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Isolation of genes differentially expressed during the fruit body development of Pleurotus ostreatus by differential display of RAPD
FEMS Microbiology Letters 246 (2005) 279–284 www.fems-microbiology.org
Isolation of genes differentially expressed during the fruit body development of Pleurotus ostreatus by differential display of RAPD Masahide Sunagawa *, Yumi Magae Department of Applied Microbiology, Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 305-8687, Japan Received 8 December 2004; received in revised form 12 April 2005; accepted 14 April 2005 First published online 27 April 2005 Edited by G.M. Gadd
Abstract To analyze genes involved in fruit body development of Pleurotus ostreatus, mRNAs from three different developmental stages: i.e., vegetative mycelium, primordium, and mature fruit body, were isolated and reverse-transcribed to cDNAs. One hundred and twenty random PCR amplifications were performed with the cDNAs, which generated 382, 394, 393 cDNA fragments from each developmental stage. From these fragments, four cDNA clones specifically expressed in primordium or mature fruit body were detected. Sequence analysis and database searches revealed significant similarity with triacylglycerol lipase, cytochrome P450 sterol 14 a-demethylase and developmentally regulated genes of other fungi. Northern blot analyses confirmed that all of the four cDNAs were unexpressed in mycelium, thus stage-specific genes for fruit body formation of P. ostreatus were successfully isolated. Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Differential display; Pleurotus ostreatus; RAPD
1. Introduction Fruit body morphogenesis is an important subject in both basic and applied fields of mycological research. The shift from vegetative growth to fruit body development is a very interesting biological phenomenon for basic research; moreover, understanding the mechanism of fruit body development will contribute to the advancement of commercial mushroom production. Several genes related to fruit body development have been identified in Coprinus cinereus [1,2], Schizophyllum commune [3,4], Tuber borchii [5,6], Agaricus bisporus [7], Agrocybe
*
Corresponding author. Tel.: +81 298 733211; fax: +81 298 743720. E-mail address: masahide@ffpri.affrc.go.jp (M. Sunagawa).
aegerita [8], Lentinula edodes [9–13], and Flammulina velutipes [14]. In Pleurotus ostreatus, Lee et al. [15] analyzed expressed sequence tags (ESTs) of cDNAs library derived from liquid-culture mycelia and fruit bodies of P. ostreatus. The method of differential mRNA display (DD) [16] has mostly been used to study differential gene expressions in plants [17–19]. In fungi, Leung et al. [11], have used this method to identify differentially expressed genes in RNA populations of four developmental stages of L. edodes: vegetative mycelium, primordium, young fruit body and mature fruit body. Also, DD has been used to identify putative genes involved in the development of fruit bodies of T. borchii [6]. In the present study, genes expressed during fruit-body development of P. ostreatus were examined by DD.
0378-1097/$22.00 Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2005.04.018
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M. Sunagawa, Y. Magae / FEMS Microbiology Letters 246 (2005) 279–284
2. Materials and methods 2.1. Strain and culture conditions
PCR amplify the second-strand cDNA. PCR was carried out as 45 cycles of the following thermal cycle: 30 s at 95 °C, 1 min at 50 °C, and 2 min at 72 °C [20].
A dikaryotic strain, P. ostreatus ASI2029, was provided by Dr. Beom-Gi Kim (National Institute of Agriculture Science and Technology, Korea). ASI2029 was cultivated in a sawdust-medium containing beech sawdust and rice bran 3:1 (v/v). The sawdust-medium was adjusted to a hydrous rate of 65% with tap water and packed into a culture bottle (850 ml). After autoclaving, 5 ml of liquid inoculum was inoculated. The cultures were grown at 20 °C for 30 days, and then the temperature was lowered to 15 °C to induce fruit body development. During the cultivation of ASI2029, samples from three stages of development, i.e., mycelium, primordium (3–7 mm in diameter), and mature fruit body (Fig. 1) were collected. These samples were immediately frozen in liquid nitrogen and stored at 80 °C until use.
2.4. Cloning and sequence of specific cDNA fragments
2.2. RNA preparation
Total RNA (20 lg) of each developmental stage used for the isolation of mRNA was fractionated in a 1.0% agarose gel containing formaldehyde, and transferred to Hybond-N (Amersham Bioscience K.K.). Northern blots were hybridized with DIG (Roche Diagnostics K.K., Tokyo, Japan) labeled cDNA at 42 °C according to the manufactureÕs instructions. As a control, Northern blots were probed with 18S rDNA fragment of the P. ostreatus (ASI2029). The 18S rDNA was isolated as described by White et al. [22].
Total RNAs were isolated using a Qiagen RNA Preparation Kit (Qiagen K.K., Tokyo, Japan). Poly(A)+– RNA was prepared by Oligotex-dT30 super (Takara Bio Co., Shiga, Japan). Both procedures were carried out according to the manufacturerÕs instructions. 2.3. Differential display of mRNA Poly(A)+–RNA (0.5 lg) was heated at 65 °C for 10 min and immediately chilled on ice. First-strand cDNA synthesis was performed in a reaction mixture containing 50 mM Tris–HCl (pH 8.5), 40 mM KCl, 5 mM MgCl2, 2 mM DTT, 850 lM each dNTP, 95 units of RNAase Inhibitor (Takara Bio Co.), 0.2 mM random primer (Takara Bio Co.), and 40 units of Superscript II Reverse Transcriptase (Invitrogen, Tokyo, Japan). The reaction was carried out for 1 h at 42 °C. After heat-denatureation of the enzyme at 95 °C for 5 min, the random primers were removed by ultrafiltration with Super-02 (Takara Bio Co.). 10-mer RAPD primers (Operon Technologies, Inc., Alameda, CA) were used to
Specific cDNA fragments found in RAPD were cut out from the agarose gel and purified with Gel purification column (Nippon Bio-Rad Lab., Tokyo, Japan). The purified DNA was cloned into vector pCR 2.1 with the TA cloning System Kit (Invitrogen). DNA sequencing of the clones were performed by Dynamic ET Terminater Sequencing Kit (Amersham Biosciences K.K., Tokyo, Japan) and Mega Base 1000 Sequencer (Amersham Biosciences K.K.). Homology search was done using BlastX program [21] for the translated protein and EST sequences in the NCBI data bank. 2.5. Northern blotting analyses
2.6. Nucleotide sequence accession numbers All the clones have been deposited with the DDBJ data banks under the Accession No. AB19629, AB196292, AB196293 and AB196294.
3. Results and discussion In the present study, four genes specifically expressed during the fruit body development of P. ostreatus were isolated by means of differential displays of randomly
Fig. 1. Samples used for the RNA preparation. A: mycelium (My), B: primordium (P), C: fruit body (F).
M. Sunagawa, Y. Magae / FEMS Microbiology Letters 246 (2005) 279–284
amplified cDNA. To detect changes in transcripts during fruit body development of P. ostreatus, mRNAs were isolated from three stages of development: mycelium, primordium, and mature fruit body (Fig. 1). Then, reverse-transcribed cDNAs were used as templates for the following PCR. A total of 120 PCR amplifications were performed with 10-mer RAPD primers. Each PCR product separated by the agarose gel was resolved into 1–9 distinct DNA bands in agarose gel. A total of 382, 394, and 393 cDNA fragments were identified in the mycelium, primordium, and mature fruit body, respectively. The electrophoresis patterns of the PCRamplified cDNA were confirmed as reproducible in three independent experiments. Of the 120 random primers tested, three primers generated cDNA fragments analyzed in the present study (Fig. 2). Ninety-five PCR amplifications (79%) gave identical cDNA patterns between the three developmental stages. In 16 PCR, the same cDNA patterns were obtained with primordium and fruit body. In one case, specific cDNA was detected only in the mycelium stage. All of the differentially expressed cDNA fragments were recovered from the agarose gel and cloned into the vector pCR2.1. As a result, two fruit body-specific and the
Fig. 2. RAPD patterns of cDNA generated from mRNA of each developmental stage. cDNA indicated by arrowheads were isolated, cloned and analyzed in this study. M: molecular standard k/HindIII, My: mycelium, P: primordium, F: fruit body.
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two primordium-specific cDNA fragments were successfully cloned. Two specifically expressed cDNA were detected in mature fruit body using primer A8 (5 0 -GTGACGTAGG-3 0 ) (Fig. 2). The sizes of the fragments were 777 and 574 bps, and were designated as A8-U and A8-D, respectively. In the cases of primer T19 (5 0 -GTCCGTATGG-3 0 ) and W3 (5 0 -GTCCGGAGTG-3 0 ), two differentially amplified cDNA fragments, 506 and 415 bps, were identified in the primordium (Fig. 2). They were designated as T19-4 and W3-7, respectively. The cDNA clones were subjected to sequence analysis. Homology of the deduced amino acid sequences of A8-U, A8-D, T19-4, and W3-7 with the database was searched using the BlastX program. When no significant homology was found with the protein databases, the homology search was performed with EST databases with the tBlastX program. The results are summarized in Table 1. Predicted protein of T19-4 (+2 frame) showed significant homology with triacylglycerol lipase and contained a conserved domain of esterase-lipase. The highest homology was found with triacylglycerol
Fig. 3. Northern analysis of four cDNA that are differentially expressed during fruit body development of P. ostreatus. My: mycelium, P: primordium, F: fruit body. Total RNA isolated from each sample was hybridized with DIG-labeled cDNA clone and as a control by DIG-labeled 18S rDNA. EtBr-stained ribosomal RNA bands are loaded as quantitative control.
Table 1 Characterization of differentially expressed cDNA clones of P. ostreatus Levels
Accession No.
Clone
Size
A8-U A8-D T19-4 W3-7
777 574 506 415
AB196291 AB196294 AB196294 AB196293
Homology
Cytochrome P450 sterol 14 a-demethylase [Aspergillus fumigatus, AAF32372] cDNA clone expressed during carbon starvation [Trichoderma reesei, CF869295] Triacylglycerol lipase (E.C.3.1.1.3) [Candida rugosa, 1LPP] Early developmental cDNA clone GM578 [Glomus mosseae, AJ315727]
E-value
0.001 2e 05 9e 26 0.008
mRNA My
P
F
+ +
+ + + +
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Fig. 4. Alignment of lipase sequences. The deduced amino acid sequence of T19-4 and three fungal lipase sequences were compared using Clustal W. Residues, which are identical to P. ostreatus T19-4 are marked by asterisks. Underline shows the conserved domain of esterase and lipase. ILPP: Triacyl glycerol Lipase (E.C.3.1.1.3) of Candida rugosa (GI:1064964), 1THG: Lipase of Galactomyces geotrichum (GI:443280), NCHP: Hypothetical protein of Neurospora crassa (XM_322879).
lipase of Candida rugosa (9e 26). The multiple alignment of the deduced T19-4 polypeptide with putative amino acid sequences of lipase from other fungi is shown in Fig. 4. Triacyl glycerol (TAG) is the predominant acyl lipid in cultures undergoing sexual development of Neurospora crassa [23]. In addition, as described with Magnaporthe grisea, triacylglycerol lipase activity increased during appressorium maturation [24]. Mass transfer of storage lipid reserve to the aspersorium occurred under the control of the MAP kinase and turgor generation proceeded under the control of protein kinase A [24]. Since TAG is known as an energy dense substance [25], it is plausible that TAG is used as an energy source for the rapid development of P. ostreatus fruit body while triacylglycerol lipase plays a role in lipid degradation. As with A8-U, similarity (51%) was found with the cytochrome P450 CYP51 (sterol 14 a-demethylase) of Aspergillus fumigatus. Gene of cytochrome P450 has been identified as involved in fruit body development of A. bisporus [7], C. cinereus [26] and L. edodes [9], but this is the first case of P450 CYP51, which is an essential enzyme required in sterol biosynthesis and primary target of azole antimycotic drugs, isolated as a gene related to fruit body development of mushroom. The multiple alignment of the deduced A8-U polypeptide with putative amino acid sequences of CYP51 from A. fumigatus and Penicillium italicum is shown in Fig. 5.
No highly significant homology was found between the function-known genes and the deduced polypeptide of A8-D and W3-7. But when they were compared with genes deposited in the EST database, high similarity was found with developmentally regulated cDNA of other fungi. W3-7 may encode a protein with 84% similarity to the early developmental gene of arbuscular mycorrhizal fungus Glomus mosseae (Table 1). On the other hand, the deduced polypeptide of A8-D showed significant homology with the predicted protein of Trichoderma reesei cDNA clone (2e 05), expressed during carbon starvation. Interestingly, A8-D codes a common gene involved in the development of T. reesei and P. ostreatus although its function is yet unknown. Northern analysis showed that T19-4 and W3-7 that were detected in primordium by the RAPD analysis, hybridized to total RNA in both primordium and mature fruit body. The fact that T19-4 and W3-7 hybridized to the RNA of both primordium and mature fruit body shows that these mRNAs encode proteins that play specific roles during both stages of fruit body development. One possible reason for why T19-4 and W3-7 were undetected in the RAPD of fruit body stage is that most of the RAPD primers were consumed for amplification of more highly expressed cDNA. In contrast, A8-U and A8-D hybridized only to the total RNA of mature fruit body indicating that
Fig. 5. The deduced amino acid sequence of A8-U was aligned with cytochrome P450 sterol 14 a-demethylase of Aspergillus fumigatus (GI:6942241) and Penicillium italicum (GI:836642). Residues, which are identical to P. ostreatus are marked by asterisks.
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they are specific genes in the latter stage of the development. None of the four cDNA isolated in the present study, hybridized to the RNA derived from mycelium. The control probe, 18S rRNA gene of the P. ostreatus, hybridized to RNA of all the stages (Fig. 3). Liang and Pardee [16] reported about differential displays of mRNA using 3 0 -anchored oligo-dT for the firststrand cDNA synthesis and 10-mer arbitrary primers for the second-strand synthesis by PCR. In their study, numerous amplified fragments were visible after autoradiography of labeled PCR products and the subsequent isolation of the specific DNA band was rather difficult. Leung et al. [11] and Zeppa et al. [6] also used 3 0 -anchored oligo-dT and various random primers for the isolation of differentially expressed gene fragments. The difference between the previous DD studies and ours is that we did not use 3 0 -anchored oligo-dT. The number of cDNA fragments obtained in one PCR reaction was not too large and each cDNA could be resolved as a separate band in agarose gel. With the basidiomycetes, not many genes related to fruit body development have been isolated but they often share common genes. For instance, Hydrophobin is one of the most abundant genes in fruit bodies of basidiomycetes, as reported by Penas et al. [27] and Asgeirsdottir et al. [28] and has been isolated as fruiting related gene with A. bisporus [7], F. velutipes [14] and L. edodes [13]. But we did not detect hydrophobin as a specific gene for fruit body development of P. ostreatus in the present study. ATPase has been detected in L. edodes [11] and T. borchii [6]. PriA was detected in L. edodes [10], Agrocybe aegerita [8] and EST of P. ostreatus [15]. In addition, Septin has been isolated as a gene related to fruit body development [15,29,6]. However, none of these genes were detected in this study. Presumably because the method used in the previous studies screen the elevated expression of genes, abundant genes instead of unique genes tended to be isolated. But by comparing RAPD patterns of ca. 1200 cDNA fragments, abundant but not stage specific gene was easily eliminated. All four cDNAs isolated in this study were novel fruiting genes for basidiomycetes. The Northern analysis confirmed that they were not expressed during the mycelium stage, but expressed after the primordium development. In conclusion, the DD technique was a simple and efficient method to detect fruit body stagespecific cDNA of P. ostreatus.
Acknowledgements We are grateful to Dr. Beom-Gi Kim of the National Institute of Agriculture Science and Technology for providing the Pleurotus ostreatus strain (ASI2029).
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[17] Stein, S., Cho, Y.J., Jackson, R.S. and Liang, P. (2003) Identification of p53 target genes by fluorescent differential display. Methods Mol. Biol. 234, 51–63. [18] Ueda, A., Shi, W., Nakamura, T. and Takabe, T. (2002) Analysis of salt-inducible genes in barley roots by differential display. J. Plant Res. 115, 119–130. [19] Wu, L.M., Ni, Z.F., Meng, F.R., Lin, Z. and Sun, Q.X. (2003) Cloning and characterization of leaf cDNAs that are differentially expressed between wheat hybrids and their parents. Mol. Genet. Genomics. 270, 281–286. [20] Sunagawa, M., Tamai, Y., Neda, H., Miyazaki, K. and Miura, K. (1995) Application of random amplified polymorphic DNA (RAPD) markers (I): analyses of fussants in edible mushrooms. Nihon Mokuzai Gakkaishi 41, 945–948. [21] Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J. (1997) Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. [22] White, T.J., Bruns, T., Lee, S. and Taylor, J. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics In: PCR Protocols (Innis, M.A., Gelfand, D.H., Sninsky, J.J. and White, T.J., Eds.), pp. 315–322. Academic Press, San Diego, CA. [23] Goodrich-Tanrikulu, M., Howe, K., Stafford, A. and Nelson, M.A. (1998) Changes in fatty acid composition of Neurospora
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Isolation of genes differentially expressed during the fruit body development of Pleurotus ostreatus by differential display of RAPD Masahide Sunagawa *, Yumi Magae Department of Applied Microbiology, Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 305-8687, Japan Received 8 December 2004; received in revised form 12 April 2005; accepted 14 April 2005 First published online 27 April 2005 Edited by G.M. Gadd
Abstract To analyze genes involved in fruit body development of Pleurotus ostreatus, mRNAs from three different developmental stages: i.e., vegetative mycelium, primordium, and mature fruit body, were isolated and reverse-transcribed to cDNAs. One hundred and twenty random PCR amplifications were performed with the cDNAs, which generated 382, 394, 393 cDNA fragments from each developmental stage. From these fragments, four cDNA clones specifically expressed in primordium or mature fruit body were detected. Sequence analysis and database searches revealed significant similarity with triacylglycerol lipase, cytochrome P450 sterol 14 a-demethylase and developmentally regulated genes of other fungi. Northern blot analyses confirmed that all of the four cDNAs were unexpressed in mycelium, thus stage-specific genes for fruit body formation of P. ostreatus were successfully isolated. Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Differential display; Pleurotus ostreatus; RAPD
1. Introduction Fruit body morphogenesis is an important subject in both basic and applied fields of mycological research. The shift from vegetative growth to fruit body development is a very interesting biological phenomenon for basic research; moreover, understanding the mechanism of fruit body development will contribute to the advancement of commercial mushroom production. Several genes related to fruit body development have been identified in Coprinus cinereus [1,2], Schizophyllum commune [3,4], Tuber borchii [5,6], Agaricus bisporus [7], Agrocybe
*
Corresponding author. Tel.: +81 298 733211; fax: +81 298 743720. E-mail address: masahide@ffpri.affrc.go.jp (M. Sunagawa).
aegerita [8], Lentinula edodes [9–13], and Flammulina velutipes [14]. In Pleurotus ostreatus, Lee et al. [15] analyzed expressed sequence tags (ESTs) of cDNAs library derived from liquid-culture mycelia and fruit bodies of P. ostreatus. The method of differential mRNA display (DD) [16] has mostly been used to study differential gene expressions in plants [17–19]. In fungi, Leung et al. [11], have used this method to identify differentially expressed genes in RNA populations of four developmental stages of L. edodes: vegetative mycelium, primordium, young fruit body and mature fruit body. Also, DD has been used to identify putative genes involved in the development of fruit bodies of T. borchii [6]. In the present study, genes expressed during fruit-body development of P. ostreatus were examined by DD.
0378-1097/$22.00 Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2005.04.018
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M. Sunagawa, Y. Magae / FEMS Microbiology Letters 246 (2005) 279–284
2. Materials and methods 2.1. Strain and culture conditions
PCR amplify the second-strand cDNA. PCR was carried out as 45 cycles of the following thermal cycle: 30 s at 95 °C, 1 min at 50 °C, and 2 min at 72 °C [20].
A dikaryotic strain, P. ostreatus ASI2029, was provided by Dr. Beom-Gi Kim (National Institute of Agriculture Science and Technology, Korea). ASI2029 was cultivated in a sawdust-medium containing beech sawdust and rice bran 3:1 (v/v). The sawdust-medium was adjusted to a hydrous rate of 65% with tap water and packed into a culture bottle (850 ml). After autoclaving, 5 ml of liquid inoculum was inoculated. The cultures were grown at 20 °C for 30 days, and then the temperature was lowered to 15 °C to induce fruit body development. During the cultivation of ASI2029, samples from three stages of development, i.e., mycelium, primordium (3–7 mm in diameter), and mature fruit body (Fig. 1) were collected. These samples were immediately frozen in liquid nitrogen and stored at 80 °C until use.
2.4. Cloning and sequence of specific cDNA fragments
2.2. RNA preparation
Total RNA (20 lg) of each developmental stage used for the isolation of mRNA was fractionated in a 1.0% agarose gel containing formaldehyde, and transferred to Hybond-N (Amersham Bioscience K.K.). Northern blots were hybridized with DIG (Roche Diagnostics K.K., Tokyo, Japan) labeled cDNA at 42 °C according to the manufactureÕs instructions. As a control, Northern blots were probed with 18S rDNA fragment of the P. ostreatus (ASI2029). The 18S rDNA was isolated as described by White et al. [22].
Total RNAs were isolated using a Qiagen RNA Preparation Kit (Qiagen K.K., Tokyo, Japan). Poly(A)+– RNA was prepared by Oligotex-dT30 super (Takara Bio Co., Shiga, Japan). Both procedures were carried out according to the manufacturerÕs instructions. 2.3. Differential display of mRNA Poly(A)+–RNA (0.5 lg) was heated at 65 °C for 10 min and immediately chilled on ice. First-strand cDNA synthesis was performed in a reaction mixture containing 50 mM Tris–HCl (pH 8.5), 40 mM KCl, 5 mM MgCl2, 2 mM DTT, 850 lM each dNTP, 95 units of RNAase Inhibitor (Takara Bio Co.), 0.2 mM random primer (Takara Bio Co.), and 40 units of Superscript II Reverse Transcriptase (Invitrogen, Tokyo, Japan). The reaction was carried out for 1 h at 42 °C. After heat-denatureation of the enzyme at 95 °C for 5 min, the random primers were removed by ultrafiltration with Super-02 (Takara Bio Co.). 10-mer RAPD primers (Operon Technologies, Inc., Alameda, CA) were used to
Specific cDNA fragments found in RAPD were cut out from the agarose gel and purified with Gel purification column (Nippon Bio-Rad Lab., Tokyo, Japan). The purified DNA was cloned into vector pCR 2.1 with the TA cloning System Kit (Invitrogen). DNA sequencing of the clones were performed by Dynamic ET Terminater Sequencing Kit (Amersham Biosciences K.K., Tokyo, Japan) and Mega Base 1000 Sequencer (Amersham Biosciences K.K.). Homology search was done using BlastX program [21] for the translated protein and EST sequences in the NCBI data bank. 2.5. Northern blotting analyses
2.6. Nucleotide sequence accession numbers All the clones have been deposited with the DDBJ data banks under the Accession No. AB19629, AB196292, AB196293 and AB196294.
3. Results and discussion In the present study, four genes specifically expressed during the fruit body development of P. ostreatus were isolated by means of differential displays of randomly
Fig. 1. Samples used for the RNA preparation. A: mycelium (My), B: primordium (P), C: fruit body (F).
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amplified cDNA. To detect changes in transcripts during fruit body development of P. ostreatus, mRNAs were isolated from three stages of development: mycelium, primordium, and mature fruit body (Fig. 1). Then, reverse-transcribed cDNAs were used as templates for the following PCR. A total of 120 PCR amplifications were performed with 10-mer RAPD primers. Each PCR product separated by the agarose gel was resolved into 1–9 distinct DNA bands in agarose gel. A total of 382, 394, and 393 cDNA fragments were identified in the mycelium, primordium, and mature fruit body, respectively. The electrophoresis patterns of the PCRamplified cDNA were confirmed as reproducible in three independent experiments. Of the 120 random primers tested, three primers generated cDNA fragments analyzed in the present study (Fig. 2). Ninety-five PCR amplifications (79%) gave identical cDNA patterns between the three developmental stages. In 16 PCR, the same cDNA patterns were obtained with primordium and fruit body. In one case, specific cDNA was detected only in the mycelium stage. All of the differentially expressed cDNA fragments were recovered from the agarose gel and cloned into the vector pCR2.1. As a result, two fruit body-specific and the
Fig. 2. RAPD patterns of cDNA generated from mRNA of each developmental stage. cDNA indicated by arrowheads were isolated, cloned and analyzed in this study. M: molecular standard k/HindIII, My: mycelium, P: primordium, F: fruit body.
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two primordium-specific cDNA fragments were successfully cloned. Two specifically expressed cDNA were detected in mature fruit body using primer A8 (5 0 -GTGACGTAGG-3 0 ) (Fig. 2). The sizes of the fragments were 777 and 574 bps, and were designated as A8-U and A8-D, respectively. In the cases of primer T19 (5 0 -GTCCGTATGG-3 0 ) and W3 (5 0 -GTCCGGAGTG-3 0 ), two differentially amplified cDNA fragments, 506 and 415 bps, were identified in the primordium (Fig. 2). They were designated as T19-4 and W3-7, respectively. The cDNA clones were subjected to sequence analysis. Homology of the deduced amino acid sequences of A8-U, A8-D, T19-4, and W3-7 with the database was searched using the BlastX program. When no significant homology was found with the protein databases, the homology search was performed with EST databases with the tBlastX program. The results are summarized in Table 1. Predicted protein of T19-4 (+2 frame) showed significant homology with triacylglycerol lipase and contained a conserved domain of esterase-lipase. The highest homology was found with triacylglycerol
Fig. 3. Northern analysis of four cDNA that are differentially expressed during fruit body development of P. ostreatus. My: mycelium, P: primordium, F: fruit body. Total RNA isolated from each sample was hybridized with DIG-labeled cDNA clone and as a control by DIG-labeled 18S rDNA. EtBr-stained ribosomal RNA bands are loaded as quantitative control.
Table 1 Characterization of differentially expressed cDNA clones of P. ostreatus Levels
Accession No.
Clone
Size
A8-U A8-D T19-4 W3-7
777 574 506 415
AB196291 AB196294 AB196294 AB196293
Homology
Cytochrome P450 sterol 14 a-demethylase [Aspergillus fumigatus, AAF32372] cDNA clone expressed during carbon starvation [Trichoderma reesei, CF869295] Triacylglycerol lipase (E.C.3.1.1.3) [Candida rugosa, 1LPP] Early developmental cDNA clone GM578 [Glomus mosseae, AJ315727]
E-value
0.001 2e 05 9e 26 0.008
mRNA My
P
F
+ +
+ + + +
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Fig. 4. Alignment of lipase sequences. The deduced amino acid sequence of T19-4 and three fungal lipase sequences were compared using Clustal W. Residues, which are identical to P. ostreatus T19-4 are marked by asterisks. Underline shows the conserved domain of esterase and lipase. ILPP: Triacyl glycerol Lipase (E.C.3.1.1.3) of Candida rugosa (GI:1064964), 1THG: Lipase of Galactomyces geotrichum (GI:443280), NCHP: Hypothetical protein of Neurospora crassa (XM_322879).
lipase of Candida rugosa (9e 26). The multiple alignment of the deduced T19-4 polypeptide with putative amino acid sequences of lipase from other fungi is shown in Fig. 4. Triacyl glycerol (TAG) is the predominant acyl lipid in cultures undergoing sexual development of Neurospora crassa [23]. In addition, as described with Magnaporthe grisea, triacylglycerol lipase activity increased during appressorium maturation [24]. Mass transfer of storage lipid reserve to the aspersorium occurred under the control of the MAP kinase and turgor generation proceeded under the control of protein kinase A [24]. Since TAG is known as an energy dense substance [25], it is plausible that TAG is used as an energy source for the rapid development of P. ostreatus fruit body while triacylglycerol lipase plays a role in lipid degradation. As with A8-U, similarity (51%) was found with the cytochrome P450 CYP51 (sterol 14 a-demethylase) of Aspergillus fumigatus. Gene of cytochrome P450 has been identified as involved in fruit body development of A. bisporus [7], C. cinereus [26] and L. edodes [9], but this is the first case of P450 CYP51, which is an essential enzyme required in sterol biosynthesis and primary target of azole antimycotic drugs, isolated as a gene related to fruit body development of mushroom. The multiple alignment of the deduced A8-U polypeptide with putative amino acid sequences of CYP51 from A. fumigatus and Penicillium italicum is shown in Fig. 5.
No highly significant homology was found between the function-known genes and the deduced polypeptide of A8-D and W3-7. But when they were compared with genes deposited in the EST database, high similarity was found with developmentally regulated cDNA of other fungi. W3-7 may encode a protein with 84% similarity to the early developmental gene of arbuscular mycorrhizal fungus Glomus mosseae (Table 1). On the other hand, the deduced polypeptide of A8-D showed significant homology with the predicted protein of Trichoderma reesei cDNA clone (2e 05), expressed during carbon starvation. Interestingly, A8-D codes a common gene involved in the development of T. reesei and P. ostreatus although its function is yet unknown. Northern analysis showed that T19-4 and W3-7 that were detected in primordium by the RAPD analysis, hybridized to total RNA in both primordium and mature fruit body. The fact that T19-4 and W3-7 hybridized to the RNA of both primordium and mature fruit body shows that these mRNAs encode proteins that play specific roles during both stages of fruit body development. One possible reason for why T19-4 and W3-7 were undetected in the RAPD of fruit body stage is that most of the RAPD primers were consumed for amplification of more highly expressed cDNA. In contrast, A8-U and A8-D hybridized only to the total RNA of mature fruit body indicating that
Fig. 5. The deduced amino acid sequence of A8-U was aligned with cytochrome P450 sterol 14 a-demethylase of Aspergillus fumigatus (GI:6942241) and Penicillium italicum (GI:836642). Residues, which are identical to P. ostreatus are marked by asterisks.
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they are specific genes in the latter stage of the development. None of the four cDNA isolated in the present study, hybridized to the RNA derived from mycelium. The control probe, 18S rRNA gene of the P. ostreatus, hybridized to RNA of all the stages (Fig. 3). Liang and Pardee [16] reported about differential displays of mRNA using 3 0 -anchored oligo-dT for the firststrand cDNA synthesis and 10-mer arbitrary primers for the second-strand synthesis by PCR. In their study, numerous amplified fragments were visible after autoradiography of labeled PCR products and the subsequent isolation of the specific DNA band was rather difficult. Leung et al. [11] and Zeppa et al. [6] also used 3 0 -anchored oligo-dT and various random primers for the isolation of differentially expressed gene fragments. The difference between the previous DD studies and ours is that we did not use 3 0 -anchored oligo-dT. The number of cDNA fragments obtained in one PCR reaction was not too large and each cDNA could be resolved as a separate band in agarose gel. With the basidiomycetes, not many genes related to fruit body development have been isolated but they often share common genes. For instance, Hydrophobin is one of the most abundant genes in fruit bodies of basidiomycetes, as reported by Penas et al. [27] and Asgeirsdottir et al. [28] and has been isolated as fruiting related gene with A. bisporus [7], F. velutipes [14] and L. edodes [13]. But we did not detect hydrophobin as a specific gene for fruit body development of P. ostreatus in the present study. ATPase has been detected in L. edodes [11] and T. borchii [6]. PriA was detected in L. edodes [10], Agrocybe aegerita [8] and EST of P. ostreatus [15]. In addition, Septin has been isolated as a gene related to fruit body development [15,29,6]. However, none of these genes were detected in this study. Presumably because the method used in the previous studies screen the elevated expression of genes, abundant genes instead of unique genes tended to be isolated. But by comparing RAPD patterns of ca. 1200 cDNA fragments, abundant but not stage specific gene was easily eliminated. All four cDNAs isolated in this study were novel fruiting genes for basidiomycetes. The Northern analysis confirmed that they were not expressed during the mycelium stage, but expressed after the primordium development. In conclusion, the DD technique was a simple and efficient method to detect fruit body stagespecific cDNA of P. ostreatus.
Acknowledgements We are grateful to Dr. Beom-Gi Kim of the National Institute of Agriculture Science and Technology for providing the Pleurotus ostreatus strain (ASI2029).
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