BACKGROUND
the last eukaryotic common ancestor  was facultatively sexual and evolved nearly  <dig>  billion years ago in the proterozoic eon  <cit> .
sexual reproduction  shares common features across the eukaryotic lineage such as ploidy changes, meiotic recombination and cell-cell recognition between gametes followed by cellular fusion and zygote formation  <cit> .
an increase in the genetic diversity of the population, making it more adaptable to changing environmental conditions, as well as the dilution of deleterious mutations out of the gene pool are the most remarkable evolutionary innovations achieved by sr  <cit> .
despite all these clear benefits, sex is energetically expensive and entails a higher chance of genetic and organelle conflicts  <cit> .
during sexual reproduction, fungi undergo dramatic morphological changes driven by environmental conditions such as light, nutrient availability and grazing by predators  <cit> .
in basidiomycetes, mushroom development starts with intense localized hyphal branching leading to the formation of hyphal knots.
these branching hyphae further aggregate to form 1–2 mm secondary nodules where cell differentiation leads to the establishment of bipolar primordia containing all the tissues observed in the mature fruiting body  <cit> .
as a last step, primordia develop to mature fruiting bodies mainly by cellular expansion  <cit> .
due to their hyphal density, primordia and fruiting bodies are attractive to predators including mollusks, arthropods and nematodes  <cit> .
coprinopsis cinerea has been used as a model basidiomycete since the mid-1950s  <cit>  due to its saprobic lifestyle, its rapid growth and the feasibility of producing fruiting bodies under defined laboratory conditions  <cit> .
in nature, c. cinerea grows on horse dung  <cit> , a eutrophic substrate rich in competing microorganisms, such as firmicutes, bacteroidetes and proteobacteria  <cit> .
the recent sequencing of the c. cinerea genome  <cit>  allows the study of this organism gene expression on transcriptome and proteome level at different developmental stages or under a variety of environmental settings.
morphological changes and environmental signals during fruiting body formation in c. cinerea are well described  <cit> ; nonetheless, comparably little is known about the molecular machinery driving sexual reproduction processes in this basidiomycete.
recently, mutations blocking fruiting body development at different stages or altering mushroom morphology were identified .
in addition, dst <dig> and dst <dig>  encoding a blue-light photoreceptor and a flavin adenine dinucleotide-binding protein, were shown to play a role in blue light sensing.
in agreement with previous experiments, strains defective in these two proteins were unable to form fruiting bodies, showing that blue light is an essential environmental trigger of mushroom development  <cit> .
most recently, c. cinerea strains carrying mutations in the putative component of the swi/snf chromatin remodeling complex snf <dig>  were shown to be defective in fruiting initiation, suggesting that epigenetic reprogramming of loci occurs during fruiting body formation  <cit> .
aerial fruiting bodies are an attractive prey for predators and thus are protected by a battery of defense molecules  including proteins , peptides  <cit>  and secondary metabolites .
some of these toxins are known to be specifically produced in the fruiting body and not in the vegetative mycelium  <cit> .
for instance, cytoplasmic lectins showing a broad range of non-self-carbohydrate specificities, also referred to as fruiting body lectins due to their specific expression pattern, have been shown to exert toxicity to nematodes, insect larvae and amoeba  <cit> .
protein-mediated inhibition of serine proteases  <cit> , proteolytic degradation of predator-derived proteins  <cit>  and sequestration of biotin  <cit>  are other strategies of basidiomycetous fruiting bodies to dissuade predators.
vegetative mycelium, in contrast, digests extracellular carbon macromolecules in the growth substrate, such as cellulose and lignin, into smaller degradation products which are absorbed by the growing hyphae.
at the same time, these smaller molecules become available for competing bacteria that profit from the fungal enzymatic machinery  <cit> .
as a response to bacterial competitors, fungi have evolved secreted antimicrobial proteins.
mygind and colleagues presented recent evidence that vegetative mycelia of fungi secrete cysteine-stabilized antibacterial peptides which play a role in the arms race with competing bacteria  <cit> .
using rna-seq in c. cinerea, we show evidence suggesting a role of the velvet protein regulon in sexual development of basidiomycetes.
our data supports the existence of a conserved transcriptional circuitry in basidiomycetes fruiting bodies consisting of at least  <dig> orthologous genes probably involved in mushroom development and function.
in addition, our data reveals the existence of two different sets of fungal defense proteins in vegetative and sexual organs matching the type of competitors and predators by which these structures are challenged in nature.
the transcriptome data is supported by the first partial shotgun mass spectrometry catalog of proteins present in c. cinerea stage  <dig> primordia  and vegetative mycelium .
RESULTS
differential gene expression during fruiting body development in c. cinerea
four different cdna libraries were sequenced with a final  <dig>  gb mapped data output.
approximately 95% of the open reading frames  in the genome of c. cinerea a43mutb43mut  were transcribed using five reads/orf as the minimal threshold for a locus to be considered as expressed .
differential gene expression at the rna level between s1p and vm was examined .
eleven percent of the annotated orfs in the c. cinerea genome were found to be differentially expressed,  <dig>  and  <dig> loci  in vm and s1p, respectively, using fold change  <dig> and fisher’s exact test p-value ≤  <dig>  as thresholds .
the number of differentially transcribed loci in these two developmental stages increased to  <dig> in vm and  <dig> in s1p when a fold change threshold of  <dig> was set, corresponding to approximately 45% annotated orfs in the genome of c. cinerea.table  <dig>
general features of the
c. cinerea
s1p and vm transcriptomes
s1p
vm
average solid read size
sequenced material
*mean percentage of annotated orfs detected to be expressed using a minimal threshold  <dig> reads/orf.
differential gene expression during early fruiting body development in
c. cinerea
.
volcano plot illustrating the relative expression ) and statistical significance of the  <dig> loci in the genome of c. cinerea between vm and s1p.
fisher’s exact test -log <dig> p-value ≥  <dig>   and log <dig> +/-  <dig>  were used as thresholds of differential gene expression.
grey boxes comprise genes significantly up-regulated in s1p  or vm .
tag clouds showing enriched psi-blast functional annotation terms from up-regulated loci in s1p  and vm  were produced in wordle  after removing frequently appearing tags.
to validate these data, qrt-pcr of four selected loci  was performed.
although s1p/vm expression ratios did not exactly match those observed by rna-seq, qrt-pcr results showed the same trend of differential gene expression for these loci during c. cinerea development as observed by solid rna-seq .
in addition, we verified that the expression values of reported housekeeping loci commonly used in qrt-pcr normalization were in a range indicative of constitutive expression  ≤ 2) .
enrichment of psi-blast-derived functional annotation terms was visualized using wordle   <cit>  after excluding frequent non-informative terms.
by far, the most commonly assigned annotation term was “hypothetical protein” in s1p  and vm ; nonetheless, there was a significant enrichment of n-methyltransferase  tags in s1p .
enrichment of functional annotation terms also shows that different sets of cytochromes, kinases, dehydrogenases, transporters and hydrophobins are specifically expressed in vegetative mycelium or young fruiting bodies .
functional annotation clustering using david bioinformatics resources  <dig>   <cit>  confirmed the enrichment of protein methyltransferases and hydrophobins in s1p with  <dig> and  <dig> fold enrichment compared to the occurrence of these functional categories in the genome of c. cinerea .
tissue-specific expression of c. cinereadefense proteins
several cytoplasmic defense lectins and protease inhibitors such as c. cinerea lectin  <dig>   <cit> , coprinopsis galectin  <dig>   <cit>  and two paralogous serine protease inhibitors from coprinopsis   <cit>  were found to be highly up-regulated in c. cinerea s1p .
out of these previously characterized genes coding for proteins with nematotoxic and insecticidal activity, cospin and ccl <dig> were found to be among the top  <dig> most highly transcribed and differentially expressed loci in s1p.
among these, two loci, cc1g_ <dig> and cc1g_ <dig>  encoding homologous proteins with a predicted aerolysin/etx pore-forming domain as well as loci encoding proteins with suspected antibacterial and antifungal function including two peptidoglycan binding proteins, the toxin component of a bacterial toxin-antitoxin system and a thaumatin-like protein were found.
in contrast to s1p-specific defense proteins, defense proteins specifically up-regulated in vm were mainly secreted.
these proteins included three putative lysozymes, several proteins containing a cfem domain  whose structure resembles cysteine-stabilized antibacterial peptides, and two representatives of the cerato-platanin family of secreted proteins .
latter protein family is expanded in basidiomycetes and has recently been implicated in interactions of dikaryotic fungi with other organisms  <cit> .
during sexual development on herbivore dung, c. cinerea is exposed to a succession of antagonists  colonizing this substrate.
the differential expression of cytoplasmic and secreted defense proteins in s1p and vm, respectively, might reflect the prevalent types of antagonists with which these tissues of c. cinerea are confronted.table  <dig>
s1p-specific defense loci
*fisher’s exact test.
†n or y indicates the lack or presence of a signal peptide.
‡n indicates a lack of transmembrane helices, while a number corresponds to the amount of transmembrane helices predicted in the orf.
vm-specific defense loci
*fisher’s exact test.
†n or y indicates the lack or presence of a signal peptide.
‡n indicates a lack of transmembrane helices, while a number corresponds to the amount of transmembrane helices predicted in the orf.
the s1p-specific aerolysin/etx pore-forming domain-containing protein cc1g_ <dig> is toxic to nematode and insect larvae
cc1g_ <dig>  cc1g_ <dig> and cc1g_ <dig> encode three homologous 30–40 kda proteins containing a predicted aerolysin/etx pore-forming domain .
this domain is homologous to the one present in the insecticidal epsilon toxin  from clostridium perfringens
<cit>  and distantly related to aerolysin pore-forming toxins  <cit> .
in order to test the significance of the up-regulation of cc1g_ <dig> and cc1g_ <dig> in s1p with regard to fungal defense, we cloned and recombinantly expressed cc1g_ <dig> in escherichia coli and assessed the toxicity of the protein by feeding the recombinant bacteria to nematode and insect larvae as described previously  <cit> .
results in figure 2b show that cc1g_ <dig> was expressed in soluble form in e. coli.
feeding of cc1g_11805-expressing bacteria to l <dig> larvae of the mosquito aedes aegypti lead to their death after 96 h .
vector control-containing and fungal lectin-expressing e. coli were used as negative and positive controls, respectively, in these experiment  <cit> .
similarly, feeding of cc1g_11805-expressing bacteria was found to significantly impair larval development of the c. elegans n <dig> wildtype and pmk- <dig> mutant strains ; in agreement with the previously reported higher susceptibility of latter strain to different kinds of abiotic and biotic stresses including other nematotoxic fungal defense proteins  <cit> , cc1g_ <dig> was shown to be more toxic to c. elegans pmk- <dig> than to n <dig>  these results support previous observations  <cit>  showing that the expression of defense proteins directed against nematodes and insects is significantly increased in c. cinerea sexual organs.figure  <dig>
the s1p-specific aerolysin/etx pore-forming domain-containing protein cc1g_ <dig> is toxic for
c. elegans
and
a. aegypti
.
multiple sequence alignment of the aerolysin/etx pore-forming domain-containing proteins c. perfringens epsilon toxin , cc1g_ <dig>  cc1g_ <dig> and cc1g_ <dig>   expression of soluble cc1g_ <dig> in soluble form in e. coli bl <dig>  whole cell protein extracts in pbs were produced from non-induced  and induced  e. coli bl21/pet24-cc1g_ <dig> cultures.
supernatants from consecutive low  and high  speed centrifugations were collected and run on a 12% sds-page to evaluate cc1g_ <dig> expression and solubility.
cc1g_11805-mediated toxicity for a. aegypti
and c. elegans
larvae was assessed as described previously  <cit> .
e. coli bl <dig> expressing the previously characterized fungal lectin cgl <dig> was used as positive control for toxicity against a. aegypti rockefeller.
iptg-induced e. coli bearing an empty pet <dig> vector  was used as a negative control.
columns represent the mean percentage of surviving insect larvae , or worms either dying or reaching the indicated developmental stage  from  <dig> replicates each.
sds are shown as error bars.
dunn’s multiple comparisons were used to test the statistical significance of the toxicity observed in the a. aegypti assay.
mann whitney test was used to compare the percentage of worms reaching each developmental stage when treated with ev or cc1g_ <dig>  *:  <dig>  < p-value <  <dig> ; **:  <dig>  < p-value <  <dig> .
differential expression of loci involved in sexual development of filamentous fungi
various genes were recently described as playing a role at different stages of fruiting body formation of c. cinerea
.
with the exception of the cyclopropane fatty acid synthase cfs <dig>  none of these loci were found to be differentially transcribed during c. cinerea sexual development .
the velvet protein regulon plays a major role in the control of sexual vs. asexual development in the ascomycete aspergillus nidulans
<cit> ; therefore, we took a closer look at the expression pattern of c. cinerea orthologs of the velvet protein family and the proteins regulated by it  <cit> .
cc1g_ <dig>  a velvet domain-containing protein bearing an nls is highly induced in s1p .
in total, three out of six velvet domain-containing proteins encoded in the c. cinerea genome  were found to be developmentally regulated .
among the other members of the velvet protein regulon, two homologs of the vea-regulated activator nsdd  were also up-regulated in s1p .
moreover, the c. cinerea homeodomain transcription factor ste- <dig>  showed a moderate up-regulation in primordia .
finally, cc1g_ <dig>  an ortholog of the a. nidulans repressor of sexual development rosa, was  <dig> fold up-regulated in vm compared to s1p.
taken together, the differential expression of several members of the velvet protein regulon suggests that these genes may play a role in sexual development of basidiomycetes.table  <dig>
most genes necessary for fruiting body formation in
c. cinerea
are not up-regulated in s1p
rmt1
ubc2
snf5
cfs1
dst2
dst1
eln3
eln3
eln3
exp1
*fisher’s exact test.
conserved transcriptional circuitry during fruiting body development among basidiomycetous fungi
in order to explore a hypothetical conserved gene expression circuitry during the formation of sr structures in basidiomycetes, a comparative transcriptomic analysis by hierarchical clustering including c. cinerea, s. commune
<cit>  and l. bicolor
<cit>  was performed.
orthologous genes in s. commune and l. bicolor were assigned from a psi-blast search  to  <dig> and  <dig> up- and down-regulated loci in c. cinerea s1p, respectively.
massively parallel signature sequencing tags/million from s. commune monokaryotic mycelium and stage  <dig> primordia ; as well as l. bicolor free-living monokaryotic mycelium  and young fruiting bodies  robust multichip average values were retrieved from original studies  <cit> .
log <dig> expression ratios were calculated using monokaryotic mycelium expression values from these conserved loci as denominators and a centroid linkage hierarchical clustering analysis was computed .
four clusters corresponding to  <dig> up-regulated loci in early sexual development in basidiomycetes were identified .
in addition, two different gene groups  were found to be down-regulated in early stages of fruiting body formation when compared to vegetative mycelium of the three species  .figure  <dig>
conserved expression pattern of some gene clusters during fruiting body formation among three different basidiomycetous species.
comparative transcriptome analysis of early sexual development in s. commune 4-39/4- <dig>  c. cinerea ab and l. bicolor s238n-h <dig> was performed.
orthologous genes  to only  <dig> and  <dig> up- and down-regulated loci in s1p, respectively, were found in s. commune 4-39/4- <dig> and l. bicolor s238n-h <dig>   fruiting body transcriptome data were retrieved in the form of log <dig> ratios for these loci from the raw data sets and analyzed by centroid-linkage hierarchical clustering.
the red sidelines mark the presence of  <dig> loci clusters which are consistently up - or down -regulated in young fruiting bodies  and stage  <dig> primordia  of the three species compared.
close-up view of the up- and down-regulated clusters highlighted by red bars in .
orthologous genes in c. cinerea, s. commune and l. bicolor corresponding to the numbered clusters, as well as the functional annotation of these loci and the cluster-associated pearson correlation coefficients can be found in table  <dig>
up- and down-regulation of orthologous loci is conserved in stage  <dig> primordia and young fruiting bodies of three basidiomycete species
*functional annotation as determined by psi-blast.
†pearson correlation coefficient.
this hierarchical clustering approach of gene expression in orthologous loci during sexual development showed that loci involved in core meiotic functions, such as mei <dig>  and spo <dig> , are up-regulated during the early stages of fruiting body formation in basidiomycetes.
given the up-regulation of a conserved velvet-domain containing protein in primordia of the analyzed basidiomycetes, the conservation and expression pattern of velvet-interacting proteins previously characterized in aspergillus
<cit>  was examined.
with the exception of crya, orthologs to these velvet-interacting proteins can be found in c. cinerea, l. bicolor and s. commune .
contrary to the conserved transcriptional regulation observed for genes encoding velvet domain-containing proteins, little conservation in expression is evident for the orthologs to rosa/nosa, stua, nsdd, ppoa, laea or fpha .
nevertheless, conserved down-regulation during sexual development is shown for velb and kapa, suggesting that a fraction of the velvet-associated regulon described for ascomycetes might be playing a role during sexual development in basidiomycetes .
similarly, down-regulation of transcription factors such as flbc , nrg <dig> and bas <dig> during early mushroom formation was conserved between the three basidiomycetous species.
transcription factor mutants  altering normal sexual development in s. commune have been described  <cit> .
although orthologs of these transcription factors are present in c. cinerea and l. bicolor, the respective genes do not show a conserved expression pattern, indicating that there might be differences in the interplay between these factors during fruiting body formation between different basidiomycetes .
shotgun ms analysis of pbs-soluble vm and s1p proteins
in order to confirm protein expression of some of the genes found to be differentially expressed by rna-seq, we assessed the pbs-soluble c. cinerea proteome in vm and s1p by lc-ms. the analysis detected peptides corresponding to a total of  <dig> proteins in the samples, including  <dig>   <dig> and  <dig> proteins in s1p, vm or both, respectively .
this analysis is highly biased towards abundant and soluble proteins and is likely to have failed to detect peptides from most of the loci identified by rna-seq.
nevertheless, this method allowed us to confirm the translation of  <dig> transcripts up-regulated in s1p and  <dig> transcripts up-regulated in vm into proteins in the respective tissues .
discussion
our data shows a transcriptional switch during the differentiation of primordia in c. cinerea comprising 11% of the protein-encoding genome being up- or down-regulated.
differential transcription analyses carried out in other basidiomycetous fungi, such as agrocybe aegerita, cordyceps militaris and ganoderma lucidum, have revealed similar or even more extensive transcriptional switches .
taking the differential expression threshold used in the present study  ≥ 3), 25% and 30% out of  <dig> a. aegerita loci are up-regulated in fruiting bodies and vegetative mycelium, respectively  <cit> .
similarly, c. militaris up-regulates 40% loci during fruiting body formation  <cit> , while g. lucidum boosts the transcription of at least 27% measured loci in its sexual organs  <cit> .
the enrichment among the up-regulated loci in s1p of the functional annotation tag “n-methyltransferase”  suggests that epigenetic regulation of gene expression or chromosome dynamics may play an important role during sexual development in c. cinerea.
in saccharomyces cerevisiae, histone h <dig> lysine  <dig> trimethylation marks the sites where double-strand breaks, the first events during interhomolog recombination, occur  <cit> .
up-regulation of histone-lysine n-methyltransferases in s1p is in accordance with karyogamy and early meiotic phases taking place at stage  <dig> primordia during fruiting body formation  <cit> .
several previously characterized nematotoxic and insecticidal proteins  <cit>  were found to be up-regulated in s1p.
among these proteins, lectins appear to be most abundant.
the fruiting body-specific ricin b-like lectins ccl <dig> and ccl <dig> were previously shown to be toxic to c. elegans due to the binding to α <dig> -fucosylated n-glycan cores in the intestine of l <dig> worms  <cit> .
in accordance to a previous study  <cit> , we found that the tetrameric galectin cgl <dig>  was highly induced in c. cinerea fruiting bodies.
similar to ccl <dig> and ccl <dig>  cgl <dig> and its isogalectin cgl <dig>  showed toxicity against c. elegans which was dependent on binding to a galβ <dig> fucα <dig> -epitope on n-glycan cores present in the worm intestine  <cit> .
protease inhibition is another defense strategy against predation in c. cinerea fruiting bodies.
we found the locus encoding the serine protease inhibitor cospin <dig>  and its isoprotein cospin <dig>  among the top  <dig> most highly transcribed and differentially expressed loci in s1p.
cospin <dig> was previously shown to be toxic against drosophila melanogaster larvae indicative of its role in fruiting body defense against arthropod predation  <cit> .
in this study, we demonstrate that cc1g_ <dig> is toxic for c. elegans and a. aegypti larvae, analogous to the toxicity of aerolysin/etx pore-forming domain-containing bacillus sphaericus protein towards larvae of the mosquito culex quinquefasciatus
<cit> , suggesting that pore formation is another defense strategy of c. cinerea primordia against predators.
the high absolute transcription of these defense loci in s1p can be explained by the extensive resource allocation to these organs and the significance of these organs for reproduction of the fungus.
the expression of putative secreted and antibacterial proteins in c. cinerea vegetative mycelium, on the other hand, probably reflects the confrontation of this tissue with bacterial competitors although the antibacterial activity of these proteins has still to be demonstrated.
in summary, the differential expression of defense proteins in the different tissues of c. cinerea is an adaptation of the fungus to the different environmental challenges with which these tissues are confronted.
surprisingly, with the exception of cfs <dig>  none of the genes previously shown to play a role in c. cinerea fruiting body formation were differentially expressed in stage  <dig> primordia.
mutations in rmt <dig>  ubc <dig> and snf5
<cit>  block development before initials are formed .
in contrast, dst <dig>  dst <dig>  eln <dig> and exp <dig> are involved in processes taking place in stage  <dig> primordia , immature fruiting bodies  and decaying fruiting bodies   <cit> .
thus, a possible explanation of our results is that these genes might be regulated before or after the formation of s1p takes place.
intriguingly, the mutation of cfs <dig> blocks development right at the transition between initials and s1p.
this observed up-regulation of cfs <dig> in s1p supports a function for this cyclopropane fatty acid synthase in the development of s1p from initials.
the velvet protein regulon, including genes nsdd, rosa, vea and stua among others, coordinates sexual development and secondary metabolism in filamentous ascomycetes  <cit> .
velvet domains structurally resemble the rhd-like fold present in the transcription factor nf-κβ that plays a central role in animal immunity, suggesting a common evolutionary origin for these two protein families  <cit> .
overexpression of vea and nsdd in a. nidulans induces the formation of nursing hülle cells surrounding the cleistothecia  <cit> .
homologous genes in c. cinerea, cc1g_ <dig> and cc1g_ <dig>  showed high expression in s1p suggesting a function of these loci in the gene circuitry involved in fruiting body development in this fungus.
on the other hand, rosa, an a. nidulans transcription factor inhibiting sexual development in low-carbon culture, is expressed in a. nidulans asexual hyphae where it represses the transcription of sexual development regulators such as nsdd, vea and stua
<cit> .
similarly, rosa-homologous c. cinerea gene cc1g_ <dig>  is expressed in vegetative mycelium where it might be inhibiting sexual development.
taken together, these results suggest a conserved role of the velvet protein regulon during sexual development in the ascomycete a. nidulans and the basidiomycete c. cinerea.
genes encoding velvet domain-containing proteins were also found to be up-regulated in fruiting bodies of s. commune and l. bicolor
<cit> .
however,  genes encoding velvet-associated proteins such as rosa/nosa, stua, nsdd, ppoa, laea, fpha or crya  <cit> , do not show inter-species conservation of differential expression.
similar lack of transcriptional conservation between the three analyzed basidiomycete species was observed for orthologs to transcription factors shown to be involved in s. commune sexual development  <cit> .
taken together, this evidence suggests that basidiomycetes show species-specific divergence of transcriptional regulation in orthologous genes similar to plants  <cit> .
a broader comparison of the transcriptomes of the three analyzed basidiomycete species, comprising all the genes differentially expressed in c. cinerea during sexual development, revealed the presence of a conserved gene regulation circuitry among basidiomycetes during fruiting body formation.
as previously observed in fruiting bodies of ascomycetes  <cit> , our orthology analysis showed that a large fraction of genes differentially expressed in s1p corresponds to loci which are not present in the basidiomycetes l. bicolor and s. commune.
these results are in agreement with previous observations in plants and animals that genes associated with sexual reproduction rapidly evolve  <cit> .
the existence of clusters comprising conserved up- or down-regulated loci with little inter-species expression variability, suggests that regulation of these loci evolved under stabilizing selection  <cit> .
sequence and expression conservation might imply an essential role of these genes in fruiting body development and sexual reproduction in basidiomycetes.
interestingly, l. bicolor and c. cinerea orthologs to transcription factors described previously as important for sexual development in s. commune, such as bri <dig>  hom <dig>  gat <dig>  fst <dig>  c2h <dig>  fst <dig> or hom <dig>  <cit> , do not show conserved transcriptional conservation in basidiomycetes, suggesting that novel regulatory pathways related to sexual development did evolve once speciation occurred.
as a proof of concept, traeger and collaborators found that the fruiting body specific transcription factor pro <dig>  in the ascomycetes sordaria macrospora and pyronema confluens is a core regulator of perithecia maturation.
s. macrospora deficient in pro <dig> was shown to be sterile and unable to produce mature perithecia  <cit> .
in addition to a velvet domain-containing protein, the expression of the rna binding protein mei <dig> , a master meiosis regulator in yeast and plants  <cit>  protecting meiosis-specific transcripts from degradation by the dsr-mmi system, was increased in s1p or yfb of c. cinerea, s. commune and l. bicolor.
similarly, the transcriptional regulation of spo <dig>  encoding a protein inducing meiotic recombination in s. cerevisiae and c. cinerea
<cit> , was also conserved.
this induction of meiosis regulators in multiple species reflects the role of mushrooms in the production and dispersal of basidiospores.
with regard to the comparative transcriptome analysis of the three basidiomycete species, it should be noted that in case of s. commune and l. bicolor, only data of monokaryotic vegetative mycelia was available  <cit> , whereas in case of c. cinerea, the transcriptome of an isogenic homodikaryotic mycelium was determined.
thus, the degree of conserved transcriptional regulation during sexual development between c. cinerea, l. bicolor and s. commune is potentially larger than observed.
lastly, the proteome of an organism provides a more direct image of its phenotype than the transcriptome  <cit> .
detection of proteins using label-free shotgun mass spectrometry fails to detect low abundance proteins in complex total extracts and allows only semi-quantitative estimation of relative protein amounts.
these properties are in contrast to the superior standardization and sensitivity achieved by state of the art nucleotide sequencing technologies.
nevertheless, lc-ms spectra showed the presence of peptides derived from the nematotoxic lectin ccl <dig>  and the nematotoxic/insecticidal aerolysin/etx pore-forming domain-containing protein cc1g_ <dig> in the pbs-soluble protein extract of c. cinerea stage  <dig> primordia, indicating that these cytoplasmic toxins are indeed expressed at protein level in these organs.
the recent development of gene targeting tools in c. cinerea
<cit> , will help to test whether some of the genes identified as differentially regulated during sexual development in multiple basidiomycete species, play a role in this process.
CONCLUSIONS
in this work, we show that sexual reproduction in c. cinerea a43mutb43mut involves the differential transcription of at least 11% of its protein-coding genome.
differentially transcribed genes include several genes coding for defense proteins that protect fruiting structures and vegetative mycelia from predators and competitors, respectively.
moreover, our data infers a role of the velvet protein family during fruiting body formation in basidiomycetes and thus, a conserved role of this protein family during sexual development in dikaryotic filamentous fungi.
finally, the result of the comparative transcriptome analysis of c. cinerea, s. commune and l. bicolor suggest that a conserved set of orthologous genes regulates sexual development in the phylum basidiomycota.
additional experiments addressing the function of the differentially expressed gene products are required to confirm these hypotheses.

 the last eukaryotic common ancestor was facultatively sexual and evolved nearly billion years ago in the proterozoic eon. sexual reproduction shares common features across the eukaryotic lineage such as ploidy changes, meiotic recombination and cell-cell recognition between gametes followed by cellular fusion and zygote formation. an increase in the genetic diversity of the population, making it more adaptable to changing environmental conditions, as well as the dilution of deleterious mutations out of the gene pool are the most remarkable evolutionary innovations achieved by sr. despite all these clear benefits, sex is energetically expensive and entails a higher chance of genetic and organelle conflicts. during sexual reproduction, fungi undergo dramatic morphological changes driven by environmental conditions such as light, nutrient availability and grazing by predators. in basidiomycetes, mushroom development starts with intense localized hyphal branching leading to the formation of hyphal knots. these branching hyphae further aggregate to form 1–2 mm secondary nodules where cell differentiation leads to the establishment of bipolar primordia containing all the tissues observed in the mature fruiting body. as a last step, primordia develop to mature fruiting bodies mainly by cellular expansion. due to their hyphal density, primordia and fruiting bodies are attractive to predators including mollusks, arthropods and nematodes. coprinopsis cinerea has been used as a model basidiomycete since the mid-1950s due to its saprobic lifestyle, its rapid growth and the feasibility of producing fruiting bodies under defined laboratory conditions. in nature, c. cinerea grows on horse dung, a eutrophic substrate rich in competing microorganisms, such as firmicutes, bacteroidetes and proteobacteria. the recent sequencing of the c. cinerea genome allows the study of this organism gene expression on transcriptome and proteome level at different developmental stages or under a variety of environmental settings. morphological changes and environmental signals during fruiting body formation in c. cinerea are well described; nonetheless, comparably little is known about the molecular machinery driving sexual reproduction processes in this basidiomycete. recently, mutations blocking fruiting body development at different stages or altering mushroom morphology were identified. in addition, dst and dst encoding a blue-light photoreceptor and a flavin adenine dinucleotide-binding protein, were shown to play a role in blue light sensing. in agreement with previous experiments, strains defective in these two proteins were unable to form fruiting bodies, showing that blue light is an essential environmental trigger of mushroom development. most recently, c. cinerea strains carrying mutations in the putative component of the swi/snf chromatin remodeling complex snf were shown to be defective in fruiting initiation, suggesting that epigenetic reprogramming of loci occurs during fruiting body formation. aerial fruiting bodies are an attractive prey for predators and thus are protected by a battery of defense molecules including proteins, peptides and secondary metabolites. some of these toxins are known to be specifically produced in the fruiting body and not in the vegetative mycelium. for instance, cytoplasmic lectins showing a broad range of non-self-carbohydrate specificities, also referred to as fruiting body lectins due to their specific expression pattern, have been shown to exert toxicity to nematodes, insect larvae and amoeba. protein-mediated inhibition of serine proteases, proteolytic degradation of predator-derived proteins and sequestration of biotin are other strategies of basidiomycetous fruiting bodies to dissuade predators. vegetative mycelium, in contrast, digests extracellular carbon macromolecules in the growth substrate, such as cellulose and lignin, into smaller degradation products which are absorbed by the growing hyphae. at the same time, these smaller molecules become available for competing bacteria that profit from the fungal enzymatic machinery. as a response to bacterial competitors, fungi have evolved secreted antimicrobial proteins. mygind and colleagues presented recent evidence that vegetative mycelia of fungi secrete cysteine-stabilized antibacterial peptides which play a role in the arms race with competing bacteria. using rna-seq in c. cinerea, we show evidence suggesting a role of the velvet protein regulon in sexual development of basidiomycetes. our data supports the existence of a conserved transcriptional circuitry in basidiomycetes fruiting bodies consisting of at least orthologous genes probably involved in mushroom development and function. in addition, our data reveals the existence of two different sets of fungal defense proteins in vegetative and sexual organs matching the type of competitors and predators by which these structures are challenged in nature. the transcriptome data is supported by the first partial shotgun mass spectrometry catalog of proteins present in c. cinerea stage primordia and vegetative mycelium.  differential gene expression during fruiting body development in c. cinerea four different cdna libraries were sequenced with a final gb mapped data output. approximately 95% of the open reading frames in the genome of c. cinerea a43mutb43mut were transcribed using five reads/orf as the minimal threshold for a locus to be considered as expressed. differential gene expression at the rna level between s1p and vm was examined. eleven percent of the annotated orfs in the c. cinerea genome were found to be differentially expressed, and loci in vm and s1p, respectively, using fold change and fisher’s exact test p-value ≤  as thresholds. the number of differentially transcribed loci in these two developmental stages increased to in vm and in s1p when a fold change threshold of was set, corresponding to approximately 45% annotated orfs in the genome of c. cinerea.table general features of the c. cinerea s1p and vm transcriptomes s1p vm average solid read size sequenced material *mean percentage of annotated orfs detected to be expressed using a minimal threshold reads/orf. differential gene expression during early fruiting body development in c. cinerea. volcano plot illustrating the relative expression ) and statistical significance of the loci in the genome of c. cinerea between vm and s1p. fisher’s exact test -log p-value ≥  and log +/- were used as thresholds of differential gene expression. grey boxes comprise genes significantly up-regulated in s1p or vm. tag clouds showing enriched psi-blast functional annotation terms from up-regulated loci in s1p and vm were produced in wordle after removing frequently appearing tags. to validate these data, qrt-pcr of four selected loci was performed. although s1p/vm expression ratios did not exactly match those observed by rna-seq, qrt-pcr results showed the same trend of differential gene expression for these loci during c. cinerea development as observed by solid rna-seq. in addition, we verified that the expression values of reported housekeeping loci commonly used in qrt-pcr normalization were in a range indicative of constitutive expression  ≤ 2). enrichment of psi-blast-derived functional annotation terms was visualized using wordle after excluding frequent non-informative terms. by far, the most commonly assigned annotation term was “hypothetical protein” in s1p and vm; nonetheless, there was a significant enrichment of n-methyltransferase tags in s1p. enrichment of functional annotation terms also shows that different sets of cytochromes, kinases, dehydrogenases, transporters and hydrophobins are specifically expressed in vegetative mycelium or young fruiting bodies. functional annotation clustering using david bioinformatics resources confirmed the enrichment of protein methyltransferases and hydrophobins in s1p with and fold enrichment compared to the occurrence of these functional categories in the genome of c. cinerea. tissue-specific expression of c. cinereadefense proteins several cytoplasmic defense lectins and protease inhibitors such as c. cinerea lectin, coprinopsis galectin and two paralogous serine protease inhibitors from coprinopsis were found to be highly up-regulated in c. cinerea s1p. out of these previously characterized genes coding for proteins with nematotoxic and insecticidal activity, cospin and ccl were found to be among the top most highly transcribed and differentially expressed loci in s1p. among these, two loci, cc1g_ and cc1g_ encoding homologous proteins with a predicted aerolysin/etx pore-forming domain as well as loci encoding proteins with suspected antibacterial and antifungal function including two peptidoglycan binding proteins, the toxin component of a bacterial toxin-antitoxin system and a thaumatin-like protein were found. in contrast to s1p-specific defense proteins, defense proteins specifically up-regulated in vm were mainly secreted. these proteins included three putative lysozymes, several proteins containing a cfem domain whose structure resembles cysteine-stabilized antibacterial peptides, and two representatives of the cerato-platanin family of secreted proteins. latter protein family is expanded in basidiomycetes and has recently been implicated in interactions of dikaryotic fungi with other organisms. during sexual development on herbivore dung, c. cinerea is exposed to a succession of antagonists colonizing this substrate. the differential expression of cytoplasmic and secreted defense proteins in s1p and vm, respectively, might reflect the prevalent types of antagonists with which these tissues of c. cinerea are confronted.table s1p-specific defense loci *fisher’s exact test. †n or y indicates the lack or presence of a signal peptide. ‡n indicates a lack of transmembrane helices, while a number corresponds to the amount of transmembrane helices predicted in the orf. vm-specific defense loci *fisher’s exact test. †n or y indicates the lack or presence of a signal peptide. ‡n indicates a lack of transmembrane helices, while a number corresponds to the amount of transmembrane helices predicted in the orf. the s1p-specific aerolysin/etx pore-forming domain-containing protein cc1g_ is toxic to nematode and insect larvae cc1g_ cc1g_ and cc1g_ encode three homologous 30–40 kda proteins containing a predicted aerolysin/etx pore-forming domain. this domain is homologous to the one present in the insecticidal epsilon toxin from clostridium perfringens and distantly related to aerolysin pore-forming toxins. in order to test the significance of the up-regulation of cc1g_ and cc1g_ in s1p with regard to fungal defense, we cloned and recombinantly expressed cc1g_ in escherichia coli and assessed the toxicity of the protein by feeding the recombinant bacteria to nematode and insect larvae as described previously. results in figure 2b show that cc1g_ was expressed in soluble form in e. coli. feeding of cc1g_11805-expressing bacteria to l larvae of the mosquito aedes aegypti lead to their death after 96 h. vector control-containing and fungal lectin-expressing e. coli were used as negative and positive controls, respectively, in these experiment. similarly, feeding of cc1g_11805-expressing bacteria was found to significantly impair larval development of the c. elegans n wildtype and pmk- mutant strains; in agreement with the previously reported higher susceptibility of latter strain to different kinds of abiotic and biotic stresses including other nematotoxic fungal defense proteins, cc1g_ was shown to be more toxic to c. elegans pmk- than to n these results support previous observations showing that the expression of defense proteins directed against nematodes and insects is significantly increased in c. cinerea sexual organs.figure the s1p-specific aerolysin/etx pore-forming domain-containing protein cc1g_ is toxic for c. elegans and a. aegypti. multiple sequence alignment of the aerolysin/etx pore-forming domain-containing proteins c. perfringens epsilon toxin, cc1g_ cc1g_ and cc1g_ expression of soluble cc1g_ in soluble form in e. coli bl whole cell protein extracts in pbs were produced from non-induced and induced e. coli bl21/pet24-cc1g_ cultures. supernatants from consecutive low and high speed centrifugations were collected and run on a 12% sds-page to evaluate cc1g_ expression and solubility. cc1g_11805-mediated toxicity for a. aegypti and c. elegans larvae was assessed as described previously. e. coli bl expressing the previously characterized fungal lectin cgl was used as positive control for toxicity against a. aegypti rockefeller. iptg-induced e. coli bearing an empty pet vector was used as a negative control. columns represent the mean percentage of surviving insect larvae, or worms either dying or reaching the indicated developmental stage from replicates each. sds are shown as error bars. dunn’s multiple comparisons were used to test the statistical significance of the toxicity observed in the a. aegypti assay. mann whitney test was used to compare the percentage of worms reaching each developmental stage when treated with ev or cc1g_ *:  < p-value < ; **:  < p-value < . differential expression of loci involved in sexual development of filamentous fungi various genes were recently described as playing a role at different stages of fruiting body formation of c. cinerea. with the exception of the cyclopropane fatty acid synthase cfs none of these loci were found to be differentially transcribed during c. cinerea sexual development. the velvet protein regulon plays a major role in the control of sexual vs. asexual development in the ascomycete aspergillus nidulans; therefore, we took a closer look at the expression pattern of c. cinerea orthologs of the velvet protein family and the proteins regulated by it. cc1g_ a velvet domain-containing protein bearing an nls is highly induced in s1p. in total, three out of six velvet domain-containing proteins encoded in the c. cinerea genome were found to be developmentally regulated. among the other members of the velvet protein regulon, two homologs of the vea-regulated activator nsdd were also up-regulated in s1p. moreover, the c. cinerea homeodomain transcription factor ste- showed a moderate up-regulation in primordia. finally, cc1g_ an ortholog of the a. nidulans repressor of sexual development rosa, was fold up-regulated in vm compared to s1p. taken together, the differential expression of several members of the velvet protein regulon suggests that these genes may play a role in sexual development of basidiomycetes.table most genes necessary for fruiting body formation in c. cinerea are not up-regulated in s1p rmt1 ubc2 snf5 cfs1 dst2 dst1 eln3 eln3 eln3 exp1 *fisher’s exact test. conserved transcriptional circuitry during fruiting body development among basidiomycetous fungi in order to explore a hypothetical conserved gene expression circuitry during the formation of sr structures in basidiomycetes, a comparative transcriptomic analysis by hierarchical clustering including c. cinerea, s. commune and l. bicolor was performed. orthologous genes in s. commune and l. bicolor were assigned from a psi-blast search to and up- and down-regulated loci in c. cinerea s1p, respectively. massively parallel signature sequencing tags/million from s. commune monokaryotic mycelium and stage primordia; as well as l. bicolor free-living monokaryotic mycelium and young fruiting bodies robust multichip average values were retrieved from original studies. log expression ratios were calculated using monokaryotic mycelium expression values from these conserved loci as denominators and a centroid linkage hierarchical clustering analysis was computed. four clusters corresponding to up-regulated loci in early sexual development in basidiomycetes were identified. in addition, two different gene groups were found to be down-regulated in early stages of fruiting body formation when compared to vegetative mycelium of the three species.figure conserved expression pattern of some gene clusters during fruiting body formation among three different basidiomycetous species. comparative transcriptome analysis of early sexual development in s. commune 4-39/4- c. cinerea ab and l. bicolor s238n-h was performed. orthologous genes to only and up- and down-regulated loci in s1p, respectively, were found in s. commune 4-39/4- and l. bicolor s238n-h fruiting body transcriptome data were retrieved in the form of log ratios for these loci from the raw data sets and analyzed by centroid-linkage hierarchical clustering. the red sidelines mark the presence of loci clusters which are consistently up - or down -regulated in young fruiting bodies and stage primordia of the three species compared. close-up view of the up- and down-regulated clusters highlighted by red bars in. orthologous genes in c. cinerea, s. commune and l. bicolor corresponding to the numbered clusters, as well as the functional annotation of these loci and the cluster-associated pearson correlation coefficients can be found in table  up- and down-regulation of orthologous loci is conserved in stage primordia and young fruiting bodies of three basidiomycete species *functional annotation as determined by psi-blast. †pearson correlation coefficient. this hierarchical clustering approach of gene expression in orthologous loci during sexual development showed that loci involved in core meiotic functions, such as mei and spo, are up-regulated during the early stages of fruiting body formation in basidiomycetes. given the up-regulation of a conserved velvet-domain containing protein in primordia of the analyzed basidiomycetes, the conservation and expression pattern of velvet-interacting proteins previously characterized in aspergillus was examined. with the exception of crya, orthologs to these velvet-interacting proteins can be found in c. cinerea, l. bicolor and s. commune. contrary to the conserved transcriptional regulation observed for genes encoding velvet domain-containing proteins, little conservation in expression is evident for the orthologs to rosa/nosa, stua, nsdd, ppoa, laea or fpha. nevertheless, conserved down-regulation during sexual development is shown for velb and kapa, suggesting that a fraction of the velvet-associated regulon described for ascomycetes might be playing a role during sexual development in basidiomycetes. similarly, down-regulation of transcription factors such as flbc, nrg and bas during early mushroom formation was conserved between the three basidiomycetous species. transcription factor mutants altering normal sexual development in s. commune have been described. although orthologs of these transcription factors are present in c. cinerea and l. bicolor, the respective genes do not show a conserved expression pattern, indicating that there might be differences in the interplay between these factors during fruiting body formation between different basidiomycetes. shotgun ms analysis of pbs-soluble vm and s1p proteins in order to confirm protein expression of some of the genes found to be differentially expressed by rna-seq, we assessed the pbs-soluble c. cinerea proteome in vm and s1p by lc-ms. the analysis detected peptides corresponding to a total of proteins in the samples, including and proteins in s1p, vm or both, respectively. this analysis is highly biased towards abundant and soluble proteins and is likely to have failed to detect peptides from most of the loci identified by rna-seq. nevertheless, this method allowed us to confirm the translation of transcripts up-regulated in s1p and transcripts up-regulated in vm into proteins in the respective tissues. discussion our data shows a transcriptional switch during the differentiation of primordia in c. cinerea comprising 11% of the protein-encoding genome being up- or down-regulated. differential transcription analyses carried out in other basidiomycetous fungi, such as agrocybe aegerita, cordyceps militaris and ganoderma lucidum, have revealed similar or even more extensive transcriptional switches. taking the differential expression threshold used in the present study  ≥ 3), 25% and 30% out of a. aegerita loci are up-regulated in fruiting bodies and vegetative mycelium, respectively. similarly, c. militaris up-regulates 40% loci during fruiting body formation, while g. lucidum boosts the transcription of at least 27% measured loci in its sexual organs. the enrichment among the up-regulated loci in s1p of the functional annotation tag “n-methyltransferase” suggests that epigenetic regulation of gene expression or chromosome dynamics may play an important role during sexual development in c. cinerea. in saccharomyces cerevisiae, histone h lysine trimethylation marks the sites where double-strand breaks, the first events during interhomolog recombination, occur. up-regulation of histone-lysine n-methyltransferases in s1p is in accordance with karyogamy and early meiotic phases taking place at stage primordia during fruiting body formation. several previously characterized nematotoxic and insecticidal proteins were found to be up-regulated in s1p. among these proteins, lectins appear to be most abundant. the fruiting body-specific ricin b-like lectins ccl and ccl were previously shown to be toxic to c. elegans due to the binding to α -fucosylated n-glycan cores in the intestine of l worms. in accordance to a previous study, we found that the tetrameric galectin cgl was highly induced in c. cinerea fruiting bodies. similar to ccl and ccl cgl and its isogalectin cgl showed toxicity against c. elegans which was dependent on binding to a galβ fucα -epitope on n-glycan cores present in the worm intestine. protease inhibition is another defense strategy against predation in c. cinerea fruiting bodies. we found the locus encoding the serine protease inhibitor cospin and its isoprotein cospin among the top most highly transcribed and differentially expressed loci in s1p. cospin was previously shown to be toxic against drosophila melanogaster larvae indicative of its role in fruiting body defense against arthropod predation. in this study, we demonstrate that cc1g_ is toxic for c. elegans and a. aegypti larvae, analogous to the toxicity of aerolysin/etx pore-forming domain-containing bacillus sphaericus protein towards larvae of the mosquito culex quinquefasciatus, suggesting that pore formation is another defense strategy of c. cinerea primordia against predators. the high absolute transcription of these defense loci in s1p can be explained by the extensive resource allocation to these organs and the significance of these organs for reproduction of the fungus. the expression of putative secreted and antibacterial proteins in c. cinerea vegetative mycelium, on the other hand, probably reflects the confrontation of this tissue with bacterial competitors although the antibacterial activity of these proteins has still to be demonstrated. in summary, the differential expression of defense proteins in the different tissues of c. cinerea is an adaptation of the fungus to the different environmental challenges with which these tissues are confronted. surprisingly, with the exception of cfs none of the genes previously shown to play a role in c. cinerea fruiting body formation were differentially expressed in stage primordia. mutations in rmt ubc and snf5 block development before initials are formed. in contrast, dst dst eln and exp are involved in processes taking place in stage primordia, immature fruiting bodies and decaying fruiting bodies. thus, a possible explanation of our results is that these genes might be regulated before or after the formation of s1p takes place. intriguingly, the mutation of cfs blocks development right at the transition between initials and s1p. this observed up-regulation of cfs in s1p supports a function for this cyclopropane fatty acid synthase in the development of s1p from initials. the velvet protein regulon, including genes nsdd, rosa, vea and stua among others, coordinates sexual development and secondary metabolism in filamentous ascomycetes. velvet domains structurally resemble the rhd-like fold present in the transcription factor nf-κβ that plays a central role in animal immunity, suggesting a common evolutionary origin for these two protein families. overexpression of vea and nsdd in a. nidulans induces the formation of nursing hülle cells surrounding the cleistothecia. homologous genes in c. cinerea, cc1g_ and cc1g_ showed high expression in s1p suggesting a function of these loci in the gene circuitry involved in fruiting body development in this fungus. on the other hand, rosa, an a. nidulans transcription factor inhibiting sexual development in low-carbon culture, is expressed in a. nidulans asexual hyphae where it represses the transcription of sexual development regulators such as nsdd, vea and stua. similarly, rosa-homologous c. cinerea gene cc1g_ is expressed in vegetative mycelium where it might be inhibiting sexual development. taken together, these results suggest a conserved role of the velvet protein regulon during sexual development in the ascomycete a. nidulans and the basidiomycete c. cinerea. genes encoding velvet domain-containing proteins were also found to be up-regulated in fruiting bodies of s. commune and l. bicolor. however, genes encoding velvet-associated proteins such as rosa/nosa, stua, nsdd, ppoa, laea, fpha or crya, do not show inter-species conservation of differential expression. similar lack of transcriptional conservation between the three analyzed basidiomycete species was observed for orthologs to transcription factors shown to be involved in s. commune sexual development. taken together, this evidence suggests that basidiomycetes show species-specific divergence of transcriptional regulation in orthologous genes similar to plants. a broader comparison of the transcriptomes of the three analyzed basidiomycete species, comprising all the genes differentially expressed in c. cinerea during sexual development, revealed the presence of a conserved gene regulation circuitry among basidiomycetes during fruiting body formation. as previously observed in fruiting bodies of ascomycetes, our orthology analysis showed that a large fraction of genes differentially expressed in s1p corresponds to loci which are not present in the basidiomycetes l. bicolor and s. commune. these results are in agreement with previous observations in plants and animals that genes associated with sexual reproduction rapidly evolve. the existence of clusters comprising conserved up- or down-regulated loci with little inter-species expression variability, suggests that regulation of these loci evolved under stabilizing selection. sequence and expression conservation might imply an essential role of these genes in fruiting body development and sexual reproduction in basidiomycetes. interestingly, l. bicolor and c. cinerea orthologs to transcription factors described previously as important for sexual development in s. commune, such as bri hom gat fst c2h fst or hom, do not show conserved transcriptional conservation in basidiomycetes, suggesting that novel regulatory pathways related to sexual development did evolve once speciation occurred. as a proof of concept, traeger and collaborators found that the fruiting body specific transcription factor pro in the ascomycetes sordaria macrospora and pyronema confluens is a core regulator of perithecia maturation. s. macrospora deficient in pro was shown to be sterile and unable to produce mature perithecia. in addition to a velvet domain-containing protein, the expression of the rna binding protein mei, a master meiosis regulator in yeast and plants protecting meiosis-specific transcripts from degradation by the dsr-mmi system, was increased in s1p or yfb of c. cinerea, s. commune and l. bicolor. similarly, the transcriptional regulation of spo encoding a protein inducing meiotic recombination in s. cerevisiae and c. cinerea, was also conserved. this induction of meiosis regulators in multiple species reflects the role of mushrooms in the production and dispersal of basidiospores. with regard to the comparative transcriptome analysis of the three basidiomycete species, it should be noted that in case of s. commune and l. bicolor, only data of monokaryotic vegetative mycelia was available, whereas in case of c. cinerea, the transcriptome of an isogenic homodikaryotic mycelium was determined. thus, the degree of conserved transcriptional regulation during sexual development between c. cinerea, l. bicolor and s. commune is potentially larger than observed. lastly, the proteome of an organism provides a more direct image of its phenotype than the transcriptome. detection of proteins using label-free shotgun mass spectrometry fails to detect low abundance proteins in complex total extracts and allows only semi-quantitative estimation of relative protein amounts. these properties are in contrast to the superior standardization and sensitivity achieved by state of the art nucleotide sequencing technologies. nevertheless, lc-ms spectra showed the presence of peptides derived from the nematotoxic lectin ccl and the nematotoxic/insecticidal aerolysin/etx pore-forming domain-containing protein cc1g_ in the pbs-soluble protein extract of c. cinerea stage primordia, indicating that these cytoplasmic toxins are indeed expressed at protein level in these organs. the recent development of gene targeting tools in c. cinerea, will help to test whether some of the genes identified as differentially regulated during sexual development in multiple basidiomycete species, play a role in this process.  in this work, we show that sexual reproduction in c. cinerea a43mutb43mut involves the differential transcription of at least 11% of its protein-coding genome. differentially transcribed genes include several genes coding for defense proteins that protect fruiting structures and vegetative mycelia from predators and competitors, respectively. moreover, our data infers a role of the velvet protein family during fruiting body formation in basidiomycetes and thus, a conserved role of this protein family during sexual development in dikaryotic filamentous fungi. finally, the result of the comparative transcriptome analysis of c. cinerea, s. commune and l. bicolor suggest that a conserved set of orthologous genes regulates sexual development in the phylum basidiomycota. additional experiments addressing the function of the differentially expressed gene products are required to confirm these hypotheses. 
it is well known that mushrooms produce defense proteins and secondary metabolites against predators and competitors; however, less is known about the correlation between the tissue-specific expression and the target organism specificity of these molecules. our data show that many genes encoding predicted and already characterized defense proteins are differentially expressed in these tissues. the pbs-soluble proteome of c. cinerea primordia and mycelium was analyzed by shotgun lc-ms. this proteome data confirmed the presence of intracellular defense proteins in primordia. in order to characterize the regulation in the expression of defense loci and the transcriptional circuitries controlling sexual reproduction in basidiomycetes, we sequenced the poly -positive transcriptome of stage primordia and vegetative mycelium of c. cinerea a43mutb43mut. this study shows that the exposure of different tissues in fungi to different types of antagonists shapes the expression pattern of defense loci in a tissue-specific manner. furthermore, we identify a transcriptional circuitry conserved among basidiomycetes during fruiting body formation that involves, amongst other transcription factors, the up-regulation of a velvet domain-containing protein. the predicted specificity of these proteins with regard to target organisms suggests that their expression pattern correlates with the type of antagonists these tissues are confronted with. the aims of this study were: first, to evaluate the tissue specificity of defense gene expression in the model mushroom coprinopsis cinerea and, second, to assess the degree of conservation in transcriptional regulation during sexual development in basidiomycetes. comparison of our data to analogous data from laccaria bicolor and schizophyllum commune revealed that the transcriptional regulation of nearly loci is conserved and probably subjected to stabilizing selection. in addition, conserved transcriptional circuitries involved in developing sexual organs in fungi are not characterized, despite the growing number of gene expression datasets available from reproductive and vegetative tissue. a velvet domain-containing protein was found to be up-regulated in all three fungi, providing preliminary evidence of a possible role of the velvet protein family in sexual development of basidiomycetes.
 it is well known that mushrooms produce defense proteins and secondary metabolites against predators and competitors; however, less is known about the correlation between the tissue-specific expression and the target organism specificity of these molecules. in addition, conserved transcriptional circuitries involved in developing sexual organs in fungi are not characterized, despite the growing number of gene expression datasets available from reproductive and vegetative tissue. the aims of this study were: first, to evaluate the tissue specificity of defense gene expression in the model mushroom coprinopsis cinerea and, second, to assess the degree of conservation in transcriptional regulation during sexual development in basidiomycetes.  in order to characterize the regulation in the expression of defense loci and the transcriptional circuitries controlling sexual reproduction in basidiomycetes, we sequenced the poly -positive transcriptome of stage primordia and vegetative mycelium of c. cinerea a43mutb43mut. our data show that many genes encoding predicted and already characterized defense proteins are differentially expressed in these tissues. the predicted specificity of these proteins with regard to target organisms suggests that their expression pattern correlates with the type of antagonists these tissues are confronted with. accordingly, we show that the stage primordium-specific protein cc1g_ is toxic to insects and nematodes. comparison of our data to analogous data from laccaria bicolor and schizophyllum commune revealed that the transcriptional regulation of nearly loci is conserved and probably subjected to stabilizing selection. a velvet domain-containing protein was found to be up-regulated in all three fungi, providing preliminary evidence of a possible role of the velvet protein family in sexual development of basidiomycetes. the pbs-soluble proteome of c. cinerea primordia and mycelium was analyzed by shotgun lc-ms. this proteome data confirmed the presence of intracellular defense proteins in primordia.  this study shows that the exposure of different tissues in fungi to different types of antagonists shapes the expression pattern of defense loci in a tissue-specific manner. furthermore, we identify a transcriptional circuitry conserved among basidiomycetes during fruiting body formation that involves, amongst other transcription factors, the up-regulation of a velvet domain-containing protein. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords fungal defensesexual developmentc. cinereaprimordiarna-seqcomparative transcriptomicstissue-specific lc-ms proteomeissue-copyright-statement© the author 2014 