Identification and characterization of the ergochrome gene cluster in the plant pathogenic fungus Claviceps purpurea
© Neubauer et al. 2016
Received: 21 December 2015
Accepted: 16 February 2016
Published: 22 March 2016
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.
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.
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.
The biotrophic ascomycete Claviceps purpurea infects a broad range of grasses including economically important cereal crop plants like rye, wheat and barley . The fungus infects exclusively the young ovaries of the host plants. After successful colonization the ovary is replaced by fungal mycelium and production of conidia begins. The infection cycle ends with the formation of a sclerotium, the resting structure of the fungus [2, 3]. Ergot alkaloids, the best characterized secondary metabolites of C. purpurea, are produced exclusively in the sclerotial tissue. These toxins are historically important as they affect the central nervous system of mammalians and were the reason for severe intoxications in the past, caused by consumption of contaminated bread . Sclerotia are shaped like a grain but are usually larger (2–25 mm) and the hard outer cortex is pigmented purple-black. Sclerotia contain 1–2 % (w/w) of pigments belonging to different structural groups .
The yellow ergochromes are dimers of tetrahydroxanthone units . Four different xanthone derivatives were described as ergochrome units in C. purpurea and all possible combinations of two of these units occur in nature . Their concentration in the sclerotia is considerably higher (5 g/kg) than that of endocrocin and clavorubin (40 mg/kg) . Ergochromes are known for their biological activity. Many of them show anti-inflammatory, cytostatic and anti-tumor activity or a neuroprotective effect [9–11]. Due to the structural similarity of the ergochromes to the anthraquinone pigments (Fig. 1) it is likely that both pigments are products of the same biosynthetic pathway. In Aspergillus nidulans it has already been shown that endocrocin is a shunt product during the production of xanthones [12, 13]. Nevertheless, the biosynthesis of the ergochromes in C. purpurea remains unclear, although some intermediates like emodin have been described .
Endocrocin could be identified in extracts of various other fungi [14–16] but its biosynthesis is best characterized in Aspergillus fumigatus where a cluster of four genes has been identified . The key enzyme of the cluster is a nonreducing polyketide synthases (NR-PKS) lacking the thioesterase (TE) domain usually necessary for releasing the polyketide product from the enzyme. In these special types of NR-PKSs the polyketide is released from the PKS by a metallo-β-lactamase-type thioesterase (MβL-TE) . Recently, in A. fumigatus a second gene cluster has been identified which also contributes to the formation of endocrocin as a shunt product from production of the anthraquinone-derivative trypacidin . Both clusters show homologies to clusters responsible for the formation of related anthraquinone-derivatives such as geodin in Aspergillus terreus  and the xanthones in A. nidulans [12, 13].
This paper reports the identification of a gene cluster in C. purpurea which shows high homology to these gene clusters and is involved in ergochrome biosynthesis. Besides the classical ergot alkaloid cluster, this is the first functional analysis of a secondary metabolite gene cluster in the ergot fungus.
Characteristics of the gene cluster
Predicted functions and homologs of the pigment gene cluster in C. purpurea
In planta expression
Gene expression in axenic culture and optimization of the culture conditions
Overexpression of the transcription factor and the PKS
To verify if the PKS CPUR_05437 gene cluster is responsible for the pigment synthesis in C. purpurea we overexpressed the Zn2Cys6 transcription factor CPUR_05433 by introducing an additional copy under the control of the strong constitutive A. nidulans oliC (mitochondrial ATP synthase subunit 9 gene) promoter (Additional file 1: Figure S1A). For three independent transformants the overexpression of the transcription factor as well as of nine further cluster genes could be confirmed by northern blot analyses (Fig. 5b). These results show that the genes CPUR_05425, CPUR_05426, CPUR_05427, CPUR_05428, CPUR_05429, CPUR_05434, CPUR_05435, CPUR_05436, as well as CPUR_05437 are regulated by CPUR_05433. As expression of the other cluster genes is very low, the co-regulation by the transcription factor could not be confirmed by northern analyses.
To get further insight into the biosynthesis of the pigments, a strain where only the PKS CPUR_05437 is overexpressed (OE PKS) was generated (Additional file 1: Figure S1B). Overexpression of the gene was confirmed by northern blot analyses (Additional file 1: Figure S1C) and the strain was cultured under growth conditions unfavorable for the pigment production. After 7 days of cultivation the culture was clearly pigmented. However, in contrast to the OE TF cultures the color was more purple red (Fig. 6). The UV signal of clavorubin at 22.8 min in Fig. 9 highlights the considerable production of the red pigments in the OE PKS strain (Fig. 9b) compared to the wild type (Fig. 9a). The UV intensity indicates an approximately 10 times higher production of clavorubin in the OE PKS mutant compared to the wild type and a two times greater production compared to the OE TF. In addition, Fig. 9d, e illustrates the increased mass spectrometric signal intensity of endocrocin, as well also of clavorubin, between the wild type and the OE PKS mutant.
Taken together, chemical analyses of the OE TF and the OE PKS cultures clearly show that activation of the whole cluster primarily leads to increased production of the yellow ergochrome dimers, whereas overexpression of the PKS merely increases occurrence of red anthraquinones.
PKS CPUR_05437 is responsible for pigmentation in axenic culture and in planta
To show that the PKS is also responsible for the typical pigmentation of the ergot sclerotia in planta, rye plants were infected with the C. purpurea wild type as well as the PKS knock out mutants. The pathogenicity assay shows that the mutants were able to infect the plants normally. First signs of successful infection (honeydew production) were evident 7–8 days post-inoculation. Approximately 15 days post-inoculation sclerotia were visible. After about 3 weeks the sclerotia of the wild type were pigmented purple black. In contrast, sclerotia of the ∆PKS mutants were colorless (Fig. 11b). Notably, sclerotia of all groups were of similar size and consistency. Complementation of the knock out with a PKS overexpression construct restored the pigmentation in axenic culture as well as in planta (Additional file 3: Figure S3).
Taken together the results clearly show that the PKS CPUR_5437 gene cluster is responsible for the formation of both kinds of ergot pigments; this applies both to the red anthraquinones and to the yellow ergochromes which are more complex xanthone derivatives.
Pigments are produced by a variety of filamentous fungi. In Aspergillus flavus or Alternaria alternata, for example, pigments such as melanin and asparasone play a protective role against abiotic stresses, e.g. UV radiation [24, 25]. In other fungi like the human pathogen A. fumigatus and the plant pathogen Magnaporthe oryzae melanin is required for virulence . Pigmentation is often associated with developmental structures like spores or sclerotia. C. purpurea produces pigments mainly in the sclerotia. Sclerotia are resting structures and thus there is a strong requirement for them to be able to overcome biotic and abiotic stresses over a longer period in the field. Sclerotia of A. flavus asparasone minus mutants, for example, were significantly less resistant to insect predation and more susceptible to ultraviolet light and heat . According to the reported functions of pigments in other fungi, it could be assumed that the ergot pigments are also important for the survival of the C. purpurea sclerotia. However, there is no difference in the consistency of the albino sclerotia in comparison to the wild type assuming that they are not more sensitive to mechanical damage. Interestingly, it has been shown that knock out of members of the NADPH oxidase (Nox) complex, usually involved in infection processes of pathogenic fungi, leads to the formation of white but small and immature pseudosclerotia in C. purpurea [27, 28]. qRT-PCR revealed a reduced PKS Cpur_05437 gene expression in the Δcpnox2 sclerotia  showing the complexity of pathogenic development and secondary metabolism in C. purpurea. Furthermore, the formation of the ergot pigments is contemporary with ergot alkaloid synthesis in planta. As the ergot alkaloids are light sensitive, pigmentation of the sclerotia is important for the protection of these toxins . Surprisingly, in axenic culture, the expression of the pigment cluster and the alkaloid cluster are regulated contrary. While for the alkaloid production low levels of phosphate and a high sucrose concentration are necessary, the pigment production is increased by high levels of phosphate and repressed by high levels of sucrose. Apparently, there are different signaling pathways regulating the secondary metabolite production in planta and in axenic culture. Nevertheless, phosphate seems to be an important factor influencing the secondary metabolism of C. purpurea. As the alkaloid biosynthesis , the pigment biosynthesis is regulated by phosphate on a transcriptional level, but the molecular mechanism is still unknown. Generally, only little is known about the phosphate control of secondary metabolism. There are some examples that high concentrations of phosphate interfere with secondary metabolism of microorganisms . Inorganic phosphate affects enzyme activities such as kinases and phosphatases, directly required in secondary metabolite biosynthesis or involved in signal transduction cascades, regulating e.g. fungal development, differentiation and other processes [32, 33]. Usually secondary metabolite production is repressed by high levels of phosphate like the production of the antifungal protein (AFP) by Aspergillus giganteus , the production of bikaverin by Fusarium oxysporum  or the aflatoxin production Aspergillus parasiticus. To our knowledge, the pigment cluster in C. purpurea is the first fungal secondary metabolite cluster which is induced by high levels of phosphate.
Interestingly, there are also differences when you compare between the expression levels of the cluster genes in the wild-type under inducing conditions (Fig. 5a) and the OE_TF strain under non-inducing culture conditions (Fig. 5b). The key gene, the PKS CPUR_5437 is highly expressed in both strains. However, some cluster genes like CPUR_5431, CPUR_5434 or CPUR_5435 show a considerable higher expression in the wild-type under inducing conditions than in the OE_TF strain under non-inducing conditions. Regulation of secondary metabolite clusters usually occurs on several levels. These results might indicate that at least for some of the pigment cluster genes, regulation by the culture conditions occurs on a higher level than the regulation by the transcription factor. However, it should be taken into account that Northern blots in Fig. 5a, b are two different experiments and therefore cannot be directly compared.
Most likely endocrocin and clavorubin are shunt products in the pathway of xanthone biosynthesis. In the next steps dimerization of different xanthone units would lead to the formation of ergochromes. Genes CPUR_05425, CPUR_05426 and CPUR_05431 are unique to Claviceps in comparison to the A. nidulans xanthone cluster. Thus it is likely that these genes are involved in further modification of xanthone units or in their dimerization. Moreover, the metabolite profile of the OE TF strain (Fig. 7) shows several new peaks where the corresponding m/z value does not fit with any known metabolite. Further investigations of these peaks may lead to the discovery of so far unknown ergochromes of C. purpurea.
Another interesting aspect is that single overexpression of the cluster key gene, the PKS CPUR_5437, is sufficient to induce production of the red anthraquinone pigments. As the cluster is not completely silent under these culture conditions, the expression level of the other cluster genes seems to be enough for the production of the simple pigments endocrocin and clavorubin but not for the complex structures of the ergochromes. It also might be that the increased through-put of the first pathway intermediate leads to a feedback loop and an up-regulation of other pathway genes.
Activation of the pigment cluster in axenic culture opens up new possibilities for uncovering the full biosynthetic pathway and for genetic engineering of the metabolic pathway to improve pigment production, or even to obtain modified molecules with novel bioactivity. Pigments are used as dyes for textiles, in cosmetics and as food colorants. Anthraquinone pigments, like the ones produce by C. purpurea, have several advantages; for example, they are relatively stable and have good light-fastness and brightness . There is increasing commercial interest in the production of pigments by filamentous fungi as sources of cheaper, more ecologically friendly alternatives to existing dyes. Thus fungal pigment synthesis has several advantages over chemical methods [36, 41]. Besides their role as colorants, there is also a pharmaceutical value of ergot pigments, especially of the ergochromes. Ergoflavin, for example, has anti-inflammatory and anticancer properties . Secalonic acid A shows cytostatic and anti-tumor activity  and, additionally, has a neuroprotective effect, making it to an interesting possibility for the treatment of neurodegenerative diseases .
This study reports the identification and characterization of the ergot pigment gene cluster in C. purpurea. This gene cluster has been shown to be responsible for the typical purple-black color of ergot sclerotia but is not required for pathogenicity of the fungus. Furthermore, knock out of the pathway key enzyme, a NR-PKS, finally proved that both groups of ergot pigments, the red anthraquinonecarboxylic acids and the yellow ergochromes, are products of the same biosynthetic pathway. Fungal pigments gain increasing interest as ecologically friendly dyes or for the development of new pharmaceuticals. Activation of the C. purpurea pigment gene cluster in axenic culture and optimization of the culture conditions opens up new possibilities for biotechnological applications.
Strains and culture conditions
Claviceps purpurea strain Ecc93 has been described previously . Mycelia were grown on BII medium  for maintenance and DNA isolation or on Mantle medium for conidia harvesting . For secondary metabolite production strains were cultivated on a rotary shaker at 26 °C in modified media according to Amici  with sucrose and PO4 concentrations as indicated.
Yeast strains FY834  used for the yeast recombinational cloning method were incubated at 30 °C in yeast extract-peptone-dextrose (YPD) or in synthetic dextrose (SD) medium lacking the selecting amino acids.
Chemical and materials
All chemicals were purchased from Sigma-Aldrich GmbH (Seelze, Germany), Carl Roth GmbH + Co. KG (Karlsruhe, Germany), or VWR International GmbH (Darmstadt, Germany). Solvents were obtained in gradient grade quality. Water for HPLC was purified by a Milli-Q Gradient A 10 system (Millipore, Schwalbach, Germany).
Nucleic acid extraction and analysis
Genomic DNA from C. purpurea was isolated as described by Cenis . For Southern blot analysis, 5–10 μg of digested genomic DNA were separated via gel electrophoresis in a 1 % agarose gel with salt-free buffer  and transferred to a nylon membrane (Nytran SPC; Whatman). For the isolation of RNA, the RNAgents total RNA isolation kit (Promega GmbH, Mannheim, Germany) was used. For northern blotting, 20 mg RNA were used for the separation on a 1 % (w/v) agarose gel containing 1 % (v/v) formaldehyde and afterwards transferred to a nylon membrane (Nytran SPC; Whatman). For southern as well as northern hybridization, 32P-labeled probes were generated using the random oligomer-primer method, and hybridized to the membranes. PCR reactions were performed using either the BioTherm Taq DNA Polymerase (GeneCraft, Germany) or the proof reading Phusion DNA polymerase (Finnzymes, Finland). Primers were synthesized by Biolegio (Nijmegen, Netherlands).
Vectors were constructed using the yeast recombinational cloning method , based on the described vector system [49, 50]. The sequences of all primers used are listed in Additional file 4: Table S1. For construction of the cp5433 overexpression vector, the cp5433 gene was amplified with Phusion polymerase with the primers OE_Cp5433_F and OE_Cp5433_R from genomic DNA and recombined with the NotI-NcoI-digested pNAH-OGG vector . For construction of the cp5437 overexpression vector, the gene cp5437 was amplified with the primers OE_PKS4_F and OE_PKS4_R1 and OE_PKS4_F1 and OE_PKS4_R from genomic DNA using Phusion polymerase and recombined with the NotI-NcoI-digested pNAH-OGG vector .
For construction of the cp5437 replacement vector, the flanking regions of cp5437 were amplified with the primers PKS4_5F and PKS4_5R for the 5′ flank as well as PKS4_3F and PKS4_3R for the 3′ flank. Primers contain overlapping sequences toward the yeast shuttle-vector pRS426 or the phleomycin resistance cassette. The phleomycin resistance cassette was amplified with the primers CpBle1F and CpBle1R from pRS426CpBle. The yeast shuttle vector pRS426  was linearized by restriction with XhoI and EcoRI.
For homologous recombination the vector fragments were transformed into yeast strain FY834. The resulting vectors were selected on SD medium lacking uracil. DNA was isolated from yeast cells using the SpeedPrep yeast plasmid isolation kit (DualSystems) and transformed into Escherichia coli TOP10’ for amplification.
Protoplasts of C. purpurea were generated with lysing enzymes from Trichoderma harzianum (Sigma-Aldrich, St. Louis) and transformed with 10 μg of vector DNA as described by Jungehülsing et al. . For selection, either phleomycin was directly applied to the protoplasts (33 μg/mL) or hygromycin was applied to regenerated protoplasts 24 h after transformation by overlay agar (1.5 mg/mL). Resistant colonies were transferred to fresh selective medium (BII, pH 8, 100 μg/mL phleomycin or 0.5 mg/mL hygromycin). By PCR using specific primers as indicated, resistant transformants were checked for the integration of the vector.
Male sterile rye plants (Secale cereale) were cultivated in growth chambers as described by Smit and Tudzynski . Florets of blooming ears (30–40 florets per ear) were inoculated with 5 µL of a suspension containing 2 × 106 conidia/mL as described by Tenberge et al. . Afterwards, the ears were covered with paper bags equipped with cellophane windows to avoid cross contamination.
For reverse transcription of the RNA template, Superscript II reverse transcriptase (Invitrogen, Darmstadt, Germany) was used. Real-time qPCR reactions were performed with the Bio-Rad iQ SYBR Green Supermix and the iCycler Thermal Cycler (Bio-Rad, Hercules, CA, USA). iCycler iQ Real-Time Detection System Software (version 3.0; Bio-Rad) was used for programming, data collection, and analyses. Expression of cp5436 was detected by the primers RTq_LN4_F and RTq_LN4_R and normalized to the expression of the housekeeping genes β-tubulin (CCE34429.1), γ-actin (AEI72275.1), and glyceraldehyde-3-phosphate dehydrogenase (X73282.1)  using primers Actin_uni and Actin_rev, Tub_uni and Tub_rev, and Gpd_uni and Gpd_rev.
Analysis of fungal mycelium
The mycelium was extracted with a mixture of acidified ethyl acetate and water. Water (3 mL) and of organic solvent (4 mL) were added to the mycelia in a 15 mL tube and shaken for 45 min. In the next step, phases were separated and the organic solvent evaporated under nitrogen at 30 °C. Residue was dissolved in 700 µL acetonitrile/water 1/9 (v/v) and 15 µL used for HPLC injection.
In order to identify the pigments, the extracted mycelia were measured by RP–HPLC–DAD–HRMS. For the HRMS measurement, an Accela LC 60057-60010 system (Thermo Fisher Scientific, Bremen, Germany) was linked to a LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific). A SPD-M20A Shimadzu PDA Detector (Shimadzu, Duisburg, Germany) was coupled to the MS spectrometer. Data acquisition was performed with Xcalibur 2.07 SP1 (Thermo Scientific). Separation was carried out on a 150 × 2 i.d., 3 μm, ReproSil-Pur C18-AQ (Dr. Maisch GmbH, Amerbuch, Germany) using a binary gradient at a column temperature of 40 °C. The injection volume was 15 µL and the autosampler was cooled to 7 °C. The flow rate was set to 260 µL/min. Solvent A was acetonitrile with 0.1 % of formic acid (v/v) and solvent B was water with 0.1 % of formic acid (v/v). The HPLC was programmed as follows: in the first 4 min isocratic 15 % of A, afterwards a binary gradient to 40 % in 21 min and next during 16 min up to 100 % of A. Then the column was washed with 100 % A and equilibrated at starting conditions. For the detection of the expected pigments a total ion scan of a mass range from m/z 165–950 with a resolution of 60,000 in the negative ion mode was used. To confirm the substances, subsequent mass spectrometric fragmentation experiments in the negative mode were used. The experiments included high-energy collision dissociation (HCD) with a relative energy of 40–85 %, depending on the ionization and an isolation width of m/z 1.5 with and activation time of 30 ms. The fragments were analyzed with the Orbitrap detector at a resolution of 30,000.
Mass spectrometer and DAD parameters
The LTQ Orbitrap XL was used with a heated electrospray ionization technique. The sheat gas flow was 30 arbitrary units, the aux gas flow 15 arbitrary units and the sweep gas flow 10 arbitrary units. In the negative mode, vaporizer temperature was set to 300 °C and capillary temperature to 270 °C. The source voltage was 3.0 kV, capillary voltage −33 V and Tube Lens −160 V.
The Shimadzu PDA-Detector had the following parameters: starting wavelength 200 nm, ending wavelength 700 nm, with a wavelength step of 4 nm. The sampling frequency was consequently 4.16 Hz.
Availability of supporting data
The data sets supporting the results of this article are included within the article and its additional files. Gene sequences are available online: http://pedant.gsf.de/pedant3htmlview/pedant3view?Method=analysis&Db=p3_p76493_Cla_purpu.
LN, JD, H-UH, PT: conceptualization and methodology; LN: generation and analyses of mutants, gene expression studies; JD: HPLC–MS measurements; LN, JD: writing the manuscript, LN, JD, H-UH, PT: review and editing; PT, H-UH funding acquisition, resources and supervision. All authors read and approved the final manuscript.
We thank the Deutsche Forschungsgemeinschaft (DFG) for funding (Grants Tu50/18-1 and Hu730/11-1) and Peter G. Mantle for critical reading of the manuscript.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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