in other fungi. By overexpressing the transcription factor, we were able to activate the cluster in the standard C. purpurea strain 20.1. Although all necessary genes for the formation of the characteristic disulfide bridge were expressed in the overexpression mutants, the fungus did not produce any ETPs. Isolation of pathway intermediates showed that the common biosynthetic pathway stops after the first steps. Our results demonstrate that hydroxylation of the diketopiperazine backbone is the critical step during the ETP biosynthesis. Due to a dysfunctional enzyme, the fungus is not able to produce toxic ETPs. Instead, the pathway end-products are new unusual metabolites with a unique nitrogen-sulfur bond. By heterologous expression of the Leptosphaeria maculans cytochrome P450 encoding gene sirC, we were able to identify the end-products of the ETP cluster in C. purpurea. The thioclapurines are so far unknown ETPs, which might contribute to the toxicity of other C. purpurea strains with a potentially intact ETP cluster.
| S-EPMC4938161 | biostudies-literature