Genes Expressed During Fruiting Body Formation of Agrocybe cylindracea.

Agrocybe cylindracea, an edible mushroom belonging to Bolbitiaceae, Agaricales, is widely used as invaluable medicinal material in the oriental countries. This study was initiated to find the genes expressed during the fruiting body formation of A. cylindracea. The cDNAs expressed differentially during fruiting body morphogenesis of A. cylindracea were isolated through subtractive hybridization between vegetative mycelia and fruiting bodies. The cDNAs expressed in the fruiting body morphogenesis of A. cylindracea were cloned and twenty genes were identified. Eleven were homologous to genes of known functions, three were homologous to genes in other organism without any function known. Six were completely novel genes specific to A. cylindracea so far examined. Some genes with known functions were a pleurotolysin, a self-assembling poreforming cytolysins; Aa-Pri1 and Pir2p, specifically induced genes during fruiting initiation of other mushroom, Agrocybe aegerita; an amino acid permease; a cytochrome P450; a MADS-box gene; a peptidylprolyl isomerase; and a serine proteinase. For other clones, no clear function was annotated so far. We believe the first report of the differentially expressed genes in fruiting process of A. cylindracea will be great helps for further research.

A. cylindracea is an edible mushroom widely cultivated and consumed all over the world. Recently its consumption is sharply increasing due to its delicious taste and unique texture. In addition to its nutritional value, it has long been used for folk remedies even without any knowledge of which component are responsible. The scientific evidences of this mushroom as multi-purpose medicines on various human diseases have been accumulated. Especially the fruiting bodies of A. cylindracea are popularly used to combat human diseases. It has anti-tumor (Kiho et al., 1989), decreasing sugar level in blood (Kiho et al., 1989), immuno-stimulating (Yoshida et al., 1996), and lipid peroxidation inhibitory activities (Lee et al., 1998). Its polysaccharide extracts have anti-mutagenic activity against direct-acting mutagens such as [4-nitro-o-phenylenediamine (NPD) and sodium azide (NaN(3))] and indirect-acting mutagens [2-aminofluorene (2-AF) and benzo[a]pyrene (B[a]P)]. And its anti-mutagenic activity is co-related with anti-tumor activity by inducing expression of detoxifying enzymes such as quinone reductase and glutathione S-transferase (Shon and Nam, 2001). In addition to exopolysaccharides which are responsible for anti-tumor and immuno-stimulating activities, antifungal proteins can be used for medical use. An antifungal protein named as agrocybin has been isolated in A. cylindracea by Ngai et al. (2005). The agrocybin isolated from fruiting bodies further shows the inhibiting activity of HIV-1 reverse transcriptase (Ngai et al., 2005), possible being used for retarding or healing AIDS disease.

Due to multiple medicinal effects of A. cylindracea on human diseases, efforts getting mycelia and exopolysaccharide without inducing fruiting bodies has also been tried to get much more biomass in short time compared to induction of fruiting bodies (Kim et al., 2005). But still the fruiting bodies of A. cylindracea are widely consumed as health food and therapeutic medicines for various diseases. Thus, study on its differentiating process is worthy for further research.

Until now, no molecular or genetic datum is available about this important mushroom. Although the genetics on the formation of fruiting bodies in other mushroom have been studied in Schizophyllum commune, Pleurotus ostreatus and Lentinus edodes (Hoge et al., 1982; Mulder and Wessels, 1986; Endo et al., 1994; Kajiwara et al., 1992), no one ever tried to find genes involved in fruiting body differentiation. In Lentinus edodes, expressions of several genes which are developmentally regulated during formation of fruiting body are elucidated (Endo et al., 1994; Kajiwara et al., 1992). An EST database containing more that 2000 clones was recently set up for the other edible mushroom, Pleurotus ostreatus with the aim of providing information about transition of vegetative mycelium to fruiting body (Lee et al., 2002). In other basidiomycetes, cDNAs which show down- or up-regulation during the development of fruiting body have been characterized for the Agrocybe aegerita and Agaricus bisporus (Salvado and Labarere 1991; De Groot et al., 1997).

In this research, we tried to identify the genes expressed during the developmental processes of fruiting body in A. cylindracea using suppression subtractive hybridization. We found some important genes specifically induced during the process of fruiting body differentiation. We expect these findings should contribute to a better understanding of gene function in the developmental process of fruiting body as well as in the process of useful metabolite formation.

Materials and Methods

Materials and culture condition

The A. cylindracea culture stock was obtained from Gyeyang mushroom cultivation company (Korea). An agar disk of 5 mm in diameter was cut from the culture that was grown on potato dextrose agar (potato 200 g, dextrose 20 g and agar 20 g/distilled water 1 l) medium at 25℃ in the dark. To get mycelia, the disk was inoculated in potato dextrose broth and cultured with shaking at 25℃ for 7 days. To get fruiting bodies, the culture was placed in a sawdust and wheat bran (8 : 2) medium at 65% absolute humidity and 25℃ for 45 days in dark, then transferred to a culture room that was under continuous illumination by a fluorescent lamp at 15℃ to induce fruiting bodies. The mycelium and fruiting bodies (Fig. 1) of A. cylindracea were harvested separately and kept in liquid nitrogen for RNA extraction.

Fig. 1

Mycelia and fruiting bodies of Agrocybe cylindracea used for SSH.

RNA extraction

Total RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer's protocol and the polysaccharides were removed from the crude RNA by the method of Asif et al. (2000). Poly A+ RNA was isolated from the mycelia and fruiting body using the PolyATtract mRNA Kit (Promega).

Suppression subtractive hybridization (SSH)

SSH was performed with 2 µg of poly A+ RNA using the PCR-SelectTM cDNA Subtraction Kit (Clontech). Mycelial mRNA was used as a driver, and the mRNA of fruiting bodies was used as a tester. The PCR-amplified DNAs selected as differentially expressed in fruiting body were cloned into the TA vector (Invitrogen).

Reverse Northern blot analyses

The subtracted cDNA clones were digested with EcoRI and separated on a 1% agarose gel. The gel was blotted onto duplicate Hybond-N+ nylon membranes (Amersham Pharmacia Biotech), and each blot was screened with alkaline phosphatase-labeled (Amersham Pharmacia Biotech) cDNA probes prepared from the total RNAs of mycelia and fruiting bodies, respectively. The hybridization was carried out at 55℃ in a hybridization buffer (Amersham Pharmacia Biotech). The membranes were washed under highly stringent conditions (0.2 × SSC at 65℃ for 30 min) as described by Sambrook et al. (1989) and exposed to X-ray film for autoradiography.

Reverse transcript-mediated polymerase chain reaction (RT-PCR)

Total RNA was isolated using an RNeasy kit (Qiagen) according to the manufacturer's guidelines. The extracted RNA was treated with DNase to completely remove residual genomic DNA contamination. First strand cDNA was synthesized from total RNA (5 µg) by reverse transcription using the oligo (dT)-primer as the antisense primer. The first strand reaction was used for subsequent PCR reactions to detect gene expression using gene-specific primers designed from the coding sequence of each gene. RT-PCR was performed for 15 to 40 cycles at 5-cycle intervals, and the best RT-PCR result showing non-saturating levels of amplified gene expression was chosen to quantify the transcript level of each gene. The rRNA RT-PCR was used as the internal standard. The primer sets for RT-PCR were shown in Table 1.

Table 1

The primer sets used for RT-PCR of cDNA clones

DNA sequence analyses

Sequences were determined using a DNA sequencer (Applied Biosystems). DNA and predicted amino acid sequences were searched against DNA and protein databases using the BLAST program available at the NCBI website.

Results and Discussion

Isolation and sequence analysis of differentially up-regulated genes during fruiting body formation

To clone genes differentially expressed in fruiting bodies including stipes and pilei of A. cylindracea, a subtraction cDNA library was constructed from fruiting bodies and mycelia by SSH. Approximately 100 cDNA clones were isolated after subtraction (Fig. 2). The cDNA clones were subsequently screened by reverse Northern analysis (Fig. 3). The 20 clones that exhibited differential expression in the fruiting bodies were isolated, and their nucleotide sequences were determined by single-run sequencing. The sequence information was searched against the database using the BLASTn and BLASTx programs. The differentially expressed cDNA clones are summarized in Table 2.

Fig. 2

Examples of putative fruiting body-specific genes cloned by SSH. All genes represented as PCR bands in this figure were used subsequently for reverse Northern analyses. Each numbered lane contains PCR product from positive clones selected by SSH. M; 100 bp DNA size marker.

Fig. 3

Identification of differentially expressed genes by reverse Northern analysis. Arrows indicate differentially expressed genes in the fruiting bodies. Numbers on the panels indicate clone numbers. rRNA were used an internal control to check equal loading.

Table 2

Partial cDNA clones of genes up-regulated in the fruiting bodies of A. cylindracea

Of the 20 cDNA clones, 11 were homologous to genes with known function, three were homologous to genes which have been cloned from other organisms but whose functions have been unknown. Six were revealed as novel genes.

Among 11 genes with known function, the translated polypeptides of Agf 10 and Agf 64 were highly homologous to the pleurotolysin, a self-assembling pore-forming cytolysins (Bernheimer and Avigad, 1979). Agf 19 and Agf 20 were homologues of Aa-Pri1 and Pir2p, respectively. Both Aa-Pri1 and Pir2p are known to be specifically expressed during fruiting initiation of a basidiomycete, Agrocybe aegerita (Fernandez Espinar and Labarere, 1997; Salvado and Labarere, 1991).

Agf 38 showed a characteristic peptide of a general amino acid permease, which is assumed to play a role in uptake of organic nitrogen from the soil. Agf 40, a homologue of a cytochrome P450 was also cloned. The cytochrome P450 is a member of xenobiotic-metabolizing enzyme family (Choudhary et al., 2004). They are believed to metabolize most foreign compounds including carcinogens and drug (Choudhary et al., 2004). Agf 46 was found to belong to a MADS-box gene family. It showed high similarity to APETALA3 and PISTILLATA, which are required for petal and stamen identity in Arabidopsis (Krizek and Meyerowitz, 1996). Agf 51 was identified as a peptidylprolyl isomerase that catalyzes the isomerization of proline residues. Agf 53, a serine-arginine (SR)-rich splicing factors and Agf 66, a serine proteinase were cloned.

Expression pattern of early fruit development-regulated mRNAs

We examined the expression of cloned genes in mycelia and fruiting bodies using semi-quantative RT-PCR (Fig. 4). The genes whose expressions were more than 4 times up-regulated in fruiting bodies compared to the mycelia were as follows; Acf 10, 19, 20, 23, 38, 46, 64, and 66. The moderately up-regulated genes were Acf 2, 7, 16, 25, 34, 37, 40, 51, 53, 56, 62, and 63.

Fig. 4

RT-PCR showing up-regulation of gene expression in the fruiting bodies. Numbers over the panels indicate clone numbers. The rRNA RT-PCR was used as the internal standard. 1, Mycelia; 2, Fruiting bodies.

Agf 10 and Agf 64, homologues of a pleurotolysin, a self-assembling pore-forming cytolysins were firstly found to be up-regulated in fruiting bodies developing process. Considering pleurotolysin has been isolated from fruiting bodies of another edible mushroom, Pleurotus ostreatus (Bernheimer and Avigad 1979), the up-regulation of the gene in Agrocybe cylindracea indicates the induction of pleurotolysin gene expression and protein are not confined in Pleurotus ostreatus. It might be produced in the fruiting process of Agrocybe cylindracea and play a certain role common in the process of fruiting in both types of mushrooms. Agf 19 and Agf 20 whose functions are unknown, were already known to be specifically expressed during fruiting initiation of a basidiomycete, A. aegerita (Fernandez Espinar and Labarere, 1997). Thus, its up-regulation in A. cylindracea in fruiting initiation indicates that 1) this gene expression is related with the fruiting process in both mushrooms and 2) the SSH methods applied in this study and RT-PCR were correctly done. We think a few multiple bands in RT-PCR (Fig. 4) may indicate that some genes belong to gene family in A. cylindracea. The similar intensity of RT-PCR bands of rRNA from mycelia and fruiting bodies shows that RTPCR method for detecting the differences of gene expression in both samples was reliable.

In conclusion, we identified 20 genes differentially expressed in the process of fruiting in A. cylindracea. Among them, 6 were completely novel genes and specific to this mushroom based on the DNA sequence data so far deposited. We believe that these data will provide basic molecular information about further physiological and biochemical changes during fruiting bodies formation in A. cylindracea. Although functions of each up-regulated gene during fruiting process can not be predicted by the data we provided, we believe we provide the first molecular and genetic data of this important mushroom. Further cloning of the full size cDNAs and functional characterization of each gene will be followed based on the data presented in this study.