Purpurea Strains Research Articles

Chromosome-level draft genome sequences of three isolates of the toxigenic fungus Claviceps purpurea showing structural rearrangements.

The whole genomes of three Claviceps purpurea strains were sequenced using Oxford Nanopore Technologies' MinION and assembled into complete, chromosome-level assemblies. The C. purpurea genome consists of eight conserved chromosomes, with evidence of inter-chromosomal structural rearrangements between strains.

Evolution of the Ergot Alkaloid Biosynthetic Gene Cluster Results in Divergent Mycotoxin Profiles in Claviceps purpurea Sclerotia.

Research into ergot alkaloid production in major cereal cash crops is crucial for furthering our understanding of the potential toxicological impacts of Claviceps purpurea upon Canadian agriculture and to ensure consumer safety. An untargeted metabolomics approach profiling extracts of C. purpurea sclerotia from four different grain crops separated the C. purpurea strains into two distinct metabolomic classes based on ergot alkaloid content. Variances in C. purpurea alkaloid profiles were correlated to genetic differences within the lpsA gene of the ergot alkaloid biosynthetic gene cluster from previously published genomes and from newly sequenced, long-read genome assemblies of Canadian strains. Based on gene cluster composition and unique polymorphisms, we hypothesize that the alkaloid content of C. purpurea sclerotia is currently undergoing adaptation. The patterns of lpsA gene diversity described in this small subset of Canadian strains provides a remarkable framework for understanding accelerated evolution of ergot alkaloid production in Claviceps purpurea.

Manipulation of cytokinin level in the ergot fungus Claviceps purpurea emphasizes its contribution to virulence.

Pathogen-derived cytokinins (CKs) have been recognized as important virulence factor in several host-pathogen interactions and it was demonstrated multiple times that phytopathogenic fungi form CKs via the tRNA degradation pathway. In contrast to previous studies, the focus of this study is on the second step of CK formation and CK degradation to improve our understanding of the biosynthesis in fungi on the one hand, and to understand CK contribution to the infection process of Claviceps purpurea on the other hand. The ergot fungus Claviceps purpurea is a biotrophic phytopathogen with a broad host range including economically important crops causing harvest intoxication upon infection. Its infection process is restricted to unfertilized ovaries without causing macroscopic defense symptoms. Thus, sophisticated host manipulation strategies are implicated. The cytokinin (CK) plant hormones are known to regulate diverse plant cell processes, and several plant pathogens alter CK levels during infection. C. purpurea synthesizes CKs via two mechanisms, and fungus-derived CKs influence the host-pathogen interaction but not fungus itself. CK deficiency in fungi with impact on virulence has only been achieved to date by deletion of a tRNA-ipt gene that is also involved in a process of translation regulation. To obtain a better understanding of CK biosynthesis and CKs' contribution to the plant-fungus interaction, we applied multiple approaches to generate strains with altered or depleted CK content. The first approach is based on deletion of the two CK phosphoribohydrolase (LOG)-encoding genes, which are believed to be essential for the release of active CKs. Single and double deletion strains were able to produce all types of CKs. Apparently, log gene products are dispensable for the formation of CKs and so alternative activation pathways must be present. The CK biosynthesis pathway remains unaffected in the second approach, because it is based on heterologous overexpression of CK-degrading enzymes from maize (ZmCKX1). Zmckx1 overexpressing C. purpurea strains shows strong CKX activity and drastically reduced CK levels. The strains are impaired in virulence, which reinforces the assumption that fungal-derived CKs are crucial for full virulence. Taken together, this study comprises the first analysis of a log depletion mutant that proved the presence of alternative cytokinin activation pathways in fungi and showed that heterologous CKX expression is a suitable approach for CK level reduction.

The Epipolythiodiketopiperazine Gene Cluster in Claviceps purpurea: Dysfunctional Cytochrome P450 Enzyme Prevents Formation of the Previously Unknown Clapurines.

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.

Ergot cluster-encoded catalase is required for synthesis of chanoclavine-I in Aspergillus fumigatus

Genes required for ergot alkaloid biosynthesis are clustered in the genomes of several fungi. Several conserved ergot cluster genes have been hypothesized, and in some cases demonstrated, to encode early steps of the pathway shared among fungi that ultimately make different ergot alkaloid end products. The deduced amino acid sequence of one of these conserved genes (easC) indicates a catalase as the product, but a role for a catalase in the ergot alkaloid pathway has not been established. We disrupted easC of Aspergillus fumigatus by homologous recombination with a truncated copy of that gene. The resulting mutant (ΔeasC) failed to produce the ergot alkaloids typically observed in A. fumigatus, including chanoclavine-I, festuclavine, and fumigaclavines B, A, and C. The ΔeasC mutant instead accumulated N-methyl-4-dimethylallyltryptophan (N-Me-DMAT), an intermediate recently shown to accumulate in Claviceps purpurea strains mutated at ccsA (called easE in A. fumigatus) (Lorenz et al. Appl Environ Microbiol 76:1822-1830, 2010). A ΔeasE disruption mutant of A. fumigatus also failed to accumulate chanoclavine-I and downstream ergot alkaloids and, instead, accumulated N-Me-DMAT. Feeding chanoclavine-I to the ΔeasC mutant restored ergot alkaloid production. Complementation of either ΔeasC or ΔeasE mutants with the respective wild-type allele also restored ergot alkaloid production. The easC gene was expressed in Escherichia coli, and the protein product displayed in vitro catalase activity with H(2)O(2) but did not act, in isolation, on N-Me-DMAT as substrate. The data indicate that the products of both easC (catalase) and easE (FAD-dependent oxidoreductase) are required for conversion of N-Me-DMAT to chanoclavine-I.

Variation in Karyotype and Ploidy Level Among Field Isolates of Claviceps purpurea

Karyotype analysis by pulsed‐field gel electrophoresis of several field isolates of Claviceps purpurea showed extensive variation in size and number of chromosomes. Analysis of F1‐ascospore‐lines of a cross between two field isolates showing chromosome‐length polymorphisms (CLPs) indicate that recombination during meiosis contributes considerably to this variability. In addition, cytofluorometrical estimation of DNA content of single nuclei indicate that the C. purpurea strains vary in their ploidy level: most strains probably are ± diploid, some appear to be haploid, others ± triploid (with varying degrees of aneuploidy). Benomyl treatment (as used, e.g. for ‘haploidization’ in the parasexual cycle of imperfect fungi) of some field isolates confirmed that the differences in 4′‐6‐diamidino‐2‐phenylindol (DAPI)fluorescence correspond to different ploidy levels: derivatives with lower nuclear DNA content could be isolated from the putatively nonhaploid strains; a minimal value (even after repeated benomyl treatment) was obtained, corresponding to the lowest value of field isolates, probably representing the haploid status. Most of these strains are not impaired in fitness and aggressiveness and therefore can be used for the selection of recessive mutants and for functional molecular studies.

D-Lysergyl Peptide Synthetase from the Ergot Fungus Claviceps purpurea

The ergot fungus Claviceps purpurea produces the medically important ergopeptines, which consist of a cyclol-structured tripeptide and D-lysergic acid linked by an amide bond. An enzyme activity capable of non-ribosomal synthesis of D-lysergyl-L-alanyl-L-phenylalanyl-L-proline lactam, the non-cyclol precursor of the ergopeptine ergotamine, has been purified about 18-fold from the ergotamine-producing C. purpurea strain D1. Analysis of radioactively labeled enzyme-substrate complexes revealed a 370-kDa lysergyl peptide synthetase 1 (LPS 1) carrying the amino acid activation domains for alanine, phenylalanine, and proline. The activation of D-lysergic acid is catalyzed by a 140-kDa peptide synthetase (LPS 2) copurifying with LPS 1. LPS 1 and LPS 2 contain 4'-phosphopantetheine and bind their substrates covalently by thioester linkage. Kinetic analysis of the synthesis reaction revealed a Km of approximately 1.4 microM for both D-lysergic acid and its structural homolog dihydrolysergic acid, which is one to two orders of magnitude lower than the Km values for the other amino acids involved. The Km values for the amino acids reflect their relative concentrations in the cellular pool of C. purpurea. This may indicate that in in vivo conditions D-lysergyl peptide formation is limited by the D-lysergic acid concentration in the cell. In vitro, the multienzyme preparation catalyzes the formation of several different D-lysergyl peptide lactams according to the amino acids supplied. Specific antiserum was used to detect LPS 1 in various C. purpurea strains. In C. purpurea wild type, the enzyme was expressed at all stages of cultivation and in different media, suggesting that it is produced constitutively.

Strain improvement of Claviceps purpurea by protoplast fusion without introducing auxotrophic markers

Protoplasts of morphologically and biochemically different Claviceps purpurea strains producing ergotoxins were fused without introducing selective auxotrophic markers. Fused strains thus obtained differed significantly in biosynthetic activity and morphology from the prototrophic isolates obtained after fusion of the same parent strains marked by auxotrophy. Comparison of the two types of fused strains showed about tenfold higher alkaloid production in fusants obtained from prototrophic strains. Selected stable prototrophic isolates also showed a significant productivity improvement in comparison with the original parent strains.

Activation of ergot alkaloid biosynthesis in prototrophic isolates by Claviceps purpurea protoplast fusion

Intraspecific protoplast fusions were carried out with active ergocornine-ergokryptine and inactive ergocristine Claviceps purpurea strains and vice versa. The isolated prototrophic strains from both types of crossings produced all three alkaloid types, showing that biosynthesis of distinct alkaloid was activated in an inactive partner strain. The prototrophic isolates were stable on minimal medium but they segregated by subculturing on complete medium. In comparison with the original partner strains, differences in morphological and cytological characteristics were also established.

Parasitic interactions between Claviceps purpurea strains in wheat and an acute necrotic host response

Concurrent inoculation of wheat florets with pairs of distinctive strains of Claviceps purpurea gave ergot sclerotia in which both strains co-existed as indicated by their alkaloid content, the ability to produce the sexual stage and sclerotial morphology. Production of heterogeneous sclerotia may facilitate hybridization of strains during subsequent ascogeny. A strain notable for its parasitic vigour and failure to biosynthesize ergot alkaloids displaced another strain when the second inoculum was introduced around infected ovaries 7 days after initial inoculation. The necrosis of affected spikelets which occurred in response to the interparasitic competition is recognized as a novel phenomenon in ergot-host relationships.

Acetohydroxyacid synthase from Claviceps purpurea: Partial purification and characterization

AbstractAn acetohydroxyacid synthase (EC 4.1.3.18) which synthesizes α‐acetolactate from pyruvate has been isolated from two different Claviceps purpurea strains. A purification of about 142‐fold was achieved by ammonium sulfate fractionation and the use of Sepharose 6B and DEAE‐Sepharose CL‐6B columns. The purified enzyme requires thiamine pyrophosphate and a divalent metal ion (Mn2 + or Mg2 +) for maximum activity but no FAD. The optimum pH is about 6.0 and the optimum temperature is 40 °C. The enzyme is not inhibited by branched‐chain amino acids neither singly nor in combination. AHAS is strongly inhibited by p‐chloromercuribenzoate and N‐ethylmaleimide. The apparent Km values for pyruvate and TPP are 1.7 × 10−2 M and 1.2 × 10−6 M, respectively.

Physiological control and process kinetics of clavine alkaloid production byClaviceps purpurea

Kinetic parameters of production of clavine alkaloids were evaluated in twoClaviceps purpurea strains. Mutagenesis brought about enhanced resistance of the biosynthetic system towards alkaloids. Addition of glucose into the fermentation medium altered the zero order kinetics of production to activation-inhibition kinetics. The glucose treatment allowed performance of both “elymoclavine-inhibitionless” and “clavine alkaloid-decompositionless” fermentations if a combination of fermentation and separation units in a closed loop was used.

Effect of clomiphene on fatty acids, sterols and membrane fluidity in clavine producing claviceps purpurea strains

Clomiphene depressed the growth and enhanced clavine production of Claviceps purpurea strains 129,35 and 59. Mycelial content of 18:2 and 16:0 fatty acids decreased, whereas that of 18:1 and 18:0 acids increased. In the mutant strain 59 clomiphene, triadimefon and ergosterol stimulated the impaired function of chanoclavine cyclase. Their effect was counteracted by plant oil. Clomiphene decreased the content of total lipids (44%), triglycerides (32%), sterols (22%) and sterol/phospholipid molar ratio. The PC/PE ratio was 9X increased. Clomiphene and triadimefon enhanced membrane fluidity of protoplasts, ergosterol and oil reverted their effect.

Ultrastructural characteristics of Claviceps purpurea seed cultures

AbstractUltrastructural characteristics of optimally developed seed cultures of two morphologically and biochemically different Claviceps purpurea strains were established. The vegetative mycelium of both conidial (L‐17) and mycelial (L‐18) strains was filamentous and consisted of cells with a polynuclear state. Differences between the strains were more expressed in the ultrastructure of the cytoplasm which contained different and numerically more organelles in the strain L‐17 in comparison with the strain L‐18. This conidial strain developed from a parasitic strain retained in submerged seed cultures the ultrastructural characteristics of sphacelia. Seed cultures of both strains were also characterized by large mitochondria with the dense matrix and numerous cristae, which usually did not appeared before the alkaloid production.

Developmental studies of Claviceps paspali seed cultures for the submerged production of lysergic acid derivatives

Metabolic pattern of mycelial Claviceps paspali seed cultures during the submerged cultivation was established. By comparing it with conidial and mycelial Claviceps purpurea strains it was found that the biosynthesis of RNA, DNA, and proteins followed a similar course in all Claviceps strains, so the fall of RNA content in mycelium may be considered a general biochemical indicator for optimally developed inoculum. But, two different patterns of carbohydrate and lipid metabolism were observed one for conidial and one for mycelial strains.

Influence of tryptophan and related compounds on ergot alkaloid formation in Claviceps purpurea (FR.) Tul.

L-Tryptophan did not exert any influence on peptide alkaloid formation in an ergotamine and in an ergosine-accumulating C. purpurea strain. A different picture was observed in a series of related C. purpurea strains. Tryptophan showed a slight stimulatory effect on the ergotoxine producer Pepty 695/S. A blocked mutant of it, designated as Pepty 695/ch which was able to accumulate secoclavines gave similar results. In a high-yielding elymoclavine strain Pepty 695/e, the progeny of the former one, tryptophan up to a concentration of 25 mM stimulated remarkably clavine biosynthesis. Furthermore, tryptophan could overcome the block of synthesis by inorganic phosphate. Increased specific activities of chanoclavine cyclase but not DMAT synthetase were observed in cultures of strain Pepty 695/e supplemented with tryptophan. 5-Methyltryptophan and bioisosteres of tryptophan were ineffective in alkaloid stimulation. These results are compared with those obtained with the grass ergot strain SD 58 and discussed with the relation to other induction phenomena.

Influence of tryptophan and related compounds on ergot alkaloid formation in Claviceps purpurea (Fr.) Tul.

AbstractL‐Tryptophan did not exert any influence on peptide alkaloid formation in an ergotamine and in an ergosine‐accumulating C. purpurea strain. A different picture was observed in a series of related C. purpurea strains. Tryptophan showed a slight stimulatory effect on the ergotoxine producer Pepty 695/S. A blocked mutant of it, designated as Pepty 695/ch which was able to accumulate secoclavines gave similar results. In a high‐yielding elymoclavine strain Pepty 695/e, the progeny of the former one, tryptophan up to a concentration of 25 mm stimulated remarkably clavine biosynthesis. Furthermore, tryptophan could overcome the block of synthesis by inorganic phosphate. Increased specific activities of chanoclavine cyclase but not DMAT synthetase were observed in cultures of strain Pepty 695/e supplemented with tryptophan. 5‐Methyltryptophan and bioisosteres of tryptophan were ineffective in alkaloid stimulation. These results are compared with those obtained with the grass ergot strain SD 58 and discussed with the relation to other induction phenomena.

Biosynthesis of Ergot Alkaloids by Protoplasts of Various Claviceps purpurea Strains1

Protoplasts of various peptide type alkaloids producing Claviceps purpurea strains were prepared. In a sucrose ammonium citrate medium suspended protoplasts were able to synthesize de novo ergosine, ergotomine and ergotoxine using 14C-labelled amino acids as precursors. The incorporation rate of the amino acids was remarkably increased if the incubation mixture was supplemented with d-lysergic acid.

Über die chemische Zusammensetzung der Zellwand von Claviceps-Arten

Summary Hyphal walls were isolated from Claviceps purpurea strains (alkaloid producing; alkaloid non-producing) and Claviceps fusiformis. The fungi were cultivated under submerged conditions in different culture media. The main neutral sugars were glucose, mannose and galactose. The principal sugar accounting for more than half of this particular fraction was glucose. The amino sugar fraction predominantly glucosamine varied in different strains, e.g. in the ergotoxine producer Pepty 695 between 20–37%. The content of the uronic acid fraction amounted to about 5–8% of the wall. Lipids comprise a relative high amount of wall material depending on the strain. The main fatty acids are lauric acid, myristic acid and palmitic acid. Claviceps cell wall material was found to have about 5–10% protein. Protein hydrolysis gave 15 amino acids, predominantly threonine, serine, aspartic and glutamic acid.

Variability of Requirements to Fermentation Media III Claviceps purpurea Strains

The alkaloid-synthesizing activity of the saprophytic Claviceps purpurea strains was variable. This fact was partly due to different requirements of the cultures to nutritional conditions. Not only single strains, but also formation of intra- and extracellular alkaloids differed in this respect. In case of extracellular alkaloids, the influence of medium was more expressive. A positive correlation between accumulation of cell polysaccharides and the level of intracellular alkaloids was evident. Constant or decreasing cell proteins were favourable for alkaloid formation.