Comparative Gene Expression Analysis of Fruiting Body Development in Two ¢Lamentous Fungi Minou Nowrousian & Ulrich Kuck¨

Comparative gene expression analysis of fruiting body development in two ¢lamentous fungi Minou Nowrousian & Ulrich Kuck¨

Lehrstuhl fur ¨ Allgemeine und Molekulare Botanik, Ruhr-Universitat¨ Bochum, Bochum, Germany

Correspondence: Minou Nowrousian, Abstract Lehrstuhl fur ¨ Allgemeine und Molekulare Botanik, Ruhr-Universitat¨ Bochum ND 7/130, The ascomycete Pyronema confluens is a member of the Pezizales and readily forms Universitatsstr.¨ 150, 44780 Bochum, fruiting bodies under laboratory conditions. Here, we report the first molecular Germany. Tel.: 149 234 3224588; analysis of fruiting body development in this filamentous fungus. Two P. confluens fax: 149 234 3214184; e-mail: cDNA libraries were generated, one derived from mycelium undergoing sexual [email protected] development, and the other from vegetative mycelium. From each library, 96 clones were end-sequenced, resulting in the identification of 132 different genes. Received 10 January 2006; revised 8 February Expression studies of 10 P. confluens genes by quantitative real-time PCR identified 2006; accepted 9 February 2006. seven genes that are transcriptionally up- or downregulated during sexual First published online March 2006. development when compared with vegetative growth. As a first step towards a doi:10.1111/j.1574-6968.2006.00192.x comparison of gene expression during fruiting body development in different filamentous fungi, transcript levels of the corresponding homologs from the Editor: Reinhard Fischer distantly related ascomycete Sordaria macrospora were analyzed by quantitative real-time PCR. The analyses revealed similar expression patterns during sexual Keywords development for several of the S. macrospora genes when compared with fruiting body development; real-time PCR; P. confluens. Sordaria macrospora; Pyronema confluens.

importance to the process of interest. One way to circum- Introduction vent this problem is to compare gene expression in different Many filamentous ascomycetes form complex three-dimen- species. Genes with expression patterns that are conserved sional fruiting bodies for the protection and dispersal of across species would be candidates of choice for detailed their sexual spores. It is generally agreed that filamentous analysis because evolutionary conservation is a powerful ascomycetes (Pezizomycotina) form a monophyletic group criterion to identify genes that might be functionally im- (Lumbsch, 2000) and that their fruiting bodies constitute portant in contrast to just coregulated (Stuart et al., 2003). homologous structures. However, even though genes in- For a comparison of gene expression patterns during volved in fruiting body formation have been characterized fruiting body formation in fungi, it would be desirable to from a number of different species, a unified model for the use organisms that are not too closely related but never- genetic control of this developmental process has yet to theless have similar life cycles. Two such fungi are the emerge (Poggeler¨ et al., 2006). ascomycetes Sordaria macrospora and Pyronema confluens One avenue towards a deeper understanding of fungal (syn. Pyronema omphalodes (Bull.) Fuckel 1869). Pyronema fruiting body development is the identification of genes that confluens belongs to the Pezizales, a basal group of ascomy- are differentially expressed during this process. On the one cetes, and forms apothecia as fruiting bodies; whereas hand, such genes are candidates for further analysis; on the S. macrospora is a member of the derived group of Sordar- other hand, an overview of genes expressed during develop- iales forming perithecia. Both are homothallic and therefore ment will give an improved picture of the cellular processes able to form fruiting bodies without the need for a partner underlying the morphological changes. In recent years, large of opposite mating type. Moreover, neither of them pro- scale expression studies with e.g. microarrays have been duces any asexual spores. Under laboratory conditions, both widely used to identify genes with differential expression species are able to complete their life cycles within 6 days. patterns correlated with certain biological phenomena Sordaria macrospora has been used extensively as a model (Nowrousian et al., 2004a). The drawback of this type of organism for the molecular analysis of fruiting body study is that one often identifies large numbers of differen- development (Poggeler¨ et al., 2006). Pyronema confluens tially regulated genes many of which might not be of direct was a model organism for the analysis of fruiting body

c 2006 Federation of European Microbiological Societies FEMS Microbiol Lett 257 (2006) 328–335 Published by Blackwell Publishing Ltd. All rights reserved Gene expression during fungal fruiting body development 329 development in higher ascomycetes during the first half of to the Neurospora crassa open reading frame number in the last century (Claussen, 1912; Gwynne-Vaughan & Wil- the genome annotation (Galagan et al., 2003); additionally, liamson, 1931; Kerl, 1937; Wilson, 1952; Moore & Korf, the provisional S. macrospora gene names as annotated in 1963), but has not yet been characterized at the molecular the EMBL nucleotide database are given. level. However, the fact that P. confluens forms fruiting bodies in the laboratory distinguishes this species from Preparation and analysis of RNA many other members of the Pezizales that do not propagate sexually under laboratory conditions. This and the fact that Sordaria macrospora and P. confluens RNA were prepared as the Pezizales are a basal group of filamentous ascomycetes, described previously (Yarden et al., 1992), or, in the case of which is relevant for the comparative aspect of our study, P. confluens, using the RNeasy lipid tissue mini kit (Qiagen, made P. confluens a suitable candidate to be included in this Hilden, Germany) according to a modified protocol from analysis. Thus, the objectives of this study were (i) an the manufacturer (mycelium was ground in liquid nitrogen, approach towards the molecular analysis of P. confluens and incubated for 10 min at room temperature with 1 mL of (ii) a first comparative analysis of gene expression during Qiazol reagent, centrifuged for 10 min at 15 000 g and the fruiting body formation in P. confluens and S. macrospora. supernatant was treated as described in the protocol starting with the addition of chloroform). Poly(A) RNA was ex- Materials and methods tracted from P. confluens total RNA with a polyATtract kit according to the manufacturer’s protocol (Promega, Man- nheim, Germany). Strains and growth conditions

Sordaria macrospora S48977 (wild type) is kept in our Quantitative real-time PCR laboratory collection. For RNA extraction, S. macrospora was grown at 25 1C in constant light as described previously Reverse transcription of total RNA and quantitative real-time (Nowrousian et al., 2005). For fruiting body development, PCR were done as described previously (Nowrousian et al., an agar plug (0.7 cm in diameter) was placed in the center of 2005). For details and primer sequences, see Supplement 1. a Petri dish with 20 mL of medium. For vegetative growth, a mycelial plug of 0.7 cm in diameter from a Petri dish with Preparation of two Pyronema confluens cDNA liquid medium was inoculated into an Erlenmeyer flask with libraries and expressed sequence tag (EST) 100 mL of liquid medium and shaken at 130 r.p.m. Pyrone- sequencing and analysis ma confluens CBS 100304 (wild type) was obtained from the Two cDNA libraries from P. confluens were prepared from CBS. Strain N1-3 used for the experiments described here is RNA derived from mycelia grown vegetatively or under a second-generation single spore isolate from CBS 100304. conditions that allow fruiting body formation, respectively. Pyronema confluens was grown at 25 1C in the following Construction of a directional library from 2 mg of poly(A) medium derived from synthetic crossing medium (Davis & RNA was performed with the Superscript Plasmid System deSerres, 1970): 1 g L1 KNO ,1gL1 KH PO , 0.5 g L1 3 2 4 with Gateway Technology for cDNA-Synthesis and Cloning MgSO 7HO, 0.1 g L1 NaCl, 0.1 g L1 CaCl , 0.1 mL L1 4 2 2 (Invitrogen, Carlsbad, CA) according to the manufacturer’s trace elements, 2 g L1 glucose. For inoculation, an agar plug protocol. cDNAs were ligated into vector pSPORT1 and (0.7 cm in diameter) was placed in the center of a Petri dish transformed into electrocompetent Escherichia coli cells with 20 mL of medium, incubation was in constant light for (Electromax) as recommended in the manufacturer’s in- fruiting body development or in constant darkness or red light structions (Invitrogen). Plasmid DNA from 96 clones from (Philips PF712E darkroom safe light) for vegetative growth. each P. confluens cDNA library was sequenced at GATC Biotech AG (Konstanz, Germany) using the T7 primer PCR amplification and cloning of Sordaria (sequences from from the 50 end). Sequences were organized macrospora gene fragments into contigs using the Phrap software (P. Green, copyright Parts of S. macrospora genes were amplified from genomic 1994–1996, http://bozeman.mbt.washington.edu/phrap. DNA as described previously (Nowrousian et al., 2004b, docs/phrap.html). The EST sequences (singletons and con- 2005). Sequences for partial S. macrospora genes have been tigs from Phrap analysis, without poly(A) tails) have been deposited in the EMBL database under the following deposited in the EMBL EST database (Accession numbers accession numbers: AM042541 (SMU0195, trp), AM042542 AM041272–AM041407). Contig names start with ‘PcCon- (SMU0443, sbp), AM042543 (SMU0467, kbp), AM042544 tig’, whereas singletons have ‘Pc1.1’ or ‘Pc2.1’ at the start of (SMU1545, cdp), AM042545 (SMU2252, pgm), AM042546 their name indicating the vegetative and sexual libraries, (SMU4170, egl), and AM042547 (SMU4803, ndo). Gene respectively. For details on library construction and sequen- names start with SMU followed by a number corresponding cing, see Supplement 2.

UMA Basidiomycetes 99.3/100.0 CCI

AGO Saccharomycetes 100.0/100.0 SCE

PCO Pezizomycetes

SSC Leotiomycetes

99.8/100.0 96.9/100.0 FGR

100.0/100.0 MGR Sordariomycetes

86.5/96.6 100.0/100.0 NCR

SNO Dothideomycetes

83.5/89.4 ANI Eurotiomycetes 99.8/100.0 CIM

Fig. 1. Phylogenetic analysis of the combined sequences from 15 (partial) proteins from 12 fungi. Numbers at branches indicate bootstrap support (10 000 bootstrap replications) in % for maximum parsimony/neighbor joining trees, respectively. The basidiomycetes Ustilago maydis and Coprinus cinereus were used as outgroups. Classes given on the right correspond to the taxonomy used by Liu & Hall (2004), and in the NCBI Entrez Taxonomy Database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Taxonomy). AGO, Ashbya gossypii; ANI, Aspergillus nidulans; CCI, Coprinus cinereus; CIM, Coccidioides immitis;FGR, Fusarium graminearum; MGR, Magnaporthe grisea; NCR, Neurospora crassa; PCO, Pyronema conﬂuens;SCE, Saccharomyces cerevisiae; SNO, Stagonospora nodorum; SSC, Sclerotinia sclerotiorum; UMA, Ustilago maydis. Sequences for A. gossypii were obtained by BLASTP search at the Duke CGT Ashbya website (http://ashbya.genome.duke.edu/blast.html) and downloading protein sequences from the Ashbya genome database (http://agd.unibas.ch/), and for all other fungi from the Fungal Genome Initiative of the Broad Institute at (http://www.broad.mit.edu/ annotation/fungi/fgi/index.html).

Phylogenetic analysis from P. confluens, and to our knowledge, these are the first sequences available from this fungus. Therefore, we have Multiple alignments were created in CLUSTALX (Thompson performed phylogenetic analyses to confirm the position of et al., 1997) and the same alignment was used for analysis by P. confluens within the phylogenetic tree using derived distance-matrix (DM) and maximum parsimony (MP) Ã amino-acid sequences from 15 full or partial genes from methods. Phylogenetic analyses were made with PAUP P. confluens and their corresponding orthologs from the fully version 4.0b10 for Windows (D.L. Swofford, distributed sequenced genomes of nine ascomycetes and two basidio- by Sinauer Associates, copyright 2001 Smithsonian mycetes (Fig. 1, Supplement 3). The sequences were com- Institution). DM and MP analyses were performed as bined and a multiple alignment containing 3018 residues described (Hall, 2004) using 10 000 bootstrap replicates. was used for neighbor joining and MP analyses. With both Consensus trees were graphically displayed with TREEVIEW methods, the same tree topology was obtained with high (Page, 1996). bootstrap support (Fig. 1): P. confluens is at the base of the filamentous ascomycetes that are well separated from the Results and discussion Saccharomycetes. The remaining ascomycetes represented in this tree cluster in two groups: one encompassing Leotiomy- Phylogenetic analysis places Pyronema cetes and Sordariomycetes, and the other Dothideomycetes confluens at the base of the filamentous and Eurotiomycetes. This tree topology is similar to the one ascomycetes previously obtained using RPB2 sequences with the excep- Pyronema confluens has been described as a member of the tion of the position of the Dothideomycetes (Liu & Hall, Pezizales based on its morphology; and a close relative, 2004). The analysis of Liu and Hall places them basal to the P. domesticum, groups with that order in phylogenetic ana- Sordariomycetes and Eurotiomycetes. However, in an 18S lyses using small subunit rRNA gene sequences (Landvik rRNA gene analysis, the Dothideomycetes were identified as et al., 1997). In this study, we have obtained cDNA sequences a sister group to the Eurotiomycetes (Berbee, 1996), and this

c 2006 Federation of European Microbiological Societies FEMS Microbiol Lett 257 (2006) 328–335 Published by Blackwell Publishing Ltd. All rights reserved Gene expression during fungal fruiting body development 331 is also the position that was found in our analysis (Fig. 1). A From the 132 genes, 45 genes (34.1%) did not show final conclusion about the correct tree topology with respect homology to any gene with known or putative function. to the Dothideomycetes has yet to be reached as our tree has The remaining genes were sorted into functional categories good bootstrap support but includes only one member of (Supplement 5). In the vegetative library, more than 40% of this fungal group. With respect to the phylogenetic position all genes code for proteins in connection with metabolism or of P. confluens, our analysis agrees with previously published protein synthesis; whereas in the sexual library, these genes trees that the Pezizales are at the base of the filamentous amount to less than 20%. Conversely, less than 2% of the ascomycetes; whereas the Sordariomycetes, of which Sordaria genes from the vegetative library are involved in DNA macrospora is a member, are a rather derived group (Berbee, synthesis, chromosome structure maintenance, or cell sig- 1996; Tehler et al., 2003; Liu & Hall, 2004). This is important naling and communication; whereas in the sexual library, with respect to the comparison of gene expression because these genes make up nearly 10%. These data confirm this makes it likely that any conserved expression patterns in previous findings in other fungi that differentiating mycelia these two distantly related fungi will be of functional express a greater variety of genes (Nelson et al., 1997; Trail significance. et al., 2003).

Construction of two Pyronema confluens cDNA Identification of genes differentially expressed libraries specific for vegetative growth and during fruiting body development in Pyronema sexual development confluens Pyronema confluens was used as a model organism for Ten P. confluens genes were chosen for which expression was fruiting body development during the first decades of the analyzed using quantitative real-time PCR (Fig. 2). The last century (Gwynne-Vaughan & Williamson, 1931). Under genes include six found only in the sexual library and four laboratory conditions on minimal media and in constant from the vegetative library. Several of the genes have homo- white light, apothecia with eight-spored asci are mature after logs in other organisms for which evidence for their cellular 5–6 days. It was noted early on that P. confluens requires light functions has been obtained experimentally or can be for fruiting body production; in constant darkness, it only inferred from annotations in the public databases; these grows vegetatively (Claussen, 1912; Kerl, 1937; Wilson, genes are involved e.g. in signaling, cell cycle, cell wall 1952). This phenomenon was found to be reproducible with formation or metabolism (Table 1). Thus, the ten genes strain CBS100304 and its derivatives used in this study. We were chosen to represent a cross-section of functional found that under illumination with red light, apothecia are categories found in the cDNA libraries. Two genes with also not formed. This makes it possible to harvest dark- homologs to known fungal developmental genes were grown mycelia in red light without induction of fruiting included in the analysis; for the other eight genes, it is not body development. We made use of the fact that P. confluens known whether they are involved in developmental pro- produces fruiting bodies only in the light to establish two cesses. By not including more previously characterized different cDNA libraries: The first library (called vegetative developmental genes, we tried to avoid a bias towards library) is derived from mycelium grown in constant dark- known developmental genes as this would not enable us to ness and therefore not producing any apothecia. The second draw more general conclusions on conserved patterns of library (called sexual library) is derived from light-induced gene expression as described in the next section. mycelia. The two different libraries were used because this Expression analysis of the 10 genes revealed that six of should increase the number of individual genes represented them are upregulated and one is downregulated more than in the libraries, as some genes might be expressed at low twofold during sexual development in P. confluens (Fig. 2a). levels in one but not the other condition. To obtain As this was the first time that expression analyses were P. confluens cDNA sequences for further investigations, 96 performed with P. confluens, the results for two of the genes, clones from each library were end-sequenced from the 50 Pc2.1B01 and Pc2.1G07, were additionally verified by end and organized into contigs. The contig analysis revealed Northern blot analysis (Fig. 2b). Pc2.1B01 is not differen- the presence of 132 different genes among the 192 clones, tially expressed, whereas Pc2.1G07 is highly upregulated among these genes were 21 contigs and 111 singletons. Of during fruiting body formation, and as expected, these these, 58 genes were found only in the vegetative library, 61 findings were confirmed by Northern blot analysis. genes only in the sexual library and 13 genes were repre- The most strongly upregulated genes are encoded by sented in both libraries (Appendix S2). The cDNA sequences Pc2.1G07, Pc2.1A03, PcContig10, and Pc2.1A02, all of or derived amino-acid sequences were compared with the which represent clones that were found only in the cDNA public databases and the fully sequenced genomes from library derived from mycelium with fruiting bodies. Con- eight ascomycetes and three basidiomycetes (Appendix S4). versely, the only gene that was found to be downregulated is

(a) 14 (a) P. confluens * 13 *

* rged ged 12 * g * 11

10 DD floatin LL floating DD subme LL submer 9 Pc2.1G07 8 7 rRNA 6 5 4 (b) S. macrospora 3 7 * * SMU4170 2 * * * 1 6

log2 ratio sexual development/vegetative growth 0 5 –1 –2 4 * –3 * 3

2 PcContig10 Pc2.1A02 Pc2.1A03 Pc2.1B01 Pc2.1B06 Pc2.1G07 PcContig2 Pc1.1A09 Pc1.1C03 Pc1.1H09

log2 ratio vs. DD submerged 1 (b) 0 Vegetative Sex.dev.

ged Pc2.1G07 rged

* LL floating * DD floating rRNA LL submer DD subme

Fig. 3. Comparison of transcript levels under different growth condi- Pc2.1B01 tions for the Pyronema confluens gene Pc2.1G07 (a) and its Sordaria macrospora ortholog SMU4170 (b). DD, growth in darkness; LL, growth rRNA in constant light; conditions that allow fruiting body formation are indicated by an asterisk. (a) Northern blot analysis for Pc2.1G07 was performed with 20 mg of total RNA per lane. (b) Quantitative real-time Fig. 2. Comparison of transcript levels between vegetative growth and PCR analysis for SMU4170. Values given are log2 ratios of means sexual development for 10 Pyronema confluens genes. (a) Quantitative calculated with relative expression software tool analysis for all growth real-time PCR analysis with two to six biological replicates (n = 2–6) for conditions compared with submerged growth in the dark. each gene. Values given are log2 ratios of mean expression ratios as calculated with relative expression software tool (Pfaffl et al., 2002) for sexual development vs. vegetative growth. Ã, indicates that a gene was differentially regulated in at least 60% of all independent experiments; Comparative analysis of gene expression in ÃÃ, indicate differential expression in at least 80% of experiments. Genes Sordaria macrospora and Pyronema confluens that were found in the sexual or vegetative library are shaded in light or identifies a set of genes with similar expression dark gray, respectively. (b) Northern blot analysis for genes correspond- patterns in both fungi ing to expressed sequence tag (EST) clones Pc2.1B01 and Pc2.1G07 was performed with 20 mg of total RNA per lane. The analysis was performed at least twice and representative results are shown. Pyronema confluens needs light to form fruiting bodies. In this respect, it differs from S. macrospora as the latter forms fruiting bodies irrespective of illumination. Both fungi, encoded by Pc1.1C03, a clone from the vegetative library. however, do not form fruiting bodies under submerged This might indicate that the libraries are enriched for genes conditions; similar to most other filamentous ascomycetes, expressed preferentially during sexual growth or vegetative they need air contact to produce fruiting bodies (Poggeler¨ development. et al., 2006). Table 2 summarizes the growth conditions that

Table 1. Pyronema conﬂuens and Sordaria macrospora genes used for expression analyses P. conﬂuens S. macrospora Closest homolog / closest homolog with known or Functional Accession e-value gene ortholog putative function category number (BLASTX) PcContig10 (sex). SMU0195 Hypothetical protein MG04927.4 (Magnaporthe grisea) – gb|EAA52235.1 4 1017 putative MFS (major facilitator superfamily) transporter – emb|CAE47906.1 9 1011 (Aspergillus fumigatus) Pc2.1A02 (sex.) SMU0467 Hypothetical protein MG05274.4 (Magnaporthe grisea) – gb|EAA52582.1 3 1091 COP9 signalosome subunit 5 (Aspergillus nidulans) Protein turnover, gb|AAM95164.1 1089 development Pc2.1A03 (sex.) – – – – – Pc2.1B01 (sex.) SMU4202 Predicted nucleoside diphosphate kinase (Aspergillus – gb|AAP85295.1 5 1070 fumigatus) Signaling sp|Q9UUY8 3 10-66 nucleoside diphosphate kinase (Neurospora crassa) Pc2.1B06 (sex.) SMU0443 Hypothetical protein AN0084.2 (Aspergillus nidulans) – gb|EAA65262.1 6 1079 spi1-GTP-binding protein (Schizosacchromyces pombe) Cell cycle pir|T51307 3 1064 Pc2.1G07 (sex.) SMU4170 Hypothetical protein (Neurospora crassa) – ref|XP_323510.1 7 1036 b-1,3 exoglucanase precursor (Trichoderma harzianum) – (cell wall?) sp|O14402 1028 PcContig2 (veg.) SMU4803 Hypothetical protein AN8801.2 (Aspergillus nidulans) – gb|EAA60594.1 10138 putative 2-nitropropane dioxygenase – (Metabolism?) ref|NP_947286.1 10104 (Rhodopseudomonas palustris) Pc1.1A09 (veg.) SMU1545 IDI-7 (Podospora anserina) Incompatibility, gb|AAN41258.1 1058 development Pc1.1C03 (veg.) SMU3600 Predicted protein (Neurospora crassa) – ref|XP_322902.1 8 1019 ESDC (Aspergillus nidulans) – gb|AAM95965.1 2 1018 Pc1.1H09 (veg.) SMU2252 Putative phosphoglyceromutase (Aspergillus oryzae) – dbj|BAB12237.1 10111 cofactor-independent phosphoglycerate mutase Metabolism gb|AAT01444.1 2 1081 (Caenorhabditis elegans)

For each P. conﬂuens gene, the library (sexual or vegetative) in which the gene was found is provided in brackets after the gene name. Functional categories are given for genes in cases where experimental evidence is published, putative functions are given in brackets.

Table 2. Growth conditions for vegetative growth and sexual develop- similar morphological structures should limit the number of ment for Sordaria macrospora and Pyronema confluens genes that are regulated as a response to specific growth Illumination/culture DD floating LL floating DD shaken LL shaken conditions and are not necessarily connected to develop- P. confluens VFVV ment. An analysis of Pc2.1G07 and its S. macrospora S. macrospora FFVV ortholog SMU4170 under all possible combinations of growth conditions showed that at least for this gene, Growth conditions used for comparisons of gene expression are shaded expression in both fungi is only upregulated under condi- in gray. V, vegetative growth; F, fruiting body development; DD, growth tions that allow fruiting body formation (Fig. 3). Further in darkness; LL, growth in constant light. analyses on a larger scale will be necessary to investigate whether this is true for the majority of genes found by allow fruiting body development in S. macrospora and comparative expression analysis. P. confluens, respectively. For comparative analysis of gene One of the genes expression of which was investigated in expression during development, P. confluens was grown in P. confluens does not have a homolog in any of the sequenced floating culture in the dark for vegetative growth and in the fungal genomes (Appendix S4). For the other nine genes, light for sexual development; whereas S. macrospora was orthologs were isolated from S. macrospora as described grown submerged for vegetative growth and in floating previously (Nowrousian et al., 2004b). Expression of the culture for fruiting body formation both under constant nine S. macrospora genes was determined by quantitative illumination as described (Nowrousian & Cebula, 2005). real-time PCR and compared with that of P. confluens Therefore, any differential gene expression observed in both (Fig. 4). Comparison of the expression patterns of both fungi could be attributed to light regulation in the case of P. ascomycetes revealed strong similarities in that the three confluens and air contact in S. macrospora; however it is genes that were found to be most strongly upregulated more likely to be due to the common factor in both cases during sexual development in P. confluens and have homo- which is the development of fruiting bodies. Thus, choosing logs in S. macrospora are also upregulated in the latter different growth conditions that lead to development of (Fig. 4). Another gene that is upregulated in P. confluens,

P.c. S.m. protein encoded by PcContig10/SMU0195 is homologous Pc1.1C03/SMU3600 –2.43** 3.89** to a putative major facilitator superfamily transporter from Aspergillus fumigatus (Table 1), and searches for conserved Pc2.1B01/SMU4202 –0.57 –0.18 domains in the predicted protein identify a putative sugar Pc1.1H09/SMU2252 0.86 0.98 phosphate permease domain (COG2271|11977). Thus, Pc2.1B06/SMU0443 1.00 0.20 PcContig10/SMU0195 could be involved in carbon metabo- PcContig2/SMU4803 1.36* –0.16 lism; however, proteins of this class of transporters might also be involved in regulation of fungal development. An Pc1.1A09/SMU1545 1.41* 0.91 example is the N. crassa glucose transporter RCO-3 that was Pc2.1A02/SMU0467 1.73** 1.45** shown not only to be involved in glucose transport, but also PcContig10/SMU0195 2.06* 1.20* in the regulation of carbon metabolism and the develop- Pc2.1G07/SMU4170 12.75** 5.10** ment of conidia in this fungus (Madi et al., 1997). In general, this first approach towards comparative expression Fig. 4. Comparison of gene expression during sexual development for analysis in fungi indicates that it is possible to identify genes nine Pyronema confluens genes and their Sordaria macrospora ortho- with similar expression patterns in different species; thus, logs. Transcript levels were determined by quantitative real-time PCR future studies could involve large-scale analyses, e.g. with with two to six biological replicates (n = 2–6) for each gene in both organisms. Values given are log2 ratios of mean expression ratios as microarrays, involving additional fungal species. calculated with relative expression software tool (Pfaffl et al., 2002) for sexual development versus vegetative growth. Ã, indicates that a gene was differentially regulated in at least 60% of all independent experi- Supplementary material ÃÃ ments; , indicate differential expression in at least 80% of experi- The following supplementary material is available for this ments. Shading in red or green indicates up- or downregulation of more article online: than twofold, respectively, and genes that are not differentially regulated Appendix S1. (a) Details on experimental procedures for are shaded in gray. reverse transcription and real time PCR. (b) Table containing sequences of real time OCR primers. Pc1.1A09/SMU1545, is weakly upregulated in S. macrospora, Appendix S2. (a) Details on experimental procedures for but does not reach the threshold of a twofold difference as construction of two P. confluens cDNA libraries. (b) Details set for this study. The three genes Pc2.1B01/SMU4204, on EST sequencing and analysis. (c) Table containing an Pc2.1B06/SMU0443, and Pc1.1H09/SMU2252 were not overview of assignment of ESTs to functional categories regulated differentially in both organisms. Only one gene (details in Appendix S5). showed opposite patterns of expression in P. confluens and Appendix S3. Details on phylogenetic analysis. S. macrospora, namely Pc1.1C03/SMU3600, that is down- Appendix S4. Table containing details about EST sequences regulated during fruiting body development in P. confluens from two P. confluens cDNA libraries (clone names, putative and upregulated in S. macrospora (Fig. 4). These data functional homologs, putative orthologs in other fungi). indicate that there is a significant overlap in gene expression Appendix S5. Table containing details about sorting of EST patterns in S. macrospora and P. confluens during sexual sequences into functional categories. development. The material is available as part of the online article from The three genes that are most strongly upregulated during http://www.blackwell-synergy.com sexual development in both P. confluens and S. macrospora are candidates of choice for further functional investigation as they might constitute genes that are involved in fruiting Acknowledgements body formation. The gene that is upregulated most strongly, The authors would like to thank Swenja Ellßel for excellent Pc2.1G07/SMU4170, encodes a putative exoglucanase pre- technical assistance. This study was supported by grants cursor (Table 1) that might be involved in cell wall metabo- by the Deutsche Forschungsgemeinschaft (DFG) to U.K. lism during fruiting body formation. The other two (SFB480, project A1) and the Ruhr-Universitat¨ Bochum to upregulated genes are PcContig10/SMU0195 and M. N. (Programm zur Forderung¨ des wissenschaftlichen Pc2.1A02/SMU0467; they encode a putative transporter Nachwuchses). protein and a homolog to the A. nidulans COP9 signalosome subunit 5, respectively. The latter has been shown to be essential for sexual development in A. nidulans, and the References COP9 signalosome is a protein complex involved in regula- Berbee ML (1996) Loculoascomycete origins and evolution of tion of development in many eukaryotes by regulating filamentous ascomycete morphology based on 18S rRNA gene targeted protein degradation (Busch et al., 2003). The sequence data. Mol Biol Evol 13: 462–470.

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