5'-Serial Analysis of Gene Expression studies reveal a transcriptomic switch during fruiting body development in Coprinopsis cinerea
© Cheng et al.; licensee BioMed Central Ltd. 2013
Received: 10 October 2012
Accepted: 8 March 2013
Published: 20 March 2013
The transition from the vegetative mycelium to the primordium during fruiting body development is the most complex and critical developmental event in the life cycle of many basidiomycete fungi. Understanding the molecular mechanisms underlying this process has long been a goal of research on basidiomycetes. Large scale assessment of the expressed transcriptomes of these developmental stages will facilitate the generation of a more comprehensive picture of the mushroom fruiting process. In this study, we coupled 5'-Serial Analysis of Gene Expression (5'-SAGE) to high-throughput pyrosequencing from 454 Life Sciences to analyze the transcriptomes and identify up-regulated genes among vegetative mycelium (Myc) and stage 1 primordium (S1-Pri) of Coprinopsis cinerea during fruiting body development.
We evaluated the expression of >3,000 genes in the two respective growth stages and discovered that almost one-third of these genes were preferentially expressed in either stage. This identified a significant turnover of the transcriptome during the course of fruiting body development. Additionally, we annotated more than 79,000 transcription start sites (TSSs) based on the transcriptomes of the mycelium and stage 1 primoridum stages. Patterns of enrichment based on gene annotations from the GO and KEGG databases indicated that various structural and functional protein families were uniquely employed in either stage and that during primordial growth, cellular metabolism is highly up-regulated. Various signaling pathways such as the cAMP-PKA, MAPK and TOR pathways were also identified as up-regulated, consistent with the model that sensing of nutrient levels and the environment are important in this developmental transition. More than 100 up-regulated genes were also found to be unique to mushroom forming basidiomycetes, highlighting the novelty of fruiting body development in the fungal kingdom.
We implicated a wealth of new candidate genes important to early stages of mushroom fruiting development, though their precise molecular functions and biological roles are not yet fully known. This study serves to advance our understanding of the molecular mechanisms of fruiting body development in the model mushroom C. cinerea.
KeywordsCoprinopsis cinerea SAGE Transcriptome Microarray Transcription start site Fruiting body Mycelium Primordium
The mystery of mushroom formation has fostered a deep curiosity for understanding the molecular mechanisms underlying fruiting body initiation and development in basidiomycetes and a goal of mycological research community. Coprinopsis cinerea (previously known as Coprinus cinereus), the inky cap, is a model mushroom commonly employed to study developmental processes in agaricomycetous fungi . In addition to studies related to mating types, enzyme production, and genome manipulation, much emphasis has been put on the fruiting process, particularly on the later stages including synchronous karyogamy and meiosis. This process is the most complex, yet rapid, of the developmental events in the life cycle of C. cinereus. When nutrients are depleted, the relatively loose mesh of undifferentiated mycelium undergoes a drastic change to form a compact multihyphal structure with many different cell types holding each other through hyphal-hyphal interactions, known as the fruiting body . The fruiting process can be divided into six main developmental stages: hyphal knot, initial, stage 1 primordium, stage 2 primordium, immature fruiting body and mature fruiting body . Besides nutritional constraints, the normal day-night rhythm cycle is also critical for the transition of one stage to another [3, 4]. Under ideal conditions, the whole process takes only a few days following the first sign of fruiting .
The advancement from mycelium (Myc) into stage 1 primordium (S1-Pri) is hypothesized to show the most significant transcriptomic changes as it represents development from an undifferentiated structure to a well-organized multihyphal structure at a later stage of fruiting body formation. A number of genes have been previously characterized to be involved during this transition. These include the oligosaccharide-binding galectins cgl1 and cgl2 for potential but yet unproven mediation of hyphal interactions [6, 7]; a homolog to the bacterial cyclopropane fatty acid synthase for generating stress signals as a result of membrane alteration ; blue light photoreceptors dst1 and dst2 for photomorphogenesis [3, 4]; a Ustilago maydis adaptor protein Ubc2 homolog for filamentous growth, pheromone response and virulence  and a number of metabolic enzymes such as adenylate cyclase, phenol oxidase and glycogen phosphorylase . Nevertheless, the lack of large scale assessment of gene expression hinders the generation of a more comprehensive picture of the fruiting process. In order to better understand the molecular basis underlying this transition, it is desirable to identify the genes differentially expressed among these two developmental stages through comparing the respective transcriptomes. Such studies are especially feasible in C. cinerea because of the recent release of its 37.5 Mb genomic sequence and predicted gene set consisting of 13,342 protein-coding genes .
Serial Analysis of Gene Expression (SAGE), invented in 1995, is a digital expression tag profiling technology for high throughput genomic-level evaluation of transcriptomes . Over the past 17 years, SAGE has become a well-recognized tool and has been extensively used in human, animal, yeast, plant and fungal studies . The advancement of next-generation DNA sequencing technologies in recent years has enabled SAGE analysis to attain higher throughput, sensitivity and cost-effectiveness compared to the original cloning-based and Sanger sequencing approaches [13–15]. In addition, the SAGE protocol was improved based on a combination of 5′ RACE and SAGE, known as 5′-SAGE, which extracts the first 15–17 bp of each mRNA transcript [16, 17]. This allows simultaneous characterization of the transcriptome and transcription start site (TSS). The TSS data are invaluable for identification and analyses of promoter regions and cis-regulatory elements that contribute to better understanding of higher-order regulatory mechanisms . The >100 bp sequence reads available with 454 Life Sciences (GS20) sequence technology  is well-suited for the length of 5′-SAGE ditags. The short length, but more numerous sequences from the 454 approach allows the tedious steps of concatenation and colony picking in the original SAGE procedures to be bypassed.
Here, we report a high throughput analysis of the two 5′-SAGE libraries, supplemented by oligomicroarray data, for comprehensive assessment of the transcriptomes of C. cinereus at the Myc and S1-Pri stages. A wealth of novel candidate genes and molecular mechanisms related to fruiting body initiation and development are identified from the comparison of the gene expression profiles at these stages. The genes and pathways identified will serve as an important platform for future studies in developmental biology of basidiomycetes.
Results and discussion
Sequencing of 5′-SAGE ditags and separation into tags
The 5′-SAGE libraries were constructed from each Myc and S1-Pri mRNA preparations and we obtained a total of 198,809 valid ditags from both developmental stages. At an average read length of 95.5 bases, the sequences accounted for 94.9% of the GS20 sequencer throughput. After tag extraction and quality filtering, we obtained a total of 253,415 valid tags, with 107,046 from the Myc stage and 146,369 from the S1-Pri stage.
Tag to gene assignment
Summary of tag extraction and C. cinerea genome mapping
Total valid tags
- Unique match to genome
- Multiple matches to genome
- 2–4 matches
- 5–10 matches
- >11 matches
- No match to genome
Gene-associated positions of tags mapped to the C. cinerea genome
Putative 5′-UTR (−500, -1) a
Putative 5′-UTR (Anti-sense) b
Coding region (sense)
Coding region (Anti-sense) b
Putative 3′-UTR (+1, +500) a
Putative 3′-UTR (Anti-sense) b
Profiling and functional annotation of the differentially expressed genes (DEGs)
Statistics of the Myc and S1-Pri differentially expressed genes (DEGs)
No. of genes with detected expression
No. of differentially expressed genes (DEGs)a
No of DEGs
with homolog identified in BLASTXb
with >3-fold up-regulation
with GO term
with KEGG ortholog
unique to fruiting body-forming
unique to C. cinerea
No. of enriched protein domainsd
List of top 15 differentially expressed genes in the Myc stage
BLASTX homolog [Organism]
CoH1 [Coprinopsis cinerea]
hemerythrin HHE cation binding domain protein [Neosartorya fischeri NRRL 181]
CoH1 [Coprinopsis cinerea]
putative phosphatidic acid phosphatase [Pleurotus sp. 'Florida']
GO:0005887 GO:0008195 GO:0006644
List of top 15 differentially expressed genes in the S1 Pri stage
BLASTX homolog [Organism]
hydrophobin [Tricholoma terreum]
hydrophobin [Tricholoma terreum]
endochitinase [Amanita muscaria]
xyn11C [Chaetomium thermophilum]
GO:0005576 GO:0016798 GO:0045493
membrane transporter [Cryptococcus neoformans var. neoformans JEC21]
histidine acid phosphatase, putative [Aspergillus clavatus NRRL 1]
The DEGs were characterized by annotation using the Gene Ontology (GO) database . GO terms were assigned to the DEGs (see “Methods”). A higher proportion of DEGs in the S1-Pri stage could be assigned at least one GO term, with 530 (out of 716, 74%) compared to 146 (out of 324, 45%) as in the Myc stage (Table 3). We have also mapped all the DEGs to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways database to investigate molecular interactions and reaction networks . Consistent with our expectations, an overall up-regulation of various cellular processes between the undifferentiated mycelium and the primordium stage 2 days prior to karyogamy was observed. Analysis result from the GO and KEGG is available in the supplemental files 5, 6, 7 and subsections “More DEGs in S1-Pri were assigned a GO term in Gene Ontology analysis” and “KEGG pathways analysis revealed an increased cellular metabolism in S1-Pri” respectively.
Validations of 5′-SAGE with microarray gene expression and real-time RT-PCR
Correlation of expression of 18 randomly-selected genes among 5 ′ -SAGE, microarray and real-time RT-PCR
Thiazole biosynthetic enzyme
CMGC/MAPK protein kinase
Actin cytoskeleton protein VIP1
Ubiquitin-carboxy protein fusion
ATP synthase subunit 5
60S ribosomal protein L34-b
40S ribosomal protein S14
Basic leucine zipper & W2 domain-containing protein 2
Correlation of 5′-SAGE and real-time PCR: R=0.936
Correlation of 5′-SAGE and microarray: R=0.792
Gene expression differences among gene families
Among the 324 up-regulated genes in the mycelium stage, only 157 (49%) have a homolog identified through BLASTX search in the NCBI GenBank (e<10-5) (Table 3). Compared to 70% (500 out of 716 DEGs) as in the S1-Pri stage, this highlighted that many of the mycelial genes are actually novel. Expression of the hydrophobin 1 gene (coh1, CC1G_02183), 3 hydrophobin genes that are in tandem with coh1 (CC1G_02181, CC1G_02182, CC1G_02184) and 2 other hydrophobin genes (CC1G_04843, CC1G_10189) in Myc revealed that two abundantly expressed ones (CC1G_02183, CC1G_02184) are among the 10 most highly expressed in this stage. All 6 genes show significantly reduced expression in S1-Pri including 4 with no detectable expression. Inverse expression patterns were seen in 4 hydrophobin genes (CC1G_01230, 06484, 04060, 04061), which were highly expressed in S1-Pri but had no detectable transcripts in Myc in 5′-SAGE. The expression of the hydrophobin 2 gene (coh2, CC1G_02185) was not detected in either developmental stage. These observations are consistent with previous findings in the mushrooms Lentinula edodes and Pleurotus ostreatus which employ two different sets of hydrophobin genes during fruiting body development [34, 35]. These hydrophobins are thought to play alternative structural roles in the respective stages.
A similar phenomenon is observed for the heat shock proteins (HSP). We found 5 HSPs up-regulated in Myc (CC1G_01131, CC1G_02271, CC1G_02341, CC1G_04585, CC1G_15006), whereas 2 are up-regulated in S1-Pri together with 1 heat shock transcription factor (CC1G_04586, CC1G_13982, CC1G_10208). We speculate that the widely employed mycelial cultivation temperature of 37°C compared to the standard fruiting temperature of 25°C [8, 10, 33] may be partly responsible for this, while other stresses during fruiting development may turn on the corresponding HSPs in the S1-Pri stage . Notably, among the 30 most highly expressed DEGs in Myc, only 7 of them have a detectable homolog in BLASTX search (e<10-5). Apart from two of the aforementioned hydrophobin genes, 3 of them (CC1G_07702, CC1G_08572, CC1G_08573) correspond to “mismatched base pair and cruciform DNA recognition protein” which is highly conserved among the fungal kingdom (A. bisporus homolog, NCBI accession ID: CAB85690). Little is known of their molecular functions and biological roles in fungal development, but the observations that this family of proteins were among the 5 most highly expressed in Myc and were significantly repressed in S1-Pri highlight their potential involvement in fruiting body development.
Genes up-regulated in stage 1 primordium
Despite a larger number of genes in the S1-Pri stage for which a homolog can be identified, excluding the mating type genes , few other genes have been described as playing a role in fruiting body formation in C. cinerea. Some developmentally regulated genes previously characterized include the galectins (β-galactoside binding lectins, CGLs). We showed that the expression of cgl1 (CC1G_05003), which is highly up-regulated in S1-Pri, is in agreement with a previous report . However, the expression of cgl2 was not detected in either Myc or S1-Pri stage in our study. One potential explanation for this, as suggested by Boulianne et al. , is that cgl2 expression may be repressed by sufficient nutrient levels in the medium (as for Myc) or under light illumination (as for S1-Pri) which resemble the conditions under which our RNA samples were harvested. Our data also revealed the expression of a cgl3 gene with novel carbohydrate-binding specificities (CC1G_00723) , which is highly up-regulated in S1-Pri as well. Cgl3 is dissimilar in protein sequence to both Cgl1 and Cgl2. The most related homolog is a galectin gene from the ectomycorrhizal basidiomycete Laccaria bicolor (NCBI accession ID: XP_001877242), yet the homologs are only 58% identical with no other significantly similar sequences identified. The uniqueness and differential expression of cgl3 probably deserves a deeper investigation.
The expression of tubulin genes has been previously explored [41–43]. Examination of this gene family found three components of microtubules: alpha1 tubulin (CC1G_01375), alpha2 tubulin (CC1G_00146), beta tubulin (CC1G_04743), which are all up-regulated in S1-Pri with a similar 5-fold of change, accompanied by a 3-fold increase of a tubulin-specific chaperone (CC1G_00477) suggesting that microtubule formation is systematically regulated and necessary for early fruiting events.
More DEGs in S1-Pri were assigned a GO term in Gene Ontology analysis
Involvement of nutrient-related pathways in fruiting body development
Involvement of the MAP kinase pathway in fruiting body development
As far as the cAMP-PKA signaling pathway is concerned, we are also interested in investigating the MAP kinase (MAPK) signaling pathway, since it has been well-documented that the two pathways function in parallel with cross talk between them [46, 52, 53]. In U. maydis, the MAP kinase homolog Ubc3 is essential for pheromone responses, mating and filamentation , and similar developmental roles have also been described in budding yeast , C. neoformans and Neurospora crassa. However, the role of the MAPK cascade in fruiting body development in basidiomycetes has not been studied in great detail save for observations such as MAPK was found preferentially expressed in primordium and young fruiting bodies in L. edodes. It was also shown that mutation of the protein kinase regulator Cc.ubc2 (CC1G_00975) gene blocked phosphorylation of a presumptive MAP kinase . Nevertheless, not only was expression of the Cc.ubc2 gene not detected in either developmental stage in our 5′-SAGE data, we also showed low expression levels for the components in the MAP kinase pathway. However, real-time PCR demonstrated that two loci of MAP kinase (CC1G_07620 and CC1G_03633) were up-regulated by 0.4 fold and down-regulated by 70% in S1-Pri respectively. Consistent with such results, expression of MAP kinase kinase loci (MAPKK) (CC1G_03368 and CC1G_02205) were up-regulated by 1.2-fold and down-regulated by 78% in S1-Pri respectively. It is unclear whether the two apparent subsets of MAPK/MAPKK combinations function independently, despite the high similarity in the conserved functional domains in these kinases. Accordingly, the budding yeast Ras2 homolog (CC1G_04430), which has been implicated to function as an upstream activator of both the cAMP-PKA and MAPK signaling cascades [60, 61], was also found to be moderately up-regulated by 0.5-fold in S1-Pri. This suggests that the MAPK pathway and its activator may still be necessary although not up-regulated, for fruiting body development in C. cinerea.
KEGG pathways analysis revealed an increased cellular metabolism in S1-Pri
Among the 270 up-regulated genes (38%) in S1-Pri in which a KEGG ortholog could be identified (Table 3), 62 (23%) were annotated to be constituents of ribosome and most of the remaining were mapped to metabolic pathways involving oxidative phosphorylation (27), nucleotide metabolism (14), TCA cycle (6), sugar metabolism (7), amino acids metabolism (20) and protein degradation (12) (Additional file 6). This implies a significant higher demand for energy production, DNA synthesis, protein synthesis machinery and turnover during fruiting body development. In contrast, only 56 up-regulated genes (17%) in Myc were assigned a KEGG ortholog, and almost all of them were mapped to basic cellular metabolism pathways (Additional file 7). Furthermore, these 56 genes were assigned to as many as 69 different pathways and most of which only had one and two mapped components (47 and 19 respectively), thereby implying that few pathways are preferentially up-regulated during mycelial growth. Moreover, a remarkable 20% of Myc and 45% of S1-Pri DEGs with KEGG ortholog could not be assigned to a KEGG pathway.
Novel pathways potentially involved in fruiting body development
Furthermore, the protein riboflavin-aldehyde forming (Raf) enzyme was found to be differentially expressed in primordium in both L. edodes and Agaricus bisporus during fruiting body formation. Although our 5′-SAGE data only showed a slight increase in the expression of the raf gene, 4 DEGs (CC1G_13718, 01137, 04209, 11697) in the S1-Pri stage were mapped to the pathway riboflavin metabolism. Genes CC1G_13718 and CC1G_01137 encode the alpha and beta chain of riboflavin synthase, suggesting that an up-regulation of riboflavin synthesis may be a necessary complement to the increase in expression of the Raf enzyme in C. cinerea as well as other fruiting body-forming basidiomycetes.
Protein domain enrichment
We searched all the annotated ORFs for protein domains using NCBI Conserved Domains , NCBI Protein Clusters , Pfam  and COG  databases. A total of 5,868 conserved domains were assigned to 3,751 ORFs at an e-value threshold of e<10-20. The occurrence of conserved domains in the Myc and S1-Pri DEGs were compared against all the ORFs. A domain is defined as ‘enriched’ in the set of DEGs if it has a p-value lower than 0.05 in the Fisher’s Exact Test. We identified 118 enriched domains in the Myc stage (Additional file 8) and 115 in the S1-Pri stage (Table 3) (Additional file 9). Intriguingly, as many as 93 Myc DEGs-enriched domains are present only once across the genome, and they are all represented in the Myc DEGs but never in the S1-Pri DEGs. This suggests types of protein functions suppressed during fruiting body formation.
We observed an enrichment of PIWI domains  in the Myc DEGs. Since this family plays a role in RNA silencing, as found in piwi proteins and a large number of piwi-related nucleic acid-binding proteins, our observations may provide some insights into gene regulation as fruiting occurs . Among these 115 S1-Pri DEGs-enriched domains, 42 belong to the Ras/Rab/Ran families and 11 to the ribosomal protein domains, which are consistent with previous observations of a significant up-regulation of signal transduction and protein synthesis events. Other highly enriched domains in S1-Pri DEGs include 10 enoyl-CoA hydratase, 8 tubulin, 7 ARF (ADP ribosylation factor)/ARF-like domains found in GTP-binding proteins of the Ras superfamily , which deserve further investigation to uncover their roles during fruiting body development.
Genes unique to fruiting body-forming basidiomycetes and C. cinerea
We searched all differentially expressed genes in both Myc and S1-Pri stages against the NCBI BLAST database and specifically against our in-house sequence database which consisted of the protein sequences of Saccharomyces cerevisiae, filamentous fungi (C. neoformans, U. maydis, Aspergillus fumigatus), L. edodes, and a number of fruiting body-forming basidiomycetes including L. bicolor, Phanerochaete chrysosporium, Postia placenta, S. commune, and two others P. ostreatus and A. bisporus obtained from the JGI Genome Portal . In order to identify the DEGs that are unique to the fruiting body-forming basidiomycetes, we filtered out the genes in the blast reports that were dissimilar in sequences (e>10-2) and used a threshold of e<10-10 to indicate sequence homology among the seven basidiomycetous species. Under these criteria, we identified 22 Myc and 30 S1-Pri DEGs that are uniquely found in at least two fruiting body-forming basidiomycetes (Additional file 10). We speculate that these genes are fundamental and unique to fruiting body development in basidiomycetes despite their roles being unclear. Intriguingly, using the same criterion of e<10-10, we identified 41 Myc and 22 S1-Pri DEGs that were found only in the C. cinerea genome (Additional file 11). These genes do not bear any conserved protein domains and were not assigned to any GO terms or KEGG pathways. They may be novel and specific to fruiting body development in C. cinerea.
We have demonstrated the capability of a combined approach of the 5′-SAGE procedures and high-throughput pyrosequencing to evaluate the transcriptomes of C. cinerea at the mycelium and stage 1 primoridum stages respectively. Expression of more than 3,000 genes were assessed, and we showed that one-third of these genes were preferentially expressed in either growth stage, suggesting an overall transcriptomic switch during the transition from mycelium to primordium. Two different sets of structural and functional proteins were found to be uniquely employed in either developmental stage. We demonstrated that sensing the nutrient levels and the environment and response to these changes, in addition to an up-regulation of cellular processes including energy production, DNA/protein synthesis, protein degradation, are crucial for fruiting body development. We also hypothesize an independent regulation of the MAPK and cAMP signaling pathways and an up-regulation of the TOR signaling pathway as necessary requirements. A wealth of candidate genes and anti-sense transcripts potentially related to early fruiting events were also uncovered. The 5′-SAGE data presented in this study serves to advance our understanding of the molecular mechanisms of fruiting body development in C. cinerea, which may as well be applicable to the other basidiomycetes that form fruiting bodies.
Strains and culturing
A dikaryotic C. cinerea strain, mated from two wild-type monokaryon strains J6;5–5 and J6;5–4 , was used in this study. The two monokaryotic strains were re-generated from single spore isolates of a dikaryotic strain which had been backcrossed with the reference strain Okayama 7 #130, for which its genome sequence is available, for 5 generations. Mycelium (Myc) was grown at 37°C in darkness on solid YMG medium and harvested when the mycelium just fully covered the Petri dish (approx. 7 days). Stage 1 primordium (S1-Pri) was grown at 25°C under a light/dark regime of 14/10 hr [10, 33, 82] and harvested by picking individual primordia from the base when they were at 2 days prior to karyogamy (height of about 5 mm).
Total RNA was extracted by TRI® reagent (Molecular Research Center, Inc) and Poly(A)+ mRNA was furthered isolated using the PolyATract® mRNA isolation system (Promega). First strand cDNA was synthesized using SuperScript™ III First-Strand Synthesis System for qRT-PCR (Invitrogen). Two different template switching oligos were employed for each developmental stage. Reverse transcription was performed at 42°C for 90 minutes. Second strand cDNA was then synthesized by low cycle primer extension using Advantage® 2 polymerase (Clontech). Double-stranded cDNA was purified by QIAquick PCR purification kit (Qiagen) and checked by electrophoresis on a 1.5% agarose gel.
The cDNAs were digested by 10units of MmeI (New England Biolabs) at 37°C for 2 hours. The digested products were extracted by phenol-chloroform and precipitated by ammonium acetate and ethanol at −20°C overnight, then re-suspended at low salt TE (2.5 mM Tris HCl, 0.25 mM EDTA, pH8.0) and electrophoresed on 15% polyacrylamide gel (Bio-Rad). After electrophoresis, the ~50 bp bands were excised, disrupted and the DNA contents were eluted and pooled. Following ethanol precipitation with ammonium acetate at −70°C for 4 h, pellets were collected and resuspended in low-salt TE buffer.
The pools of MmeI-digested fragments were ligated by 600units of T4 DNA ligase at 16°C overnight to form 100 bp ditags. The ligation mixtures were divided into half and PCR-amplified. The two PCR reactions were then pooled together, extracted by phenol/chloroform and precipitated with ammonium acetate and ethanol. Pellets were collected by centrifugation and resuspended in 15 μl ultra-pure water.
Prior to pyrosequencing, the identity of the 100 bp ditags were checked by 2% agarose gel electrophoresis and TA cloning using cloning vector pMD-18 T (Takara Biotechnology). Products of PCR screening with expected size were purified and sequenced for confirmation of the identity of the ditags. The mycelial and primordial ditags were sequenced with a GS20 sequencer in two separate metrics at 454 Life Sciences (Connecticut, U.S.A.).
Tag extraction and genome mapping
The accuracy of all sequence reads were checked using a Phred-equivalent quality score. Perl scripts were written to remove low-quality sequences, extract valid ditags and subsequently extract individual tags. All tags were mapped to the C. cinerea genome sequence obtained from the Broad Institute using Perl scripts . Only tags with a single exact match were retained for further analysis. Singleton tags were also discarded.
Identification of differentially expressed genes
The expression level of individual genes was assessed by summing up the occurrence of all the tags mapped to the putative 5′-UTR and dividing this number by the total occurrence of all genome-mapped tags, then multiplying by 100,000, following which they were expressed as tags per hundred thousand in the corresponding stage. Differentially expressed genes (DEGs) between the Myc and S1-Pri stages were defined by a p-value of 0.05 in the pairwise Fisher Exact Test.
GO and KEGG analysis
Protein homologs for all C. cinerea predicted gene models were assigned according to the best hit in BLASTX  against the NCBI non-redundant protein sequence database (e<10-5). Gene Ontology (GO) terms were assigned to protein homologs through BLAST2GO (version 2.4.2) using default parameters . In subsequent GO term analysis of the differential expression data, corresponding level 2 parent terms were used in “biological process”, “molecular function” and “cellular component”. KEGG orthologs (KO) and KEGG biological pathways were also assigned through the KEGG Automatic Annotation Server (KAAS)  using the following parameters: BBH (bi-directional best hit method), and all fungal species as the representative gene set.
The microarray platform, hybridization methods and data analysis have been described [10, 33]. More than 13,000 oligonucleotides were designed using ArrayOligoSelector and printed randomly in duplicates on the slides. They were designed to minimize chances of a secondary match, and to be 3′ biased where possible. Myc and S1-Pri RNA samples from biological replicates were isolated from stages mentioned in “Strains and culturing”. First-strand cDNA was synthesized and labeled using Superscript Indirect cDNA Labeling System (Invitrogen) with alexa-fluor 635 and 532. Arrays were hybridized in four replicates. Data were captured and analyzed with GenePix 4200A scanner and GenePix Pro software (Molecular Devices). Data for a given oligonucleotide were included if two or more of the four replicates contained data for both probes. Intra-slide normalization and log2 transformation were performed using OLIN (Optimized local intensity-dependent normalization) and data were Z-adjusted to facilitate comparisons among arrays. Significance analysis of microarrays (SAM) was used to determine significant differences in gene expression between samples and the control.
Quantitative real-time RT-PCR
The reliability of 5′-SAGE data was verified by examining expression of genes showing higher expression in Myc or S1-Pri and similar expression levels. Total RNAs were isolated from Myc and S1-Pri stages mentioned in “Strains and culturing” and were DNase-treated prior to first-strand cDNA synthesis using TaqMan® Reverse Transcription (RT) reagents (Applied Biosystems). RT-PCR primers were designed using the OLIGO™ software (version 4.0) (National Biosciences). Real-time RT-PCR was performed on the MiniOpticon™ Real-Time PCR detection system (Bio-Rad) using iQ™ SYBR® Green supermix (Bio-Rad) according to the manufacturer’s instructions. Reaction volumes of 20 μl containing 2 μl of 10X diluted cDNA were used and samples were prepared from biological replicates for each developmental stage and primer pair. Gene CC1G_03085 encoding a clathrin coat assembly protein was selected as the control for normalization. Melting curve analysis was also performed according to the manufacturer’s instructions (Additional file 12 and Additional file 13).
Availability of supporting data
The data sets supporting the results of this article are available in the NCBI Gene Expression Omnibus (GEO) repository under accession GSE40722.
5'-Serial Analysis of Gene Expression
Stage 1 primordium
Differentially expressed gene
Transcription start site
Kyoto Encyclopedia of Genes and Genomes
Reverse transcription PCR
Heat shock protein
cyclic adenosine monophosphate
Mitogen-activated protein kinase
mitogen-activated protein kinase kinase
Protein kinase A
Target of rapamycin.
We thank Prof. Fred Dietrich and Zhihong Zhang for providing the detailed 5′-SAGE protocols. We also thank Dr. David Wilmshurst for the revision of the manuscript for grammar and sentence structure. This work was supported by the RGC General Research Fund CUHK466608 from the Research Grants Council of the HKSAR, PR China.
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