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Heterothallism in Cordyceps takaomontana
FEMS Microbiology Letters 250 (2005) 145–150 www.fems-microbiology.org
Heterothallism in Cordyceps takaomontana Eiji Yokoyama a
a,*
, Kenzo Yamagishi b, Akira Hara
c
The Agricultural High-Tech Research Center, Meijo University, Tempaku-ku, Nagoya 468-8502, Japan b Laboratory of Entomology, Meijo University, Tempaku-ku, Nagoya 468-8502, Japan c Laboratory of Biological Chemistry, Meijo University, Tempaku-ku, Nagoya 468-8502, Japan Received 10 May 2005; received in revised form 28 June 2005; accepted 5 July 2005 First published online 19 July 2005 Edited by M.J. Bidochka
Abstract Perithecium formation of an entomopathogenic fungus Cordyceps takaomontana was promoted by treating the mycelia with cell wall-degrading enzymes and PEG 4000. Perithecia were formed in the mixed culture of both mating-type strains MAT1 and MAT2, and not in the culture of MAT1 or MAT2 alone. The MAT1 strains did not possess a mating-type gene MAT1-1-3, but could produce perithecia. These results strongly suggested that C. takaomontana is heterothallic, and does not need MAT1-1-3 for the perithecium formation. MAT1-1-3 was also not found in another entomopathogenic fungus Cordyceps militaris. On the other hand, phytopathogenic fungi Balansia sp., Claviceps purpurea and Epichloe¨ typhina possessed MAT1-1-3. The structures of mating-type locus MAT1-1 of these phytopathogenic fungi in the family Clavicipitaceae were similar to that of a phytopathogenic fungus Gibberella fujikuroi in the family Nectriaceae, which is closely related to Clavicipitaceae. These results suggested that phytopathogen might be more ancestral group than entomopathogen in Clavicipitaceae, and that MAT1-1-3 might be lost in the course of the host shift from plants to insects. 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Balansia; Claviceps; Cordyceps; Epichloe¨; Mating type; Perithecium formation
1. Introduction Clavicipitaceae is a family of the order Hypocreales in the class Pyrenomycetes of the phylum Ascomycota. Clavicipitaceous fungi and related anamorphs contain diverse econutritional groups [1] such as pathogens of plants, insects, spiders, nematodes and fungi; endophytes of plants; yeast-like endosymbionts of insects; and saprophytes. Besides the interest in their various lifestyles, many clavicipitaceous fungi and related anamorphs are useful for industry and agriculture. They *
Corresponding author. Tel.: +81 52 832 1151; fax: +81 52 835 7450. E-mail address: [email protected] (E. Yokoyama).
are the potential sources for bioactive molecules and medicines such as ergot alkaloids from Claviceps purpurea, cordycepin from Cordyceps militaris and cyclosporin from Cordyceps subsessilis. Entomopathogenic fungi such as Cordyceps brongniartii are used as microbial insecticides. Nematophagous fungi such as Cordyceps chlamydosporia also have great potential as biocontrol agents. Anamorphic endophytes of grasses such as Ephelis japonica confer resistance to herbivory, parasitism and drought, and are expected to be valuable for golf links and parks. But the difficulties in artificially producing perithecioid ascomata and their unidentified lifecycles hinder their usefulness. Construction of the mating system would provide a clue to solving these problems and the anamorph-teleomorph connection.
0378-1097/$22.00 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2005.07.004
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Recently, the mating-type loci have been analyzed mainly in the phytopathogenic fungi belonging to the classes Discomycetes, Loculoascomycetes and Pyrenomycetes. The mating-type genes of Aspergillus species of the class Plectomycetes have been reported [2,3]. Fungal mating type and mating-type genes were reviewed by Coppin et al. [4], Kronstad and Staben [5], Po¨ggeler [6], Turgeon [7] and Turgeon and Yoder [8]. The mating types MAT1 and MAT2 are determined by the single mating-type locus MAT1. Alleles for MAT1 and MAT2 are MAT1-1 and MAT1-2, respectively. Although the flanking regions of MAT1-1 and MAT12 are homologous, the nucleotide sequences of MAT11 and MAT1-2 are highly dissimilar. The term ‘‘idiomorph’’ instead of ‘‘allele’’ is usually used for MAT1-1 and MAT1-2 [9]. Heterothallic fungi have one of the idiomorphs MAT1-1 or MAT1-2. On the other hand, homothallic fungi have both idiomorphs, namely MAT1-1/2. But there are several exceptions; the MAT A (MAT1-1) strains of some Neurospora species are homothallic [10], and the MAT1-1/2 strains of some Ceratocystis species act as MAT2 [11]. So the genotype of mating type is not always consistent with its phenotype. Entomopathogenic fungus Cordyceps takaomontana [12] (anamorph: Paecilomyces tenuipes [13]) has been used as a traditional medicine in Korea and a source of bioactive molecules [14]. Recently, we reported that the structures of mating-type loci of C. takaomontana are similar to those of other heterothallic fungi [15]. In this study, we constructed a perithecium formation system of C. takaomontana, and confirmed its heterothallism by perithecium formation ability.
2. Materials and methods 2.1. Fungal strains C. takaomontana IFO 31161, Claviceps purpurea NBRC 32971 and Epichloe¨ typhina MAFF 511345 were purchased from the Institute for Fermentation, Osaka (Japan), NITE Biological Resource Center (Japan) and Ministry of Agriculture, Forestry and Fisheries Genebank (Japan), respectively. Balansia sp. (anamorph: Ephelis japonica) NIAES 6588 was kindly provided by Dr. Takao Tsukiboshi (National Institute of Agro-Environmental Sciences, Japan). C. militaris BCMU CM03, which was collected by Mr. Nobuo Sugiyama (The entomogenous fungi club, Japan), and C. takaomontana BCMU IJ20, BCMU IJ21 and BCMU IJ25 were isolated in previous studies [1,15]. C. takaomontana BCMU IJ774-1 and BCMU IJ774-2 were re-isolated from single ascospores of a strain NIFTS HF774 (mixture of MAT1-1 and MAT1-2 cells), which was kindly provided
by Dr. Fumio Ihara (National Institute of Fruit Technology Sciences, Japan). 2.2. Perithecium formation system Mating system of the genus Cordyceps has not been established yet. Although mating type was not taken into account, perithecium production in the laboratory has been reported for some species such as C. militaris [16]. On the other hand, the characteristics of asexual synnema production were fully examined in some species, including C. takaomontana [17,18]. Light, CO2 concentration and temperature are generally important factors in both perithecium and synnema production of Cordyceps and related anamorphs. Recently, blue light was reported to promote asexual reproduction of the entomopathogenic fungus Paecilomyces fumosoroseus [19]. In preliminary experiments, we tested the mixed cultivations of the MAT1-1 and MAT1-2 strains started from conidia, colonies on agar plate or mycelia in liquid medium, but perithecia could not be formed. Cell fusion in plasmogamy is one of the most critical events in mating [20]. It occurred to us that an artificial cell fusion might trigger a teleomorphic development. So we examined the mixed cultivation started from protoplasts with PEG 4000. The MAT1-1 and MAT1-2 strains were cultivated independently in Potato Dextrose Broth (Difco) for three days. When both mating-type strains were cocultivated in a liquid medium, the population of final culture biased to either of mating-type strains by a possible mating-type associated vegetative incompatibility [21]. Mycelia were treated with the cell wall-degrading enzymes, Yatalase (2 mg ml1, Ozeki, Japan) and Glucanex (5 mg ml1, Novozyme), in 1.2 M solbitol and 10 mM sodium phosphate buffer (pH 7.0) for 2 h. In this condition, the mycelia were partially converted to protoplasts; prolonged incubation did not improve the protoplast formation. Mycelia containing protoplasts were re-suspended in 10 mM CaCl2, 1.2 M solbitol and 10 mM Tris–HCl buffer (pH 8.0). The MAT1-1 and MAT1-2 cells were mixed at the same ratio. Then three volumes of 40% PEG 4000, 10 mM CaCl2, 1.2 M solbitol and 10 mM Tris–HCl buffer (pH 8.0) were gently added. One milliliter of the protoplast mixture was inoculated on a rolled barley medium (20 g of rolled barley, Oshimugi (Aeon, Japan), was soaked in 30 ml of 0.1% Yeast Extract (Difco), and then autoclaved) in a plant culture jar, Agripot (Iwaki, Japan). The jar was incubated at 20 C and 80% relative humidity under the cycle of 16 h of light with a blue filter (No. 183, Lee) and 8 h of darkness. Perithecia appeared on stromata (citron yellow) after about five weeks (Fig. 1(a)). In our perithecium formation system, perithecia first formed as half-immersed and ovoid (Fig. 1(b)), and then they became superficial and obpyriform
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Fig. 1. Teleomorph formation of C. takaomontana (BCMU IJ25 and BCMU IJ774-2). (a) Stroma carrying perithecia; (b) young perithecium; (c) perithecium; (d) smashed perithecium and asci; (e) ascus including ascospores. Bars indicate 1 mm (a), 100 lm (b–d) and 10 lm (e).
(480–600 lm · 200–250 lm, Fig. 1(c)). Each perithecium contained abundant asci (Fig. 1(d)). Mature ascus (cylindrical, 300–350 lm · 4–6 lm) included eight ascospores (Fig. 1(e)). The shape of the ascospore was filiform to somewhat bifusiform. In the original report of C. takaomontana [12], perithecium, ascus and ascospore were described as ‘‘ovoidea 500–570 l longa 230–300 l crassa’’, ‘‘cylindracei ad 360 l longi 3.5– 4.5 l crassi’’ and ‘‘minutissimi cylindracei’’, respectively. There were some minor morphological disagreements between the original descriptions and the data obtained in this study, which were considered to be derived from the maturity of the examined samples. A possibility that morphological characters of the samples formed in laboratory might be different from those in the field could not be excluded. We do not intend to perform a taxonomic revision in this study. Extensive taxonomic work of C. takaomontana is under progress by Mr. Shigeru Uchiyama, Japan Society of Cordyceps Research (personal communication). 2.3. DNA extraction, PCR and sequencing Genomic DNA was extracted using the Isoplant II kit (Nippon Gene, Japan). PCR-based mating-type assay was performed as described previously [1]. The ITS region was amplified by PCR using Ex Taq DNA polymerase (Takara, Japan) using a primer set [5 0 -GATTGAATGGCTCAGTGAGG and 5 0 -TTACTGGGGCAATCCCTGTT]. In the amplification of mating-type locus MAT1-1 a primer set [5 0 -GATGCGGAACGTTTATCTGG and 5 0 -CCCATCTC(A/G)TC(A/C)CGGAC(A/G)AA(C/G)GA] was used. Nucleotide sequences of the PCR products were determined on the both strands using DYEnamic ET Terminator Cycle Sequencing kit (Amersham Biosciences) and ABI PRISM 310 Genetic Analyzer (Perkin–Elmer). 2.4. Phylogenetic analysis Phylogenetic analysis was performed by the heuristic search of the maximum parsimony method of PAUP ver.4.0b10 (Sinauer Associates) using the same settings in the previous study [1]. The bootstrap tests were conducted with 1000 resamplings.
2.5. Nucleotide sequence accession numbers The nucleotide sequence of the ITS region of C. takaomontana NIFTS HF774 was deposited in the DDBJ/ EMBL/GenBank databases with the accession number AB189444. The nucleotide sequences of MAT1-1 were also deposited with the accession numbers AB194984 (Balansia sp. NIAES 6588), AB194983 (C. purpurea NBRC 32971), AB194982 (C. militaris BCMU CM03), AB211979 (C. takaomontana BCMU IJ774-1) and AB194985 (E. typhina MAFF 511345).
3. Results and discussion 3.1. Perithecium formation system of C. takaomontana We succeeded in forming the perithecia of C. takaomontana by the treatment of mycelia with cell walldegrading enzymes and PEG 4000. Mating occurs between a female ascogonium and a male antheridium [20]. A possible mating-type associated vegetative incompatibility was observed in C. takaomontana, but vegetative incompatibility can be bypassed by protoplast fusion in some fungi [21], including Cordyceps bassiana [22]. It was uncertain whether the perithecia formed in this study were derived from just a cell fusion or a mating. So we used the term ‘‘perithecium formation’’ but not ‘‘mating’’ in this study. Without the treatment with the cell wall-degrading enzymes and PEG 4000, perithecium formation was hardly observed even in the combinations using progenies from ascospores. This indicated that the cell wall-degrading enzymes and PEG 4000 play an important role in the perithecium formation system. The structure of the cell wall was supposed to be one of the causes of the difficulties in perithecium formation. C. takaomontana is usually found as an entomopathogen. But the addition of pupal powder, Sanagiko (Marukyu, Japan), to the rolled barley medium increased anamorphic synnema rather than teleomorphic perithecium production. Additions of Casamino acid (Difco) or Peptone (Difco) gave similar results to that of the pupal powder. So perithecium formation does not require a nutrient-rich condition. Under our conditions, the rolled barley is the most important component
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for perithecium formation. We did not observe any perithecium in the cultures on agar-solidified media. 3.2. Perithecium formation test We performed the mixed cultivations using the strains; MAT1-1 (BCMU IJ21, BCMU IJ25 and BCMU IJ774-1) and MAT1-2 (IFO 31161, BCMU IJ20 and BCMU IJ774-2) listed in Table 1, and examined the perithecium formation ability. All strains used in this study could produce conidia and were asexually fertile. All strains except for IFO 31161 could often produce stromata by itself. Four strains BCMU IJ20, BCMU IJ25, BCMU IJ774-1 and BCMU IJ774-2 could produce perithecia with opposite mating-type strains, and their mating-type phenotypes were MAT2, MAT1, MAT1 and MAT2, respectively. Perithecia were formed in the mixed culture of both mating-type strains MAT1 and MAT2, and not in the culture of MAT1 or MAT2 alone. The combinations using these four strains gave high perithecium formation efficiency (more than 80%). Perithecium formation efficiency was calculated as (the number of mixed cultivation succeeded in perithecium formation)/(the number of mixed cultivation) · 100. Table 1 Perithecium formation test of C. takaomontana Mixed cultivation
Perithecium formationa
BCMU IJ21 (MAT1-1) and BCMU IJ25 (MAT1-1) BCMU IJ21 (MAT1-1) and BCMU IJ774-1 (MAT1-1) BCMU IJ25 (MAT1-1) and BCMU IJ774-1 (MAT1-1) BCMU IJ20 (MAT1-2) and BCMU IJ774-2 (MAT1-2) BCMU IJ20 (MAT1-2) and IFO 31161 (MAT1-2) BCMU IJ774-2 (MAT1-2) and IFO 31161 (MAT1-2) BCMU IJ21 (MAT1-1) and BCMU IJ20 (MAT1-2) BCMU IJ21 (MAT1-1) and BCMU IJ774-2 (MAT1-2) BCMU IJ21 (MAT1-1) and IFO 31161 (MAT1-2) BCMU IJ25 (MAT1-1) and BCMU IJ20 (MAT1-2) BCMU IJ25 (MAT1-1) and BCMU IJ774-2 (MAT1-2) BCMU IJ25 (MAT1-1) and IFO 31161 (MAT1-2) BCMU IJ774-1 (MAT1-1) and BCMU IJ20 (MAT1-2) BCMU IJ774-1 (MAT1-1) and BCMU IJ774-2 (MAT1-2) BCMU IJ774-1 (MAT1-1) and IFO 31161 (MAT1-2)
0/10
a
Perithecium formation was examined in 10 trials.
0/10 0/10 0/10 0/10
To confirm that the perithecia formed in the combination of BCMU IJ25 and BCMU IJ774-2 were derived from both mating-type cells, we examined the mating type of progenies. The ascus of C. takaomontana is thin and cylindrical (diameter is about 6 lm, Fig. 1(e)); we could not tear it with a micromanipulator. A stroma carrying perithecia was broken up with a needle, and then a perithecium was smashed with a cover glass. Single ascospores were transferred to Potato Dextrose Agar (Difco) plate, and then their mating-type genotypes were determined. Both mating-type ascospores were detected in all examined 10 perithecia. We could not destroy asci completely and most ascospores could not be dispersed, so the exact ratio of mating type of progenies could not be calculated. The ratios of MAT1-1 to MAT1-2 in 10 perithecia varied from 0.5 to 2. These results strongly suggested that C. takaomontana is heterothallic, consistent with our previous study which concluded that the structures of the mating-type loci of C. takaomontana are similar to those of other heterothallic fungi [15]. Teleomorphic ascospores could be formed in the combination of BCMU IJ20 and BCMU IJ25, which were derived from anamorphic conidia. This suggested that teleomorph can be artificially developed using adequate combination partners, and that the mating-type method can be applied to the elucidation of teleomorph–anamorph connection. On the other hand, the combinations using IFO 31161 or BCMU IJ21, which were also derived from conidia, could not produce perithecia. To confirm the phylogenetic positions of the strains used in this study, a phylogenetic analysis based on the nucleotide sequence of the ITS1-5.8S rDNA-ITS2 region was performed (Fig. 2). The nucleotide sequences, except for in the case of C. takaomontana NIFTS HF774, were determined in previous studies [1,15,23]. The unambiguously aligned 497 positions were analyzed. Entomopathogenic fungi Cordyceps sinclairii and Paecilomyces fumosoroseus are closely related to
0/10 0/10 0/10 0/10 8/10 10/10 0/10 8/10 10/10 0/10
Fig. 2. Phylogenetic analysis of C. takaomontana based on the ITS region. The bootstrap values (%) were indicated on each nodes. Tree length = 26, CI = 1.00, HI = 0.00, RI = 1.00, RC = 1.00. The nucleotide sequences indicated by a, b and c were determined in previous studies [1,15,23].
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Fig. 3. Structures of the mating-type locus MAT1-1 in Clavicipitaceae. The nucleotide sequences indicated by a and b were determined in previous studies [15,29].
C. takaomontana [1], and were used as outgroup. All strains used in this study were closely related. So the ability to produce perithecia can be lost in a short lineage. 3.3. MAT1-1-3 gene in the mating-type locus MAT1-1 The structure of mating-type locus MAT1-1 differs among the fungal classes [8]. Loculoascomycetes has a single conserved gene MAT1-1-1, which encodes a-box transcriptional regulator. Pyrenomycetes usually possesses two additional mating-type genes MAT1-1-2 and MAT1-1-3, which encode amphipathic-helix and HMG-box transcriptional regulators, respectively. Discomycetes has also three MAT1-1 genes like Pyrenomycetes, but MAT1-1-2 is replaced by MAT1-1-4, which encodes a metallothionein. On the other hand, the MAT1-2 structure is conserved well in the filamentous fungi [8]. All MAT1-2 loci reported so far, except for that of Neurospora crassa, have a single conserved gene MAT1-2-1, which encodes HMG-box transcriptional regulator. In Neurospora crassa, a second MAT1-2 gene, mat a-2, has been reported [24]. Previously, we reported the mating-type loci of C. takaomontana of the strains derived from anamorphic conidia [15]. MAT1-2 has a single gene MAT1-2-1 like other pyrenomycetous fungi. But MAT1-1 contains only two genes MAT1-1-1 and MAT1-1-2. MAT1-1-3 could not be found in the strains BCMU IJ21 and BCMU IJ25. In this study, the MAT1-1 sequence of BCMU IJ774-1, which was derived from a teleomorphic ascospore, was determined. The structure of MAT1-1 of BCMU IJ774-1 was similar to that of BCMU IJ25 (Fig. 3). MAT1-1-3 could also not be found in BCMU IJ774-1. BCMU IJ25 and BCMU IJ774-1 could form abundant asci containing ascospores. These results indicated that C. takaomontana does not need MAT1-1-3 for the perithecium formation. Sordaria macrospora SMT A-3 does not have an HMG-box [25], and is supposed not to be a typical MAT1-1-3. A Neurospora crassa mat A-3 (MAT1-1-3) mutant can mate and produce abundant asci, for MAT A-2 (MAT1-1-2) and MAT
A-3 (MAT1-1-3) have redundant roles [26]. On the other hand, in Podospora anserina the SMR2 (MAT1-1-3) mutant can mate but produces reduced and abnormal asci [27]. SMR1 (MAT1-1-2) and SMR2 (MAT1-1-3) are involved in the initial development of biparental ascogenous cells [27] and the internuclear recognition [28], respectively. Thus the importance of MAT1-1-3 homologues in perithecium formation is variable in Pyrenomycetes. We determined the nucleotide sequences of MAT1-1 of another entomopathogenic fungus C. militaris BCMU CM03 and of three phytopathogenic fungi Balansia sp. NIAES 6588, Claviceps purpurea NBRC 32971 and Epichloe¨ typhina MAFF 511345 (Fig. 3). Entomopathogenic fungus C. militaris BCMU CM03, which was derived from a perithecium, did not have MAT1-1-3 like C. takaomontana. On the other hand, three phytopathogenic fungi possessed MAT1-1-3 between the DNA lyase gene and MAT1-1-2. The deduced amino acid sequences of MAT1-1-3 of these three species had a high identity (more than 42% in the aligned 143 amino acid residues) with that of Gibberella fujikuroi [29]. As a result of protein profile analysis using Pfam [30], three MAT1-1-3 proteins deduced in this study belonged to the family of HMG protein. The lengths between DNA lyase gene and MAT1-1-2 of the phytopathogenic fungi were longer than those of the entomopathogenic fungi. The structures of MAT1-1 of the phytopathogenic clavicipitaceous fungi were similar to that of a phytopathogenic fungus G. fujikuroi [29] in the family Nectriaceae, which is closely related to Clavicipitaceae. These results are consistent with our previous study which reported that the phytopathogen might be more ancestral group than the entomopathogen in Clavicipitaceae as a result of the phylogenetic analysis of MAT1-2-1 [1], and suggested that MAT1-1-3 might be lost in the course of the host shift from plants to insects. But the relationship between loss of MAT11-3 and pathogeny is still unknown. The deletion of mating-type locus has been reported in some Ceratocystis species [11].
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Acknowledgements This work was supported by the Agricultural HighTech Research Center, Meijo University under the ‘‘Environmental Control through the Function of Microorganisms’’ project. We are grateful to Mr. N. Sugiyama (The entomogenous fungi club, Japan), Dr. T. Tsukiboshi (National Institute of Agro-Environmental Sciences, Japan) and Dr. F. Ihara (National Institute of Fruit Technology Sciences, Japan) for providing fungal materials.
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Heterothallism in Cordyceps takaomontana Eiji Yokoyama a
a,*
, Kenzo Yamagishi b, Akira Hara
c
The Agricultural High-Tech Research Center, Meijo University, Tempaku-ku, Nagoya 468-8502, Japan b Laboratory of Entomology, Meijo University, Tempaku-ku, Nagoya 468-8502, Japan c Laboratory of Biological Chemistry, Meijo University, Tempaku-ku, Nagoya 468-8502, Japan Received 10 May 2005; received in revised form 28 June 2005; accepted 5 July 2005 First published online 19 July 2005 Edited by M.J. Bidochka
Abstract Perithecium formation of an entomopathogenic fungus Cordyceps takaomontana was promoted by treating the mycelia with cell wall-degrading enzymes and PEG 4000. Perithecia were formed in the mixed culture of both mating-type strains MAT1 and MAT2, and not in the culture of MAT1 or MAT2 alone. The MAT1 strains did not possess a mating-type gene MAT1-1-3, but could produce perithecia. These results strongly suggested that C. takaomontana is heterothallic, and does not need MAT1-1-3 for the perithecium formation. MAT1-1-3 was also not found in another entomopathogenic fungus Cordyceps militaris. On the other hand, phytopathogenic fungi Balansia sp., Claviceps purpurea and Epichloe¨ typhina possessed MAT1-1-3. The structures of mating-type locus MAT1-1 of these phytopathogenic fungi in the family Clavicipitaceae were similar to that of a phytopathogenic fungus Gibberella fujikuroi in the family Nectriaceae, which is closely related to Clavicipitaceae. These results suggested that phytopathogen might be more ancestral group than entomopathogen in Clavicipitaceae, and that MAT1-1-3 might be lost in the course of the host shift from plants to insects. 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Balansia; Claviceps; Cordyceps; Epichloe¨; Mating type; Perithecium formation
1. Introduction Clavicipitaceae is a family of the order Hypocreales in the class Pyrenomycetes of the phylum Ascomycota. Clavicipitaceous fungi and related anamorphs contain diverse econutritional groups [1] such as pathogens of plants, insects, spiders, nematodes and fungi; endophytes of plants; yeast-like endosymbionts of insects; and saprophytes. Besides the interest in their various lifestyles, many clavicipitaceous fungi and related anamorphs are useful for industry and agriculture. They *
Corresponding author. Tel.: +81 52 832 1151; fax: +81 52 835 7450. E-mail address: [email protected] (E. Yokoyama).
are the potential sources for bioactive molecules and medicines such as ergot alkaloids from Claviceps purpurea, cordycepin from Cordyceps militaris and cyclosporin from Cordyceps subsessilis. Entomopathogenic fungi such as Cordyceps brongniartii are used as microbial insecticides. Nematophagous fungi such as Cordyceps chlamydosporia also have great potential as biocontrol agents. Anamorphic endophytes of grasses such as Ephelis japonica confer resistance to herbivory, parasitism and drought, and are expected to be valuable for golf links and parks. But the difficulties in artificially producing perithecioid ascomata and their unidentified lifecycles hinder their usefulness. Construction of the mating system would provide a clue to solving these problems and the anamorph-teleomorph connection.
0378-1097/$22.00 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2005.07.004
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Recently, the mating-type loci have been analyzed mainly in the phytopathogenic fungi belonging to the classes Discomycetes, Loculoascomycetes and Pyrenomycetes. The mating-type genes of Aspergillus species of the class Plectomycetes have been reported [2,3]. Fungal mating type and mating-type genes were reviewed by Coppin et al. [4], Kronstad and Staben [5], Po¨ggeler [6], Turgeon [7] and Turgeon and Yoder [8]. The mating types MAT1 and MAT2 are determined by the single mating-type locus MAT1. Alleles for MAT1 and MAT2 are MAT1-1 and MAT1-2, respectively. Although the flanking regions of MAT1-1 and MAT12 are homologous, the nucleotide sequences of MAT11 and MAT1-2 are highly dissimilar. The term ‘‘idiomorph’’ instead of ‘‘allele’’ is usually used for MAT1-1 and MAT1-2 [9]. Heterothallic fungi have one of the idiomorphs MAT1-1 or MAT1-2. On the other hand, homothallic fungi have both idiomorphs, namely MAT1-1/2. But there are several exceptions; the MAT A (MAT1-1) strains of some Neurospora species are homothallic [10], and the MAT1-1/2 strains of some Ceratocystis species act as MAT2 [11]. So the genotype of mating type is not always consistent with its phenotype. Entomopathogenic fungus Cordyceps takaomontana [12] (anamorph: Paecilomyces tenuipes [13]) has been used as a traditional medicine in Korea and a source of bioactive molecules [14]. Recently, we reported that the structures of mating-type loci of C. takaomontana are similar to those of other heterothallic fungi [15]. In this study, we constructed a perithecium formation system of C. takaomontana, and confirmed its heterothallism by perithecium formation ability.
2. Materials and methods 2.1. Fungal strains C. takaomontana IFO 31161, Claviceps purpurea NBRC 32971 and Epichloe¨ typhina MAFF 511345 were purchased from the Institute for Fermentation, Osaka (Japan), NITE Biological Resource Center (Japan) and Ministry of Agriculture, Forestry and Fisheries Genebank (Japan), respectively. Balansia sp. (anamorph: Ephelis japonica) NIAES 6588 was kindly provided by Dr. Takao Tsukiboshi (National Institute of Agro-Environmental Sciences, Japan). C. militaris BCMU CM03, which was collected by Mr. Nobuo Sugiyama (The entomogenous fungi club, Japan), and C. takaomontana BCMU IJ20, BCMU IJ21 and BCMU IJ25 were isolated in previous studies [1,15]. C. takaomontana BCMU IJ774-1 and BCMU IJ774-2 were re-isolated from single ascospores of a strain NIFTS HF774 (mixture of MAT1-1 and MAT1-2 cells), which was kindly provided
by Dr. Fumio Ihara (National Institute of Fruit Technology Sciences, Japan). 2.2. Perithecium formation system Mating system of the genus Cordyceps has not been established yet. Although mating type was not taken into account, perithecium production in the laboratory has been reported for some species such as C. militaris [16]. On the other hand, the characteristics of asexual synnema production were fully examined in some species, including C. takaomontana [17,18]. Light, CO2 concentration and temperature are generally important factors in both perithecium and synnema production of Cordyceps and related anamorphs. Recently, blue light was reported to promote asexual reproduction of the entomopathogenic fungus Paecilomyces fumosoroseus [19]. In preliminary experiments, we tested the mixed cultivations of the MAT1-1 and MAT1-2 strains started from conidia, colonies on agar plate or mycelia in liquid medium, but perithecia could not be formed. Cell fusion in plasmogamy is one of the most critical events in mating [20]. It occurred to us that an artificial cell fusion might trigger a teleomorphic development. So we examined the mixed cultivation started from protoplasts with PEG 4000. The MAT1-1 and MAT1-2 strains were cultivated independently in Potato Dextrose Broth (Difco) for three days. When both mating-type strains were cocultivated in a liquid medium, the population of final culture biased to either of mating-type strains by a possible mating-type associated vegetative incompatibility [21]. Mycelia were treated with the cell wall-degrading enzymes, Yatalase (2 mg ml1, Ozeki, Japan) and Glucanex (5 mg ml1, Novozyme), in 1.2 M solbitol and 10 mM sodium phosphate buffer (pH 7.0) for 2 h. In this condition, the mycelia were partially converted to protoplasts; prolonged incubation did not improve the protoplast formation. Mycelia containing protoplasts were re-suspended in 10 mM CaCl2, 1.2 M solbitol and 10 mM Tris–HCl buffer (pH 8.0). The MAT1-1 and MAT1-2 cells were mixed at the same ratio. Then three volumes of 40% PEG 4000, 10 mM CaCl2, 1.2 M solbitol and 10 mM Tris–HCl buffer (pH 8.0) were gently added. One milliliter of the protoplast mixture was inoculated on a rolled barley medium (20 g of rolled barley, Oshimugi (Aeon, Japan), was soaked in 30 ml of 0.1% Yeast Extract (Difco), and then autoclaved) in a plant culture jar, Agripot (Iwaki, Japan). The jar was incubated at 20 C and 80% relative humidity under the cycle of 16 h of light with a blue filter (No. 183, Lee) and 8 h of darkness. Perithecia appeared on stromata (citron yellow) after about five weeks (Fig. 1(a)). In our perithecium formation system, perithecia first formed as half-immersed and ovoid (Fig. 1(b)), and then they became superficial and obpyriform
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Fig. 1. Teleomorph formation of C. takaomontana (BCMU IJ25 and BCMU IJ774-2). (a) Stroma carrying perithecia; (b) young perithecium; (c) perithecium; (d) smashed perithecium and asci; (e) ascus including ascospores. Bars indicate 1 mm (a), 100 lm (b–d) and 10 lm (e).
(480–600 lm · 200–250 lm, Fig. 1(c)). Each perithecium contained abundant asci (Fig. 1(d)). Mature ascus (cylindrical, 300–350 lm · 4–6 lm) included eight ascospores (Fig. 1(e)). The shape of the ascospore was filiform to somewhat bifusiform. In the original report of C. takaomontana [12], perithecium, ascus and ascospore were described as ‘‘ovoidea 500–570 l longa 230–300 l crassa’’, ‘‘cylindracei ad 360 l longi 3.5– 4.5 l crassi’’ and ‘‘minutissimi cylindracei’’, respectively. There were some minor morphological disagreements between the original descriptions and the data obtained in this study, which were considered to be derived from the maturity of the examined samples. A possibility that morphological characters of the samples formed in laboratory might be different from those in the field could not be excluded. We do not intend to perform a taxonomic revision in this study. Extensive taxonomic work of C. takaomontana is under progress by Mr. Shigeru Uchiyama, Japan Society of Cordyceps Research (personal communication). 2.3. DNA extraction, PCR and sequencing Genomic DNA was extracted using the Isoplant II kit (Nippon Gene, Japan). PCR-based mating-type assay was performed as described previously [1]. The ITS region was amplified by PCR using Ex Taq DNA polymerase (Takara, Japan) using a primer set [5 0 -GATTGAATGGCTCAGTGAGG and 5 0 -TTACTGGGGCAATCCCTGTT]. In the amplification of mating-type locus MAT1-1 a primer set [5 0 -GATGCGGAACGTTTATCTGG and 5 0 -CCCATCTC(A/G)TC(A/C)CGGAC(A/G)AA(C/G)GA] was used. Nucleotide sequences of the PCR products were determined on the both strands using DYEnamic ET Terminator Cycle Sequencing kit (Amersham Biosciences) and ABI PRISM 310 Genetic Analyzer (Perkin–Elmer). 2.4. Phylogenetic analysis Phylogenetic analysis was performed by the heuristic search of the maximum parsimony method of PAUP ver.4.0b10 (Sinauer Associates) using the same settings in the previous study [1]. The bootstrap tests were conducted with 1000 resamplings.
2.5. Nucleotide sequence accession numbers The nucleotide sequence of the ITS region of C. takaomontana NIFTS HF774 was deposited in the DDBJ/ EMBL/GenBank databases with the accession number AB189444. The nucleotide sequences of MAT1-1 were also deposited with the accession numbers AB194984 (Balansia sp. NIAES 6588), AB194983 (C. purpurea NBRC 32971), AB194982 (C. militaris BCMU CM03), AB211979 (C. takaomontana BCMU IJ774-1) and AB194985 (E. typhina MAFF 511345).
3. Results and discussion 3.1. Perithecium formation system of C. takaomontana We succeeded in forming the perithecia of C. takaomontana by the treatment of mycelia with cell walldegrading enzymes and PEG 4000. Mating occurs between a female ascogonium and a male antheridium [20]. A possible mating-type associated vegetative incompatibility was observed in C. takaomontana, but vegetative incompatibility can be bypassed by protoplast fusion in some fungi [21], including Cordyceps bassiana [22]. It was uncertain whether the perithecia formed in this study were derived from just a cell fusion or a mating. So we used the term ‘‘perithecium formation’’ but not ‘‘mating’’ in this study. Without the treatment with the cell wall-degrading enzymes and PEG 4000, perithecium formation was hardly observed even in the combinations using progenies from ascospores. This indicated that the cell wall-degrading enzymes and PEG 4000 play an important role in the perithecium formation system. The structure of the cell wall was supposed to be one of the causes of the difficulties in perithecium formation. C. takaomontana is usually found as an entomopathogen. But the addition of pupal powder, Sanagiko (Marukyu, Japan), to the rolled barley medium increased anamorphic synnema rather than teleomorphic perithecium production. Additions of Casamino acid (Difco) or Peptone (Difco) gave similar results to that of the pupal powder. So perithecium formation does not require a nutrient-rich condition. Under our conditions, the rolled barley is the most important component
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for perithecium formation. We did not observe any perithecium in the cultures on agar-solidified media. 3.2. Perithecium formation test We performed the mixed cultivations using the strains; MAT1-1 (BCMU IJ21, BCMU IJ25 and BCMU IJ774-1) and MAT1-2 (IFO 31161, BCMU IJ20 and BCMU IJ774-2) listed in Table 1, and examined the perithecium formation ability. All strains used in this study could produce conidia and were asexually fertile. All strains except for IFO 31161 could often produce stromata by itself. Four strains BCMU IJ20, BCMU IJ25, BCMU IJ774-1 and BCMU IJ774-2 could produce perithecia with opposite mating-type strains, and their mating-type phenotypes were MAT2, MAT1, MAT1 and MAT2, respectively. Perithecia were formed in the mixed culture of both mating-type strains MAT1 and MAT2, and not in the culture of MAT1 or MAT2 alone. The combinations using these four strains gave high perithecium formation efficiency (more than 80%). Perithecium formation efficiency was calculated as (the number of mixed cultivation succeeded in perithecium formation)/(the number of mixed cultivation) · 100. Table 1 Perithecium formation test of C. takaomontana Mixed cultivation
Perithecium formationa
BCMU IJ21 (MAT1-1) and BCMU IJ25 (MAT1-1) BCMU IJ21 (MAT1-1) and BCMU IJ774-1 (MAT1-1) BCMU IJ25 (MAT1-1) and BCMU IJ774-1 (MAT1-1) BCMU IJ20 (MAT1-2) and BCMU IJ774-2 (MAT1-2) BCMU IJ20 (MAT1-2) and IFO 31161 (MAT1-2) BCMU IJ774-2 (MAT1-2) and IFO 31161 (MAT1-2) BCMU IJ21 (MAT1-1) and BCMU IJ20 (MAT1-2) BCMU IJ21 (MAT1-1) and BCMU IJ774-2 (MAT1-2) BCMU IJ21 (MAT1-1) and IFO 31161 (MAT1-2) BCMU IJ25 (MAT1-1) and BCMU IJ20 (MAT1-2) BCMU IJ25 (MAT1-1) and BCMU IJ774-2 (MAT1-2) BCMU IJ25 (MAT1-1) and IFO 31161 (MAT1-2) BCMU IJ774-1 (MAT1-1) and BCMU IJ20 (MAT1-2) BCMU IJ774-1 (MAT1-1) and BCMU IJ774-2 (MAT1-2) BCMU IJ774-1 (MAT1-1) and IFO 31161 (MAT1-2)
0/10
a
Perithecium formation was examined in 10 trials.
0/10 0/10 0/10 0/10
To confirm that the perithecia formed in the combination of BCMU IJ25 and BCMU IJ774-2 were derived from both mating-type cells, we examined the mating type of progenies. The ascus of C. takaomontana is thin and cylindrical (diameter is about 6 lm, Fig. 1(e)); we could not tear it with a micromanipulator. A stroma carrying perithecia was broken up with a needle, and then a perithecium was smashed with a cover glass. Single ascospores were transferred to Potato Dextrose Agar (Difco) plate, and then their mating-type genotypes were determined. Both mating-type ascospores were detected in all examined 10 perithecia. We could not destroy asci completely and most ascospores could not be dispersed, so the exact ratio of mating type of progenies could not be calculated. The ratios of MAT1-1 to MAT1-2 in 10 perithecia varied from 0.5 to 2. These results strongly suggested that C. takaomontana is heterothallic, consistent with our previous study which concluded that the structures of the mating-type loci of C. takaomontana are similar to those of other heterothallic fungi [15]. Teleomorphic ascospores could be formed in the combination of BCMU IJ20 and BCMU IJ25, which were derived from anamorphic conidia. This suggested that teleomorph can be artificially developed using adequate combination partners, and that the mating-type method can be applied to the elucidation of teleomorph–anamorph connection. On the other hand, the combinations using IFO 31161 or BCMU IJ21, which were also derived from conidia, could not produce perithecia. To confirm the phylogenetic positions of the strains used in this study, a phylogenetic analysis based on the nucleotide sequence of the ITS1-5.8S rDNA-ITS2 region was performed (Fig. 2). The nucleotide sequences, except for in the case of C. takaomontana NIFTS HF774, were determined in previous studies [1,15,23]. The unambiguously aligned 497 positions were analyzed. Entomopathogenic fungi Cordyceps sinclairii and Paecilomyces fumosoroseus are closely related to
0/10 0/10 0/10 0/10 8/10 10/10 0/10 8/10 10/10 0/10
Fig. 2. Phylogenetic analysis of C. takaomontana based on the ITS region. The bootstrap values (%) were indicated on each nodes. Tree length = 26, CI = 1.00, HI = 0.00, RI = 1.00, RC = 1.00. The nucleotide sequences indicated by a, b and c were determined in previous studies [1,15,23].
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Fig. 3. Structures of the mating-type locus MAT1-1 in Clavicipitaceae. The nucleotide sequences indicated by a and b were determined in previous studies [15,29].
C. takaomontana [1], and were used as outgroup. All strains used in this study were closely related. So the ability to produce perithecia can be lost in a short lineage. 3.3. MAT1-1-3 gene in the mating-type locus MAT1-1 The structure of mating-type locus MAT1-1 differs among the fungal classes [8]. Loculoascomycetes has a single conserved gene MAT1-1-1, which encodes a-box transcriptional regulator. Pyrenomycetes usually possesses two additional mating-type genes MAT1-1-2 and MAT1-1-3, which encode amphipathic-helix and HMG-box transcriptional regulators, respectively. Discomycetes has also three MAT1-1 genes like Pyrenomycetes, but MAT1-1-2 is replaced by MAT1-1-4, which encodes a metallothionein. On the other hand, the MAT1-2 structure is conserved well in the filamentous fungi [8]. All MAT1-2 loci reported so far, except for that of Neurospora crassa, have a single conserved gene MAT1-2-1, which encodes HMG-box transcriptional regulator. In Neurospora crassa, a second MAT1-2 gene, mat a-2, has been reported [24]. Previously, we reported the mating-type loci of C. takaomontana of the strains derived from anamorphic conidia [15]. MAT1-2 has a single gene MAT1-2-1 like other pyrenomycetous fungi. But MAT1-1 contains only two genes MAT1-1-1 and MAT1-1-2. MAT1-1-3 could not be found in the strains BCMU IJ21 and BCMU IJ25. In this study, the MAT1-1 sequence of BCMU IJ774-1, which was derived from a teleomorphic ascospore, was determined. The structure of MAT1-1 of BCMU IJ774-1 was similar to that of BCMU IJ25 (Fig. 3). MAT1-1-3 could also not be found in BCMU IJ774-1. BCMU IJ25 and BCMU IJ774-1 could form abundant asci containing ascospores. These results indicated that C. takaomontana does not need MAT1-1-3 for the perithecium formation. Sordaria macrospora SMT A-3 does not have an HMG-box [25], and is supposed not to be a typical MAT1-1-3. A Neurospora crassa mat A-3 (MAT1-1-3) mutant can mate and produce abundant asci, for MAT A-2 (MAT1-1-2) and MAT
A-3 (MAT1-1-3) have redundant roles [26]. On the other hand, in Podospora anserina the SMR2 (MAT1-1-3) mutant can mate but produces reduced and abnormal asci [27]. SMR1 (MAT1-1-2) and SMR2 (MAT1-1-3) are involved in the initial development of biparental ascogenous cells [27] and the internuclear recognition [28], respectively. Thus the importance of MAT1-1-3 homologues in perithecium formation is variable in Pyrenomycetes. We determined the nucleotide sequences of MAT1-1 of another entomopathogenic fungus C. militaris BCMU CM03 and of three phytopathogenic fungi Balansia sp. NIAES 6588, Claviceps purpurea NBRC 32971 and Epichloe¨ typhina MAFF 511345 (Fig. 3). Entomopathogenic fungus C. militaris BCMU CM03, which was derived from a perithecium, did not have MAT1-1-3 like C. takaomontana. On the other hand, three phytopathogenic fungi possessed MAT1-1-3 between the DNA lyase gene and MAT1-1-2. The deduced amino acid sequences of MAT1-1-3 of these three species had a high identity (more than 42% in the aligned 143 amino acid residues) with that of Gibberella fujikuroi [29]. As a result of protein profile analysis using Pfam [30], three MAT1-1-3 proteins deduced in this study belonged to the family of HMG protein. The lengths between DNA lyase gene and MAT1-1-2 of the phytopathogenic fungi were longer than those of the entomopathogenic fungi. The structures of MAT1-1 of the phytopathogenic clavicipitaceous fungi were similar to that of a phytopathogenic fungus G. fujikuroi [29] in the family Nectriaceae, which is closely related to Clavicipitaceae. These results are consistent with our previous study which reported that the phytopathogen might be more ancestral group than the entomopathogen in Clavicipitaceae as a result of the phylogenetic analysis of MAT1-2-1 [1], and suggested that MAT1-1-3 might be lost in the course of the host shift from plants to insects. But the relationship between loss of MAT11-3 and pathogeny is still unknown. The deletion of mating-type locus has been reported in some Ceratocystis species [11].
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Acknowledgements This work was supported by the Agricultural HighTech Research Center, Meijo University under the ‘‘Environmental Control through the Function of Microorganisms’’ project. We are grateful to Mr. N. Sugiyama (The entomogenous fungi club, Japan), Dr. T. Tsukiboshi (National Institute of Agro-Environmental Sciences, Japan) and Dr. F. Ihara (National Institute of Fruit Technology Sciences, Japan) for providing fungal materials.
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