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Applied and Environmental Microbiology, August 2003, p . 5019-5022, Vol . 69, No . 8

Structures of the Mating-Type Loci of Cordyceps takaomontana

Eiji Yokoyama,1 Kenzo Yamagishi,2 and Akira Hara3*

The Agricultural High-Tech Research Center,1 Laboratory of Entomology,2 Laboratory of Biological Chemistry, Meijo University, Tempaku-ku, Nagoya 468-8502, Japan3

Received 24 February 2003/ Accepted 16 May 2003


   ABSTRACT

 
Nucleotide sequences of the mating-type loci MAT1-1 and MAT1-2 of Cordyceps takaomontana were determined, which is the first such report for the clavicipitaceous fungi . MAT1-1 contains two mating-type genes, MAT1-1-1 and MAT1-1-2, but MAT1-1-3 could not be found . On the other hand, MAT1-2 has MAT1-2-1. A pseudogene of MAT1-1-1 is located next to MAT1-2 .


   INTRODUCTION

 
Entomopathogenic fungus Paecilomyces tenuipes is an anamorph of Cordyceps takaomontana (Ascomycota: Pyrenomycetes: Hypocreales [Clavicipitales]: Clavicipitaceae [15]) . We use the holomorphic name C . takaomontana for P . tenuipes in this report . C . takaomontana has been used as a traditional medicine in Korea . Clavicipitaceous fungi are the potential sources for medicines such as ergot alkaloids from Claviceps purpurea, cordycepin from Cordyceps sinensis, and cyclosporine from Tolypocladium inflatum . C . takaomontana is also a source of bioactive compounds such as 4-acetyl-12,13-epoxyl-9-trichothecene-3,15-diol (12) . But the difficulties involved in artificially producing fruiting bodies such as perithecioid ascomata and synnemata hinder their usefulness . The analysis of mating type will shed light on the mating mechanisms and on the anamorph-teleomorph connection .

Recently the mating-type loci have been analyzed, mainly in the phytopathogenic fungi belonging to Discomycetes, Loculoascomycetes, and Pyrenomycetes (3-4, 8, 17-19) . 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 MAT1-2 are homologous, the nucleotide sequences of MAT1-1 and MAT1-2 are highly dissimilar . The term "idiomorph" is usually used instead of "allele" for MAT1-1 and MAT1-2 . Heterothallic fungi have one of the idiomorphs MAT1-1 and MAT1-2 . On the other hand, most of the homothallic fungi, except for some Neurospora species (6), have both idiomorphs .

The mating system of C . takaomontana is unknown; we determined the nucleotide sequences of the mating-type loci to investigate the molecular background of its mating system .


   Fungal strains.

 
A total of 22 isolates (BCMU IJ01 to IJ09, IJ11, IJ13 to IJ18, and IJ20 to IJ25) of C . takaomontana were obtained from the synnemata formed on the 22 lepidopteran pupae, which were collected at the Prefecture of Aichi in Japan . The nucleotide sequences of the 18S ribosomal DNA of all C . takaomontana isolates were determined . The phylogenetic analysis based on the nucleotide sequences of 18S ribosomal DNA was done using the neighbor-joining method (13) . These isolates were closely related to C . takaomontana (accession numbers AB044631 [11] and AB070372 [21]) and formed a monophyly supported by a 97.7% bootstrap value . C . takaomontana IFO 31161, which had been isolated at the Prefecture of Nara in Japan, was purchased from the Institute for Fermentation, Osaka (Japan) .


   Mating-type locus MAT1-2.

 
The HMG box of MAT1-2-1 is most conserved among the mating-type gene products of MAT1-1 and MAT1-2 . MAT1-2-1 protein belongs to the TCF/SOX family (9) of the HMG protein . Some primer sets for the amplification of DNA encoding an HMG box were reported (2), but no primer sets have been able to amplify the MAT1-2-1 gene of C . takaomontana . We synthesized a degenerate primer set (5'-GAGCCWCAYTTGTCSAAYAA and 5'-TTCTCCGACATTTCCTTGTA) based on the amino acid sequence of the conserved ascomycete HMG box (22) . PCR using the degenerate primers was able to amplify a part of the MAT1-2-1 gene of C . takaomontana BCMU IJ13 . The 6.2-kb DNA fragment containing the MAT1-2-1 gene (Fig . 1) was obtained using the cassette ligation-mediated PCR (7) and the inverse PCR . The deduced amino acid sequence of MAT1-2-1 of BCMU IJ13 has a 34.2% identity (in 196 amino acid residues) with that of Gibberella zeae (22) . MAT1-2-1 of BCMU IJ13 has two introns whose positions were confirmed by sequencing of the product of reverse transcription-PCR (RT-PCR) . The mRNA of BCMU IJ13 was prepared from the mycelia formed on the potato dextrose agar (Difco) . After the RT, PCR was performed using the primers M2RT-F (5'-ATGGATCTGCTTCTAGATCG) and M2RT-R (5'-TTAAACGACTCGGGGCTCAT) (Fig . 1) . The inserted positions of introns were same as those of the pyrenomycete fungi (22) . The MAT1-2-1 gene is expressed in vegetative mycelia . This suggests the possibility that MAT1-2-1 can operate on some events besides mating . In Neurospora crassa, the MAT a-1 (a homologue of MAT1-2-1) plays roles in both vegetative incompatibility and fertilization (16) .


FIG . 1 . Structures of the mating-type loci of C . takaomontana BCMU IJ25 and BCMU IJ13 . Numbered arrows represent positions and directions of the primers (arrow 1, Lyase-F; arrow 2, M112-R; arrow 3, Alpha-F; arrow 4, Alpha-R; arrow 5, M1R-F; arrow 6, M1R-R; arrow 7, M1RT-F; arrow 8, M1RT-R; arrow 9, HMG-R; arrow 10, HMG-F; arrow 11, M2RT-R; arrow 12, M2RT-F) . *, the boundary between MAT1-1 and the right flanking region was not able to be determined.

 
A latter part of the MAT1-1-1 gene, lacking the region encoding a conserved alpha box, was located upstream from the MAT1-2-1 gene (Fig . 1) . This pseudogene (542 bp) does not have an initiation codon and is interrupted by several termination codons . A small gap (54 bp) is present between the nucleotide sequences of MAT1-1-1 of BCMU IJ13 and BCMU IJ25 (see below) . The nucleotide sequence of the partial MAT1-1-1 of BCMU IJ13 has 91.7% identity (in 531 bp) with that of BCMU IJ25 but has little identity with those of MAT1-1-1 of other fungi reported so far . The terminal runoff of MAT1-1-1 was also reported for the flanking region of MAT1-2 of Pyrenopeziza brassicae (14) .

A putative gene encoding a DNA lyase was found downstream from the MAT1-2-1 gene . The deduced amino acid sequence of the DNA lyase of C . takaomontana has 46.3% identity (in 607 amino acid residues) with that of Mycosphaerella graminicola (20) . In M . graminicola, the DNA lyase gene is present downstream from the MAT1-2-1 (20) . Database searching by BLAST (1) revealed a DNA lyase gene next to the mat a-1 of N . crassa (accession number M54787) (16) .


   Mating-type locus MAT1-1.

 
The alpha box of MAT1-1-1 is most conserved among the mating-type gene products of MAT1-1, but the homologies of MAT1-1-1 among the filamentous ascomycetes are relatively low . In G . zeae (22) and M . graminicola (20), a DNA lyase (described as Exo1 in reference 22) gene is present next to the MAT1-1 . Database searching by BLAST (1) revealed a DNA lyase gene next to MAT1-1-3 of Cryphonectria parasitica (accession number AF380365) (10) . The DNA lyase gene might be present next to both idiomorphs . The DNA lyase gene of C . takaomontana BCMU IJ25 was amplified by PCR using the primers based on the nucleotide sequence of the DNA lyase gene of C . takaomontana BCMU IJ13 . The 10.2-kb DNA fragment containing the DNA lyase gene, MAT1-1-2, and MAT1-1-1 (Fig . 1) was obtained using the cassette ligation-mediated PCR (7) and the inverse PCR . MAT1-1 of the pyrenomycete fungi reported so far contains three genes, MAT1-1-3, MAT1-1-2, and MAT1-1-1 (10, 19) . However, the MAT1-1-3 gene could not be found in C . takaomontana BCMU IJ25 .

The deduced amino acid sequence of MAT1-1-2 of C . takaomontana has 26.8% identity (in 336 amino acid residues) with that of G . fujikuroi (22) . The deduced amino acid sequence of MAT1-1-1 of C . takaomontana has 38.4% identity (in 203 amino acid residues) with that of G . zeae (22) . MAT1-1-1 has one intron whose position was confirmed by sequencing of the product of RT-PCR . The mRNA of C . takaomontana BCMU IJ25 was prepared from the mycelia formed on the potato dextrose agar (Difco) . After the RT, PCR was performed using the primers M1RT-F (5'-CGCTTTCAGAAGTTATTATGTG) and M1RT-R (5'-TGCTGGGGACAAGAAAGACTAG) (Fig . 1) . The inserted position of intron was same as those of the hypocrealean fungi (22) . MAT1-1-1 of C . takaomontana BCMU IJ25 is expressed in vegetative mycelia . This suggests the possibility that MAT1-1-1 could operate in some events besides mating . In N . crassa, the MAT A-1 (a homologue of MAT1-1-1) plays roles in both vegetative incompatibility and fertilization (5) .


   Mating-type loci of other C . takaomontana isolates.

 
We analyzed the structures of the mating-type loci of 20 other isolates from Aichi in Japan . Three sets of degenerate primers were synthesized {Alpha-F [5'-CG(A/G)GC(A/T)AA(A/G)CG(A/G)CCATTGAA(C/T)GC] and Alpha-R [5'-CCCATCTC(A/G)TC(A/T)CGGAC(A/G)AA(G/C)GA] for the alpha box-encoding part of MAT1-1-1, M1R-F [5'-(C/T)TGA(A/G)ATCGAAAGATCTCCC] and M1R-R [5'-GACAAGAAAGACTAGAAAAC] for the latter part of MAT1-1-1, and HMG-F [5'-AAGATTCC(A/G)AG(A/G)CC(A/G)CC(G/C)AA] and HMG-R [5'-CGAGGTTGATA(C/T)TGATA(C/T)TG] for the HMG box-encoding part of MAT1-2-1} (Fig . 1) . PCRs were performed using an ExTaq DNA polymerase (Takara, Japan) according to the manufacturer's recommendations . After denaturation at 95°C for 1 min, amplification with 35 cycles of denaturation (95°C for 30 s), annealing (60°C for 30 s), and polymerization (72°C for 30 s) was done .

As the result of degenerate PCRs (Fig . 2), two isolates, BCMU IJ21 and BCMU IJ23, were found to the same organization as BCMU IJ25, with a complete MAT1-1-1 . MAT1-1-3 was not found in isolate BCMU IJ25, although other pyrenomycete fungi usually have MAT1-1-3 upstream from MAT1-1-2 (10, 19) . The lengths of the PCR products using the primers Lyase-F (5'-ACTGGCTGTGATGACAGGAC) and M112-R (5'-CTCGAGTTGCAACAGGCACG) (Fig . 1) were identical among the isolates BCMU IJ21, BCMU IJ23, and BCMU IJ25 (data not shown), so the isolates BCMU IJ21 and BCMU IJ23 might lack MAT1-1-3 . But the possibility that the arrangement of MAT1-1-3 of C . takaomontana was different from those of other fungi could not be excluded . On the other hand, the rest of the BCMU isolates had MAT1-2-1 and the latter part of MAT1-1-1 (Fig . 2) .


FIG . 2 . Results of degenerate PCRs for the alpha box-encoding part of MAT1-1-1 (A), the latter part of MAT1-1-1 (B), and the HMG box-encoding part of MAT1-2-1 (C) . PCR products were electrophoresed on a 1.5% agarose gel . Lane M is a 100-bp DNA ladder (New England BioLabs) . Lanes 1 to 25 show the PCR products from C . takaomontana BCMU IJ01 to IJ09, IJ11, IJ13 to IJ18, and IJ20 to IJ25, respectively.

 
The 9.7-kb DNA fragment of the mating-type locus of C . takaomontana IFO 31161 from Nara in Japan was determined . Strain IFO 31161 has also MAT1-2-1 and the latter part of MAT1-1-1. It can be speculated that C . takaomontana or its ancestor originally retained MAT1-1 and MAT1-2 and was homothallic (Fig . 1) . The isolates BCMU IJ21, BCMU IJ23, and BCMU IJ25 lost MAT1-2 (and MAT1-1-3) . The rest of the BCMU isolates and IFO 31161 lost MAT1-1 except for the latter part of MAT1-1-1, which was retained . The 22 BCMU isolates and IFO 31161 might have lost their homothallic nature by the partial deletions of mating-type loci .


   Nucleotide sequence accession numbers.

 
The nucleotide sequences of the 18S ribosomal DNA of C . takaomontana isolates (BCMU IJ01 to IJ09, IJ11, IJ13 to IJ18, and IJ20 to IJ25) were deposited in the DDBJ/EMBL/GenBank databases with the accession numbers AB086203 to AB086224, respectively . The nucleotide sequences of the mating-type loci of C . takaomontana were also deposited, with accession numbers AB084921 (BCMU IJ13), AB096216 (BCMU IJ25), and AB103335 (IFO 31161) .

 


   ACKNOWLEDGMENTS

 
This work was supported by the Agricultural High-Tech Research Center, Meijo University, under the "Environmental Control through the Function of Microorganisms" project .


   FOOTNOTES

 
* Corresponding author . Mailing address: Laboratory of Biological Chemistry, Meijo University, Tempaku-ku, Nagoya 468-8502, Japan . Phone: 81-52-832-1151 . Fax: 81-52-835-7450 . E-mail: hara{at}ccmfs.meijo-u.ac.jp .


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