Isolation of Genes Specifically Expressed in Different Developmental Stages of Pleurotus ostreatus Using Macroarray Analysis.

The oyster mushroom (Pleurotus ostreatus) is one of the most important edible mushrooms worldwide. The mechanism of P. ostreatus fruiting body development has been of interest both for the basic understanding of the phenotypic change of the mycelium-fruiting body and to improve breeding of the mushrooms. Based on our previous publication of P. ostreatus expressed sequence tag database, 1,528 unigene clones were used in macroarray analysis of mycelium, fruiting body and basidiospore developmental stages of P. ostreatus. Gene expression profile databases generated by evaluating expression levels showed that 33, 10, and 94 genes were abundantly expressed in mycelium, fruiting body and basidiospore developmental stages, respectively. Among them, the genes specifically expressed in the fruiting body stage were further analyzed by reverse transcription-polymerase chain reaction and Northern blot to investigate temporal and spatial expression patterns. These results provide useful information for future studies of edible mushroom development.

The oyster mushroom (Pleurotus ostreatus) is commercially important in the worldwide mushroom market. In addition to its importance in food production, P. ostreatus exhibits vigorous lignin degradation activity, which is exploited in industrial applications for biobleaching and catalysis of difficult chemical conversion, and potent immunoactivation activity that is used in pharmaceutical products (Kues and Liu, 2000; Cohen et al., 2002). Basidiomycetes, which have fleshy fruiting bodies commonly known as edible mushrooms, are an important part of many diets worldwide. Understanding the mechanism of fruiting body formation is important because it not only provides basic knowledge of basidiomycetes sexual development but also facilitates improvement of industrial mushroom cultivation.

Coprinus cinereus and Schizophyllum commune are model organisms for studying basidiomycetes mating type factors. Molecular studies on both mushrooms have provided basic understanding about mating and dikaryon formation, which are necessary but not sufficient for fruiting body formation (Casselton and Olesnicky, 1998; Kamada, 2002; Fowler et al., 2004). In addition, particular environmental conditions such as temperature, humidity and nutrient deficiency are required to induce fruiting body formation. These physiological and environmental conditions have been well-characterized, but the molecular mechanisms of fruiting body formation remain unknown despite isolation of several abnormal fruiting body mutants and their complementing genes in C. cinereus (Muraguchi and Kamada, 1998; Kues, 2000; Terashima et al., 2005; Liu et al., 2006).

Recently, several different molecular biology methods including differential display reverse transcription polymerase chain reaction (DDRT-PCR), expressed sequence tag (EST) analysis and reverse Northern blot were used to isolate developmentally expressed genes from several commercial mushrooms including Lentinus edodes and P. ostreatus, as well as from model basidiomycetes such as C. cinereus and S. commune (Dons et al., 1984; Sunagawa and Magae, 2005; Yamada et al., 2006). In addition, transformation techniques were developed to identify the function of genes in several commercial mushroom species, although the transformation efficiency was lower than that in other model basidiomycetes (Kim et al., 1999, 2003; Kuo et al., 2004). We tried to profile quantitative gene expression patterns during fruiting body development using large scale macroarray analysis in P. ostreatus. From a P. ostreatus EST database of three different developmental stages (mycelium, fruiting body and basidiospore), 1528 genes were isolated as unigenes through sequence comparison (Lee et al., 2002; Joh et al., 2007). Presently, these cDNA clones were selected as unigenes and were used in macroarray analysis to generate a gene expression profile for fruiting body development.

Materials and Methods

Strains and culture conditions

P. ostreatus strain ASI 2029, a Korean wild type strain, was obtained from the National Institute of Agricultural Science and Technology (NIAST, Suwon, Korea). ASI 2029 was grown on MCM agar plate (0.2% yeast extract, 0.2% peptone, 2% glucose, 0.05% MgSO4·7H2O, 0.05% KH2PO4 and 0.1% K2HPO4) for 1 week at 28℃. The mycelia were homogenized using a blender, inoculated into liquid MCM, and then cultured vigorously by shaking (230 rpm) at 28℃ for 1 week. For the production of fruiting bodies, ASI 2029 was inoculated in 570 g poplar sawdust 120 g rice bran and 65% water in a 1000 ml bottle. The cultures were incubated at 25℃ in the dark for 25~40 days and then transferred to conditions that induced fruiting (12~15℃, greater than 85% humidity, and light). Liquid-cultured mycelia and fruiting bodies were frozen in liquid nitrogen and stored at -80℃.

RNA and plasmid DNA extraction

For macroarray analysis and Northern blot hybridization, samples were harvested in several different stages: mycelium grown in sawdust (45 days after incubation); mycelium (9 days after cold shock); primordia, young fruiting body, mature fruiting body and basidiospore. Total RNA was extracted using TRI Reagent (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer's protocol. Plasmid DNAs were prepared using a plasmid purification kit (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol.

Macroarray analysis

Concentration-adjusted (100 ng) plasmids for 1528 unigene clones were manually deposited on positively charged nylon membranes (Amersham Biosciences, Piscataway, NJ, USA). Radiolabelled cDNA probes were synthesized by incorporating [α-33P] dCTP into primary strand cDNA. Total RNA (50 µg) was mixed with oligo (dT)18, heated to 70℃ for 10 min and chilled on ice. It was then mixed with 20 µl 5 × first strand buffer, dATP, dGTP, dTTP (333 µM, each) and dCTP (10 µM), 70 µCi of [α-33P]-dCTP and 1000 units Moloney murine leukemia virus (MMLV) reverse transcriptase (Promega, Madison, WI, USA). The reaction mixture was incubated at 37℃ for 1 h. The blots were prehybridized in 15 ml hybridization solution at 65℃ for 1 h. The entire collection of cDNA probes was added to the prehybridization solution and the filters were hybridized with probes for 20 h. The hybridized filters were washed consecutively with 2 × standard saline citrate (SSC)/0.1% sodium dodecyl sufate (SDS) for 20 min at 65℃, with 1 × SSC/0.1% SDS for 20 min at 65℃ and with 0.5 × SSC/0.1% SDS for 20 min at 65℃. The filters were exposed to BioMax MR film (Kodak, Rochester, NY, USA) for 48 h.

Data analysis

The exposed BioMax MR films were scanned on an ARCUS II apparatus (Agfa-Gevaert, Mortsel, Belgium). The resulting TIFF image files were analyzed using TotalLab software (Nonlinear Dynamics, Newcastle upon Tyne, UK) to determine the pixel density (intensity) for each spot on the filter. β-actin clone spot intensity was calculated as 100 and was used as a scaling factor for the spot intensities of genes.

RT-PCR and RNA analysis

First-strand cDNA was synthesized using MMLV reverse transcriptase (Promega) with total RNA and oligo dT primer. Second-strand primers were designed based on EST sequences to produce 300~500 bp DNA fragments. PCR was performed using a PTC-200 thermocycler (MJ Research, Waltham, MA, USA). Total RNA was fractionated on a 1% denaturing agarose gel and transferred onto nylon membranes (Amersham Biosciences). Northern blots were hybridized with 32P-labelled probe DNA, and washed consecutively with 2 × SSC/0.1% SDS for 20 min at 65℃, with 1 × SSC/0.1% SDS for 20 min at 65℃ and with 0.5 × SSC/0.1% SDS for 20 min at 65℃. The filters were exposed to BioMax MR film (Kodak).

Results

Gene expression profiles in different developmental stages of P. ostreatus

A total of 4,060 different genes (PoUnigenes) were already identified in P. ostreatus by EST studies (Lee et al., 2002; Joh et al., 2007). Of these unigene clones, 581, 592 and 355 unigenes were selected from three different cDNA libraries of liquid-cultured mycelia, fruiting bodies, and basidiospores, respectively, for gene expression profiling. Plasmids isolated from these clones were blotted on nylon membranes for macroarray analysis. Meanwhile, total RNA was isolated from three different developmental stages [mycelia cultured for 1 week after 1 week preculture in MCM liquid media (Mc) fruiting bodies sampled 20 days after cold shock (Fb) and basidiospores (Sp)]. 33P-labelled cDNA probes generated from total RNAs were hybridized with the clones fixed on membranes. All photographic values of hybridization signals were quantified. Of 1,528 genes screened, 1,036 (68%) genes showed clearly readable expression levels (expression value ≥ 50) in at least one of the three developmental stages (Fig. 1). Genes with known patterns of expression, such as HYDROPHOBIN and PRIA, served as a quality control and showed that this macroarray analysis produced reasonable gene expression data.

Fig. 1

cDNA macroarrays of 1594 unigenes of P. ostreatus hybridized with probes generated from RNA extracted from A, liquid-cultured mycelia; B, mature fruiting body; C, basidiospore.

Isolation of specifically expressed genes in mycelia and basidiospores stages

Gene expression profiles were analyzed in order to isolate genes specifically expressed at the fruiting body stage of developmental stages. Genes were selected when the expression value was above 200 in only one developmental stage and below 100 in the other developmental stages. We identified 33 genes that were expressed specifically in liquid-cultured mycelia (Table 1). Among them, the 01893LM clone encoding an unidentified protein showed the highest expression value. The hydrophobin gene (1934LM) showed specific expression in liquid-cultured mycelia, as previously reported. However, most of the genes specifically expressed in mycelia did not show significant homology with functionally characterized genes (E value < 1E-10). Ten genes were identified as genes specifically expressed in basidiospores (Table 2). Among them, two genes showed high homology with secreted glycosyl hydrolase and acyl-CoA dehydrogenase. Genes P03-E11 and P01-E06 exhibited the two highest expression values.

Table 1

Genes specifically expressed in liquid cultured mycelia

LCM (liquid cultured mycelia), SPO (basidiospores), MFB (mature fruiting body)

Table 2

Genes specifically expressed in basidiospores

LCM (liquid cultured mycelia), SPO (basidiospores), MFB (mature fruiting body)

Specific gene expression related to fruiting body formation

Using the criteria described above, we isolated 94 genes that were expressed specifically during the fruiting body stage (Table 3). Of these, 14 genes were highly expressed, exhibiting expression values > 1,000. Of these 14 genes, AaPri1 was the only gene previously reported, while the other 13 did not correspond to any previously reported gene. To confirm expression patterns of these 13 genes, RT-PCR was performed using total RNA isolated from four different developmental stages. Seven genes were successfully amplified (Fig. 2). Two genes (MFB01-B08 and MFB08-B09) were overexpressed abundantly in the fruiting body stage, although they were also expressed at much lower levels in mycelia and basidiospores. Two other genes were expressed in the fruiting body stage but also in basidiospores (MFB02-E09) or mycelia (MFB08-D01). Three genes (MFB12-B10, MFB9-A07 and MFB6-B06) were expressed only in the fruiting body stage. To investigate the temporal and spatial expression of these genes during fruiting body development, Northern blot analysis were performed using total RNA isolated from 17 dissected developmental stages and tissues. Three genes showed clear Northern hybridization signals corresponding to RT-PCR results. However MFB02-E09 showed fruiting body stage-specific expression but not fruiting body tissue-specific expression.

Table 3

Genes abundantly expressed in mature fruiting bodies

LCM (liquid cultured mycelia), SPO (basidiospores), MFB (mature fruiting body)

Fig. 2

RT-PCR analysis of genes specifically expressed in the fruiting body stage. Lane 1, liquid cultured mycelia; Lane 2, primordia; Lane 3, fruiting bodies; Lane 4, basidiospores.

Discussion

Fruiting body development from vegetative mycelia has attracted much scientific and economic interest. Many environmental factors (i.e., temperature, light, nutrients and fruiting inducing substances) and genetic factors (i.e., mating type genes and developmentally regulated genes) influence fruiting body formation (Kues and Liu, 2000; Wessels, 1993). These factors are well-characterized. However, how gene expression is regulated and how environmental signals are interpreted during fruiting body formation in the edible mushroom remains elusive. To study gene expression during fruiting body formation of P. ostreatus, expression profiles were established in three different developmental stages of the edible mushroom, Pleurotus ostreatus, using macroarray analysis. A total of 137 genes were identified as being specifically expressed in three different developmental stages. Among them, 94 genes were abundantly expressed in fruiting bodies, perhaps as part of fruiting body formation. Several of the genes encoded proteins of known function, including Aa-Pril, Hsp12, histidine kinase and glyoxal oxidase. Aa-Pri1 was previously reported to be highly expressed only in the primordial and fruiting body stages of Agrocybe aegerita (Fernandez and Labarere, 1997). Hsp12 gene was postulated to be highly expressed in fruiting bodies based on its high redundancy observed in the fruiting body EST database (Lee et al., 2002). Mutants of histidine kinase and glyoxal oxidase genes were reported to impair sexual development of Dictyostelium and Ustilago maydis, which suggests that they may play a critical role in fruiting body development in homobasidiomycetes (Leuthner et al., 2005; Thomason et al., 2006).

Fruiting body formation is induced in stress conditions such as nutrient deficiency and cold shock. Hence, it is reasonable to speculate that stress-related genes such as Hsp12, Nam9 and DNA repair helicase RAD15 are expressed specifically in fruiting bodies. These genes were not found to be directly related to fruiting body induction, but they may be necessary components in the fruiting development processes. Complex signal transduction mechanisms are required in cells to recognize, transduce and respond to environmental signals. Components of signal transduction such as calmodulin, phosphatase 2C and RAB18 were developmentally expressed during fruiting body formation, and the function of these genes would be deduced by these expression profiles even if this is a primitive clue that these genes function in fruiting body formation.

Among the 95 genes expressed abundantly in fruiting body, more than half encoded proteins of unidentified function. These functions, and their relevance to fruiting body formation, await further studies.

The expression profile data presented here represents an important contribution to the elucidation of molecular mechanisms of fruiting body development of P. ostreatus.