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Claviceps paspali is used in the pharmaceutical industry for the production of ergot alkaloids. This fungus also biosynthesizes paspalitrems, indole diterpene (IDT) mycotoxins that cause significant economic losses in agriculture and represent safety concerns for ergot alkaloid manufacture. Here, we use Agrobacterium-mediated transformation to replace the idtP and the idtF genes in the IDT biosynthetic gene cluster of C. paspali with a selectable marker gene. We show that the ΔidtP knockout mutant produces paspaline, the first IDT intermediate of the pathway. The ΔidtF strain produces unprenylated IDTs such as paspalinine and paspaline. These experiments validate the function of idtP as the gene encoding the cytochrome P450 monooxygenase that oxidizes and demethylates paspaline to produce 13-desoxypaxilline, and that of idtF as the gene that encodes the α-prenyltransferase that prenylates paspalinine at the C20 or the C21 positions to yield paspalitrems A and C, respectively. In addition, we also show that axenic cultures of the wild type, the ΔidtP and the ΔidtF mutant C. paspali strains fail to produce an assembly of IDTs that are present in C. paspali-Paspalum spp. associations.

Claviceps purpurea bifunctional Δ12-hydroxylase/desaturase, CpFAH12, and monofunctional desaturase CpFAD2, share 86% of sequence identity. To identify the underlying determinants of the hydroxylation/desaturation specificity, chimeras of these two enzymes were tested for their fatty acid production in an engineered Yarrowia lipolytica strain. It reveals that transmembrane helices are not involved in the hydroxylation/desaturation specificity whereas all cytosolic domains have an impact on it. Especially, replacing the CpFAH12 cytosolic part near the second histidine-box by the corresponding CpFAD2 part annihilates all hydroxylation activity. Further mutagenesis experiments within this domain identified isoleucine 198 as the crucial element for the hydroxylation activity of CpFAH12. Monofunctional variants performing the only desaturation were obtained when this position was exchanged by the threonine of CpFAD2. Saturation mutagenesis at this position showed modulation in the hydroxylation/desaturation specificity in the different variants. The WT enzyme was demonstrated as the most efficient for ricinoleic acid production and some variants showed a better desaturation activity. A model based on the recently discovered membrane desaturase structures indicate that these changes in specificity are more likely due to modifications in the di-iron center geometry rather than changes in the substrate binding mode., (© 2019 The Authors Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.)

In plants, cytokinins (CKs) are synthesized de novo or by the degradation of modified tRNAs. Recently, the first fungal de novo pathway was identified within the plant pathogen Claviceps purpurea. As the deletion of the de novo pathway did not lead to a complete loss of CKs, this work focuses on the tRNA-modifying protein tRNA-isopentenyltransferase (CptRNA-IPT). The contribution of this enzyme to the CK pool of Claviceps and the role of CKs in the host-pathogen interaction are emphasized. The effects of the deletion of cptRNA-ipt and the double deletion of cptRNA-ipt and the key gene of de novo biosynthesis cpipt-log on growth, CK biosynthesis and virulence were analyzed. In addition, the sites of action of CptRNA-IPT were visualized using reporter gene fusions. In addition to CK-independent functions, CptRNA-IPT was essential for the biosynthesis of cis-zeatin (cZ) and contributed to the formation of isopentenyladenine (iP) and trans-zeatin (tZ). Although ΔcptRNA-ipt was reduced in virulence, the 'CK-free' double deletion mutant was nearly apathogenic. The results prove a redundancy of the CK biosynthesis pathway in C. purpurea for iP and tZ formation. Moreover, we show, for the first time, that CKs are required for the successful establishment of a host-fungus interaction., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

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.

Ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid) has many specialized uses in bioproduct industries, while castor bean is currently the only commercial source for the fatty acid. This report describes metabolic engineering of a microbial system (Pichia pastoris) to produce ricinoleic acid using a "push" (synthesis) and "pull" (assembly) strategy. CpFAH, a fatty acid hydroxylase from Claviceps purpurea, was used for synthesis of ricinoleic acid, and CpDGAT1, a diacylglycerol acyl transferase for the triacylglycerol synthesis from the same species, was used for assembly of the fatty acid. Coexpression of CpFAH and CpDGAT1 produced higher lipid contents and ricinoleic acid levels than expression of CpFAH alone. Coexpression in a mutant haploid strain defective in the Δ12 desaturase activity resulted in a higher level of ricinoleic acid than that in the diploid strain. Intriguingly, the ricinoleic acid produced was mainly distributed in the neutral lipid fractions, particularly the free fatty acid form, but with little in the polar lipids. This work demonstrates the effectiveness of the metabolic engineering strategy and excellent capacity of the microbial system for production of ricinoleic acid as an alternative to plant sources for industrial uses., (Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.)

The recently discovered cytokinin (CK)-specific phosphoribohydrolase "Lonely Guy" (LOG) is a key enzyme of CK biosynthesis, converting inactive CK nucleotides into biologically active free bases. We have determined the crystal structures of LOG from Claviceps purpurea (cpLOG) and its complex with the enzymatic product phosphoribose. The structures reveal a dimeric arrangement of Rossmann folds, with the ligands bound to large pockets at the interface between cpLOG monomers. Structural comparisons highlight the homology of cpLOG to putative lysine decarboxylases. Extended sequence analysis enabled identification of a distinguishing LOG sequence signature. Taken together, our data suggest phosphoribohydrolase activity for several proteins of unknown function., (© 2015 Wiley Periodicals, Inc.)

The tripeptide chains of the ergopeptines, a class of pharmacologically important D-lysergic acid alkaloid peptides, are arranged in a unique bicyclic cyclol based on an amino-terminal α-hydroxyamino acid and a terminal orthostructure. D-lysergyl-tripeptides are assembled by the nonribosomal peptide synthetases LPS1 and LPS2 of the ergot fungus Claviceps purpurea and released as N-(D-lysergyl-aminoacyl)-lactams. We show total enzymatic synthesis of ergopeptines catalyzed by a Fe²⁺/2-ketoglutarate-dependent dioxygenase (EasH) in conjunction with LPS1/LPS2. Analysis of the reaction indicated that EasH introduces a hydroxyl group into N-(D-lysergyl-aminoacyl)-lactam at α-C of the aminoacyl residue followed by spontaneous condensation with the terminal lactam carbonyl group. Sequence analysis revealed that EasH belongs to the wide and diverse family of the phytanoyl coenzyme A hydroxylases. We provide a high-resolution crystal structure of EasH that is most similar to that of phytanoyl coenzyme A hydroxylase, PhyH, from human., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Although there are numerous oleochemical applications for ricinoleic acid (RA) and its derivatives, their production is limited and subject to various safety legislations. In an effort to produce RA from alternative sources, we constructed a genetically modified strain of the oleaginous yeast Yarrowia lipolytica. This strain is unable to perform β-oxidation and is invalidated for the native triacylglycerol (TAG) acyltransferases (Dga1p, Dga2p, and Lro1p) and the ∆12 desaturase (Fad2p). We also expressed the Ricinus communis ∆12 hydroxylase (RcFAH12) under the control of the TEF constitutive promoter in this strain. However, RA constituted only 7% of the total lipids produced by this modified strain. By contrast, expression of the Claviceps purpurea hydroxylase CpFAH12 in this background resulted in a strain able to accumulate RA to 29% of total lipids, and expression of an additional copy of CpFAH12 drove RA accumulation up to 35% of total lipids. The co-expression of the C. purpurea or R. communis type II diacylglycerol acyltransferase (RcDGAT2 or CpDGAT2) had negative effects on RA accumulation in this yeast, with RA levels dropping to below 14% of total lipids. Overexpression of the native Y. lipolytica PDAT acyltransferase (Lro1p) restored both TAG accumulation and RA levels. Thus, we describe the consequences of rerouting lipid metabolism in this yeast so as to develop a cell factory for RA production. The engineered strain is capable of accumulating RA to 43% of its total lipids and over 60 mg/g of cell dry weight; this is the most efficient production of RA described to date.

Claviceps purpurea is a biotrophic fungal pathogen of grasses causing the ergot disease. The infection process of C. purpurea on rye flowers is accompanied by pectin degradation and polygalacturonase (PG) activity represents a pathogenicity factor. Wheat is also infected by C. purpurea and we tested whether the presence of polygalacturonase inhibiting protein (PGIP) can affect pathogen infection and ergot disease development. Wheat transgenic plants expressing the bean PvPGIP2 did not show a clear reduction of disease symptoms when infected with C. purpurea. To ascertain the possible cause underlying this lack of improved resistance of PvPGIP2 plants, we expressed both polygalacturonases present in the C. purpurea genome, cppg1 and cppg2 in Pichia pastoris. In vitro assays using the heterologous expressed PGs and PvPGIP2 showed that neither PG is inhibited by this inhibitor. To further investigate the role of PG in the C. purpurea/wheat system, we demonstrated that the activity of both PGs of C. purpurea is reduced on highly methyl esterified pectin. Finally, we showed that this reduction in PG activity is relevant in planta, by inoculating with C. purpurea transgenic wheat plants overexpressing a pectin methyl esterase inhibitor (PMEI) and showing a high degree of pectin methyl esterification. We observed reduced disease symptoms in the transgenic line compared with null controls. Together, these results highlight the importance of pectin degradation for ergot disease development in wheat and sustain the notion that inhibition of pectin degradation may represent a possible route to control of ergot in cereals., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

We have succeeded to produce a high content of ricinoleic acid (RA), a hydroxylated fatty acid with great values as a petrochemical replacement, in fission yeast Schizosaccharomyces pombe by introducing Claviceps purpurea oleate Δ12-hydroxylase gene (CpFAH12). Although the production was toxic to S. pombe cells, we solved the problem by identifying plg7, encoding phospholipase A2, as a multicopy suppressor. Characterization of the RA-tolerant strains suggested that the removal of RA moieties from phospholipids would be the suppression mechanism by plg7. In this study, we extended our analysis and report our new discovery that the overexpression of plg7 enabled cells to secrete free RA into culture media. When the FAH12 integrant in the absence of the overexpressed plg7 was grown at 20 °C for 11 days, the amount of intracellular RA reached 200.1 μg/ml of culture and only 69.3 μg/ml of RA was detected in culture media. On the other hand, the FAH12 integrant harboring the plg7 multicopy plasmid secreted RA in the media (184.5 μg/ml) without decreasing the amount in the cells, i.e., a significantly higher total secretion and a lead to making RA by its secretory production in S. pombe.

The aromatic prenyltransferase dimethylallyltryptophan synthase in Claviceps purpurea catalyzes the normal prenylation of tryptophan at C4 of the indole nucleus in the first committed step of ergot alkaloid biosynthesis. 4-Methyltryptophan is a competitive inhibitor of the enzyme that has been used in kinetic studies. Upon investigation of background activity during incubations of 4-methyltryptophan with dimethylallyl diphosphate, we found that the analogue was an alternate substrate, which gave four products. The structures of three of these compounds were established by (1)H NMR and 2D NMR studies and revealed that dimethylallyltryptophan synthase catalyzed both normal and reverse prenylation at C3 of the indole ring and normal prenylation of N1. Similarly, 4-methoxytryptophan was an alternate substrate, giving normal prenylation at C5 as the major product. 4-Aminotryptophan, another alternate substrate, gave normal prenylation at C5 and C7. The ability of dimethylallyltryptophan synthase to prenylate at five different sites on the indole nucleus, with normal and reverse prenylation at one of the sites, is consistent with a dissociative electrophilic alkylation of the indole ring, where orientation of the substrates within the active site and substituent electronic effects determine the position and type of prenylation. These results suggest a common mechanism for prenylation of tryptophan by all of the members of the structurally related dimethylallyltryptophan synthase family.

In an effort to produce ricinoleic acid (12-hydroxy-octadeca-cis-9-enoic acid: C18:1-OH) as a petrochemical replacement in a variety of industrial processes, we introduced Claviceps purpurea oleate ∆12-hydroxylase gene (CpFAH12) to Schizosaccharomyces pombe, putting it under the control of inducible nmt1 promoter. Since Fah12p is able to convert oleic acid to ricinoleic acid, we thought that S. pombe, in which around 75% of total fatty acid (FA) is oleic acid, would accordingly be an ideal microorganism for high production of ricinoleic acid. Unfortunately, at the normal growth temperature of 30 °C, S. pombe cells harboring CpFAH12 grew poorly when the CpFAH12 gene expression was induced, perhaps implicating ricinoleic acid as toxic in S. pombe. However, in line with a likely thermoinstability of Fah12p, there was almost no growth inhibition at 37 °C or, by contrast with 30 °C and lower temperatures, ricinoleic acid accumulation. Accordingly, various optimization steps led to a regime with preliminary growth at 37 °C followed by a 5-day incubation at 20 °C, and the level of ricinoleic acid reached 137.4 μg/ml of culture that corresponded to 52.6% of total FA.

Ergot alkaloids are indole derivatives with diverse structures and biological activities. They are produced by a wide range of fungi with Claviceps purpurea as the most important producer for medical use. Chanoclavine-I aldehyde is proposed as a branch point via festuclavine or pyroclavine to clavine-type alkaloids in Trichocomaceae and via agroclavine to ergoamides and ergopeptines in Clavicipitaceae. Here we report the conversion of chanoclavine-I aldehyde to agroclavine by EasG from Claviceps purpurea, a homologue of the festuclavine synthase FgaFS in Aspergillus fumigatus, in the presence of reduced glutathione and NADPH. EasG comprises 290 amino acids with a molecular mass of about 31.9 kDa. The soluble monomeric His(6)-EasG was purified after overproduction in E. coli by affinity chromatography and used for enzyme assays. The structure of agroclavine was unequivocally elucidated by NMR and MS analyses.

Eicosapentaenoic acid (EPA, 20:5n-3) plays an important role in many aspects of human health. In our efforts towards producing high levels of EPA in plants, we investigated the effects of different host species, genes and promoters on EPA biosynthesis. Zero-erucic acid Brassica carinata appeared to be an outstanding host species for EPA production, with EPA levels in transgenic seed of this line reaching up to 25%. Two novel genes, an 18-carbon omega3 desaturase (CpDesX) from Claviceps purpurea and a 20-carbon omega3 desaturase (Pir-omega3) from Pythium irregulare, proved to be very effective in increasing EPA levels in high-erucic acid B. carinata. The conlinin1 promoter from flax functioned reasonably well in B. carinata, and can serve as an alternative to the napin promoter from B. napus. In summary, the judicious selection of host species and promoters, together with the inclusion of genes that enhance the basic very long chain polyunsaturated fatty acid biosynthetic pathway, can greatly influence the production of EPA in plants.

Ergot alkaloids are indole-derived secondary metabolites synthesized by the phytopathogenic ascomycete Claviceps purpurea. In wild-type strains, they are exclusively produced in the sclerotium, a hibernation structure; for biotechnological applications, submerse production strains have been generated by mutagenesis. It was shown previously that the enzymes specific for alkaloid biosynthesis are encoded by a gene cluster of 68.5 kb. This ergot alkaloid cluster consists of 14 genes coregulated and expressed under alkaloid-producing conditions. Although the role of some of the cluster genes in alkaloid biosynthesis could be confirmed by a targeted knockout approach, further functional analyses are needed, especially concerning the early pathway-specific steps up to the production of clavine alkaloids. Therefore, the gene ccsA, originally named easE and preliminarily annotated as coding for a flavin adenine dinucleotide-containing oxidoreductase, was deleted in the C. purpurea strain P1, which is able to synthesize ergot alkaloids in axenic culture. Five independent knockout mutants were analyzed with regard to alkaloid-producing capability. Thin-layer chromatography (TLC), ultrapressure liquid chromatography (UPLC), and mass spectrometry (MS) analyses revealed accumulation of N-methyl-dimethylallyltryptophan (Me-DMAT) and traces of dimethylallyltryptophan (DMAT), the first pathway-specific intermediate. Since other alkaloid intermediates could not be detected, we conclude that deletion of ccsA led to a block in alkaloid biosynthesis beyond Me-DMAT formation. Complementation with a ccsA/gfp fusion construct restored alkaloid biosynthesis. These data indicate that ccsA encodes the chanoclavine I synthase or a component thereof catalyzing the conversion of N-methyl-dimethylallyltryptophan to chanoclavine I.

Claviceps purpurea, the fungal pathogen that causes the cereal disease ergot, produces glycerides that contain high levels of ricinoleic acid [(R)-12-hydroxyoctadec-cis-9-enoic acid] in its sclerotia. Recently, a fatty acid hydroxylase (C. purpurea FAH [CpFAH]) involved in the biosynthesis of ricinoleic acid was identified from this fungus (D. Meesapyodsuk and X. Qiu, Plant Physiol. 147:1325-1333, 2008). Here, we describe the cloning and biochemical characterization of a C. purpurea type II diacylglycerol acyltransferase (CpDGAT2) involved in the assembly of ricinoleic acid into triglycerides. The CpDGAT2 gene was cloned by degenerate RT-PCR (reverse transcription-PCR). The expression of this gene restored the in vivo synthesis of triacylglycerol (TAG) in the quadruple mutant strain Saccharomyces cerevisiae H1246, in which all four TAG biosynthesis genes (DGA1, LRO1, ARE1, and ARE2) are disrupted. In vitro enzymatic assays using microsomal preparations from the transformed yeast strain indicated that CpDGAT2 prefers ricinoleic acid as an acyl donor over linoleic acid, oleic acid, or linolenic acid, and it prefers 1,2-dioleoyl-sn-glycerol over 1,2-dipalmitoyl-sn-glycerol as an acyl acceptor. The coexpression of CpFAH with CpDGAT2 in yeast resulted in an increased accumulation of ricinoleic acid compared to the coexpression of CpFAH with the native yeast DGAT2 (S. cerevisiae DGA1 [ScDGA1]) or the expression of CpFAH alone. Northern blot analysis indicated that CpFAH is expressed solely in sclerotium cells, with no transcripts of this gene being detected in mycelium or conidial cells. CpDGAT2 was more widely expressed among the cell types examined, although expression was low in conidiospores. The high expression of CpDGAT2 and CpFAH in sclerotium cells, where high levels of ricinoleate glycerides accumulate, provided further evidence supporting the roles of CpDGAT2 and CpFAH as key enzymes for the synthesis and assembly of ricinoleic acid in C. purpurea.

Objective: Cloning of a Homologous Gene of PMK1 Type Mitogen-Activated Protein Kinase (MAPK) from the rice false smut fungus Ustilaginoidea virens., Methods: According to the conserved amino acid sequence of several filamentous fungus MAPKs, which were homologous to Magnaporthe grisea PMKI, degenerate PCR primers were designed to amplify the MAPK internal DNA fragment from Ustilaginoidea grisea. The complete UVMK1 DNA and cDNA sequences were obtained using Thermal Asymmetric Interlaced-PCR (TAIL-PCR) and RT-PCR methods. Functional Identification was done by using the M. grisea APMKI mutant stain nn78, including appressoria differentiation assay and barley infection test., Results: The total length of UVMKJ was 1435 bp. It contained 3 introns and encoded 355 amino acids. The induced amino acid sequence showed identical to Magnaporthe grisea PMKI, Fusarium oxysporum FMKJ, Fusarium solani FsMAPK, Colletotrichum lagenarium CMKI, Botrytis cinerea BMKI, Claviceps purpurea CMPKI. After transformation of the APMK1 mutant of M. grisea using a complement vector with the complete cDNA of UVMK1 (under the M. grisea MPG1 promoter), five transformants were obtained. Furthermore, the selected two transformants fully restored their ability to form appressoria and infect a barley leaf., Conclusion: In this study, we characterized the frst MAPK protein from U. virens, and that UVMK1 is a homologue of M. grisea PMK1.

Claviceps purpurea, a fungal pathogen responsible for ergot diseases in many agriculturally important cereal crops, produces high levels of ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid) in its sclerotia. It has been believed for many years that the biosynthesis of this fatty acid in C. purpurea involves a hydration process with linoleic acid as the substrate. Using degenerate polymerase chain reaction, we cloned a gene from the sclerotia encoding an enzyme (CpFAH) that has high sequence similarity to the C. purpurea oleate desaturase, but only low similarity to plant oleate hydroxylases. Functional analysis of CpFAH in yeast (Saccharomyces cerevisiae) indicated it acted predominantly as a hydroxylase, introducing hydroxyl groups at the 12-position of oleic acid and palmitoleic acid. As well, it showed Delta(12) desaturase activities on 16C and 18C monounsaturated fatty acids and, to a much lesser extent, omega(3) desaturase activities on ricinoleic acid. Heterologous expression of CpFAH under the guidance of a seed-specific promoter in Arabidopsis (Arabidopsis thaliana) wild-type and mutant (fad2/fae1) plants resulted in the accumulation of relatively higher levels of hydroxyl fatty acids in seeds. These data indicate that the biosynthesis of ricinoleic acid in C. purpurea is catalyzed by the fungal desaturase-like hydroxylase, and CpFAH, the first Delta(12) oleate hydroxylase of nonplant origin, is a good candidate for the transgenic production of hydroxyl fatty acids in oilseed crops.

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.

Claviceps purpurea, the ergot fungus, is a highly specialized pathogen of grasses; its colonization of host ovarian tissue requires an extended period of strictly polarized, oriented growth towards the vascular tissue. To understand this process, we study the role of signalling factors affecting polarity and differentiation. We showed that the small GTPase Cdc42 is involved in polarity, sporulation and in planta growth in C. purpurea. Here we present evidence that the GTPase Rac has an even stronger and, in some aspects, inverse impact on growth and development: Deltarac mutants form coralline-like colonies, show hyper-branching, loss of polarity, sporulation and ability to penetrate. Functional analyses and yeast two-hybrid studies prove that the p21-activated kinase Cla4 is a major downstream partner of Rac. Phosphorylation assays of MAP kinases and expression studies of genes encoding reactive oxygen species (ROS)-scavenging and -generating enzymes indicate a function of Rac and Cla4 in fungal ROS homoeostasis which could contribute to their drastic impact on differentiation.

Two cDNAs with sequence similarity to fatty acid desaturase genes were isolated from the phytopathogenic fungus, Claviceps purpurea. The predicted amino acid sequences of the corresponding genes, named CpDes12 and CpDesX, share 87% identity. Phylogenetic analysis indicates that CpDes12 and CpDesX arose by gene duplication of an ancestral Delta(12)-desaturase gene after the divergence of Nectriaceae and Clavicipitaceae. Functional expression of CpDes12 and CpDesX in yeast (Saccharomyces cerevisiae) indicated that CpDes12 is primarily a "Delta(12)"-desaturase, whereas CpDesX is a novel desaturase catalyzing "Delta(12)," "Delta(15)," and "omega(3)" types of desaturation with omega(3) activity predominating. CpDesX sequentially desaturates both 16:1-9c and 18:1-9c to give 16:3-9c,12c,15c and 18:3-9c,12c,15c, respectively. In addition, it could also act as an omega(3)-desaturase converting omega(6)-polyunsaturates 18:3-6c,9c,12c, 20:3-8c,11c,14c, and 20:4-5c,8c,11c,14c to their omega(3) counterparts 18:4-6c,9c,12c,15c, 20:4-8c,11c,14c,17c, and 20:5-5c,8c,11c,14c,17c, respectively. By using reciprocal site-directed mutagenesis, we demonstrated that two residues (isoleucine at 152 and alanine at 206) are critical in defining the catalytic specificity of these enzymes and the C-terminal amino acid sequence (residues 302-477) was also found to be important. These data provide insights into the nature of regioselectivity in membrane-bound fatty acid desaturases and the relevant structural determinants. The authors suggest that the regios-electivity of such enzymes may be best understood by considering the relative importance of more than one regioselective preference. In this view, CpDesX is designated as anu + 3(omega(3)) desaturase, which primarily references an existing double bond (nu + 3 regioselectivity) and secondarily shows preference for omega(3) desaturation.

Clavines and D-lysergic acid-derived alkaloid amides and alkaloid peptides are two different families of compounds that have the indole-derived tetracyclic metergoline ring system in common. Previous work has shown that D-lysergic acid is biosynthetically derived from clavine alkaloids. Recent cloning and analysis of the ergot alkaloid biosynthesis gene cluster from the D-lysergic acid peptide (ergopeptines)-producing Claviceps purpurea, has shown that it most probably contains all genes necessary for D-lysergic acid synthesis as well as those that encode the assembly of D-lysergic acid peptides, such as ergotamine. To address the role of the oxygenase genes of alkaloid-gene clusters, the only cytochrome P450 monooxygenase gene of this cluster was inactivated by disruption. The resultant mutant accumulated agroclavine, elymoclavine, and chanoclavine in substantial amounts but not ergopeptines. Feeding the mutant with D-lysergic acid restored ergopeptine synthesis; this suggests a block in the conversion of elymoclavine to D-lysergic acid. The gene was designated cloA (for encoding a clavine oxidase, CLOA). Retransformation of the mutant with the intact cloA gene also restored ergopeptine synthesis. These data show that CLOA catalyses the conversion of clavines to D-lysergic acid, it acts as a critical enzyme in the ergot alkaloid gene cluster, and bridges the biosynthesis of the two different families of alkaloids.

The ergot alkaloids are a family of indole-derived mycotoxins with a variety of significant biological activities. Aspergillus fumigatus, a common airborne fungus and opportunistic human pathogen, and several fungi in the relatively distant taxon Clavicipitaceae (clavicipitaceous fungi) produce different sets of ergot alkaloids. The ergot alkaloids of these divergent fungi share a four-member ergoline ring but differ in the number, type, and position of the side chains. Several genes required for ergot alkaloid production are known in the clavicipitaceous fungi, and these genes are clustered in the genome of the ergot fungus Claviceps purpurea. We investigated whether the ergot alkaloids of A. fumigatus have a common biosynthetic and genetic origin with those of the clavicipitaceous fungi. A homolog of dmaW, the gene controlling the determinant step in the ergot alkaloid pathway of clavicipitaceous fungi, was identified in the A. fumigatus genome. Knockout of dmaW eliminated all known ergot alkaloids from A. fumigatus, and complementation of the mutation restored ergot alkaloid production. Clustered with dmaW in the A. fumigatus genome are sequences corresponding to five genes previously proposed to encode steps in the ergot alkaloid pathway of C. purpurea, as well as additional sequences whose deduced protein products are consistent with their involvement in the ergot alkaloid pathway. The corresponding genes have similarities in their nucleotide sequences, but the orientations and positions within the cluster of several of these genes differ. The data indicate that the ergot alkaloid biosynthetic capabilities in A. fumigatus and the clavicipitaceous fungi had a common origin.

Claviceps purpurea produces the pharmacological important ergopeptines, a class of cyclol-structured alkaloid peptides containing D-lysergic acid. These compounds are assembled from D-lysergic acid and three different amino acids by the nonribosomal peptide synthetase enzymes LPS1 and LPS2. Cloning of alkaloid biosynthesis genes from C. purpurea has revealed a gene cluster including two NRPS genes, cpps 1 and cpps 2. Protein sequence data had assigned earlier cpps1 to encode the trimodular LPS1 assembling the tripeptide portion of ergopeptines. Here, we show by transcriptional analysis, targeted inactivation, analysis of disruption mutants, and heterologous expression that cpps 2 encodes the monomodular LPS2 responsible for D-lysergic acid activation and incorporation into the ergopeptine backbone. The presence of two distinct NRPS subunits catalyzing formation of ergot peptides is the first example of a fungal NRPS system consisting of different NRPS subunits.

The lead phosphate precipitation method showed the reaction product of acid phosphatase (which reflects the presence of the enzyme glycoprotein) in peripheral cytoplasmic vesicles in the ascomycetous fungus Claviceps purpurea. The product appeared to diffuse from these vesicles (diameter 100-200 nm) towards the cell wall, usually to its sites covered by the capsular fibres exhibiting also acid phosphatase activity. This observation of diffusion of secretory glycoprotein in the cytoplasmic matrix and its orientation to the plasmalemma and capsular fibrils suggests an alternative to the well-described secretory mechanism of transport and exocytosis of glycoproteins via membrane-bound transport conveyors fusing with the cell membrane. It confirms and enlarges our previous finding of the reaction product of acid phosphatase performed by ultrastructural cytochemistry in vacuoles (lysosomes), in the growing cell septum, in cytoplasmic vesicles and in the fibres of the external capsule.

Cpmk2, encoding a mitogen-activated protein (MAP) kinase from the ascomycete Claviceps purpurea, is an orthologue of SLT2 from Saccharomyces cerevisiae, the first isolated from a biotrophic, non-appressorium-forming pathogen. Deletion mutants obtained by a gene replacement approach show impaired vegetative properties (no conidiation) and a significantly reduced virulence, although they retain a limited ability to colonize the host tissue. Increased sensitivity to protoplasting enzymes indicates that the cell wall structure of the mutants may be altered. As the phenotypes of these mutants are similar to those observed in strains of the rice pathogen, Magnaporthe grisea, that have been deprived of their MAP kinase gene mps1, the ability of cpmk2 to complement the defects of delta mps1 was investigated. Interestingly, the C. purpurea gene, under the control of its own promoter, was able to complement the M. grisea mutant phenotype: transformants were able to sporulate and form infection hyphae on onion epidermis and were fully pathogenic on barley leaves. This indicates that, despite the differences in infection strategies, which include host and organ specificity, mode of penetration and colonization of host tissue, CPMK2/MPS1 defines a second MAP kinase cascade (after the Fus3p/PMK1 cascade) essential for fungal pathogenicity.

Claviceps purpurea is a biotrophic, organ-specific pathogen of grasses and cereals, attacking exclusively young ovaries. We have previously shown that its mainly intercellular growth is accompanied by degradation of pectin, and that two endopolygalacturonase genes (cppg1/cppg2) are expressed throughout all stages of infection. We report here on a functional analysis of these genes using a gene-replacement approach. Mutants lacking both polygalacturonase genes are not affected in their vegetative properties, but they are nearly nonpathogenic on rye. Complementation of the mutants with wild-type copies of cppg1 and cppg2 fully restored pathogenicity, proving that the endopolygalacturonases encoded by cppg1 and cppg2 represent pathogenicity factors in the interaction system C. purpurea/Secale cereale, the first unequivocally identified so far in this system.

Claviceps purpurea is a common pathogen of a wide range of grasses and cereals that is able to establish a stable, balanced interaction with its host plant and is considered a biotroph. It does not form special penetration structures such as appressoria. To study the signaling processes involved in this special host-pathogen interaction, we have cloned a gene, cpmk1, encoding a mitogen-activated protein (MAP) kinase that shows significant homology to Fus3 of Saccharomyces cerevisiae and to pmk1 of Magnaporthe grisea. Using a gene-replacement approach, we isolated a Acpmk1 mutant and characterized it in detail. Loss of CPMK1 has no obvious effect on vegetative properties (such as growth rate, morphology, and conidia formation); however, infection tests on rye show that the mutant is unable to colonize rye tissue, i.e., it appears to be completely nonpathogenic. Complementation of the mutant with a wild-type copy of cpmk1 fully restores its pathogenicity, confirming that this MAP kinase is essential for infection of rye by C. purpurea. Transformation of the delta pmk1 mutant of M. grisea with a complete copy of cpmk1 (including the C. purpurea promoter) fully restored its ability to form appressoria and its pathogenicity on barley. Although both fungi drastically differ in their pathogenic strategies, this result indicates that the signal pathway involving CPMK1 is highly conserved.

From a mixture of lysergamide and its epimer isolysergamide, Rhodococcus equi A4 containing amidase preferentially hydrolyzed lysergamide into lysergic acid.

The potential of the polymerase chain reaction for the detection of ergot alkaloid producers among microscopic fungi of the genera Penicillium and Claviceps was evaluated. Twenty-three strains of various species of fungi with a previously studied capacity for alkaloid production were used. The internal fragment of the gene encoding 4-dimethylallyltryptophan synthase, the enzyme catalyzing the first step in the biosynthesis of ergot alkaloids, was amplified using degenerated primers. This approach revealed an about 1.2-kb specific DNA fragment in micromycetes synthesizing ergot alkaloids with complete tetracyclic ergoline system. Microorganisms that produce alkaloids with modified C or D ergoline rings, as well as alpha-cyclopiazonic acid, did not yield the PCR fragment of the expected size. This fragment was also not found in fungi incapable of ergot alkaloid production.

Claviceps purpurea has been shown to secrete catalases in axenic and parasitic culture. In order to determine the importance of these enzymes in the host-parasite interaction, especially their role in overcoming oxidative stress imposed on the pathogen by the plant's defense system, the catR gene from A. niger was used to isolate a putative catalase gene from a genomic library of C. purpurea, cpcat1 consists of an open reading frame of 2,148 bp that is interrupted by five introns. Its derived gene product shows significant homology to fungal catalases and contains a putative signal peptide of 19 amino acids and three putative N-glycosylation sites, which indicates that CPCAT1 is a secreted catalase. Disruption of the gene by a gene replacement approach resulted in the loss of two catalase isoforms, CATC and CATD, strongly suggesting that they are both encoded by cpcat1. CATD is the major secreted catalase of C. purpurea and is furthermore the only catalase present in the honeydew of infected rye ears. Deletion mutants of cpcat1 were inoculated on rye plants and showed no significant reduction in virulence. Ovarian tissue and honeydew of plants inoculated with the mutants lacked CATD, confirming that this catalase is not essential for colonization of the host tissue by C. purpurea.

The production of toxic ergopeptine alkaloids by the fungi Claviceps purpurea and Neotyphodium coenophialum involves the activity of one or more nonribosomal peptide synthetases. Claviceps purpurea and N. coenophialum each have several different peptide synthetase genes, fragments of which have been cloned previously. An additional Claviceps purpurea peptide synthetase gene was cloned by hydridization with one of the N. coenophialum peptide synthetase gene fragments. We detected the presence of mRNA from the peptide synthetase genes in cultures of different ages grown under conditions favorable or unfavorable for ergopeptine production. All four peptide synthetase genes from Claviceps purpurea were transcribed under at least some of the experimental conditions. Transcripts from three of the four genes were detected under conditions consistent with their potential involvement in ergopeptine biosynthesis. All three peptide synthetase genes previously identified in N. coenophialum were transcribed during symbiotic growth of this fungus with tall fescue, as well as ergopeptine-producing cultures. The data show that all of the peptide synthetase genes are transcribed, that one of the peptide synthetase genes is dissociated from ergopeptine biosynthesis, and, as a result, prioritize the remaining genes for functional analyses by transformation-mediated gene disruption.

Background: Previous analyses of the biosynthesis of the alkaloid cyclopeptides from the ergot fungus Claviceps purpurea were hampered by a lack of suitable systems for study in vitro, and this led to conflicting results concerning the mechanism of alkaloid cyclopeptide formation. Recently, D-lysergyl peptide synthetase (LPS) of the ergot fungus Claviceps purpurea, which assembles the non-cyclol precursors of the ergopeptines, has been partially purified and shown to consist of two polypeptide chains of 370 kDa (LPS 1) and 140 kDa (LPS 2); these contain all the sites necessary for the assembly of the D-lysergyl peptide backbone. The mechanism of D-lysergyl peptide synthesis remained unclear, however., Results: We have identified the obligatory peptidic intermediates in D-lysergyl peptide synthesis and the sequential order of their formation. The two LPS subunits catalyze the formation of D-lysergyl mono-, di-, and tripeptides as enzyme-thioester intermediates, the formation of which appears to be irreversible. Peptide synthesis starts when D-lysergic acid binds to the LPS 2 subunit, which most probably occurs after the previous round of synthesis has been completed by the release of the end product from the LPS enzyme., Conclusions: We have shown that the mechanism of D-lysergyl peptide synthesis is an ordered process of successive acyl transfers on a multienzyme complex. This knowledge opens the way for enzymatic and genetic investigations into the formation of novel alkaloid cyclopeptides.

At the host-pathogen interface of hyphae penetrating host cell walls in the rye ovary, a lack of cellulase-gold labeling of beta-1, 4-glucan in host cell walls indicates that enzymatic degradation of cellulose might be an important factor during the infection of rye by Claviceps purpurea. Using cbh1 from Trichoderma reesei as a probe, a putative cellulase gene (cel1) was isolated from a genomic library of the C. purpurea strain T5. The coding region of 1,616 bp contains two introns and a putative signal peptidase cleavage site, leaving a coding capacity of 437 amino acids for the mature protein. The derived amino acid sequence shares significant homology with other fungal cellobiohydrolases and lacks the substrate binding domain. Expression analysis using reverse transcriptase-polymerase chain reaction (RT-PCR) shows that cel1 is induced during the first days of infection of rye by C. purpurea. It may be involved in the penetration and degradation of host cell walls by depolymerizing plant beta-1, 4-glucan and, therefore, play a role in the infection process.

Claviceps fusiformis, an ergot pathogen of pearl millet, was equilibrated in media without and with 0.1 mM ouabain (g-strophantin). Highly significant effects of ouabain on the contents of sodium ions and water were found, suggesting that an active transport of sodium ions, similar to that in animal cells, regulates the cell volume of the fungus. The basic role of the sodium pump appears to be to decrease larger cell volumes and to increase the small ones; for this purpose it can even reverse its direction.

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.

The first pathway-specific step of ergot alkaloid biosynthesis in the fungus, Claviceps purpurea, is catalyzed by the prenyltransferase, 4-(gamma,gamma-dimethylallyl)tryptophan synthase. Partial sequence information was obtained for the purified enzyme and a degenerate oligonucleotide mixture was used to identify and amplify segments of the gene, dmaW. The complete gene and near-full-length cDNA were cloned and sequenced. The cDNA was cloned in a yeast expression vector in sense and antisense orientations relative to the inducible GAL1 promoter. Extracts of yeast transformants with the sense constructs, but not antisense constructs or cloning vector, catalyzed production of 4-(gamma,gamma-dimethylallyl)tryptophan. The sequence of dmaW and its cDNA indicated that it encoded a 455 amino acid polypeptide with a predicted molecular mass of 51,824 Da and a putative prenyl diphosphate binding motif.

The biosynthetic pathway leading from L-tryptophan, mevalonic acid and methionine to the tetracyclic ergoline ring system of the ergot alkaloids in Claviceps species is reviewed. This pathway entails many mechanistically intriguing features. Recent studies are also discussed which reveal the stereochemical course of the isoprenylation of tryptophan and of the N-methylation of dimethylallyltryptophan (DMAT) and which shed some light on the likely steps leading from the open-chain precursors, N-methyl-DMAT to the tricyclic intermediate, chanoclavine-1. Finally, some plans are outlined to probe the evolutionary relationship of ergot alkaloid biosynthesis in fungi to that in higher plants of the family Convolvulaceae.

A homologous transformation system was developed for the phytopathogenic fungus Claviceps purpurea. Orotidine-5'-monophosphate decarboxylase (OMPD)-deficient mutants were obtained by UV mutagenesis and selection for resistance against 5-fluoroorotate. These mutants could be complemented well by the corresponding genes of Aspergillus niger (pyrA) and Neurospora crassa (pyr4), yielding significantly higher transformation rates (and lower copy numbers per transformant) than the phleomycin resistance system. The homologous OMPD gene was isolated from a lambda genomic library by heterologous hybridization with the pyr4 gene of N. crassa, identified by complementation of Aspergillus and Claviceps mutants, and used to confirm homologous integration in Claviceps. The pyr transformation system also proved to be very efficient in cotransformation experiments using the bacterial beta-glucuronidase gene (uidA) as a reporter gene, which was also efficiently expressed during the parasitic cycle: honeydew produced by plants infected with pyr/uidA cotransformants was shown to contain significant levels of beta-glucuronidase activity.

Dimethylallyl tryptophan synthase (DMAT synthase) catalyzes the alkylation of L-tryptophan by dimethylallyl diphosphate to form 4-(gamma,gamma-dimethylallyl)-L-tryptophan. The enzyme from mycelia of Claviceps purpurea was purified approximately 125-fold to apparent homogeneity by chromatography on n-butyl Sepharose, Q Sepharose, phenyl Sepharose, and Protein Pak as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Analysis by gel filtration chromatography and SDS-PAGE indicated that DMAT synthase is an alpha 2 dimer with a molecular mass of 105 kDa. The purified enzyme was active in metal-free buffer containing EDTA. However, activity was enhanced upon addition of divalent calcium or magnesium ions to the buffer. Values for KM and Vmax were determined in the metal-free EDTA buffer (KMDMAPP, 14 microM; KML-tryptophan, 40 microM; Vmax, 215 nmol min-1 mg-1), 4 mM CaCl2 (KMDMAPP, 8.0 microM; KML-tryptophan, 17 microM; Vmax, 504 nmol min-1 mg-1), and 4 mM MgCl2 (KMDMAPP, 8.0 microM; KML-tryptophan, 12 microM; Vmax, 455 nmol min-1 mg-1). The product was isolated and characterized by 1H NMR, uv, and FAB mass spectrometry.

Dimethylallyl pyrophosphate: L-tryptophan dimethylallyltransferase (dimethylallyl tryptophan synthetase) has been purified from Claviceps strain SD58 to a homogeneous crystalline form. The enzyme is pure as judged by polyacrylamide gel electrophoresis and contains two similar subunits of 34,000 molecular weight as shown by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. Since the enzyme was determined to have a molecular weight of 70,000 by gel exclusion chromatography, it is dimeric. The substrates dimethylallyl pyrophosphate and tryptophan each have mixed (negative to positive) cooperativity with a minimum Hill coefficient of 0.37 and 0.73, respectively. Calcium ion is a positive allosteric effector and, at 20 mM concentration, deregulates the enzyme. The deregulated enzyme has Km values of 7.2 microM for dimethylallyl pyrophosphate and 8.8 microM tryptophan. The Vmax under deregulated conditions is 953 nmol/min/mg, giving a turnover number of 14. Ca2+ has a S[0.5] of 4.5 mM. The kinetic mechanism is random sequential.

New cytochemical method, based on biochemical experiments, was elaborated for the ultrastructural localization of phospho(enol)pyruvate carboxylase (EC 4.1.1.31). The procedure was used to study the saprophytic submerged mycelium of the ascomycetous fungus Claviceps purpurea Tul. producing clavine alkaloids. The pelleted mycelium was fixed in ice cold 3% glutaraldehyde in 50 mM cacodylate buffer pH 7.2 and washed repeatedly in the same cold buffer The reaction mixture contained 100 mM Tris-HCl buffer pH 9.0, 10 mM phospho(enol)pyruvate, 30 mM sodium potassium tartrate, 3 mM Pb(NO3)2, 60 mM MgCl2 and 30 mM NaHCO3. Enzyme activity was localized in vacuoles, particularly inside lipid globules (spherosomes) and less frequently in membranous vesicles. Acetyl-CoA activated PEP-carboxylase both in cell free extracts and in the cytochemical staining. Aspartate inhibited the enzyme in the biochemical assay with coupled malate dehydrogenase system; the cytochemical reaction was not influenced, probably due to the interference of asparagine synthase (EC 6.3.1.1).

A three-enzyme complex containing anthranilate synthetase, phosphoribosyl anthranilate isomerase and indole-3-glycerol phosphate synthetase was partially purified from Claviceps spec., strain SD 58. The anthranilate synthetase activity of the enzyme complex was quite unstable unless glutamine, MgCl2, TRIS and, most importantly, glycerol were included in the extraction buffer. The three-enzyme complex showed a molecular weight of 400,000 when estimated using Sephadex gel filtration, and a molecular weight of 200,000 when using sucrose density gradient centrifugation. At least two bands of anthranilate synthetase were detected on disc gel electrophoresis. An enzyme complex containing phosphoribosyl anthranilate synthetase and indoleglycerol phosphate synthetase, but no anthranilate synthetase, was isolated from Claviceps. This enzyme complex had an apparent molecular weight of 165,000 as estimated by sucrose gradient centrifugation. Anthranilate synthetase is inhibited by L-tryptophan and elymoclavine, the terminal ergot alkaloid produced by this strain of Claviceps. No differences could be detected between the enzyme complexes isolated from 2-day-old growing mycelia and from 6-day-old alkaloid-producing mycelia of the organism.

Extra- and intracellular proteinases from various Claviceps purpurea strains grown in submerged culture have been studied. A maximum level of intracellular proteinases was observed on the 6th day of culture growth, whereas extracellular activity continued to increase throughout the culture growth. Proteinases were purified and characterized. The ergotamine strain secreted one aspartic and two serine proteinases, whereas from the disrupted mycelium only the aspartic proteinase could be isolated. The ergocornine strain secreted the aspartic proteinase in two forms and the ergocristine strain produced an aspartic and a serine proteinase.

p-Nitrophenylphosphate in combination with lead salt technique was used for the cytochemical localization of acid phosphatase (EC 3.1.3.2) in saprophytic submerged culture of Claviceps purpurea Tul. The lead reaction product was found in capsular fibrils, in the newly formed parts of the cell wall and in the vacuoles of aged cells (autolysosomes). Phosphatase activity was present also in particulate intra-cytoplasmatic organelles. The concentric layering of lead deposits in these organelles indicates their relationship to endoplasmic reticulum membranes.

The enhancement of ergot alkaloid production by tryptophan and its analogues in both normal and high-phosphate cultures is more directly related to increased dimethylallyltryptophan (DMAT) synthetase activity rather than to a lack of regulation of the tryptophan biosynthetic enzymes. Thiotryptophan [beta-(1-benzo-thien-3-yl)-alanine] is rather ineffective in the end product regulation of tryptophan biosynthesis, whereas tryptophan and 5-methyltryptophan are potent effectors. The presence of increased levels of DMAT synthetase in ergot cultures supplemented with tryptophan or thiotryptophan, and to a lesser extent with 5-methyltryptophan, suggests that the induction effect involves de novo synthesis of the enzyme. Thiotryptophan and tryptophan but not 5-methyltryptophan can overcome the block of alkaloid synthesis by inorganic phosphate. The results with thiotryptophan indicate that the phosphate effect cannot be explained merely on the basis of a block of tryptophan synthesis.