WO2011159153A2

WO2011159153A2 - Inducible promoter and its use

Info

Publication number: WO2011159153A2
Application number: PCT/NL2011/050428
Authority: WO
Inventors: Robin Arthur Ohm; Luis Gaston Lugones; Herman Abel Bernard WÖSTEN
Original assignee: Universiteit Utrecht Holding B.V.; Stichting Voor De Technische Wetenschappen
Priority date: 2010-06-15
Filing date: 2011-06-14
Publication date: 2011-12-22
Also published as: WO2011159153A3

Abstract

The invention relates to a method of producing mushroom using a heat inducible promoter. The invention further relates to a heat inducible promoter, a nucleic acid construct, a vector and a fungus or mushroom comprising a heat inducible promoter of the invention and to methods for producing such a mushroom or fungus or a substance of interest.

Description

Inducible promoter and its use

Field of the invention

The invention relates to a method of producing mushroom using a heat inducible promoter. The invention further relates to a heat inducible promoter, a nucleic acid construct, a vector and a fungus or mushroom comprising a heat inducible promoter of the invention and to methods for producing such a mushroom or fungus or a substance of interest. Background of the invention

Formation of mushrooms is a highly complex developmental process. As an example, we describe a generalized scheme for formation of agaric fruiting bodies such as those of Agaricus bisporus (see Kiies, 2000; Umar and van Griensven, 1997). After a "critical mass" of submerged mycelium has been formed, hyphae escape the substrate to grow into the air. These hyphae form aggregates, which are called hyphal knots or nodules. Within the knots hyphae aggregate forming a fruiting body initial. Within the core of the fruiting body initial differentiation of cells occurs. The lower part will develop into the stipe, while the cap will be formed from the upper part. Within the cap different tissues develop. In the inner part of the cap the pileus trama and gills with a hymenium can be distinguished. In the hymenium different cell types are formed, among which the basidia. In the basidia karyogamy and meioses take place, ultimately resulting in basidiospores. That development of fruiting bodies is complex, is also exemplified by the fact that formation of the different tissues overlaps in time. Moreover, cells in the developing mushroom differ in diameter, length, the number of septa, nuclei and vacuoles as well as the molecular composition (e.g. the content of reserve carbohydrate).

Spores formed by A. bisporus contain two nuclei with a different mating type. Germination of these spores thus results in a self-fertile heterokaryotic mycelium, containing a variable number of both nuclear types. In contrast, the fertile stage of a majority of mushroom forming fungi results from a mating of two compatible strains with different mating type loci. During mating, partners exchange nuclei. These nuclei do not fuse but are maintained in the hyphal compartment. Such mycelia are therefore called heterokaryotic (in the case that each compartment contains one nucleus of each type it is called a dikaryon). They can form fruiting bodies under the appropriate environmental and nutritional conditions. The mating type loci are the master regulators of fruiting body development. Little is known about the mating type system of A. bisporus. It is assumed that this fungus contains a single mating type locus. The mating type loci of Schizophyllum commune and Coprinus cinereus and their role in development have been studied well (for a review see Kiies, 2000). Both S. commune and C. cinereus contain two mating type loci. The A locus encodes homeodomain proteins. These proteins function by forming heterodimers with homeodomain proteins encoded in a compatible A locus. Some of these homeodomain proteins also seem to form functional homodimers. The B locus encodes pheromones and receptors. These receptors can bind pheromones encoded by other alleles of the B locus. Both the A and the B locus regulate distinct cellular processes involved in establishing the dikaryotic mycelium. However, they co-ordinately regulate fruiting body initiation. Clearly, the presence of compatible A and B mating type loci is not sufficient for fruiting. For instance, in C. cinereus aggregates formed by a dikaryon can develop into a fruiting initial or into sclerotia. Environmental conditions such as light and nutrient availability will determine which developmental program will be switched on.

Little is known about regulatory proteins other than those encoded by the mating type loci that are involved in mushroom formation in general and fruiting initiation in particular. In contrast to A. bisporus, at least some mutants and genes have been identified in S. commune and C. cinereus. The fbf mutation is a frequently observed recessive mutation in S. commune that suppresses dikaryon specific processes in vegetative hyphae as well as formation of fruiting bodies (Springer and Wessels, 1989). The FBF gene thus seems to be an activator. On the other hand, the FRT gene of S. commune suppresses expression of dikaryon- specific genes in monokaryons (Horton et al, 1999). Other mutant strains affected in fruiting body development in S. commune have been described but the genes involved have not been identified. These mutants are affected in the morphology of the fruiting body or its sporulation (Raper and Krongelb, 1958; Bromberg and Schwalb, 1977). Recently, several genes involved in fruiting body formation have been identified in C. cinereus. The peel gene functions in A regulated development and encodes a putative DNA binding protein (Murata et al, 1998). A mutation in the gene resulted in a complete program of sexual differentiation independent of the mating type genes. The function of peel is, however, not known. The ichl and the eln2 genes are involved in differentiation of the primordium (Muraguchi et al, 1998; 2000). Mutations in these genes affect pileus and stipe formation, respectively. Clearly, these data do not present a picture how fruiting body formation is regulated at the molecular level.

So far, genes involved in regulation/initiation of mushroom production have not been described. These genes control mushroom formation and are therefore targets to improve mushroom production, to improve quality of mushrooms, to increase predictability of mushroom production and to enable production of mushrooms on substrates on which they can not yet be produced efficiently. In addition, once such a gene has been identified, there is a need for an expression system to regulate or control expression of the gene, especially in the mushroom forming fungi.

Summary of the invention

In an aspect, the present invention provides a method for the production of a mushroom, mycelium, fungal cell, a fruiting body, a spore or a gene product of interest comprising the steps of: a) culturing a fungal cell, fruiting body, spore, mycelium or mushroom comprising a nucleic acid sequence coding for a gene product of interest wherein said nucleic acid sequence is operably linked to a heat inducible promoter functional in said mushroom, mycelium, fungal cell, fruiting body and/or spore; b) treating the mushroom, mycelium, fungal cell, fruiting body, or spore with heat during part of the culturing under a); and c) optional recovery of the mushroom, mycelium, fungal cell, fruiting body, spore or gene product of interest.

In a preferred embodiment, the method according to the invention comprises the step of transforming a mushroom, mycelium or a fungal cell with an expression vector comprising a nucleic acid sequence coding for a gene product of interest operably linked to the heat inducible promoter.

Alternatively or in combination with other embodiments, the method of the invention further comprises heat treatment of the mushroom, mycelium, fungal cell, fruiting body or spore.

Alternatively or in combination with other embodiments, the heat treatment is selected from the group consisting of 2 minutes to 2 hour placement in an incubator of 37-95 °C, contacting a needle with a temperature of 37-95°C for 0.01 to 120 seconds with the culture medium of the mushroom, the mycelium, the fruiting body, the fungal cell or the spore or into the mushroom, the mycelium, the fruiting body or a colony of fungal cells or spores. Heat treatment can also be obtained by using light, preferably laser light, or another energy source. The light source may expose the whole culture to the light (e.g the whole mushroom bed), or may expose a limited area of 10 cm² or less, 1 cm² or less, or 1 mm² or less..

In a preferred method, the heat treatment is selected from the group consisting of: i) 2 minutes to 2 hour placement in an incubator of 37-95 °C;

ii) contacting a heat source, preferably a needle with a temperature of 37- 95°C for 0.01 to 120 seconds with the culture medium of the mushroom, the mycelium, the fruiting body, the fungal cell or the spore or into the mushroom, the mycelium, the fruiting body or a colony of fungal cells or spores;

iii) 0.01 sec to 16 hour exposure to light, preferably laser light and

iv) treatment i), ii) and/or iii) may be given once or at time intervals of 8 h, 16 h or 24 h.

Alternatively or in combination with other embodiments, the heat inducible promoter is preferably from a eukaryote or functional in a eukaryote, more preferably a fungus, even more preferably a Basidiomycete, even more preferably an Agaricales, even more preferably a Schizophyllaceae, even more preferably a Schizophyllum, most preferably Schizophyllum commune. Alternatively, said promoter is derived from a eukaryote, more preferably a fungus, even more preferably a Basidiomycete, even more preferably from an Agaricales, even more preferably a Schizophyllaceae, even more preferably a Schizophyllum, most preferably Schizophyllum commune. Said promoter may be derived from a native promoter by substituting, deleting and/or adding a nucleotide in order to improve the transcriptional activity of the promoter as later defined herein.

Alternatively or in combination with other embodiments, the heat inducible promoter is represented by:

- a nucleic acid sequence of 100 till 1000 bp present upstream of the start codon of a nucleic acid sequence encoding a heat shock protein having at least 60% identity or similarity with any of SEQ ID NO: 263-269;

- any of SEQ ID NO: 1-7 or has at least 60% identity with any of SEQ ID NO: 1-7; and/or - wherein the heat inducible promoter comprises the following nucleic acid sequence: GAAX1X2X3TCX4X5GX6X7 (SEQ ID NO:262), wherein X_u X₂ are A, G, C, T; X₃ is T or G; X₄ is C, T, or G, X₅ is A, G or T, X₆ is A or T and X₇ is A or C.

Alternatively or in combination with other embodiments, the gene of interest is a gene which upon expression of the gene product of interest induces mushroom formation. Alternatively or in combination with other embodiments, the gene of interest is a gene which upon expression of the gene inhibits mushroom formation.

Alternatively or in combination with other embodiments, the gene of interest is represented by a nucleotide sequence encoding a polypeptide that comprises an amino acid sequence: (a) that has at least 40 % amino acid identity or similarity with a sequence selected from SEQ ID NO: 8-207; and/or, (b) that has at least 50% amino acid identity or similarity with a sequence selected from SEQ ID NO: 208-215.

In a further aspect, the invention provides a heat inducible promoter having a length of 100 - 1000 base pairs that is represented by a nucleic acid sequence comprising any of SEQ ID NO: 1-7 or having at least 60% identity with any of SEQ ID NO: l-7.

In a further aspect, the invention provides a heat inducible promoter represented by a nucleic acid sequence of 100 till 1000 bp present upstream of the start codon of a nucleic acid sequence encoding a heat shock protein having at least 60% identity or similarity with any of SEQ ID NO: 263-269.

In a further aspect, the invention provides a heat inducible promoter having a length of 100 - 1000 base pairs, which is preferably a promoter from a basidiomycete or derived there from and/or functional therein, comprising the following nucleic acid sequence: GAAXiX₂X₃TCX₄X₅GX₆X₇ (SEQ ID NO:262), wherein X_u X₂ are A, G, C, T; X₃ is T or G; X₄ is C, T, or G, X₅ is A, G or T, X₆ is A or T and X₇ is A or C.

In a further aspect, the invention provides a nucleic acid construct comprising a promoter according to the invention. In a preferred embodiment, said nucleic acid construct further comprises a gene of interest operably linked to a heat inducible promoter.

In a further aspect, the invention provides an expression vector comprising a nucleic acid construct according to the invention. In a further aspect, the invention provides a fungal cell, a mycelium, a spore, a fruiting body or a mushroom, comprising a nucleic acid construct or an expression vector according to the invention.

Description of the invention

Inducible promoters can be used in biotechnological applications to produce proteins in a specific growth phase or in (part of) the vegetative or reproductive mycelium. Inducible promoters can also be used to study gene function at a particular condition, developmental stage or position in the colony. Many inducible promoters have been described. For instance, the promoter of hsp30 of Aspergillus oryzae, which encodes a heat-shock protein, was found to be highly induced simply by a short heat- treatment at 40 °C (Matsushita et al, 2009). Heat shock proteins are found throughout the domains of life. They function in protection of cells against stress such as that caused by high temperature. At low temperatures the expression of the heat shock genes can be low, but upon heat stress these genes are rapidly activated by the transcription factor HSF (heat shock factor) and expression increases several orders of magnitude (Santoro, 2000). Advantages of heat shock protein promoters over other inducible promoters, such as the copper-inducible promoter of Histoplasma capsulatum, the pectin-inducible promoter of Penicillium griseoroseum, the thiamine- regulatable thiA promoter (pthiA) of Aspergillus oryzae, the benzoic acid inducible promoter of Aspergillus niger, and the inducible AlcA promoter of Aspergillus nidulans are that no additives to the medium are needed to induce the promoter. Thus, the composition of the medium is not changed upon induction of the promoter.

So far, an inducible promoter system has neither been described for S. commune nor for any other mushroom forming fungus. Here, we show that S. commune contains 7 genes of the small heat shock protein family hsp26/hsp42 that are homologous to hsp30 of A. oryzae. Three of these genes, hspl-3, were found not to be expressed at 25 °C. However, expression was detected at 42 °C. The promoters of the 7 genes of the heat shock protein family hsp26/hsp42 can be used as an inducible system in S. commune. Preferably, the promoters of hspl-3 are used, as is further explained hereafter. The promoters of hspl-7 correspond to SEQ ID NO: 1-7 herein. Promoter

In an aspect, there is provided a promoter, preferably a heat inducible promoter having a length of 100 - 1000 base pairs (bp). The term "base pairs" is used herein as to depict a double stranded polynucleotide, such as DNA, in which form a promoter, gene and/or nucleic acid molecule according to the invention are generally present in genomic DNA in their indigenous situation. The person skilled in the art will comprehend that a polynucleotide, such as a promoter, gene and/or nucleic acid molecule can also be depicted by one of the two polynucleotide strands and can furthermore also be present in the form of a single stranded polynucleotide. All these forms are also within the scope of the present invention. More preferably, the promoter according to the invention is from a eukaryote and/or is functional in a eukaryote, more preferably from a fungus, more preferably from a Basidiomycete, more preferably from an Agaricales, more preferably from a Schizophyllaceae or a Agaricaceae, more preferably from a Schizophyllum or an Agaricus and most preferably from Schizophyllum commune or Agaricus bisporus. Preferably, the promoter according to the invention, or a nucleic acid molecule comprising said promoter is isolated from its indigenous situation.

In a preferred embodiment, the promoter is represented by a nucleic acid sequence comprising 100-1000 bp, or 200-800 bp, or 300-500 bp and which is found upstream of the start codon of a heat shock protein. Preferably such promoter sequence ends at the last nucleotide before the start codon. The promoter is thus in its indigenous situation preferably located immediately upstream of the start codon of a heat shock protein. Heat shock proteins are a class of functionally related proteins whose expression is increased when cells are exposed to elevated temperatures or other stress. This increase in expression is transcriptionally regulated. An up-regulation of the heat shock proteins is a key part of the heat shock response. Preferably, the heat shock protein is a heat shock protein from a eukaryote and/or functional in a eukaryote, more preferably from a fungus, more preferably from a Basidiomycete, more preferably from an Agaricales, more preferably from a Schizophyllaceae or a Agaricaceae, more preferably from a Schizophyllum or an Agaricus and most preferably from Schizophyllum commune or Agaricus bisporus. Preferably, the heat shock protein has at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% identity or similarity with any of SEQ ID NO: 263-269. In another preferred embodiment, the promoter is represented by a nucleic acid sequence comprising any of SEQ ID NO: 1-7 or having at least 60% identity with any of SEQ ID NO: l-7.

In another preferred embodiment, the promoter is represented by the following nucleic acid sequence comprising: GAAX1X2X3TCX4X5GX6X7 (SEQ ID NO:262), wherein X_{l s} X₂ are A, G, C, T; X₃ is T or G; X₄ is C, T, or G, X₅ is A, G or T, X₆ is A or T and X₇ is A or C. Most preferably, the promoter is represented by a nucleic acid sequence comprising any of SEQ ID NO: l - 3 or having at least 60% identity with any of SEQ ID NO: 1 - 3, since there is no expression of these promoters at 25°C, whereas expression is induced at a temperature of 37°C or higher. Therefore there is no leak of expression at the preferred maintenance temperature of many mushroom forming fungi or mushrooms as further described later herein.

As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more genes or nucleic acids, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is related to the binding site identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one skilled in the art to act directly or indirectly to regulate the amount of transcription from the promoter. Within the context of the invention, a promoter preferably ends at nucleotide -1 of the transcription start site (TSS).

A promoter of the invention is preferably said to be non-constitutive and/or preferably inducible. More preferably, said promoter is inducible by a change in temperature and/or is called a heat shock promoter and/or a heat inducible promoter; said are used interchangeably herein. It may mean that such promoter is active above a given temperature and inactive below a given temperature. One speaks also of the activity or transcriptional activity of said promoter. Within this context, "active" means that said promoter is able to induce the expression of a nucleotide sequence operably linked thereto in a given expression assay. Within this context, "inactive" means that said promoter is not able to induce the expression of a nucleotide sequence operably linked thereto in the same expression assay. Preferably, a promoter according to the invention exhibits in its inactive situation (i.e. not induced) less than 80%, more preferably less than 70%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.01% of the transcriptional activity the promoter exhibits in its active form as determined under identical conditions (except for induction), preferably determined using the assay described in example 2 herein. Most preferably, the promoter according to the invention exhibits no transcriptional activity in its inactive situation. Example 2 illustrates a way of testing the functionality (active/inactive) of the promoter. This assay is preferred to determine transcriptional activity of a promoter according to the invention. In a preferred embodiment, said promoter is inactive at a temperature below 36°C, 35°C, 34°C, 33°C, 32°C, 31°C, 30°C, 29°C, 28°C, 27°C, 26°C, 25°C, 24°C, 23°C, 22°C, 21°C, 20°C, 19°C, 18°C, 17°C, 16°C or 15°C or a temperature of 15 °C. In a preferred embodiment, said promoter is active at a temperature of at least 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, or a temperature of 42 °C. In a more preferred embodiment, said promoter is inactive at a temperature of 25°C and is active at a temperature of 42°C. Such a promoter is able to induce a heat shock. Temperature can be determined using conventional means in the art, such as for example by using a conventional thermometer or a thermograph. Temperature can be determined in the medium or soil, in the air surrounding the fungi/mushroom/fruiting body/spore/mycelium, or within a fungus colony/mushroom/fruiting body/mycelium.

In a preferred embodiment, the heat induciblity of a promoter is tested using Northern blotting, qPCR, or a reporter system. For instance, a promoter to be tested is cloned in front of the coding sequence of red fluorescent protein dTomato and is then introduced in the monokaryotic strain 4-8 of S. Commune (FGSC #9210). Transformed S. commune is grown on solid minimal medium (Dons et al, 1979) for 5 days at 25°C. Then the 5 day old colonies are incubated for 1 hour at either 25°C, 37°C or 42°C, after which they are placed back at 25°C. If more fluorescence is observed after incubation at 42°C (and preferably also after incubation at 37°C) as compared to the colonies that were incubated at 25°C, the promoter is said to be a heat inducible promoter. Preferably, more fluorescence means at least 2 times more, at least 4 times more, at least lOx, at least lOOx, at least lOOOx or at least lOOOOx more in the clones that were incubated at 42°C (and preferably also in the clones that were incubated at 37°C) as compared to the clones that were incubated at 25°C. Preferably, more fluorescence means an infinite induction (if there is no fluorescence at all at 25°C). More preferably, fluorescence intensity increases with increasing incubation temperature. Fluorescence can be measured using conventional means in the art, such as fluorescence microscopy using e.g. dsRED3 filters.

In a preferred embodiment, a promoter according to the invention has a length of

100 - 1000 base pairs or comprises 100-1000 base pairs. Said promoter may also have a length of 100-750, 100-500, 100-400, 100-250 base pairs. The length of the promoter is not critical as long as it exhibits a transcriptional activity as defined above, preferably an inducible transcriptional activity, more preferably a transcriptional activity which is inducible by a change in temperature as defined above.

The origin of the promoter and the way the promoter has been identified are also not critical as long as it is functional in a desired fungal cell, mycelium, fruiting body, spore and/or a mushroom as identified herein. Functional means that this promoter may exhibit a transcriptional activity as defined herein, preferably an inducible transcriptional activity, more preferably a transcriptional activity which is inducible by a change of temperature or induce a heat shock as defined above. In a preferred embodiment, the promoter is represented by any of SEQ ID NO: 1-7; i.e. SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO; 5, SEQ ID NO; 6 and/or SEQ ID NO: 7 or has at least 60% identity with at least one of SEQ ID NO: 1-7, i.e. SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO; 5, SEQ ID NO; 6 and/or SEQ ID NO: 7. Said promoter also preferably exhibits a transcriptional activity as defined above, preferably an inducible transcriptional activity, more preferably a transcriptional activity which is inducible by a change in temperature as defined above. More preferably, the promoter is represented by any of SEQ ID NO: l - 3, i.e. SEQ ID NO: 1, SEQ ID NO: 2 and/or SEQ ID NO: 3or has at least 60% identity with at least one of SEQ ID NO: 1 - 3, for the same reason as explained earlier herein.

Each promoter sequence described herein by virtue of its identity percentage (at least 60%)) with a given nucleotide sequence (i.e. any of SEQ ID NO: 1-7) respectively has in a further preferred embodiment an identity of at least 70%>, 75%>, 80%>, 82%>, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the given nucleotide sequence. In a preferred embodiment, sequence identity is determined by comparing the whole length of the sequences as identified herein. A promoter according to the invention may be a mutant, truncated or hybrid promoter derived from a promoter according to the invention.

Nucleic acid construct

In a further aspect there is provided a nucleic acid construct comprising a promoter as defined in the previous section entitled "promoter". In a preferred embodiment a nucleic acid construct of the invention further comprises a gene of interest. More preferably, a gene of interest is operably linked to a promoter of the invention.

A nucleic acid construct is defined as a nucleic acid molecule, which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acids which are combined or juxtaposed in a manner which would not otherwise exist in nature. A nucleic acid molecule is represented by a nucleotide sequence. Optionally, a nucleotide sequence present in a nucleic acid construct is operably linked to one or more control sequences, which direct the production of said polypeptide in a fungus or in a mushroom or in any suitable system, in vitro or in vivo, to produce the encoded gene product.

Optionally, a promoter represented by a nucleotide sequence present in a nucleic acid construct is operably linked to another nucleotide sequence encoding a gene of interest. A gene of interest codes for a polypeptide or for another gene product of interest (as defined elsewhere herein). The promoter according to the invention may be natively associated with the gene of interest or may be foreign to the gene of interest, i.e. in its indigenous from the promoter is not natively associated with the gene of interest.

"Polypeptide" as used herein refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. A polypeptide is comprised of consecutive amino acids. The term "polypeptide" encompasses naturally occurring or synthetic molecules.

"Operably linked" is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to the nucleotide sequence coding for the polypeptide of the invention such that the control sequence directs the production of the polypeptide of the invention in a fungal cell and/or in a mushroom. Expression will be understood to include any step involved in the production of the polypeptide including, but not limited to transcription, post-transcriptional modification, translation, post-translational modification and secretion.

Control sequence is defined herein to include all components, which are necessary or advantageous for the expression of a polypeptide. At a minimum, the control sequences include a promoter and transcriptional and trans lational stop signals.

The invention also relates to an expression vector comprising a nucleic acid construct of the invention. Preferably, an expression vector comprises a nucleotide sequence of the invention, which is operably linked to one or more control sequences, which direct the production of the encoded polypeptide in a fungal cell and/or in a mushroom. An expression vector may be seen as a recombinant expression vector. An expression vector may be any vector which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of a nucleotide sequence encoding a polypeptide of the invention in a fungus and/or in a mushroom. Depending on the identity of the fungus/mushroom wherein this expression vector will be introduced and on the origin of the nucleotide sequence of the invention, the skilled person will know how to choose the most suited expression vector and control sequences.

Single or multiple copies of a nucleic acid construct according to the invention may be introduced into a fungal cell, a mycelium and/or a mushroom. A nucleic acid construct may be maintained episomally and thus comprises a sequence for autonomous replication, such as an ARS sequence. Suitable episomal nucleic acid constructs may e.g. be based on the yeast 2μ or pKDl (Fleer et al, 1991) plasmids. Alternatively, a nucleic acid construct is integrated in one or more copies into the genome of a fungal cell, a mycelium and/or a mushroom. Integration into a fungal cell, a mycelium and/or a mushroom genome may occur at random by illegitimate recombination or via homologous recombination at a targeted integration site. Preferably, a nucleic acid construct integrates into the genome of a fungus, a mycelium and/or a mushroom. This type of nucleic acid construct may comprise a bacterial cloning vehicle, a nucleotide sequence encoding a gene product of interest and a selection marker. A selection marker may confer antibiotic resistance or be an auxotrophic marker. Such markers are known to the skilled person. Nucleic acid constructs comprising a bacterial cloning vehicle and a selection marker are for example disclosed in Schuren et al (1994) and Munoz-Rivas et al (1986). Alternatively, this type of nucleic acid construct may be synthesised using techniques such as for example PCR.

If the expression level of a polypeptide of the invention is to be increased, a nucleotide sequence encoding said polypeptide is introduced into an expression construct. If the expression level of a polypeptide of the invention is to be decreased, a nucleotide sequence encoding said polypeptide may be introduced into an inactivation construct. An inactivation construct is known to the skilled person. Such construct may comprise a nucleotide sequence encoding a mutated polypeptide or containing the flanking sequences of a nucleotide sequence encoding said polypeptide. Such a construct should integrate at the endogenous locus of said polypeptide to replace the endogenous gene and inactivate it. Alternatively, the inactivation construct may contain a sequence inducing RNAi. RNAi techniques are known to the skilled person (De Jong et al, 2006).

Inactivation of the polypeptide may be due to the inactivation of the corresponding gene or nucleotide sequence. In the case of an RNAi like inactivation, mRNA levels are reduced. The inactivation construct may also result in mRNA levels similar to that observed in the wild-type. In this case, the encoded mutated polypeptide has a decreased activity, wherein said decreased activity is assessed by comparison with the activity of the polypeptide the mutated polypeptide originates or derives from. An activity of a polypeptide may be assessed using an assay known to the skilled person. Such assay may include the introduction of said mutated polypeptide into a fungus and compare an activity of said expressed mutated polypeptide with corresponding activity of the polypeptide the mutated polypeptide originates from. An activity of a mutated polypeptide may be compared with the activity of a control polypeptide. If a fungus is a Schizophyllum, a control activity of a control polypeptide may be an activity as present in the strain Schizophyllum commune strain 4-8 (FGSC #9210). Preferably a mutated polypeptide has no detectable activity. An activity has the same meaning as a transcriptional activity or being functional as earlier defined herein.

Suitable procedures for transformation of a fungus are well known to the skilled person. Such procedures may involve a process comprising protoplast formation, transformation of the protoplast, and regeneration of the cell wall in a manner known to the skilled person. Suitable transformation procedures for Aspergillus are described in Yelton et al (1984), whereas transformation of a mushroom forming fungus could be done using protoplasts using for instance procedures according to van Peer et al (2009).

The invention is not limited to a specific gene product of interest, such as for example a polypeptide. In this section entitled nucleic acid construct, several polypeptides are identified. Depending on the method of the invention the identity of the polypeptide may be distinct. In a preferred embodiment, if a fungus is a fungus which is able to produce a mushroom, a polypeptide is encoded by a gene of interest as defined below.

Preferred are gene products involved in timing of mushroom formation and/or involved in morphology and/or yield (i.e., for mushrooms that are currently produced commercially, including but not limited to the common white button mushroom [the "champignon"], the oyster mushroom and shiitake) or to enable (i.e. for mushrooms that are currently not produced in a commercial setting) commercial mushroom formation. We have identified 200 genes in the genome of S. commune that encode putative regulators whose expression change during mushroom formation. These genes could be involved in mushroom formation. We have found that inactivation of eight of these genes affected mushroom production. The production was either promoted or decreased, if present at all. Homologues of these putative regulatory genes can be found in other mushroom forming fungi. These genes which encode putative transcriptional regulators and whose expression change during mushroom formation may be used in combination with a promoter of the present invention in order to 1) enable commercial production of mushrooms that can not yet be produced in a commercial setting, 2) improve yield of mushrooms, 3) improve quality (e.g. shape and homogeneity of morphology), 4) improve predictability of the process of mushroom formation and/or 5) enable production of mushrooms on substrates that can not yet be used for commercial mushroom production. Thus, alternatively or in combination with another preferred embodiment, in a preferred embodiment of the invention, there is provided a nucleic acid construct comprising a nucleotide sequence encoding a polypeptide that comprises an amino acid sequence: (a) that has at least 40 % amino acid identity or similarity with an amino acid sequence selected from SEQ ID NO: 8-207 (Tables 1 and 8); and/or,

(b) that has at least 50 % amino acid identity or similarity with an amino acid sequence selected from SEQ ID NO:208-215 (Tables 7 and 8);

wherein the nucleotide sequence is operably linked to a promoter of the invention.

In a preferred embodiment, there is provided a nucleic acid construct comprising a nucleotide sequence encoding a polypeptide that comprises an amino acid sequence that has at least 40% amino acid identity or similarity with a sequence selected from the group consisting of SEQ ID NO: 36; 184; 27; 63; 11; and/or that has at least 50% amino acid identity or similarity with a sequence selected from the group consisting of SEQ ID NO: 211; 214; 215; 209; 208. In another preferred embodiment, there is provided a nucleic acid construct comprising a nucleotide sequence encoding a polypeptide that comprises an amino acid sequence that has at least 40% amino acid identity or similarity with a sequence selected from the group consisting of SEQ ID NO: 62; 28; 61; and/or that has at least 50% amino acid identity or similarity with a sequence selected from the group consisting of SEQ ID NO: 213; 212; 210.

Preferably, the promoter of the invention is capable of driving expression of the nucleotide sequence in a fungus and/or in a mushroom or in another system suitable to produce the product encoded by the nucleotide sequence..

Table 8 links the name of each polypeptide to a given SEQ ID NO corresponding to an amino acid sequence. Each of these polypeptides is suspected to be involved in the regulation of the production of a mushroom. These polypeptides are further identified in Table 1, Table 7 and Table 8 and are also named Transcription Factor (TF). These polypeptides are available in a public database (htt ://j gi.doe.gov/Scommune). Therefore using a promoter of the invention operably linked to a nucleotide sequence encoding a TF involved in mushroom formation allows to develop an inducible method for producing a mushroom.

Each amino acid sequence described herein by virtue of its amino acid identity or similarity percentage (at least 40% identity or similarity for SEQ ID NO's 8 - 207; at least 50% identity or similarity for SEQ ID NO's 208 - 215) with a amino acid sequence respectively has in a further preferred embodiment an identity of at least 42%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity or similarity with the given polypeptide. In a preferred embodiment, sequence identity or similarity is determined by comparing the whole length of the sequences as identified herein. More preferably, an amino acid sequence described herein by virtue of its amino acid identity or similarity percentage (at least 40% identity or similarity for SEQ ID NO's 8 - 207; at least 50% identity or similarity for SEQ ID NO's 208 - 215, respectively) has an identity of at least 42%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID NO: 8-207 or has an identity of at least 52%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID NO: 208-215, respectively.

Each nucleotide sequence encoding a polypeptide as described herein may encode a fungal polypeptide, i.e. a polypeptide with an amino acid sequence that is identical to that of a polypeptide that naturally occurs in a fungal or a mushroom organism. The functionality of such polypeptide depends on the relatedness (identity or similarity percentage) of the amino acid sequence compared to that of the corresponding identified SEQ ID NO.

A transcription factor or TF is preferably said to be functional when said TF has a detectable transcriptional activity during at least part of the life cycle of a mushroom. Preferably a mushroom is a Schizophyllum. More preferably strain 4-8 (FGSC#9210) of Schizophyllum commune (Fungal Genetic Stock Center, Missouri, USA). The presence of an activity, preferably a transcriptional activity, is preferably assessed by inactivating a nucleotide sequence encoding said TF in said fungus or mushroom and analysing whether a mushroom will be produced compared to the mushroom production of a control mushroom wherein said nucleotide sequence has not been inactivated. If a mushroom is not produced or if less or more mushroom is produced, said TF is said to exhibit an activity, preferably a transcriptional activity, and therefore to be functional. Less or more mushroom are later defined herein.

Alternatively or in combination with a previous embodiment, a polypeptide of the invention may be a natural polypeptide or it may be a polypeptide that does not occur naturally. A polypeptide that does not occur naturally may be a polypeptide encoded by a nucleic acid sequence that is mutated for example by using site directed mutagenesis or a mutation prone PCR.

In order to select a TF with a desired function, a skilled person may select a TF that has a detectable transcriptional activity during at least part of the life cycle of a mushroom. Preferably a mushroom is a Schizophyllum. More preferably strain 4-8 (FGSC#9210) of Schizophyllum commune. The presence of an activity, preferably a transcriptional activity is preferably assessed by inactivating a nucleotide sequence encoding said TF in said fungus or mushroom and analysing whether a mushroom will be produced compared to the mushroom production of a control mushroom wherein said nucleotide sequence has not been inactivated.

This inactivation or down regulation may have been achieved by deletion of one or more nucleotides in the encoding gene. Alternatively, it may have been caused by an R Ai-like mechanism. It is also possible to introduce a mutation in the gene or nucleotide encoding said polypeptide. This may be done by introducing a replacement or inactivation vector into a fungus/mushroom by transformation. The skilled person knows how to construct such a vector. For example such vector may comprise flanking regions of a nucleotide sequence coding for a polypeptide with a selection marker gene present in between said flanking regions.

If a mushroom is not produced or if less mushroom is produced, said TF is said to exhibit an activity, preferably a transcriptional activity and therefore said TF can be used as a gene product of interest in the invention. The meaning of less mushroom is preferably at least 3%, 6%, 10% or 15% less of the mushroom than the parental mushroom the transformed mushroom derives from will produce when both types of cells (parental and transformed cells) are cultured under the same conditions. Also at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or 150% less of said mushroom than the parental mushrooms are preferred. A preferred parental mushroom is strain 4-8 (FGSC#9210) of Schizophyllum commune. Thus, a TF, the absence of which results in less or no mushroom being produced, can be used in a method to produce mushrooms. The advantage is that more mushroom can be produced by regulating the production of the TF using the promoter of the invention, thereby increasing the yield.

If more mushroom is produced, said TF is said to exhibit an activity, preferably a transcriptional activity and therefore said TF can be used as a gene product of interest in the invention. The meaning of more mushroom is preferably at least 1%, 3%, 6%, 10% or 15% more of the mushroom than the parental mushroom the transformed mushroom derives from will produce when both types of cells (parental and transformed cells) are cultured under the same conditions. Also at least 20%>, 30%>, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 500%, 1000%, 10.000% or more of said mushroom than the parental mushrooms are preferred. In a preferred embodiment, strain 4-8 (FGSC#9210) of Schizophyllum commune is a parental mushroom. It is also encompassed by the present invention that the increased mushroom production is detectable by eye. Thus, a TF, the absence of which results in more mushroom being produced, can be used in a method to produce mushrooms. The advantage is that less mushroom can be produced by regulating the production of the TF using the promoter of the invention, thereby facilitating easy harvesting of the mushrooms and /or better conditions for the mushrooms to grow (e.g. more space and more nutrients per mushroom). For example, when it is desired that a mushroom develops that is not hindered by other, perhaps smaller, mushrooms in their vicinity that do not fully develop. For example, the inventors found that inactivation of SEQ ID NO: 28, 61, 62 led to more mushroom production. Therefore, in order to produce more mushrooms, it is preferred to reduce the expression of a polypeptide comprising an amino acid sequence with identity or similarity to SEQ ID NO: 28, 61, or 62 as further defined elsewhere herein. However, should fewer but larger mushrooms be desired, it is preferred to over-express a polypeptide comprising an amino acid sequence with identity or similarity to SEQ ID NO: 28, 61, or 62.

Detection of mushroom is preferably done visually, such as for example by determining the number of mushrooms in a petri dish, a container with substrate or on a mushroom bed.

Fungal cell/mycelium/mushroom/fruiting body/spore

In an aspect, there is provided a fungus, preferably a fungus which is able to form a mushroom, a mycelium, a fruiting body, a spore, or a mushroom comprising a nucleic acid construct or an expression vector as defined earlier herein. Said fungus or mushroom comprising a nucleic acid construct or an expression vector as defined earlier herein, are herein referred to as fungus according to the invention and mushroom according to the invention.

A fungus according to the invention may be any fungus. Said fungus is preferably a fungus which is able to form or produce a mycelium/mushroom, more preferably, a filamentous fungus, a Basidiomycete or an Ascomycete as described elsewhere herein. The choice of the fungus, mycelium and/or mushroom will to a large extent depend upon the source of the nucleic acid sequence of the invention. Depending on the identity of the fungus, the skilled person would know how to transform it with the construct or vector of the invention.

A mycelium is the vegetative part of a fungus, consisting of a mass of branching, thread-like hyphae. Fungal colonies composed of mycelia are found in soil and on or within many other substrates. A mycelium may be minute and forming a colony that is too small to see by eye, or it may be extensive. The mycelium is involved in absorption of nutrients from its environment by first secreting enzymes which break down biological polymers into smaller units (e.g. monomers) and subsequently absorbing the smaller units by facilitated diffusion and active transport.

A mushroom may be defined as a fleshy, spore-bearing fruiting body of a fungus, typically produced above ground on soil or on its food source. Preferably, a mushroom is defined as a fleshy, spore-bearing fruiting body of a fungus, typically produced above ground on soil or on its food source. The standard for the name "mushroom" is the cultivated white button mushroom, Agaricus bisporus, hence the word mushroom is most often applied to those fungi (Basidiomycota, Agaricomycetes) that have a stem (stipe), a cap (pileus), and gills (lamellae, sing, lamella) on the underside of the cap, just as do store-bought white mushrooms. Mushrooms may also have pores in stead of lamellae. The word "mushroom" can also be used for a wide variety fungal fruiting bodies that produce sexual spores and that either or not have stems, and the term is used even more generally, to describe both the fleshy fruiting bodies of some Ascomycota and the woody or leathery fruiting bodies of some Basidiomycota. Forms deviating from the standard morphology usually have more specific names, such as "puffball", "stinkhorn", and "morel", and gilled mushrooms themselves are often called "agarics" in reference to their similarity to Agaricus or their place Agaricales. By extension, the term "mushroom" can also designate the entire fungus when in culture or the thallus (called a mycelium) of species forming the fruiting bodies called mushrooms, or the species itself.

A fruiting body (also known as sporocarp or fruit body) in fungi is a multicellular structure on which spore-producing structures, such as basidia in basidiomycetes, are born. The fruiting body is part of the sexual phase of a fungal life cycle, with the rest of the life cycle being characterized by vegetative mycelial growth and asexual spore production. A fruiting body of a basidiomycete is also known as a basidiocarp. A significant range of different shapes and morphologies is found in basidiocarps, which features play an important role in the identification and taxonomy of fungi.

Fruiting bodies may be either epigeous (if they grow on the ground as with ordinary mushrooms) or hypogeous (if they grow underground). Epigeous fruiting bodies that are visible to the naked eye, especially fruiting bodies of a more or less agaricoid morphology (i.e., a fungal fruiting body characterized by the presense of a pileus [cap] that is clearly differentiated from the stipe [stalk], with lamellae [gills] on the underside of the pileus; often also refers to a basidiomycete species characterized by an agaric-type fruiting body), are often referred to as mushrooms, while hypogeous fungi are usually called truffles or false truffles. During their evolution truffles lost the ability to disperse their spores via air currents, instead opting for animal consumption and subsequent dispersal of their spores.

A spore is a reproductive structure that is formed during a a-sexual or sexual process and that are adapted for dispersal and surviving for extended periods of time in unfavourable conditions. Spores form part of the life cycles of many bacteria, plants, algae, fungi and some protozoans. Spores may be dispersed by forcible ejection from their reproductive structures. This ejection ensures exit of the spores from the reproductive structures as well as travelling through the air over long distances. Many fungi thereby possess specialized mechanical and physiological mechanisms as well as spore-surface structures, such as hydrophobins. In the present application, "spore" or "spores" is used to mean a spore of a fungus, preferably of a fungus that is able to form a mushroom, more preferably of a basidiomycete (i.e. a basidiospore).

In an embodiment, a fungus/mycelium/fruiting body/spore/mushroom according to the invention is preferably not (of) a yeast.

In an embodiment, a fungus/mycelium/fruiting body/spore/mushroom according to the invention is (of) a basidiomycete or an bscomycete, preferably a basidiomycete, preferably an Agaricales, more preferably a Schizophyllaceae and even more preferably a Schizophyllum. More preferred is a Schizophyllum commune. Even more preferably Schizophyllum commune strain 4-8 (FGSC#9210). Preferred Agaricales are for example Agaricus bisporus, Pleurotus ostreatus and Lentius edodus. Other preferred fungal organisms are fungi which are not able to produce a mushroom. Such fungi include filamentous fungi such as those belonging to Aspergillus, Trichoderma, Penicillium, Chrysosporium, Fusarium. Preferred species include: Aspergillus fumigatus,

Aspergillus flavus, Aspergillus parasiticus, Aspergillus nidulans, Aspergillus oryzae, Penicilium chrysogenum, Neurospora crassa, Trichoderma reesei, Trichoderma viridie, Chrysosporium lucknowense, Gibber ella zeae (anamorph Fusarium

graminarium), Cryptococcus neoformans, Coccidioides immitis, Magneporthe grisae, Ustilago maydis. Such fungus/my celium/fruiting body/spore/mushroom according to the invention is attractive to be used in many different applications as illustrated in next section.

Preferably, if a fungus according to the invention is a fungus which is able to form or produce a mycelium/mushroom, said fungus comprises a nucleic acid construct comprising a promoter of the invention as earlier defined herein, wherein said promoter is operably linked with a nucleotide sequence that encodes for a polypeptide that is represented by an amino acid sequence having or comprising at least 40% amino acid identity or similarity with SEQ ID NO: 8-207 as identified earlier herein or for a polypeptide that is represented by an amino acid sequence having or comprising at least 50% amino acid identity or similarity with SEQ ID NO: 208-215 as identified earlier herein in the section entitled nucleic acid construct. More preferably, a mushroom that is attractive to be produced or which is suspected to be attractive to produce a gene product of interest. It may be due to commercial reasons. Such fungus/my celium/fruiting body/spore/mushroom is attractive to be used in many different applications. These procedures would improve/enable mushroom production. Improvement or enabling commercial mushroom production may result from

-growth on substrates that can not yet be used to grow mushrooms commercially -controlled formation of mushrooms in time and space

-increased production levels

-increased quality (e.g. more homogeneity in size and outgrowth)

In another embodiment, our invention may enable to improve growth of mushrooms in a commercial setting, or to allow commercial production of species that can not yet be produced, thereby creating opportunities to produce edible fungi in a cheaper way or to produce (or improve production of) pharmaceuticals or proteins that are of interest for agriculture, food, feed or non-food or non-feed applications. These proteins may either or not originate from the mushroom forming fungus. Some possible applications of a fungus of the invention are below presented. A gene product of interest, e.g. preferably a polypeptide, that is expressed in a fungus, mycelium and/or a mushroom, as is described above, may be a heterologous polypeptide or an endogenous polypeptide, as is further defined below. Preferably, a polypeptide that is expressed in a fungus, mycelium and/or a mushroom is a heterologous polypeptide, for example when a polypeptide of the invention is used to improve the production of an edible mushroom. Where herein it is said that a fungus, mycelium and/or a mushroom has an "increased expression level of a polypeptide", this includes expression of a heterologous polypeptide, although as a definition the heterologous polypeptide is not naturally expressed by a fungus, mycelium and/or a mushroom. However, the "increased" expression level in this situation is construed to mean that there is a detectable expression of the heterologous polypeptide whereas a control fungus and/or mushroom does not have detectable expression of the heterologous polypeptide.

The increased expression of a gene product of interest occurs during and/or after heat treatment of the fungus/my celium/fruiting body/spore/mushroom. "Heat treatment of a fungus/mycelium/fruiting body/spore/mushroom" as used herein is defined as subjecting the fungus/my celium/fruiting body/spore/mushroom or a culture comprising said fungus/mycelium/fruiting body/spore/mushroom to a higher temperature than the temperature wherein the mushroom or fungus is normally maintained. "Heat treatment" may also mean subjecting a fungus/mycelium/fruiting body/spore/mushroom or a culture comprising said fungus/mycelium/fruiting body/spore/mushroom to a heat source. Heat treatment may be either systemically, such as for example by placing the fungal cells/mushroom/mycelium/spore/fruiting body in an incubator or by using (laser) light, or it may be locally, such as for example by inserting a needle/rod/pole/stick/stave of the desired temperature into the mycelium/mushroom or by locally exposure to (laser) light. The period of heat treatment depends on the temperature that is used and on the method that is used and can be determined by the skilled person in the art. The higher the temperature, the shorter the time needed to obtain a detectable induction of the heat shock promoter. Also, the higher the temperature, the higher the induction of the promoter as compared to the same period of treatment at a lower temperature. A needle may be seen as a heat source. For example, a needle, or a hot needle (or rod/pole/stick/stave) (e.g. held several minutes in boiling water or heated using heating elements) is used to locally treat a mycelium or a mushroom, preferably a mycelium. Such needle may contact or may be inserted into the mycelium or mushroom for at least 0.01, 0.5, 1, 5, 10, 11 , 12, 13, 14, 15, 16, 17 seconds and up to 10 minutes. In this embodiment, a mycelium or a mushroom, preferably a mycelium is preferably contacted with a needle with a temperature of 37-95°C for 0.01 to 120 seconds. Said needle may also be called a hot needle A needle may comprise or consist of a metal, plastic, ceramic.

A needle that has been held in boiling water for several minutes is thought to rapidly cool when exposed to the humid environment of the mushroom/mycelium/culture medium. It is probable that the needle will be fully back to the temperature of the environment after about a minute. However, if the needle is continued to be heated while being inserted, the maximum time may be reduced depending on the temperature used in order to prevent cells from dying. For example, Aspergillus niger cells die if they are placed in water of 65°C for 10 minutes. Further, and without wishing to be bound to any theory, it is thought that even a few seconds of insertion of said needle are sufficient to provide heat induction of the promoter. Therefore, in a preferred embodiment, the needle is inserted into or contacted with the mycelium or mushroom for less than 120, preferably 100, more preferably 80, 60, 40, 35, 30, 25 or 20, 17, 16, 15, 14, 13, 12, 11, 10, 5, 1, 0.5, 0.1, 0.01 seconds. In another example, an array of needles is used to give a heat pulse in the culture medium in order to start the induction.

Alternatively, a light source may act as a heat source. The light source may expose a limited area of the whole culture, i.e. 10 cm² or less, 1 cm² or less , or 1 mm² or less or large areas to the light (i.e. more than 10 cm², more than 1 m², more than 10 m², more than 100 m²). The light of the light source may be exposed to the fungal cells, the mycelium, the fruiting body, the spore or the mushrooms for at least 0.01 , 0.5, 1, 5, 10, 1 1, 12, 13, 14, 15, 16, 17 seconds, up to 10 minutes, up to 1 h, 2h, 5h, and up to 16 h.

Another example is when the fungal cells, the mycelium, the fruiting body, the spore or the mushrooms or culture comprising said fungal cells, the mycelium, the fruiting body, the spore or the mushrooms are placed in an incubator, which may be done for at least 10, 20, 30, 40, 50, 60, 90, 120, 150 or 180 minutes, but preferably not longer than 16 hours, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 hours. Preferably heat treatment is done for a period of 2 minutes to 4 hours, more preferably of 2 minutes to 2 hours, more preferably 5 minutes to 1.5 hours, most preferably 1 hour or 40, 50, 60 minutes. The heat treatment is preferably done at a temperature of more than 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45°C. Heat treatment is preferably done at a temperature of less than 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 49, 48, 47, 46°C. More preferably, heat treatment is done at a temperature of 37-95°C, more preferably 40-90°C, more preferably 42-70°C, more preferably 42-60°C, more preferably 42- 55°C, more preferably 42-50°C.

In yet another example the fungal cells, the mycelium, the fruiting body, the spores or the mushroom are exposed to light or another energy source as a heat source, preferably laser light. Preferably, the light is of the visible spectrum (about 390-750 nm), of the infrared spectrum (about 0.7-300μιη). Exposure time to light depends on the intensity of the light source and treatment could therefore take 0.01, 0.1, 0.5, 1, 5, 10, 11 , 12, 13, 14, 15, 16, 17 seconds and up to 10 minutes. Alternatively, the treatment could take 10, 20, 30, 40, 50, 60, 90, 120, 150 or 180 minutes, but preferably not longer than 16 hours, 15, 14, 13, 12, 1 1, 10, 9, 8, 7, 6, 5 hours. Preferably the fungal cells, the mycelium, the fruiting body, the spores or the mushroom are exposed to light for less than a minute, more preferable for less than 10 seconds, most preferably for less than a second.

The type of heat treatment used, the way it is applied and its duration are not critical as long as said treatment can induce a heat shock in a fungal cell, mycelium, fruiting body, spore and/or mycelium of the invention. A heat shock is preferably defined as being a transcriptional activation of the promoter of the invention as earlier defined herein.

A treatment with a needle or a light source with a narrow beam is preferred since by applying this treatment, one can specifically define where a mushroom will appear or will be formed and will grow. Accordingly, a method of the invention is wherein the heat treatment is selected from the group consisting of:

i) 2 minutes to 2 hour placement in an incubator of 37-95 °C;

iii) 0.01 sec to 16 hour exposure to light, preferably laser light and

Increased expression of a gene product of interest is detectable, e.g. using RT- PCR or Northern blot, preferably between several minutes to an hour after heat treatment. The inventors have observed that RNA of the gene product of interest sometimes has disappeared within an hour after heat treatment, whereas RNA of other genes was still present. This is related to the stability of the RNA.

The maintenance temperature depends on the fungus/mycelium/mushroom that is used and is for example between 15 and 30°C.

Method for production

In an aspect of the invention, there is provided a method for the production of a mushroom, mycelium, a fungal cell, a fruiting body or a spore as identified in the previous section. Preferably, the method comprises the steps of culturing a fungal cell comprising a nucleic acid molecule which is represented by a nucleic acid sequence coding for a gene product of interest as defined above under circumstances that are conducive for the generation of the gene product of interest, wherein the nucleic acid sequence is operably linked to a heat inducible promoter which is functional in a eukaryote, preferably a basidiomycete. In a preferred embodiment, the fungal cell is a mushroom forming fungal cell, preferably as defined earlier herein. Preferably, the heat inducible promoter is a promoter as defined earlier herein.

In a preferred embodiment, the present invention provides a method for the production of a mushroom, mycelium, fungal cell, a fruiting body, a spore or a gene product of interest comprising the steps of: a) culturing a fungal cell, fruiting body, spore, mycelium or mushroom comprising a nucleic acid molecule represented by a nucleic acid sequence coding for a gene product of interest, wherein said nucleic acid sequence is operably linked to a heat inducible promoter which is from a basidiomycete and/or which is functional in a basidiomycete; b) treating the mushroom, mycelium, fungal cell, fruiting body, or spore with heat during part of the culturing under a); and c) optional recovery of the mushroom, mycelium, fungal cell, fruiting body, spore or gene product of interest.

The skilled person knows how to carry out a method for production of a mushroom for instance described in Stamets and Chilton (1983) and van Griensven (1988). Such production includes colonization of a substrate (with or without a casing layer) followed by a phase where fruiting bodies are produced. The production may be carried out at a commercial scale with an optimal production level and/or quality level (e.g. more homogeneity in size and outgrowth). This method is preferably carried out using a fungus able to produce or form a mushroom, wherein said mushroom comprises a nucleic acid construct comprising a promoter of the invention as earlier defined herein, wherein said promoter is operably linked with a nucleotide coding for a polypeptide represented by an amino acid sequence having at least 50% amino acid identity or similarity with SEQ ID NO: 8-207 as identified earlier herein or having at least 40% amino acid identity or similarity with SEQ ID NO:208-215 as earlier defined herein in the section entitled nucleic acid construct. More preferably, said promoter is operably linked to a nucleotide sequence encoding a polypeptide that comprises an amino acid sequence that has at least 40% amino acid identity or similarity with a sequence selected from the group consisting of SEQ ID NO: 36; 184; 27; 63; 11; and/or that has at least 50% amino acid identity or similarity with a sequence selected from the group consisting of SEQ ID NO: 211; 214; 215; 209; 208; and/or said promoter is operably linked to a sequence selected from the group consisting of SEQ ID NO: 62; 28; 61; and/or that has at least 50% amino acid identity or similarity with a sequence selected from the group consisting of SEQ ID NO: 213; 212; 210; as earlier defined herein in the section entitled nucleic acid construct.

The term "culturing a fungal cell, fruiting body, spore, mycelium or mushroom" is herein understood to mean growing and/or maintenance of a fungal cell, mycelium or mushroom in medium, e.g. culture medium. The temperature depends on the fungal cell, mycelium or mushroom that is cultured, but is typically between 15 and 30°C.

The term "treating with heat" is herein used interchangeably with "heat treatment" and is intended to mean exposure of the fungal cell, mycelium, fruiting body, spore or mushroom to a temperature that is higher than the growth/maintenance temperature and is preferably between 37° and 97°C as is further defined elsewhere herein.

The term "during part of the culturing" is herein understood to mean as during part of the time of the culturing for the durations as indicated above in the section entitled "Fungal cell/mycelium/mushroom/fruiting body/spore". Part is preferably at least 0.01% of the duration of the culturing step a), or at least, 0.05%, 0.1%>, 0.5%>, 1%, 1.5%, 2%, 5% or more. It is preferred that after heat treatment the fungal cell/mycelium/mushroom/fruiting body/spore is maintained at the maintenance temperature that is suitable for the specific species. Heat treatment during part of the culturing can be performed without interruption the process of culture, but can also be performed during an interruption of the culture.

Preferably, a method of the invention comprises the step of transforming a mushroom, mycelium or a fungal cell with an expression vector comprising a nucleic acid sequence coding for a gene product of interest operably linked to a heat inducible promoter.More preferably, a method of the invention further comprises heat treatment of the mushroom, mycelium, fungal cell, fruiting body or spore as further discussed earlier herein. In a preferred embodiment, the heat treatment is selected from the group consisting of at least 2 minutes to 4 hour, more preferably 2 minutes to 2 hour, placement in an incubator of 37 - 95 °C, inserting a needle with a temperature of 37 - 95 °C for 0.5 to 120 seconds into culture medium of the mushroom, the mycelium, the fruiting body, the fungal cell or the spore or into the mushroom, the mycelium, the fruiting body or a colony of fungal cells or spores.

In a preferred embodiment, the gene of interest is a gene which upon expression of the gene product of interest induces mushroom formation. In another preferred embodiment, the gene of interest is a gene which upon expression of the gene inhibits mushroom formation. Preferably, the gene of interest is represented by a nucleotide sequence encoding a polypeptide that comprises an amino acid sequence: (a) that has at least 40 % amino acid identity or similarity with a sequence selected from SEQ ID NO: 8-207, preferably as defined earlier herein; and/or,

(b) that has at least 50% amino acid identity or similarity with a sequence selected from SEQ ID NO: 208-215, preferably as defined earlier herein.

The term "induces mushroom formation" is herein understood to encompass both direct and indirect induction. It can be used interchangeably with the term "stimulation of mushroom formation" and it is used to indicate that the number of mushrooms is increased as compared to a control (e.g. the same species not comprising that gene of interest and/or the same species comprising that gene of interest but not subjected to heat treatment).

The invention also relates to a method for producing a gene product of interest or a substance of interest using a fungal cell, a mycelium or a mushroom of the invention. In this method, a fungus/mushroom may have been modified in order to be able to produce said gene product of interest or substance of interest. A gene product of interest may be any gene product that could be produced by a fungus/mycelium/mushroom/fruiting body. Such substance includes a nucleic acid sequence, a protein, a polypeptide, a metabolite. A protein or polypeptide in this context may be a pharmaceutical protein or polypeptide and/or a protein or polypeptide for interest for food, feed, or non-food, non-feed applications. Such gene product of interest may be endogenous for a fungus/mushroom or not. Preferably, the method comprises the steps of: culturing of a fungus/mycelium/mushroorn/fruiting body/spore of the invention that is able to produce a substance of interest under circumstances that are conducive for the generation of the substance of interest; and optional recovery of the substance of interest. Preferably, the circumstances that are conducive for the generation of the gene product of interest, comprise a heat treatment as defined earlier herein.

The assessment of the production level of the polypeptide may be performed at the mPvNA level by carrying out a Northern Blot, qPCR or an array analysis and/or at the polypeptide level by carrying out SDS PAGE or a Western blot. All these methods are well known to the skilled person.

According to a more preferred embodiment, a wild type or native fungus/mycelium/mushroom/fruiting body/spore does not produce any detectable amounts of the polypeptide and/or does not exhibit any detectable activity of said polypeptide under maintenance conditions of the fungus/mushroom/mycelium/fruiting body/spore. Preferably, a native or wild type fungus/mycelium/mushroom does not produce or produces substantially no polypeptide under maintenance conditions.

In an embodiment, a fungus/mycelium/mushroom/fruiting body/spore comprises a nucleic acid construct comprising a nucleotide encoding a protein/polypeptide to be produced. In this case, a substance of interest may be such protein/polypeptide. Alternatively, said protein/polypeptide may be involved in the production or synthesis of such substance of interest. Each feature of said nucleic acid construct has been earlier defined herein. It is also encompassed by the present invention to use a fungus/mushroom which has been further modified by increasing/decreasing the expression level of a protein/polypeptide known to be involved in the method of production of said substance. The term "endogenous" when used with respect to a nucleic acid or polypeptide molecule refers to a nucleic acid or polypeptide as natively expressed in a fungus/mushroom/mycelium/fruiting body/spore, preferably in a wild type state.

The term "heterologous" is used as opposite of "endogenous". The term "heterologous" when used with respect to a nucleic acid or polypeptide molecule refers to a nucleic acid or polypeptide from a foreign fungus/mushroom/mycelium/fruiting body/spore which does not occur naturally as part of a given fungus/mushroom/mycelium/fruiting body/spore (genome or DNA or RNA from said fungus) or which is found in a fungus/mushroom or location or locations in the genome or DNA or RNA sequence that differ from that in which it is found in nature. Heterologous nucleic acids or proteins are not endogenous to the fungus/mushroom/mycelium/fruiting body/spore into which they are introduced, but have been obtained from another fungus/mushroom/mycelium/fruiting body/spore or synthetically or recombinantly produced. Generally, though not necessarily, such nucleic acids encode proteins or polypeptides that are not normally produced by the fungus in which the DNA is transcribed or expressed, similarly exogenous RNA codes for proteins not normally expressed in the fungus/mushroom/mycelium/fruiting body/spore in which the exogenous RNA is present. Furthermore, it is known that a heterologous protein or polypeptide can be composed of homologous elements arranged in an order and/or orientation not normally found in a fungus/mushroom/mycelium/fruiting body/spore in which it is transferred, i.e. the nucleotide sequence encoding said protein or polypeptide originates from the same species but is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. Heterologous nucleic acids and proteins may also be referred to as foreign nucleic acids or proteins. Any nucleic acid or protein that one of skill in the art would recognize as heterologous or foreign to a fungus/mushroom/mycelium/fruiting body/spore in which it is expressed is herein encompassed by the term heterologous nucleic acid or protein. The term heterologous also applies to non-natural combinations of nucleic acid or amino acid sequences, i.e. combinations where at least two of the combined sequences are foreign with respect to each other.

In an embodiment, a fungal cell, mycelium or mushroom according to the invention may further over-express one or more other gene products of interest under a promoter of the invention or under a promoter that is not a promoter of the invention. Said other gene products of interest may either be endogenous or heterologous. Alternatively or in combination with the previous embodiment, in another embodiment a fungal cell, mycelium or mushroom according to the invention may further comprise an inactivated endogenous gene (product). This inactivation or down regulation may have been achieved by deletion of one or more nucleotides in the encoding gene. Alternatively, it may have been caused by an R Ai-like mechanism.

Sequence identity

"Sequence identity" is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (nucleotide, polynucleotide) sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. In a preferred embodiment, identity or similarity is calculated over the whole SEQ ID NO as identified herein. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988).

Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include e.g. the GCG program package (Devereux, J., et al, Nucleic Acids Research 12 (1): 387 (1984)), BestFit, BLASTP, BLASTN, and FASTA (Altschul, S. F. et al, J. Mol. Biol. 215:403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al, NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al, J. Mol. Biol. 215:403-410 (1990). The well-known Smith Waterman algorithm may also be used to determine identity.

Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89: 10915-10919 (1992); Gap Penalty: 12; and Gap Length Penalty: 4. A program useful with these parameters is publicly available as the "Ogap" program from Genetics Computer Group, located in Madison, WI. The aforementioned parameters are the default parameters for amino acid comparisons (along with no penalty for end gaps).

Preferred parameters for nucleic acid comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: matches=+10, mismatch=0; Gap Penalty: 50; Gap Length Penalty: 3. Available as the Gap program from Genetics Computer Group, located in Madison, Wis. Given above are the default parameters for nucleic acid comparisons.

Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called "conservative" amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide- containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine- valine, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to ser; Arg to lys; Asn to gin or his; Asp to glu; Cys to ser or ala; Gin to asn; Glu to asp; Gly to pro; His to asn or gin; He to leu or val; Leu to ile or val; Lys to arg; gin or glu; Met to leu or ile; Phe to met, leu or tyr; Ser to thr; Thr to ser; Trp to tyr; Tyr to trp or phe; and, Val to ile or leu.

The term "medium" or "culture medium" are used interchangeably herein. (Culture) medium should not be construed narrowly, but encompasses any medium on which a fungus/mushroom/spore/mycelium/fruiting body can develop/grow. As an example, the medium preferably is a solid minimal medium (Dons et al. 1979). However, it may also be soil. In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb "to consist" may be replaced by "to consist essentially of meaning that a product or a composition as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

Description of the figures

Figure 1. A. Consensus sequence found in all 7 promoters of the predicted heat shock genes. B. Locations of the identified sequences on the promoters.

Figure 2. Fluorescence induced by the promoter of the gene encoding heat shock protein 3 (hsp3). At 25 °C (A, E) and 30 °C (B, F) no fluorescence was visible. At 37 °C (C, G) weak fluorescence and at 42 °C (D, H) clear fluorescence was observed. (A- D) represent light microscopy pictures. (E-H) represent fluorescence microscopy pictures of the same areas as depicted in (A-D). Figure 3. Local induction of dTomato expression from the hsp3 promoter using a hot needle. Light (above) and fluorescence (below) microscopy of a wild-type S. commune colony (A) and a colony expressing dTomato from the hsp3 promoter (B).

Figure 4. Colonies were grown continuously at 25 °C (A, B, C) or incubated at 42 °C for 1 hour a day (D, E, F). Wild type forms mushrooms (A, D), the Awc2Awc2 strain does not form mushrooms (B, E), the Awc2Awc2 strain complemented with hsp3_Prom-wc2 only forms mushrooms at 42 °C (C, F).

Examples

Example 1: identification of regulators of mushrooms

A/Identification of putative transcription factors in the genome of Schizophyllum commune Automatic gene calling of the genome of the Schizophyllum commune strain 4-8 (FGSC #9210) (8.29X coverage) by the Joint Genome Institute resulted in 13181 predicted genes (see http://jgi.doe.gov/Scommune). These genes were automatically annotated using GO (Gene Ontology) (Ashburner et al, 2000), KOG (euKaryotic Orthologous Groups) (Koonin et al, 2004) and PFAM algorithms (Finn et al, 2008). This automatic annotation was used as a starting point to identify transcription factors (TFs):

• Based on known DNA binding domains, 190 genes were placed in the GO accession term "Transcription Factor Activity" (GO:0003700).

· The KOG annotation algorithm predicted 569 genes to be generally involved in transcription (Function ID: "K"). From these, 205 transcription factors were manually identified.

• A total of 151 POLYPEPTIDES were identified based on the presence of PFAM domain PF00096/IPR007087 (Zinc finger, C2H2-type).

All putative TFs were manually inspected and gene model or annotation were adjusted when necessary. Duplicate entries in the list were removed. To identify TFs that were missed during the automatic annotation, the identified TFs were used in a BLASTp (to the database of predicted proteins) and BLASTn (to the genomic database) analysis. Resulting hits were manually inspected and newly identified TFs were added to the list. These procedures led to the identification of 472 putative TFs in the genome of S. commune.

Expression profile of the putative transcription factors

In order to determine the expression profile of the putative TFs during fruiting body development, we used the technique MPSS (massive parallel signature sequencing). Total RNA was isolated from the monokaryotic strain 4-40 (CBS 340.81) and from the dikaryon resulting from a cross between 4-40 and 4-39 (CBS 341.81). A 7-day-old colony grown on solid minimal medium (MM; Dons et al, 1979) at 30°C in the dark was homogenized in 200 ml MM using a Waring blender for 1 min at low speed. 2 ml of the homogenized mycelium was spread out over a polycarbonate membrane that was placed on top of solidified MM. Vegetative monokaryotic mycelium was grown for 4 days in the light. The dikaryon was grown for 2 and 4 days in the light to isolate mycelium with stage I aggregates and stage II primordia, respectively. Mature mushrooms of 3 days old were picked from dikaryotic cultures that had grown for 8 days in the light. R A was isolated as described (van Peer et al, 2009). MPSS was performed essentially as described (Brenner et al, 2000) except that after DpnII digestion Mmel was used to generate 20 bp tags. Tags were sequenced using the Clonal Single Molecule Array technique (Illumina, Hayward, CA, US). Programs were developed in the programming language Python to analyze the data. Tag counts were normalized to transcripts per million (TPM). Tags with a maximum of < 4 TPM were removed from the data set. TPM values of tags originating from the same transcript were summed to assess their expression levels. If the gene of a putative TF did not contain a known 5' or 3 ' UTR, then 200 bp of genomic DNA was added to the respective end of the coding sequence of the gene. MPSS expression analysis agreed with expression studies that have been performed in the past (for a review see Wosten, H.A.B. & Wessels, 2006). Identification of interesting expression profiles

A TF is considered to be potentially involved in regulation of fruiting body formation when it is either up or down regulated in one of the developmental stages compared to the sterile monokaryon. There are 75 TFs which were at least two fold-up regulated, and 155 at least two-fold down regulated. More interestingly, 29 of these were at least four fold up-regulated and 77 at least 4 fold down-regulated. Even more interestingly, 9 were at least ten fold up-regulated and 27 at least ten-fold down regulated.

The proteinlDs and the MPSS values of these interesting TFs are indicated in Table 1.

_{i PI}D_tro_en

Table 1 A. Transcription factor genes (first column) that are up regulated at least 2 times compared to monokaryons during some stage o fruiting body formation. Expression of the transcription factor genes was assessed by MPSS and is expressed in tags per millio Homoloqu_{k lih (} Me_tonoa_ryon_gis of the transcription factors in L. bicolor and C. cinereus are indicated in the last two columns.

k_{dk (} Monoa_ryona_ri Laccaria bicolor Coprinus cinereus l_{ih (}) _{S Itt}a_gegs _d ^{k ()} _{S It}a_gea^rs ^{lih ()} _S ^IItta^gegs

84275 356 356 28 794 679 148 3.51123e-028 1.05479e-024 (CC1G 00475.1 | Coprinus cinereu h⁽⁾ Mu^sroom^s- (jgi|Lacbil |301245|eu2.LbsclO001g06940) predicted protein (translation) (288 aa))

112067 4 7 2 20 18 8 2.16056e-042 8.29072e-040 (CC1G_09213.1 | Coprinus cinereu

(jgi|Lacbil |165205|gwhl.50.46.1) hypothetical protein (translation) (163 aa))

75142 23 25 96 46 46 26 1.45116e-045 8.20197e-022 (CC1G 06695.1 | Coprinus cinereu

(jgi|Lacbil |149718|gwwl.48.44.1) hypothetical protein (translation) (71 aa))

255701 16 17 9 73 56 23 7.91122e-122 8.46417e-123 (CC1G_03649.1 | Coprinus cinereu

(jgi|Lacbil |300797|eu2.LbsclO001g02460) hypothetical protein (translation) (804 aa))

17463 4 4 2 10 7 2 7.57488e-029 6.88671e-012 (CC1G 03947.1 | Coprinus cinereu

(jgi|Lacbil |311419|eu2.LbsclO004g03750) hypothetical protein (translation) (433 aa))

80413 75 142 767 1373 3741 548 4.37799e-016 1.50246e-015 (CC1G 00158.1 | Coprinus cinereu

(jgi|Lacbil |307632|eu2.LbsclO036g00800) predicted protein (translation) (207 aa))

269940 0 5 0 8 10 0 1.3221 le-040 4.43575e-036 (CC1G 01926.1 | Coprinus cinereu

(jgi|Lacbil |298063|eu2.Lbscf0013g01370) predicted protein (translation) (594 aa))

103341 17 15 18 105 46 31 2.56311e-110 8.69424e-108 (CC1G_08208.1 | Coprinus cinereu

(jgi|Lacbil 1303733 |eu2.LbscfD025g00680) hypothetical protein (translation) (750 aa))

68168 42 53 12 102 224 80 1.39087e-047 3.83544e-048 (CC1G_11894.1 | Coprinus cinereu

(jgi|Lacbil |296675|eu2.LbscfD010g04840) hypothetical protein (translation) (392 aa))

80935 20 11 8 13 15 48 6.82767e-162 2.38275e-161 (CC1GJ 1233.1 | Coprinus cinereu

(jgi|Lacbil 1181470|estExt_GeneWisePlus_h hypothetical protein (translation) (1884 aa)) uman.C_10729)

65208 0 4 0 8 9 2 8.54145e-080 9.87732e-070 (CC1G_08279.1 | Coprinus cinereu

(jgi|Lacbil |300068|eu2.LbscfD018g01550) hypothetical protein (translation) (947 aa))

269941 49 43 31 185 155 269 2.3621 le-049 3.76822e-021 (CC1G 10208.1 | Coprinus cinereu

(jgi|Lacbil | 150048|gwwl .2.776.1) hypothetical protein (translation) (658 aa))

81364 29 16 11 28 16 62 7.0499e-024 2.26843e-015 (CC1G 10373.1 | Coprinus cinereu

(jgi|Lacbil |302141 |eu2.LbscfD020g00560) predicted protein (translation) (473 aa))

236086 1 0 0 9 15 7 3.49156e-039 1.24586e-029 (CC1G 09355.1 | Coprinus cinereu

(jgi|Lacbil |296378|eu2.LbscfD010g01870) predicted protein (translation) (493 aa))

258244 0 2 0 1 5 0 7.71139e-017 1.17097e-018 (CC1G 13487.1 | Coprinus cinereu

(jgi|Lacbil |299352|eu2.LbscfD016g01460) hypothetical protein (translation) (421 aa))

233954 83 40 230 83 52 160 5.4199 5.96533e-005 (CC1G 13389.1 | Coprinus cinereu

(jgi|Lacbi 112933381 estExt_fgenesh2_pg.C_ predicted protein (translation) (358 aa))

60273)

81115 0 0 5 1 0 3 0.0 0.0 (CC1G 10583.1 Coprinus cinereus hypothet

(jgi|Lacbil |307431 |eu2.LbscfD035g00900) protein (translation) (1281 aa))

50846 0 2 0 8 11 0 6.36781e-097 4.90667e-098 (CC1G 12965.1 | Coprinus cinereu

(jgi|Lacbil |161020|gwhl .2.487.1) hypothetical protein (translation) (379 aa))

269944 38 58 7 116 106 53 5.19099e-020 1.15336e-014 (CC1G_06364.1 | Coprinus cinereu

(jgi|Lacbil |297310|eu2.Lbscf0011g04670) predicted protein (translation) (1332 aa))

11907 2 2 1 5 12 5 1.20451e-031 5.5527e-016 (CC1G 01569.1 | Coprinus cinereus

(jgi|Lacbil |227966|e_gwhl .1.1408.1) hypothetical protein (translation) (878 aa))

13988 19 10 18 34 32 42 4.82221e-058 1.32299e-023 (CC1G_01095.1 | Coprinus cinereu

(jgi|Lacbil |306097|eu2.LbscfD002gl 1550) hypothetical protein (translation) (249 aa))

255004 73 51 17 149 328 95 1.75263e-038 1.13601e-033 (CC1G_01461.1 | Coprinus cinereu

(jgi Lacbil 292733 estExt_fgenesh2_pg.C_ hypothetical protein (translation) (568 aa)) 40260)

85539 42 35 21 36 30 92 No Hit 3.09688 (CC1G_06401.1 | Coprinus cinereus predicted protein (translation) (274 aa))

83110 21 19 36 33 17 42 No Hit 9.01414 (CC1G_05428.1 | Coprinus cinereus predicted protein (translation) (819 aa))

16376 10 18 35 42 28 49 4.29556e-008 4.57134e-015 (CC1G 11831.1 | Coprinus cinereu

(jgi|Lacbil |296276|eu2.LbscfD010g00850) predicted protein (translation) (230 aa))

53446 0 0 0 1 5 0 4.91911e-127 4.92489e-101 (CC1G_04979.1 | Coprinus cinereu

(jgi|Lacbil |309166|eu2.LbscfD003g08050) predicted protein (translation) (927 aa))

67562 1 2 2 3 4 7 2.09205e-113 0.0 (CC1G 00774.1 | Coprinus cinereus hypothet

(jgi|Lacbil |244290|e_gwwl .2.539.1) protein (translation) (937 aa))

54452 57 45 170 61 37 31 2.56697e-016 3.47804e-015 (CC1G 00943.1 | Coprinus cinereu

(jgi|Lacbil |305501 |eu2.LbscfD002g05590) predicted protein (translation) (76 aa))

86194 21 18 1 73 50 4 2.45579e-025 2.79927e-031 (CC1G 08665.1 | Coprinus cinereu

(jgi|Lacbil |247901 |e_gwwl .7.313.1) hypothetical protein (translation) (632 aa))

269936 5 11 17 215 239 141 1.90329e-165 1.006e-155 (CC1G 11764.1 | Coprinus cinereus

(jgi|Lacbil |230018|e_gwhl .2.213.1) hypothetical protein (translation) (684 aa))

66861 58 62 55 136 195 258 0.0 1.3136e-162 (CC1G 05035.1 | Coprinus cinereus

(jgi|Lacbil |308722|eu2.LbscfD003g03610) predicted protein (translation) (743 aa))

255327 2 10 1 12 20 3 5.60605e-005 1.50915e-080 (CC1G 00489.1 | Coprinus cinereu

(jgi|Lacbil |255877|e_gwwl .68.5.1) hypothetical protein (translation) (874 aa))

84267 43 46 11 102 85 46 6.77968e-054 9.86192e-019 (CC1G_00539.1 | Coprinus cinereu

(jgi|Lacbil |301338|eu2.LbscfD001g07870) predicted protein (translation) (203 aa))

84657 4 12 1 35 36 5 1.34045e-080 2.71068e-039 (CC1G_09309.1 | Coprinus cinereu

(jgi|Lacbil |293353|estExt_fgenesh2_pg.C_ predicted protein (translation) (553 aa))

60305)

102836 2 3 2 6 13 3 0.00197812 0.0384765 (CC1G 09335.1 | Coprinus cinereus

(jgi|Lacbil |310368|eu2.LbscfD045g00870) hypothetical protein (translation) (761 aa))

102971 20 9 5 14 11 40 1.20314e-114 9.33746e-095 (CC1G 01274.1 | Coprinus cinereu

(jgi|Lacbil |305200|eu2.LbscfD002g02580) hypothetical protein (translation) (503 aa))

103145 39 9 159 33 23 45 1.13092e-017 2.79273 (CC1G 06961.1 | Coprinus cinereus

(jgi|Lacbil |163053|gwhl .2.659.1) hypothetical protein similar to b-glucan synthase

(translation) (1778 aa))

105299 0 0 2 5 13 5 1.91552e-124 4.89067e-121 (CC1G 05112.1 | Coprinus cinereu

(jgi|Lacbil |293478|estExt_fgenesh2_pg.C_ predicted protein (translation) (856 aa))

60495)

108072 2 1 7 3 8 11 0.000119896 0.00172153 (CC1G_04096.1 | Coprinus cinereus

(jgi|Lacbil |305624|eu2.LbscfD002g06820) predicted protein (translation) (846 aa))

269950 6 17 1 22 40 7 4.75256e-040 2.63605e-025 (CC1G 12565.1 | Coprinus cinereu

(jgi|Lacbil |297262|eu2.LbscfD011 g04190) hypothetical protein (translation) (1114 aa))

110010 8 7 3 9 7 19 1.02481e-134 5.44366e-097 (CC1G 09476.1 | Coprinus cinereu

(jgi|Lacbil |254611 |e_gwwl .48.60.1) hypothetical protein (translation) (495 aa))

110310 8 2 2 1 5 17 8.89088e-067 6.27856e-058 (CC1G_00878.1 | Coprinus cinereu

(jgi|Lacbil |298403|eu2.Lbscf0014g00160) hypothetical protein (translation) (632 aa))

110595 3 4 1 5 8 2 5.00291e-006 4.42266e-006 (CC1G 05683.1 | Coprinus cinereu

(jgi|Lacbil |292128|estExt_fgenesh2_pg.C_ predicted protein (translation) (900 aa))

20415)

269932 14 12 6 37 80 79 2.4932e-022 9.12302e-015 (CC1G_00448.1 | Coprinus cinereu

(jgi|Lacbil |311840|eu2.LbscfD004g07960) predicted protein (translation) (853 aa))

111234 49 43 46 135 61 32 1.14196e-068 1.11396e-038 (CC1G_01352.1 | Coprinus cinereu

(jgi|Lacbil 1330180|fgenesh3_pg.C_scaffold predicted protein (translation) (537 aa))

_25000163)

269957 2 5 0 3 5 10 2.56747 0.208227 (CC1G 11229.1 | Coprinus cinereus

(jgi Lacbil 327452 fgenesh3_pg.C_scaffold hypothetical protein similar to RNA polymerase I _12000342) largest subunit (translation) (1643 aa))

113625 0 1 1 5 2 1 5.11144e-017 2.91369e-020 (CC1G_03384.1 | Coprinus cinereu

(jgi Lacbil 324266 fgenesh3_pg.C_scaffold predicted protein (translation) (472 aa))

_5000373)

1 14874 1 1 14 13 55 35 9 3.1 1655e-015 2.85953e-014 (CC1G 07059.1 | Coprinus cinereu

(jgi|Lacbil |296989|eu2.Lbscf001 lg01460) predicted protein (translation) (636 aa))

230844 20 15 12 20 46 15 3.39291e-005 6.53481e-100 (CC1G 01258.1 | Coprinus cinereu

(jgi|Lacbil |242593 |e_gwwl .1.1018.1) hypothetical protein (translation) (456 aa))

233513 0 2 0 4 5 0 1.90507e-008 1.59982e-006 (CC1G 04980.1 | Coprinus cinereu

(jgi|Lacbil |309166|eu2.Lbscf0003g08050) predicted protein (translation) (546 aa))

236631 37 33 51 64 28 74 1.31226e-037 5.4302e-029 (CC1G 08004.1 | Coprinus cinereus

(jgi|Lacbil |316437|eu2.Lbscf0073g00530) predicted protein (translation) (433 aa))

255185 6 10 2 37 54 21 0.0 0.0 (CC1G 00087.1 | Coprinus cinereus hypothet

(jgi|Lacbil |292149|estExt_fgenesh2_pg.C_ protein (translation) (946 aa))

20489)

255490 16 17 2 21 52 14 2.13217e-043 2.32009e-033 (CC1G 06721.1 | Coprinus cinereu

(jgi|Lacbil |291485|estExt_fgenesh2_pg.C_ predicted protein (translation) (1 168 aa)) 10083)

255941 4 4 3 23 64 12 3.39452e-034 1.14738e-038 (CC1G 04277.1 | Coprinus cinereu

(jgi Lacbil 292246 estExt_fgenesh2_pg.C_ predicted protein (translation) (444 aa))

20664)

256135 16 13 2 33 10 2 6.41127e-146 8.05896e-081 (CC1G 02690.1 | Coprinus cinereu

(jgi|Lacbil |305928|eu2.LbscfD002g09860) hypothetical protein (translation) (801 aa))

257422 66 48 23 158 165 236 0.0 0.0 (CC1G 02690.1 | Coprinus cinereus hypothet

(jgi|Lacbil |307309|eu2.LbscfD034g01740) protein (translation) (801 aa))

257652 64 105 39 540 282 541 2.23702e-022 3.07303e-013 (CC1G 01991.1 | Coprinus cinereu

(jgi|Lacbil |324166|fgenesh3_pg.C_scaffold predicted protein (translation) (269 aa))

_5000273)

112780 1 0 20 0 1 1 3.30746e-008 8.63879e-006 (CC1G_05981.1 | Coprinus cinereu

(jgi|Lacbil |312052|eu2.Lbscf0004gl0080) predicted protein (translation) (278 aa))

114363 11 24 10 113 247 166 4.54998e-018 9.482e-011 (CC1G 02553.1 | Coprinus cinereus

(jgi|Lacbil |295582|estExt_fgenesh2_pg.C_ predicted protein (translation) (203 aa))

480074)

103949 1 1 29 1 1 89 52 23 5.98193e-020 8.48828e-013 (CC1GJ 1310.1 | Coprinus cinereu

(jgi|Lacbil |298392|eu2.Lbscf0014g00050) hypothetical protein (translation) (318 aa))

250177 3 8 0 16 12 3 3.40735e-081 3.015e-078 (CC1 G 06686.1 | Coprinus cinereus

(jgi|Lacbil |307141 |eu2.LbscfD034g00060) hypothetical protein (translation) (876 aa))

258543 0 1 0 2 4 0 8.22696e-058 9.35938e-049 (CC1G_03556.1 | Coprinus cinereu

(jgi|Lacbil |297675|eu2.Lbscf0012g02300) predicted protein (translation) (932 aa))

85474 0 2 1 9 8 15 1.0596 0.00216492 (CC1G 02553.1 | Coprinus cinereus

480074)

11542 28 4 72 13 8 18 4.78687e-046 4.28541e-046 (CC1G_03228.1 | Coprinus cinereu

(jgi|Lacbil |313756|eu2.Lbscf0005g03450) hypothetical protein (translation) (226 aa))

110354 4 9 1 26 29 7 1.31693e-049 4.87952e-040 (CC1G_04483.1 | Coprinus cinere

(jgi|Lacbil |313784|eu2.LbscfD005g03730) hypothetical protein (translation) (373 aa))

230646 3 12 5 10 36 2 0.632651 0.837813 (CC1G 02232.1 | Coprinus cinereus

(jgi|Lacbil |297378|eu2.Lbscf001 1g05350) hypothetical protein (translation) (1133 aa))

110178 3 0 6 2 1 7 0.344423 2.44418 (CC1G 07066.1 | Coprinus cinereus

(jgi|Lacbil |335236|fgenesh3_pg.C_scaffold hypothetical protein (translation) (858 aa))

_79000041)

237000 0 4 1 9 5 1 1.6891 le-029 1.08788e-025 (CC1G 11340.1 | Coprinus cinereu

(jgi|Lacbil |293969|estExt_fgenesh2_pg.C_ predicted protein (translation) (845 aa))

90377)

110445 5 5 14 11 17 1894 0.00110609 0.0455875 (CC1G 06494.1 | Coprinus cinereus

(jgi|Lacbil |305984|eu2.Lbscf0002gl0420) hypothetical protein (translation) (456 aa))

269975 13 57 5 169 140 37 6.5175e-037 4.8057e-035 (CC1G 04895.1 | Coprinus cinereus

(jgi Lacbil 333777 fgenesh3_pg.C_scaffold hypothetical protein (translation) (1005 aa)) _55000060)

256910 14 14 4 44 34 14 0.000486722 5.13982e-010 (CC1G 02553.1 | Coprinus cinereu

(jgi|Lacbil |305505|eu2.LbscfD002g05630) predicted protein (translation) (203 aa))

84085 4 11 1 22 15 4 2.07695e-073 1.7064e-051 (CC1G 04390.1 | Coprinus cinereus

(jgi|Lacbil |301763|eu2.LbscfD001gl2120) hypothetical protein (translation) (342 aa))

104304 34 29 81 11 11 21 6.67277e-027 2.56769e-026 (CC1G 08390.1 | Coprinus cinereu

(jgi Lacbil 174921 estExt Genewisel hum hypothetical protein (translation) (152 aa)) an.C_380088)

111555 1 1 9 36 50 9 3.36395e-014 0.000545947 (CC1G 03253.1 | Coprinus cinereu

(jgi|Lacbil |296536|eu2.LbscfD010g03450) hypothetical protein (translation) (638 aa))

258217 0 2 0 3 9 0 2.4074e-129 3.69617e-130 (CC1G_11608.1 | Coprinus cinereu

(jgi|Lacbil |293964|estExt_fgenesh2_pg.C_ hypothetical protein (translation) (1093 aa)) 90364)

Table IB.. Transcription factor genes (first column) that are down regulated at least 2 times compared to monokaryons during some stag_i _PID_tro_en

of fruiting body formation. Expression of the transcription factor genes was assessed by MPSS and is expressed in tags per millio Homoloques of the transcription factors in L. bicolor and C. cinereus are indicated in the last two columns.

k_{lih (} M_tonoa_ryon_gi

Laccaria bicolor Coprinus cinereus

k_{dk (} Monoa_ryona_rs _{lih (} ⁾ _{S Itt}a_gegs d^{k ()} _{S It}a_gea^rs ^{lih ()} S ^IItta^gegs

257931 2 15 1 5 10 0 1.38426e-012 2.11163e-015 (CC1G_03237.1 | Coprinus cine h⁽⁾ Mu^sroom^s (jgi|Lacbil |313869|eu2.LbscfD005g04580) predicted protein (translation) (427 aa))

84275 356 356 28 794 679 148 3.51123e-028 1.05479e-024 (CC1G_00475.1 | Coprinus cine

(jgi|Lacbil |301245|eu2.Lbscf0001g06940) predicted protein (translation) (288 aa))

269938 25 27 12 39 23 27 1.18154e-009 1.56532e-010 (CC1G_02553.1 | Coprinus cine

(jgi|Lacbil |305984|eu2.LbscfD002gl0420) predicted protein (translation) (203 aa))

112067 4 7 2 20 18 8 2.16056e-042 8.29072e-040 (CC1G_09213.1 | Coprinus cine

(jgi|Lacbil |165205|gwhl.50.46.1) hypothetical protein (translation) (163 aa))

110229 11 22 1 21 28 2 3.72591 7.58724e-022 (CC1G_07415.1 | Coprinus cine

(jgi|Lacbil |293031 |estExt_fgenesh2_pg.C_ hypothetical protein (translation) (319 aa)) 50093)

112017 60 46 22 55 44 75 0.0 (jgi|Lacbil |248902|e_gwwl.9.20.1) 0.0 (CC1G 11339.1 Coprinus cinereus hypothe protein (translation) (927 aa))

110458 137 152 6 86 46 28 0.0 6.14873e-173 (CC1G_01924.1 | Coprinus cine

(jgi|Lacbil |313737|eu2.LbscfD005g03260) hypothetical protein (translation) (1195 aa))

255386 8 15 3 11 7 10 0.0 4.74314e-148 (CC1G_04393.1 | Coprinus cine

(jgi|Lacbil |301759|eu2.LbscfD001gl2080) hypothetical protein (translation) (1072 aa))

14572 21 16 5 9 9 15 3.66387e-074 9.7012e-097 (CC1G 05086.1 | Coprinus cine

(jgi|Lacbil |293468|estExt_fgenesh2_pg.C_ hypothetical protein (translation) (1212 aa)) 60477)

81262 127 142 17 112 147 91 0.0 (jgi|Lacbil |141130|gwwl .6.14.1) 0.0 (CC1G 05143.1 Coprinus cinereus hypothe protein (translation) (2340 aa))

269939 61 20 4 15 12 13 0.000340456 0.0207699 (CC1G 03679.1 | Coprinus cine

(jgi|Lacbil |300830|eu2.LbscfD001g02790) predicted protein (translation) (172 aa))

48318 11 18 6 22 16 3 4.00071e-135 1.00096e-128 (CC1G_00244.1 | Coprinus cine

(jgi|Lacbil |231004|e_gwhl .4.426.1) hypothetical protein (translation) (290 aa))

257380 2 7 1 9 11 2 6.56874e-035 1.17834e-035 (CC1G 04758.1 | Coprinus cine

(jgi|Lacbil |294744|estExt_fgenesh2_pg.C_ predicted protein (translation) (982 aa))

200137)

17463 4 4 2 10 7 2 7.57488e-029 6.88671e-012 (CC1G 03947.1 | Coprinus cine

(jgi|Lacbil |311419|eu2.LbscfD004g03750) hypothetical protein (translation) (433 aa))

112634 10 9 19 2 2 7 2.32919e-007 0.00611536 (CC1G_02682.1 | Coprinus cine

(jgi|Lacbil 1303693 |eu2.LbscfD025g00280) predicted protein (translation) (126 aa))

255183 16 19 20 31 20 3 1.98491e-040 4.57118e-031 (CC1GJ2420.1 | Coprinus cine

(jgi|Lacbil |327087|fgenesh3_pg.C_scaffold predicted protein (translation) (642 aa))

_11000389)

80526 180 96 13 101 38 88 3.1483e-035 6.97275e-024 (CC1G 00462.1 | Coprinus cine

(jgi|Lacbil |312016|eu2.Lbscf0004g09720) predicted protein (translation) (486 aa))

107138 8 14 5 11 9 1 2.73236e-145 1.20794e-144 (CC1G 11705.1 | Coprinus cine

(jgi|Lacbil |309262|eu2.Lbscf0003g09010) hypothetical protein (translation) (788 aa))

236743 16 26 5 36 8 3 2.36001e-047 1.14005e-051 (CC1G 04606.1 | Coprinus cine

(jgi|Lacbil |298274|eu2.Lbscf0013g03480) predicted protein (translation) (429 aa))

68168 42 53 12 102 224 80 1.39087e-047 3.83544e-048 (CCIG I 1894.1 | Coprinus cine

(jgi|Lacbil |296675 |eu2.Lbscf0010g04840) hypothetical protein (translation) (392 aa))

105290 13 27 4 27 27 18 No Hit 8.40552 (CC1G 05500.1 | Coprinus cine predicted protein (translation) (552 aa))

84273 342 338 49 169 104 77 1.45714e-024 4.26924e-021 (CC1G 00473.1 | Coprinus cine

(jgi|Lacbil 1320903 |fgenesh3_pg.C_scaffold predicted protein (translation) (149 aa))

J000473)

254923 27 28 9 20 13 14 5.60843e-042 4.1397e-022 (CC1G 00307.1 | Coprinus cine

(jgi|Lacbi 1 1 1793 |eu2.Lbscf0004g07490) hypothetical protein (translation) (874 aa))

71685 158 312 16 192 128 46 0.0 0.0 (CC1 G 0031 1.1 | Coprinus cinereus predi

(jgi|Lacbil |31 1799|eu2.Lbscf0004g07550) protein (translation) (804 aa))

237374 22 12 11 1 1 5 14 3.10486e-007 1.76255e-007 (CC1G 07890.1 | Coprinus cine

(jgi|Lacbil |327532|fgenesh3_pg.C_scaffold predicted protein (translation) (610 aa))

J3000055)

255836 69 119 6 57 42 22 5.64052e-064 5.42325e-060 (CC1G 10208.1 | Coprinus cine

(jgi|Lacbil | 147701 |gwwl .7.218.1) hypothetical protein (translation) (658 aa))

255161 287 213 15 31 1 106 65 2.23106e-051 1.76778e-050 (CC1G 01262.1 | Coprinus cine

(jgilLacbil |311992|eu2.LbscfD004g09480) hypothetical protein (translation) (436 aa))

62967 6 3 0 2 2 4 1.32474e-023 4.58036e-027 (CC1G 00316.1 | Coprinus cine

(jgi|Lacbil |311809|eu2.LbscfD004g07650) predicted protein (translation) (190 aa))

79748 175 117 79 46 77 53 9.92764e-018 1.57881e-057 (CC1G 08635.1 | Coprinus cine

(jgilLacbil |242593 |e_gwwl .1.1018.1) hypothetical protein (translation) (549 aa))

82694 91 147 18 166 239 59 4.7646e-026 1.79347e-021 (CC1G_02249.1 | Coprinus cine

(jgi|Lacbil |293210|estExt_fgenesh2_pg.C_ predicted protein (translation) (460 aa))

50421)

112825 33 35 8 61 66 12 4.78753e-049 1.71008e-050 (CC1G 01657.1 | Coprinus cine

(jgi|Lacbil |318917|eu2.LbscfD009g02770) hypothetical protein (translation) (372 aa))

257915 256 224 7 156 170 31 7.94765e-037 0.0677954 (CC1G 13487.1 | Coprinus cine

(jgi|Lacbil |294648|estExt_fgenesh2_pg.C_ hypothetical protein (translation) (421 aa)) 180094)

103232 9 29 4 13 12 3 0.0 0.0 (CC1G 01322.1 Coprinus cinereus hypothe

(jgi|Lacbil |305781 |eu2.LbscfD002g08390) protein (translation) (3270 aa))

81412 25 52 6 22 29 13 0.00052408 1.3798 (CC1G 11549.1 | Coprinus cine

(jgi|Lacbil |301200|eu2.LbscfD001g06490) hypothetical protein (translation) (413 aa))

269943 20 6 4 2 2 1 2.03637e-014 4.5042e-010 (CC1G 09698.1 | Coprinus cine

(jgilLacbil |318938|eu2.LbscfD009g02980) predicted protein (translation) (370 aa))

102719 2 4 1 1 1 0 6.69498e-017 1.69905e-020 (CC1G_06239.1 | Coprinus cine

(jgi|Lacbil |164524|gwhl .30.48.1) hypothetical protein (translation) (470 aa))

255207 47 22 6 49 48 28 7.09912e-042 1.41019e-038 (CC1G_00022.1 | Coprinus cine

(jgi|Lacbil |305751 |eu2.LbscfD002g08090) predicted protein (translation) (777 aa))

17379 9 20 3 15 15 12 0.0 2.61612e-172 (CC 1G 08756.1 | Coprinus cine

(jgi|Lacbil |318801 |eu2.Lbscf0009g01610) predicted protein (translation) (923 aa))

73063 5 22 2 1 1 15 6 0.0 0.0 (CC1 G_01760.1 | Coprinus cinereus hypothe

(jgi|Lacbil |301025|eu2.LbscfD001g04740) protein (translation) (999 aa))

104375 5 9 1 7 9 5 0.0 0.0 (CC1 G_00528.1 | Coprinus cinereus hypothe

(jgi|Lacbil |301359|eu2.LbscfD001g08080) protein (translation) (1155 aa))

85886 43 116 4 118 73 33 1.11822e-113 2.51255e-110 (CC1G 07649.1 | Coprinus cine

(jgi|Lacbil |294384|estExt_fgenesh2_pg.C_ hypothetical protein (translation) (1828 aa)) 120202)

232127 13 22 3 22 17 14 3.20966e-024 1.37253e-020 (CC 1G 01030.1 | Coprinus cine

(jgi|Lacbil |307389|eu2.Lbscf0035g00480) predicted protein (translation) (971 aa))

257445 317 359 22 255 172 75 4.5863 l e-076 4.2098 l e-055 (CC1 G 05561.1 | Coprinus cine

(jgi|Lacbil |295409|estExt_fgenesh2_pg.C_ hypothetical protein (translation) (641 aa)) 400037)

104344 6 16 0 4 2 0 9.3631e-021 9.96152e-01 1 (CC1G 01834.1 | Coprinus cine

(jgi|Lacbil |301103|eu2.LbscfD001g05520) hypothetical protein similar to a 1-2 pr

(translation) (614 aa))

81107 46 63 4 69 58 57 0.647628 0.0585053 (CC1G 12431.1 | Coprinus cine

(jgi|Lacbil |327452|fgenesh3_pg.C_scaffold hypothetical protein (translation) (479 aa)) J2000342)

269944 38 58 7 1 16 106 53 5.19099e-020 1.15336e-014 (CC1 G 06364.1 | Coprinus cine

(jgi|Lacbil |297310|eu2.LbscfD01 lg04670) predicted protein (translation) (1332 aa))

255863 11 75 0 49 42 2 2.47285e-020 1.16841 e-018 (CC1G 04121.1 | Coprinus cine

(jgi|Lacbil |327972|fgenesh3_pg.C_scaffold hypothetical protein (translation) (434 aa))

J4000219)

232771 13 10 16 5 4 11 3.09919e-085 4.1061e-076 (CC1G 12018.1 | Coprinus cine

(jgi|Lacbil |306006|eu2.LbscfD002gl0640) predicted protein (translation) (551 aa))

233370 7 11 7 5 3 12 1.28465 1.27315 (CC 1G 04251.1 | Coprinus cine

(jgi Lacbil 319363 fgenesh3j3m.C_scaffol hypothetical protein (translation) (790 aa)) d_2000088)

256320 8 19 3 23 18 12 2.27359e-033 5.39458e-017 (CC1G 04874.1 | Coprinus cine

(jgi|Lacbil |315442|eu2.LbscfD006g01260) predicted protein (translation) (541 aa))

109936 13 54 2 35 18 7 1.35204e-078 1.762e-066 (CC1G 04598.1 | Coprinus cine

(jgi|Lacbil |294487|estExt_fgenesh2_pg.C_ predicted protein (translation) (1238 aa)) 130174)

114988 37 47 17 33 24 21 2.26887e-016 2.54978e-016 (CC1G 07470.1 | Coprinus cine

(jgi|Lacbil |327972|fgenesh3_pg.C_scaffold predicted protein (translation) (672 aa))

J4000219)

1 1 1683 10 16 2 21 17 5 7.20095e-007 7.38744e-007 (CC1 G 06427.1 | Coprinus cine

(jgi|Lacbil |298964|eu2.LbscfD015g01100) hypothetical protein (translation) (390 aa))

230584 2 4 1 5 4 3 8.39844e-013 5.00166e-014 (CC1G 09178.1 | Coprinus cine

(jgi|Lacbil |293340|estExt_fgenesh2_pg.C_ predicted protein (translation) (213 aa))

60280)

232060 5 7 0 7 7 7 1.63353e-006 5.78542e-008 (CC1G 00255.1 | Coprinus cine

(jgi|Lacbil |31 1738 |eu2.LbscfD004g06940) predicted protein (translation) (480 aa))

1 1907 2 2 1 5 12 5 1.20451e-031 5.5527e-016 (CC1G 01569.1 | Coprinus cine

66326 54 234 3 166 122 26 5.1963e-042 4.54227e-039 (CC1G 01569.1 | Coprinus cine

(jgi|Lacbil |293949|estExt_fgenesh2_pg.C_ hypothetical protein (translation) (878 aa)) 90336)

78316 2 37 0 55 66 4 2.2508e-121 6.95698e-097 (CC1G 09316.1 | Coprinus cine

(jgi|Lacbil |313622|eu2.LbscfD005g02110) hypothetical protein (translation) (2012 aa))

86018 84 212 3 114 70 33 4.04465e-108 4.52565e-090 (CC1G 01569.1 | Coprinus cine

255004 73 51 17 149 328 95 1.75263e-038 1.13601e-033 (CC1G_01461.1 | Coprinus cine

257247 26 21 4 30 26 15 5.18591e-110 8.40596e-086 (CC1G 06391.1 | Coprinus cine

(jgi|Lacbil |297145|eu2.LbscfD01 lg03020) hypothetical protein (translation) (544 aa))

232448 30 43 10 75 42 41 1.3602e-024 6.09293e-023 (CC1G 05197.1 | Coprinus cine

(jgi Lacbil 325071 fgenesh3_pg.C_scaffold hypothetical protein (translation) (220 aa)) _6000565)

232514 14 9 5 6 3 11 No Hit 9.72573 (CC1G 07424.1 | Coprinus cine hypothetical protein (translation) (828 aa))

63699 75 588 5 351 224 48 2.15172e-056 1.81485e-037 (CC1G 00101.1 | Coprinus cine

(jgi|Lacbil |305681 |eu2.LbscfD002g07390) predicted protein (translation) (504 aa))

111405 25 40 2 37 42 26 2.56691e-008 1.81797e-010 (CC1 G O 1905.1 | Coprinus cine

(jgi|Lacbil |293995|estExt_fgenesh2_pg.C_ predicted protein (translation) (479 aa))

100036)

231700 6 7 2 13 6 8 0.73058 1.01715 (CC1G 01826.1 | Coprinus cine

(jgi|Lacbil |237398|e_gwhl .25.72.1) predicted protein (translation) (217 aa))

256693 2 4 0 0 0 1 9.74717 No Hit

(jgi|Lacbil |310048|eu2.Lbscf0043g00860)

257455 16 20 1 18 14 14 3.47293e-019 1.835e-018 (CC1G 06938.1 | Coprinus cine

(jgi|Lacbil |317361 |eu2.LbscfD007g04890) predicted protein (translation) (1514 aa))

258832 5 2 1 3 4 3 2.5967e-006 2.99814e-006 (CC1G 03237.1 | Coprinus cine

(jgi|Lacbil |313869|eu2.LbscfD005g04580) predicted protein (translation) (427 aa))

52392 29 11 1 1 6 1 3.02104e-139 3.53753e-119 (CC1G 02915.1 | Coprinus cine

(jgi|Lacbil |309008|eu2.LbscfD003g06470) predicted protein (translation) (730 aa))

257495 85 39 4 23 9 8 0.0 0.0 (CC1G 09834.1 | Coprinus cinereus predi

(jgi|Lacbil |317073 |eu2.Lbscf0007g02010) protein (translation) (1043 aa))

57298 9 7 1 7 5 7 3.19925e-023 1.54075e-028 (CC1G 01588.1 | Coprinus cine

(jgi|Lacbil |293198|estExt_fgenesh2_pg.C_ predicted protein (translation) (293 aa))

50395)

57817 20 22 6 25 24 21 2.83971e-153 1.57392e-008 (CC1G 05375.1 | Coprinus cine

(jgi|Lacbil |296434|eu2.Lbscf0010g02430) predicted protein (translation) (188 aa))

83015 30 15 4 16 13 5 0.0 0.0 (CC1G 09335.1 | Coprinus cinereus hypothe

(jgi|Lacbil |313642|eu2.LbscfD005g02310) protein (translation) (761 aa))

269928 3 187 1 131 1 18 6 4.24134e-074 4.35046e-082 (CC1G 02207.1 | Coprinus cine

(jgi|Lacbil |162920|gwhl .36.39.1) hypothetical protein (translation) (1068 aa))

86194 21 18 1 73 50 4 2.45579e-025 2.79927e-031 (CC1G 08665.1 | Coprinus cine

63410 12 38 4 52 35 29 0.0 0.0 (CC1G 00218.1 Coprinus cinereus predi

(jgi|Lacbil |311698|eu2.LbscfD004g06540) protein (translation) (653 aa))

66095 35 41 10 34 28 28 1.3252e-166 1.4852 le- 120 (CC 1 G O 1157.1 | Coprinus cine

(jgi|Lacbil |158182|gwhl .2.262.1) hypothetical protein (translation) (515 aa))

66586 185 294 47 243 215 75 5.64877e-028 5.04929e-033 (CC1G 05406.1 | Coprinus cine

(jgi|Lacbil |293242|estExt_fgenesh2_pg.C_ hypothetical protein (translation) (337 aa)) 50565)

73210 181 89 34 137 72 228 0.0 0.0 (CC1G 06540.1 Coprinus cinereus predi

(jgi|Lacbil |301157|eu2.LbscfD001g06060) protein (translation) (813 aa))

74309 11 27 2 19 12 3 4.04216e-160 2.70609e-057 (CC1G 05174.1 | Coprinus cine

(jgi|Lacbil |316098|eu2.LbscfD006g07820) hypothetical protein (translation) (449 aa))

74719 21 45 5 21 15 13 0.0 0.0 (CC1G 01048.1 | Coprinus cinereus hypothe

(jgi|Lacbil |305811 |eu2.LbscfD002g08690) protein (translation) (1017 aa))

77191 183 219 47 232 239 131 2.11426e-048 8.95851e-044 (CC1G 01665.1 | Coprinus cine

(jgilLacbil 1144806|gwwl .1.428.1) hypothetical protein (translation) (460 aa))

78089 24 39 1 22 16 12 3.02159e-142 4.92773e-124 (CC1G 09309.1 | Coprinus cine

(jgi|Lacbil |313614|eu2.LbscfD005g02030) predicted protein (translation) (553 aa))

255327 2 10 1 12 20 3 5.60605e-005 1.50915e-080 (CC1G 00489.1 | Coprinus cine

81726 14 32 3 28 33 20 0.0 0.0 (CC1G 02934.1 | Coprinus cinereus hypothe

(jgi|Lacbil |309255|eu2.LbscfD003g08940) protein (translation) (703 aa))

81806 267 183 39 483 322 515 1.51176e-061 4.60296e-072 (CC1G 08004.1 | Coprinus cine

(jgi|Lacbil |312043|eu2.LbscfD004g09990) predicted protein (translation) (433 aa))

84267 43 46 11 102 85 46 6.77968e-054 9.86192e-019 (CC1G_00539.1 | Coprinus cine

84657 4 12 1 35 36 5 1.34045e-080 2.71068e-039 (CC1G_09309.1 | Coprinus cine

60305)

84684 20 95 1 37 13 6 3.11314e-009 9.49477e-009 (CC1 G O 1752.1 | Coprinus cine

(jgi|Lacbil |301023|eu2.LbscfD001g04720) predicted protein (translation) (176 aa))

269945 8 9 1 15 14 6 0.558256 8.39053e-005 (CC1G 04998.1 | Coprinus cine

(jgilLacbil |297319|eu2.LbscfD011g04760) predicted protein (translation) (540 aa))

254988 5 21 2 37 37 7 0.0 7.68764e-091 (CC1G 00348.1 | Coprinus cine

(jgilLacbil |311840|eu2.LbscfD004g07960) hypothetical protein (translation) (727 aa))

255385 29 24 1 37 18 31 6.12672e-005 1.35469e-005 (CC1G 09834.1 | Coprinus cine

(jgi|Lacbil |317073 |eu2.LbscfD007g02010) predicted protein (translation) (1043 aa))

104000 5 14 2 13 10 4 7.64306e-036 4.79579e-029 (CC1G 06539.1 | Coprinus cine

(jgi|Lacbil |301156|eu2.LbscfD001g06050) hypothetical protein (translation) (408 aa))

269948 8 29 0 39 52 9 1.45195e-130 1.67247e-052 (CC1G 11570.1 | Coprinus cine

(jgi|Lacbil |305537|eu2.LbscfD002g05950) hypothetical protein (translation) (349 aa))

269949 16 4 2 26 18 20 0.0 0.0 (CC1G 09022.1 Coprinus cinereus predi

(jgi|Lacbil |302769|eu2.LbscfD022g00590) protein (translation) (850 aa))

256993 45 23 7 16 12 32 2.07879e-014 6.92699e-008 (CC1G 01056.1 | Coprinus cine

(jgi|Lacbil |307391 |eu2.LbscfD035g00500) hypothetical protein (translation) (848 aa))

108591 5 7 4 6 2 2 0.0253515 0.0887384 (CC1G_06409.1 | Coprinus cine

(jgi|Lacbil |309226|eu2.Lbscf0003g08650) hypothetical protein (translation) (962 aa))

109190 134 19 28 6 3 12 2.72907e-094 1.3879e-073 (CC1G 02275.1 | Coprinus cine

(jgi|Lacbil |315220|eu2.Lbscf0069g00020) hypothetical protein (translation) (960 aa))

269950 6 17 1 22 40 7 4.75256e-040 2.63605e-025 (CC1G 12565.1 | Coprinus cine

(jgi|Lacbil |297262|eu2.Lbscf0011 g04190) hypothetical protein (translation) (1114 aa))

269952 5 7 1 13 9 9 2.91537e-009 1.36788e-007 (CC1G 06668.1 | Coprinus cine

(jgi|Lacbil |296436|eu2.Lbscf0010g02450) hypothetical protein (translation) (648 aa))

110010 8 7 3 9 7 19 1.02481e-134 5.44366e-097 (CC1G 09476.1 | Coprinus cine

110136 134 79 15 266 187 131 2.67901e-009 1.42573e-016 (CC1G 07536.1 | Coprinus cine

(jgi|Lacbil |310623 |eu2.Lbscf0047g00510) predicted protein (translation) (262 aa))

110310 8 2 2 1 5 17 8.89088e-067 6.27856e-058 (CC1G_00878.1 | Coprinus cine

110416 115 53 2 6 3 5 3.57277e-013 0.000851334 (CC1G_11170.1 | Coprinus cine

(jgi|Lacbil |313811 |eu2.Lbscf0005g04000) hypothetical protein (translation) (429 aa))

110595 3 4 1 5 8 2 5.00291e-006 4.42266e-006 (CC1G 05683.1 | Coprinus cine

20415)

269932 14 12 6 37 80 79 2.4932e-022 9.12302e-015 (CC1G_00448.1 | Coprinus cine

(jgi|Lacbil |311840|eu2.Lbscf0004g07960) predicted protein (translation) (853 aa))

250298 16 6 10 3 2 9 0.0246277 0.0265601 (CC1G 06409.1 | Coprinus cine

(jgi Lacbil 324266 fgenesh3_pg.C_scaffold hypothetical protein (translation) (962 aa)) _5000373)

111623 5 2 2 1 2 1 0.0353287 0.0118479 (CC1G_02934.1 | Coprinus cine

(jgi|Lacbil |309226|eu2.Lbscf0003g08650) hypothetical protein (translation) (703 aa))

269956 222 76 19 55 19 122 2.71131e-047 9.94127e-007 (CC1G 00937.1 | Coprinus cine

(jgi|Lacbil |299004|eu2.Lbscf0015g01500) hypothetical protein (translation) (473 aa))

269957 2 5 0 3 5 10 2.56747 0.208227 (CC1G 11229.1 | Coprinus cine

(jgi Lacbil 327452 fgenesh3_pg.C_scaffold hypothetical protein similar to RNA polymeras J2000342) largest subunit (translation) (1643 aa))

231698 10 4 0 3 2 1 2.12687e-008 0.00144182 (CC1G_01833.1 | Coprinus cine

(jgi|Lacbil |243459|e_gwwl .1.1010.1 ) hypothetical protein (translation) (449 aa))

234557 9 2 0 1 1 3 0.019675 0.0217871 (CC1G 06409.1 | Coprinus cine

(jgi|Lacbil |254568|e_gwwl .48.59.1) hypothetical protein (translation) (962 aa))

234560 27 16 5 13 7 15 0.0290787 0.106428 (CC1G_09318.1 | Coprinus cine

(jgi|Lacbil |311707|eu2.Lbscf0004g06630) predicted protein (translation) (411 aa))

255185 6 10 2 37 54 21 0.0 0.0 (CC1G 00087.1 | Coprinus cinereus hypothe

(jgi|Lacbil |292149|estExt_fgenesh2_pg.C_ protein (translation) (946 aa))

20489)

269958 140 95 8 64 61 19 9.22881e-032 2.43397e-026 (CC1G_06624.1 | Coprinus cine

(jgi|Lacbil |320896|fgenesh3_pg.C_scaffold predicted protein (translation) (615 aa))

_1000466)

255490 16 17 2 21 52 14 2.13217e-043 2.32009e-033 (CC1G_06721.1 | Coprinus cine

(jgi|Lacbil |291485|estExt_fgenesh2_pg.C_ predicted protein (translation) (1168 aa)) 10083)

255656 41 27 7 21 11 26 5.05368e-116 5.01661e-093 (CC1G 02275.1 | Coprinus cine

(jgi|Lacbil |307141 |eu2.LbscfD034g00060) hypothetical protein (translation) (960 aa))

255852 14 21 2 35 25 22 0.0 0.0 (CC1G 01056.1 Coprinus cinereus hypothe

(jgi|Lacbil |307391 |eu2.LbscfD035g00500) protein (translation) (848 aa))

256135 16 13 2 33 10 2 6.41127e-146 8.05896e-081 (CC1G 02690.1 | Coprinus cine

256706 149 61 19 91 89 40 1.54206e-024 9.4943e-025 (CC1G 08430.1 | Coprinus cine

(jgi|Lacbil |295375|estExt_fgenesh2_pg.C_ predicted protein (translation) (551 aa))

380074)

257056 11 6 2 7 12 8 8.35596e-125 5.37092e-068 (CC1G 07060.1 | Coprinus cine

(jgi|Lacbil |296988|eu2.LbscfD01 lg01450) hypothetical protein (translation) (662 aa))

257422 66 48 23 158 165 236 0.0 0.0 (CC1G 02690.1 Coprinus cinereus hypothe

(jgi|Lacbil |307309|eu2.LbscfD034g01740) protein (translation) (801 aa))

257622 103 91 23 130 85 93 8.27689e-140 9.46138e-139 (CC1G_03210.1 | Coprinus cine

(jgi|Lacbil |313780|eu2.LbscfD005g03690) hypothetical protein (translation) (542 aa))

257926 180 333 36 412 478 51 5.27989e-010 8.2724e-009 (CC1G 09318.1 | Coprinus cine

(jgi|Lacbil |304198|eu2.Lbscf0271g00010) predicted protein (translation) (411 aa))

257987 37 142 1 79 59 15 3.52426e-038 1.82065e-018 (CC1 G O 1962.1 | Coprinus cine

(jgi|Lacbi 112939881 estExt_fgenesh2_pg.C_ predicted protein (translation) (560 aa))

100022)

269960 4 15 0 15 17 1 1.24493e-036 8.86947e-028 (CC1G 05468.1 | Coprinus cine

(jgi|Lacbil |294583|estExt_fgenesh2_pg.C_ hypothetical protein (translation) (1311 aa)) 160148)

250177 3 8 0 16 12 3 3.40735e-081 3.015e-078 (CC1G 06686.1 | Coprinus cine

257265 134 283 4 77 60 24 6.99718e-008 1.62257e-006 (CC1G 07030.1 | Coprinus cine

(jgi|Lacbil |298964|eu2.LbscfD015g01100) predicted protein (translation) (468 aa))

269961 78 24 17 7 3 14 0.0309308 2.19663 (CC1G_03809.1 | Coprinus cine

(jgi|Lacbil |311738|eu2.LbscfD004g06940) hypothetical protein (translation) (2341 aa))

248401 21 15 29 10 6 10 0.000176154 2.31883e-005 (CC1G 12125.1 | Coprinus cine

(jgi|Lacbil |318427|eu2.LbscfD090g00220) predicted protein (translation) (575 aa))

82883 11 9 3 8 4 16 2.13804e-008 6.40863e-017 (CC1G 02553.1 | Coprinus cine

110354 4 9 1 26 29 7 1.31693e-049 4.87952e-040 (CC1G 04483.1 | Coprinus cine

84749 138 115 13 28 12 2 1.54768e-012 0.00102858 (CC1G_04483.1 | Coprinus cine

(jgi|Lacbil |296536|eu2.LbscfD010g03450) hypothetical protein (translation) (373 aa))

110478 10 10 5 2 2 12 3.08918e-006 6.85377e-012 (CC1G 02553.1 | Coprinus cine

480074)

254870 9 41 2 18 13 15 4.85676e-019 5.50242e-009 (CC1G 13435.1 | Coprinus cine

(jgi|Lacbil |294684|estExt_fgenesh2_pg.C_ predicted protein (translation) (477 aa))

190064)

258883 8 7 0 8 9 1 6.57999e-035 2.04345e-039 (CC1G 00671.1 | Coprinus cine

(jgi|Lacbil |301563|eu2.LbscfD001gl0120) hypothetical protein (translation) (594 aa))

108216 9 5 2 9 7 3 1.77166e-005 0.000296472 (CC1G_09111.1 | Coprinus cine

(jgi|Lacbil |297378|eu2.LbscfD01 lg05350) hypothetical protein (translation) (549 aa))

269975 13 57 5 169 140 37 6.5175e-037 4.8057e-035 (CC1G 04895.1 | Coprinus cine

108605 16 6 1 10 4 3 0.000123481 0.000230165 (CC1G 00937.1 | Coprinus cine

(jgi|Lacbil | 150072|gwwl .21.88.1) hypothetical protein (translation) (473 aa))

102516 16 8 3 17 13 7 1.238e-005 1.02461 (CC1G_01048.1 | Coprinus cine

(jgi|Lacbil |311605|eu2.LbscfD004g05610) hypothetical protein (translation) (1017 aa))

256910 14 14 4 44 34 14 0.000486722 5.13982e-010 (CC1G 02553.1 | Coprinus cine

256746 262 109 4 3 3 4 0.012 0.0235741 (CC1G 00609.1 | Coprinus cine

(jgi|Lacbil |305505|eu2.LbscfD002g05630) hypothetical protein (translation) (294 aa))

258642 48 30 5 22 11 21 0.137871 0.00187319 (CC1G_11643.1 | Coprinus cine

(jgi|Lacbil |301087|eu2.LbscfD001g05360) predicted protein (translation) (361 aa))

112405 20 8 4 6 3 5 0.00325358 0.00251037 (CC1G 06477.1 | Coprinus cine

(jgi|Lacbil | 144261 |gwwl .5.220.1) predicted protein (translation) (257 aa))

83895 31 51 1 15 13 10 9.3958e-012 1.69333e-016 (CC1G_01345.1 | Coprinus cine

(jgi|Lacbil |305984|eu2.LbscfD002gl0420) predicted protein (translation) (468 aa))

84085 4 11 1 22 15 4 2.07695e-073 1.7064e-051 (CC1G 04390.1 | Coprinus cine

(jgi|Lacbil |301763|eu2.Lbscf0001gl2120) hypothetical protein (translation) (342 aa))

109596 6 48 2 76 22 8 1.55855e-022 0.00342346 (CC1G_03235.1 | Coprinus cine

(jgi|Lacbil |296536|eu2.LbscfD010g03450) predicted protein (translation) (254 aa))

269979 3 28 0 13 9 4 4.39042e-035 5.28352e-029 (CC1G 10841.1 | Coprinus cine

(jgi|Lacbil |293360|estExt_fgenesh2_pg.C_ predicted protein (translation) (798 aa))

60316)

77161 491 584 170 733 191 762 6.13943e-061 6.14124e-024 (CC1G_00609.1 | Coprinus cine

(jgi|Lacbil |301873|eu2.LbscfD001gl3220) hypothetical protein (translation) (294 aa))

233354 4 3 2 1 1 1 7.38778e-011 0.00482856 (CC1G_04483.1 | Coprinus cine

104304 34 29 81 11 11 21 6.67277e-027 2.56769e-026 (CC1G 08390.1 | Coprinus cine

233946 9 7 1 4 5 5 2.70722e-007 1.06564e-006 (CC1G_09111.1 | Coprinus cine

(jgi|Lacbil |150072|gwwl.21.88.1) hypothetical protein (translation) (549 aa))

12349 63 26 14 66 10 12 6.07215e-025 1.98713e-013 (CC1G_00609.1 | Coprinus cine

250247 16 17 5 16 19 15 0.0443209 0.0554149 (CC1G 03253.1 | Coprinus cine

(jgi|Lacbil |299004|eu2.LbscfD015g01500) hypothetical protein (translation) (638 aa))

Table 1C. Transcription factor genes (first column) that are up regulated at least 4 times compared to monokaryons during some stage o fruiting body formation. Expression of the transcription factor genes was assessed by MPSS and is expressed in tags per millio Homoloques of the transcription factors in L. bicolour and C. cinereus are indicated in the last two columns.

Laccaria bicolor Coprinus cinereus

i P_lD_troen k ^lih) M ^(tonoa_iyon^gs ^{k dk)} M ⁽onoaryonar^s

255701 16 17 9 ^{lih ()} ^{S Itt}a^ge^gs 73 56 23 7.91122e-122 8.46417e-123 (CC1G 03649.1 | Coprinus cine

(jgi|Lacbil |300797|eu2.Lbscf0001g02460) hypothetical protein (translation) (804 aa)) d^{k ()} ^{S It}a^gear^s

80413 75 142 767 1373 3741 548 4.37799e-016 1.50246e-015 (CC1G 00158.1 | Coprinus cine

(jgi|Lacbil |307632|eu2.LbscfD036g00800) predicted protein (translation) (207 aa)) l^{ih (} ^{S II)tt}a^ge^gs

103341 17 15 18 105 46 31 2.5631 le-1 10 8.69424e-108 (CC1G 08208.1 | Coprinus cine h⁽ M⁾u^sroom^s (jgi|Lacbil |303733|eu2.Lbscf0025g00680) hypothetical protein (translation) (750 aa))

68168 42 53 12 102 224 80 1.39087e-047 3.83544e-048 (CC1G 11894.1 | Coprinus cine

(jgi|Lacbil |296675|eu2.Lbscf0010g04840) hypothetical protein (translation) (392 aa))

269941 49 43 31 185 155 269 2.3621 le-049 3.76822e-021 (CC1G 10208.1 | Coprinus cine

236086 1 0 0 9 15 7 3.49156e-039 1.24586e-029 (CC1G 09355.1 | Coprinus cine

(jgi|Lacbil |296378|eu2.Lbscf0010g01870) predicted protein (translation) (493 aa))

81 1 15 0 0 5 1 0 3 0.0 0.0 (CC1G 10583.1 | Coprinus cinereus hypothe

(jgi|Lacbil |307431 |eu2.Lbscf0035g00900) protein (translation) (1281 aa))

50846 0 2 0 8 1 1 0 6.36781e-097 4.90667e-098 (CC1G 12965.1 | Coprinus cine

(jgi|Lacbil |161020|gwhl .2.487.1) hypothetical protein (translation) (379 aa))

1 1907 2 2 1 5 12 5 1.20451e-031 5.5527e-016 (CC1G 01569.1 | Coprinus cine

53446 0 0 0 1 5 0 4.91911e-127 4.92489e-101 (CC1G_04979.1 | Coprinus cine

269936 5 11 17 215 239 141 1.90329e-165 1.006e-155 (CC1G 11764.1 | Coprinus cine

66861 58 62 55 136 195 258 0.0 1.3136e-162 (CC1G 05035.1 | Coprinus cine

102836 2 3 2 6 13 3 0.00197812 0.0384765 (CC1G 09335.1 | Coprinus cine

103145 39 9 159 33 23 45 1.13092e-017 2.79273 (CC1G 06961.1 | Coprinus cine

(jgi|Lacbil |163053|gwhl .2.659.1) hypothetical protein similar to b-glucan synt

(translation) (1778 aa))

105299 0 0 2 5 13 5 1.91552e-124 4.89067e-121 (CC1G 05112.1 | Coprinus cine

60495)

108072 2 1 7 3 8 11 0.000119896 0.00172153 (CC1G_04096.1 | Coprinus cine

269932 14 12 6 37 80 79 2.4932e-022 9.12302e-015 (CC1G_00448.1 | Coprinus cine

113625 0 1 1 5 2 1 5.11144e-017 2.91369e-020 (CC1G_03384.1 | Coprinus cine

(jgi|Lacbil |324266|fgenesh3_pg.C_scaffold predicted protein (translation) (472 aa))

_5000373)

255185 6 10 2 37 54 21 0.0 0.0 (CC1G 00087.1 | Coprinus cinereus hypothe

(jgi|Lacbil |292149|estExt_fgenesh2_pg.C_ protein (translation) (946 aa))

20489)

255941 4 4 3 23 64 12 3.39452e-034 1.14738e-038 (CC1G 04277.1 | Coprinus cine

20664)

257652 64 105 39 540 282 541 2.23702e-022 3.07303e-013 (CC1G 01991.1 | Coprinus cine

_5000273)

1 12780 1 0 20 0 1 1 3.30746e-008 8.63879e-006 (CC1G 05981.1 | Coprinus cine

1 14363 11 24 10 113 247 166 4.54998e-018 9.482e-011 (CC1G 02553.1 | Coprinus cine

(jgi Lacbil 295582 estExt_fgenesh2_pg.C_ predicted protein (translation) (203 aa))

480074)

258543 0 1 0 2 4 0 8.22696e-058 9.35938e-049 (CC1G 03556.1 | Coprinus cine

85474 0 2 1 9 8 15 1.0596 0.00216492 (CC1G 02553.1 | Coprinus cine

480074)

1 10445 5 5 14 1 1 17 1894 0.001 10609 0.0455875 (CC1G 06494.1 | Coprinus cine

111555 1 1 9 36 50 9 3.36395e-014 0.000545947 (CC1G 03253.1 | Coprinus cine i _PID_tro_en

258217 0 2 0 3 9 0 2.4074e-129 3.69617e-130 (CC1G_11608.1 | Coprinus cine k_{lih ()} M_tonoa_ryon_gs (jgi|Lacbil |293964|estExt_fgenesh2_pg.C_ hypothetical protein (translation) (1093 aa))

90364)

k_{dk ()} Monoa_ryona_rs _{lih ()} _{S Itt}a_gegs

Table ID. Transcription fact _{dk ()} _{S It}a_gea_rsor genes (first column) that are down regulated at least 4 times compared to monokaryons during some stag of fruiting body formation. Expression of the transcription factor genes was assessed by MPSS and is expressed in tags per millio 5 Homoloques of the transcription fac _{lih ()} _{S IItt}ta_gegsors in L. bicolor and C. cinereus are indicated in the last two columns. h₍₎ Mu_sroom_s Laccaria bicolor Coprinus cinereus

84275 356 356 28 794 679 148 3.51123e-028 1.05479e-024 (CC1G 00475.1 | Coprinus cine

(jgi|Lacbil |301245|eu2.LbscfD001g06940) predicted protein (translation) (288 aa))

110229 11 22 1 21 28 2 3.72591 7.58724e-022 (CC1G_07415.1 | Coprinus cine

110458 137 152 6 86 46 28 0.0 6.14873e-173 (CC1G_01924.1 | Coprinus cine

(jgi|Lacbil |313737|eu2.Lbscf0005g03260) hypothetical protein (translation) (1195 aa))

269939 61 20 4 15 12 13 0.000340456 0.0207699 (CC1G 03679.1 | Coprinus cine

(jgi|Lacbil |300830|eu2.Lbscf0001g02790) predicted protein (translation) (172 aa))

112634 10 9 19 2 2 7 2.32919e-007 0.00611536 (CC1G_02682.1 | Coprinus cine

(jgi|Lacbil 1303693 |eu2.Lbscf0025g00280) predicted protein (translation) (126 aa))

255183 16 19 20 31 20 3 1.98491e-040 4.57118e-031 (CC1GJ2420.1 | Coprinus cine

_11000389)

107138 8 14 5 11 9 1 2.73236e-145 1.20794e-144 (CC1G 11705.1 | Coprinus cine

236743 16 26 5 36 8 3 2.36001e-047 1.14005e-051 (CC1G 04606.1 | Coprinus cine

84273 342 338 49 169 104 77 1.45714e-024 4.26924e-021 (CC1G_00473.1 | Coprinus cine

J000473)

71685 158 312 16 192 128 46 0.0 0.0 (CC1G 00311.1 Coprinus cinereus predi

(jgi|Lacbil |311799|eu2.Lbscf0004g07550) protein (translation) (804 aa))

(jgi|Lacbil 1147701 |gwwl .7.218.1) hypothetical protein (translation) (658 aa))

255161 287 213 15 311 106 65 2.23106e-051 1.76778e-050 (CC1G 01262.1 | Coprinus cine

(jgi|Lacbil |311992|eu2.Lbscf0004g09480) hypothetical protein (translation) (436 aa))

82694 91 147 18 166 239 59 4.7646e-026 1.79347e-021 (CC1G 02249.1 | Coprinus cine

(jgi Lacbil 293210 estExt_fgenesh2_pg.C_ predicted protein (translation) (460 aa))

50421)

112825 33 35 8 61 66 12 4.78753e-049 1.71008e-050 (CC1G 01657.1 | Coprinus cine

81412 25 52 6 22 29 13 0.00052408 1.3798 (CC1G 11549.1 | Coprinus cine

(jgi|Lacbil |301200|eu2.Lbscf0001g06490) hypothetical protein (translation) (413 aa))

269943 20 6 4 2 2 1 2.03637e-014 4.5042e-010 (CC1G 09698.1 | Coprinus cine

(jgi|Lacbil |318938|eu2.LbscfD009g02980) predicted protein (translation) (370 aa))

104375 5 9 1 7 9 5 0.0 0.0 (CC1G 00528.1 | Coprinus cinereus hypothe

(jgi|Lacbil |301359|eu2.LbscfD001g08080) protein (translation) (1155 aa))

232127 13 22 3 22 17 14 3.20966e-024 1.37253e-020 (CC1 G O 1030.1 | Coprinus cine

257445 317 359 22 255 172 75 4.58631e-076 4.2098 le-055 (CC1G 05561.1 | Coprinus cine

(jgi|Lacbil |295409|estExt_fgenesh2_pg.C_ hypothetical protein (translation) (641 aa))

400037)

104344 6 16 0 4 2 0 9.3631e-021 9.96152e-011 (CC1G_01834.1 | Coprinus cine

(translation) (614 aa))

81107 46 63 4 69 58 57 0.647628 0.0585053 (CC1G 12431.1 | Coprinus cine

(jgi Lacbil 327452 fgenesh3_pg.C_scaffold hypothetical protein (translation) (479 aa)) J2000342)

269944 38 58 7 116 106 53 5.19099e-020 1.15336e-014 (CC1G_06364.1 | Coprinus cine

(jgi|Lacbil |297310|eu2.LbscfD011g04670) predicted protein (translation) (1332 aa))

255863 11 75 0 49 42 2 2.47285e-020 1.16841e-018 (CC1G_04121.1 | Coprinus cine

(jgi Lacbil 327972 fgenesh3_pg.C_scaffold hypothetical protein (translation) (434 aa)) J4000219)

109936 13 54 2 35 18 7 1.35204e-078 1.762e-066 (CC1G 04598.1 | Coprinus cine

111683 10 16 2 21 17 5 7.20095e-007 7.38744e-007 (CC1G_06427.1 | Coprinus cine

232060 5 7 0 7 7 7 1.63353e-006 5.78542e-008 (CC1G 00255.1 | Coprinus cine

(jgi|Lacbil |311738|eu2.LbscfD004g06940) predicted protein (translation) (480 aa))

257247 26 21 4 30 26 15 5.18591e-110 8.40596e-086 (CC1G 06391.1 | Coprinus cine

111405 25 40 2 37 42 26 2.56691e-008 1.81797e-010 (CC1G 01905.1 | Coprinus cine

100036)

257455 16 20 1 18 14 14 3.47293e-019 1.835e-018 (CC1G 06938.1 | Coprinus cine

(jgi|Lacbil |317361 |eu2.Lbscf0007g04890) predicted protein (translation) (1514 aa))

52392 29 11 1 1 6 1 3.02104e-139 3.53753e-119 (CC1G 02915.1 | Coprinus cine

257495 85 39 4 23 9 8 0.0 0.0 (CC1G 09834.1 | Coprinus cinereus predi

(jgi|Lacbil |317073 |eu2.LbscfD007g02010) protein (translation) (1043 aa))

57298 9 7 1 7 5 7 3.19925e-023 1.54075e-028 (CC1G 01588.1 | Coprinus cine

50395)

86194 21 18 1 73 50 4 2.45579e-025 2.79927e-031 (CC1G 08665.1 | Coprinus cine

74309 1 1 27 2 19 12 3 4.04216e-160 2.70609e-057 (CC1G 05174.1 | Coprinus cine

(jgi|Lacbil |316098|eu2.Lbscf0006g07820) hypothetical protein (translation) (449 aa))

74719 21 45 5 21 15 13 0.0 0.0 (CC1G 01048.1 | Coprinus cinereus hypothe

(jgi|Lacbil |305811 |eu2.Lbscf0002g08690) protein (translation) (1017 aa))

78089 24 39 1 22 16 12 3.02159e-142 4.92773e-124 (CC1 G 09309.1 | Coprinus cine

(jgi|Lacbil |313614|eu2.Lbscf0005g02030) predicted protein (translation) (553 aa))

81726 14 32 3 28 33 20 0.0 0.0 (CC1G 02934.1 | Coprinus cinereus hypothe

(jgi|Lacbil |309255|eu2.Lbscf0003g08940) protein (translation) (703 aa))

81806 267 183 39 483 322 515 1.51 176e-061 4.60296e-072 (CC1G 08004.1 | Coprinus cine

(jgi|Lacbil |312043|eu2.Lbscf0004g09990) predicted protein (translation) (433 aa))

84657 4 12 1 35 36 5 1.34045e-080 2.71068e-039 (CC1 G 09309.1 | Coprinus cine

60305)

84684 20 95 1 37 13 6 3.11314e-009 9.49477e-009 (CC1 G O 1752.1 | Coprinus cine

(jgi|Lacbil |301023|eu2.Lbscf0001g04720) predicted protein (translation) (176 aa))

269945 8 9 1 15 14 6 0.558256 8.39053e-005 (CC1G 04998.1 | Coprinus cine

(jgi|Lacbi 11297319|eu2.LbscfD01 1 g04760) predicted protein (translation) (540 aa))

255385 29 24 1 37 18 31 6.12672e-005 1.35469e-005 (CC1 G 09834.1 | Coprinus cine

(jgi|Lacbil |317073 |eu2.Lbscf0007g02010) predicted protein (translation) (1043 aa))

269948 8 29 0 39 52 9 1.45195e-130 1.67247e-052 (CC1 G 11570.1 | Coprinus cine

(jgi|Lacbil |305537|eu2.Lbscf0002g05950) hypothetical protein (translation) (349 aa))

109190 134 19 28 6 3 12 2.72907e-094 1.3879e-073 (CC1G_02275.1 | Coprinus cine

(jgi|Lacbil |315220|eu2.LbscfD069g00020) hypothetical protein (translation) (960 aa))

269950 6 17 1 22 40 7 4.75256e-040 2.63605e-025 (CC1G 12565.1 | Coprinus cine

(jgi|Lacbil |297262|eu2.Lbscf001 1g04190) hypothetical protein (translation) (1114 aa))

269952 5 7 1 13 9 9 2.91537e-009 1.36788e-007 (CC 1G 06668.1 | Coprinus cine

1 10136 134 79 15 266 187 131 2.67901 e-009 1.42573e-016 (CC1 G 07536.1 | Coprinus cine

110416 115 53 2 6 3 5 3.57277e-013 0.000851334 (CCIG I 1170.1 | Coprinus cine

(jgi|Lacbil |31381 1 |eu2.Lbscf0005g04000) hypothetical protein (translation) (429 aa))

231698 10 4 0 3 2 1 2.12687e-008 0.00144182 (CC1G 01833.1 | Coprinus cine

(jgi|Lacbil |243459|e_gwwl .1.1010.1) hypothetical protein (translation) (449 aa))

269958 140 95 8 64 61 19 9.22881e-032 2.43397e-026 (CC 1G 06624.1 | Coprinus cine

_1000466)

255490 16 17 2 21 52 14 2.13217e-043 2.32009e-033 (CC1G 06721.1 | Coprinus cine

255852 14 21 2 35 25 22 0.0 0.0 (CC1G_01056.1 | Coprinus cinereus hypothe

(jgi|Lacbil |307391 |eu2.Lbscf0035g00500) protein (translation) (848 aa))

256135 16 13 2 33 10 2 6.41127e-146 8.05896e-081 (CC 1G 02690.1 | Coprinus cine

(jgi|Lacbil |305928|eu2.Lbscf0002g09860) hypothetical protein (translation) (801 aa))

257926 180 333 36 412 478 51 5.27989e-010 8.2724e-009 (CC1 G 09318.1 | Coprinus cine

(jgi|Lacbil |293988|estExt_fgenesh2_pg.C_ predicted protein (translation) (560 aa))

100022)

269960 4 15 0 15 17 1 1.24493e-036 8.86947e-028 (CC1G 05468.1 | Coprinus cine

(jgi|Lacbil |294583|estExt_fgenesh2_pg.C_ hypothetical protein (translation) (131 1 aa)) 160148)

269961 78 24 17 7 3 14 0.0309308 2.19663 (CC1G 03809.1 | Coprinus cine

1 10354 4 9 1 26 29 7 1.31693e-049 4.87952e-040 (CC1G 04483.1 | Coprinus cine

(jgi|Lacbil |313784|eu2.Lbscf0005g03730) hypothetical protein (translation) (373 aa))

84749 138 1 15 13 28 12 2 1.54768e-012 0.00102858 (CC1G 04483.1 | Coprinus cine

(jgi|Lacbil |296536|eu2.Lbscf0010g03450) hypothetical protein (translation) (373 aa))

1 10478 10 10 5 2 2 12 3.08918e-006 6.85377e-012 (CC1G 02553.1 | Coprinus cine

480074)

254870 9 41 2 18 13 15 4.85676e-019 5.50242e-009 (CC1G 13435.1 | Coprinus cine

190064)

258883 8 7 0 8 9 1 6.57999e-035 2.04345e-039 (CC1G 00671.1 | Coprinus cine

108605 16 6 1 10 4 3 0.000123481 0.000230165 (CC1G 00937.1 | Coprinus cine

256746 262 109 4 3 3 4 0.012 0.0235741 (CC1G 00609.1 | Coprinus cine

258642 48 30 5 22 11 21 0.137871 0.00187319 (CC1G_11643.1 | Coprinus cine

83895 31 51 1 15 13 10 9.3958e-012 1.69333e-016 (CC1G_01345.1 | Coprinus cine

84085 4 11 1 22 15 4 2.07695e-073 1.7064e-051 (CC1G 04390.1 | Coprinus cine

233946 9 7 1 4 5 5 2.70722e-007 1.06564e-006 (CC1G_09111.1 | Coprinus cine

(jgi|Lacbil |150072|gwwl.21.88.1) hypothetical protein (translation) (549 aa))

Table IE. Transcription factor genes (first column) that are up regulated at least 10 times compared to monokaryons during some stage o fruiting body formation. Expression of the transcription factor genes was assessed by MPSS and is expressed in tags per millio Homoloques of the transcription factors in L. bicolor and C. cinereus are indicated in the last two columns.

(jgi|Lacbil |307632|eu2.LbscfD036g00800) predicted protein (translation) (207 aa))

236086 1 0 0 9 15 7 3.49156e-039 1.24586e-029 (CC1G 09355.1 | Coprinus cine

105299 0 0 2 5 13 5 1.91552e-124 4.89067e-121 (CC1G 05112.1 | Coprinus cine

60495)

255941 4 4 3 23 64 12 3.39452e-034 1.14738e-038 (CC1G 04277.1 | Coprinus cine

(jgi|Lacbil |292246|estExt_fgenesh2_pg.C_ predicted protein (translation) (444 aa))

20664)

112780 1 0 20 0 1 1 3.30746e-008 8.63879e-006 (CC1G 05981.1 | Coprinus cine

(jgi|Lacbil |312052|eu2.LbscfD004gl0080) predicted protein (translation) (278 aa))

114363 11 24 10 113 247 166 4.54998e-018 9.482e-011 (CC1G 02553.1 | Coprinus cine

480074)

110445 5 5 14 11 17 1894 0.00110609 0.0455875 (CC1G 06494.1 | Coprinus cine

(jgi|Lacbil |305984|eu2.LbscfD002gl0420) hypothetical protein (translation) (456 aa))

111555 1 1 9 36 50 9 3.36395e-014 0.000545947 (CC1G 03253.1 | Coprinus cine

Table IF. Transcription factor genes (first column) that are down regulated at least 10 times compared to monokaryons during some stag of fruiting body formation. Expression of the transcription factor genes was assessed by MPSS and is expressed in tags per millio Homoloques of the transcription factors in L. bicolor and C. cinereus are indicated in the last two columns.

(jgi|Lacbil |294648|estExt_fgenesh2_pg.C_ hypothetical protein (translation) (421 aa))

180094)

85886 43 116 4 118 73 33 1.1 1822e-113 2.51255e-110 (CC1G 07649.1 | Coprinus cine

81 107 46 63 4 69 58 57 0.647628 0.0585053 (CC1G 12431.1 | Coprinus cine

(jgi|Lacbil |327452|fgenesh3_pg.C_scaffold hypothetical protein (translation) (479 aa)) _12000342)

255863 1 1 75 0 49 42 2 2.47285e-020 1.16841 e-018 (CC1G 04121.1 | Coprinus cine

(jgi|Lacbil |327972|fgenesh3_pg.C_scaffold hypothetical protein (translation) (434 aa)) J4000219)

(jgi|Lacbil 1305681 |eu2.Lbscf0002g07390) predicted protein (translation) (504 aa))

1 1 1405 25 40 2 37 42 26 2.56691 e-008 1.81797e-010 (CC1G 01905.1 | Coprinus cine

100036)

257455 16 20 18 14 14 3.47293e-019 1.835e-018 (CC1G 06938.1 | Coprinus cine

¹

52392 29 11 1 6 1 3.02104e-139 3.53753e-119 (CC1G 02915.1 | Coprinus cine

¹

86194 21 18 73 50 4 2.45579e-025 2.79927e-031 (CC1G 08665.1 | Coprinus cine

¹

78089 24 39 22 16 12 3.02159e-142 4.92773e-124 (CC1G 09309.1 | Coprinus cine

¹

84684 20 95 37 13 6 3.11314e-009 9.49477e-009 (CC1 G O 1752.1 | Coprinus cine

¹

255385 29 24 37 18 31 6.12672e-005 1.35469e-005 (CC1G 09834.1 | Coprinus cine

¹

110416 115 53 2 6 3 5 3.57277e-013 0.000851334 (CC1G_11170.1 | Coprinus cine

(jgi|Lacbil |313811 |eu2.LbscfD005g04000) hypothetical protein (translation) (429 aa))

_1000466)

100022)

(jgi|Lacbil |298964|eu2.Lbscf0015g01100) predicted protein (translation) (468 aa))

84749 138 115 13 28 12 2 1.54768e-012 0.00102858 (CC1G_04483.1 | Coprinus cine

256746 262 109 4 3 3 4 0.012 0.0235741 (CC1G 00609.1 | Coprinus cine

83895 31 51 1 15 13 10 9.3958e-012 1.69333e-016 (CC1G_01345.1 | Coprinus cine

(jgi|Lacbil |305984|eu2.Lbscf0002gl0420) predicted protein (translation) (468 aa))

Conservation of POLYPEPTIDES in the fungal kingdom

In order to determine the level of conservation of the TFs throughout the fungal kingdom, protein sequences were blasted against the protein databases of Laccaria bicolor, Coprinus cinereus, Phanerochaete chrysosporium, Cryptococcus neoformans (H99), Cryptococcus neoformans (JEC21), Ustilago maydis, Aspergillus niger (DSM sequence), Aspergillus niger (Broad Institute), Magnaporthe grisea and Neurospora crassa.

The expect value and the name of the highest hit with the protein databases of the mushroom forming fungi Coprinus cinereus and Laccaria bicolor are indicated in Table 1.

B/Proof of principle: a knockout of fst3 (proteinID 257422) affects mushroom development

A knock-out was made of the putative transcription factor gene fst3 (proteinID 257422). To this end, vector pDelcas was used as described in Ohm et al. (2010). Primers that were used to create the knock out construct are indicated in Table 2. This knock out construct called pRO097 consists of the flanking regions of the coding sequence of fst3 in between which the nourseothricin resistance cassette is situated. The phleomycin resistance cassette is present elsewhere in the construct (for details see Ohm et al., 2010). Transformation of S. commune strain H4-8 was done as described (van Peer et al., 2009). Regeneration medium contained no antibiotic, whereas selection plates contained 20 μg ml^"1 nourseothricin. Deletion of the target gene was confirmed by PCR (for procedure see Ohm et al, 2010). Compatible monokaryons with a gene deletion were selected from spores originating from a cross of the mutant strains with wild-type strain 4-8.3 (L.G. Lugones, unpublished). Monokaryons with an inactivated fst3 gene showed no phenotype when compared to the wild type. On the other hand, Afst3Afst3 dikaryons showed clear differences in mushroom development compared to the wild-type. When grown from a point inoculum, mushrooms grew in the same location as in the wild type, but the number of mushrooms was increased and the size decreased (data not shown). When grown as a synchronized colony (by plating out homogenized mycelium), the number of mushrooms increased (data not shown). From these data we conclude that Fst3 inhibits formation of clusters of mushrooms. This regulation may be important in a natural environment to ensure sufficient energy is available for full fruiting body development.

CI Proof of principle: a knockout of fst4 (proteinID 66861) affects mushroom development

A knock-out was made of the putative transcription factor gene fst4 (proteinID 66861). To this end, vector pDelcas was used as described in Ohm et al. (2010). Primers that were used to create the knock out construct are indicated in Table 2. The knock out construct called pR0191 consists of the flanking regions of the coding sequence of fst4 in between which the nourseothricin resistance cassette is situated. The phleomycin resistance cassette is present elsewhere in the construct (for details see Ohm et al, 2010). Transformation of S. commune strain H4-8 was done as described (van Peer et al, 2009). Regeneration medium contained no antibiotic, whereas selection plates contained 20 μg ml^"1 nourseothricin. Deletion of the target gene was confirmed by PCR (for procedure see Ohm et al, 2010). Compatible monokaryons with a gene deletion were selected from spores originating from a cross of the mutant strains with wild-type strain 4-8.3. The Afst4 monokaryon showed no phenotypic differences when compared to the wild-type. In contrast, the Afst4Afst4 dikaryon did not fruit but produced more aerial hyphae when compared to the wild-type (data not shown). Apparently, Fst4 is involved in the switch between the vegetative phase and the reproductive phase.

Table 2: primers used to inactivate putative transcription factors

Gene Deletion construct (pROxxxx) with primers used to SEQ ID NO:

(ProteinID) amplify flanking sequences

c2h2 (114363) pRO103

dC2H2UpFw GGCCTAATAGGCCCGGATGCTTTCTCGGAGAGG 216

dC2H2UpRev GGCCTCGCAGGCCGAGCAGATGCTTCGCTCCGG 217

dC2H2DwFw GGCCTGCGAGGCCCCAGTCGACCTCAATTAGCC 218

dC2H2DwRev GGCCTATTAGGCCGCCCCTCACCCGTGTACCCG 219

Gatl (255004) pRO190

dGATAlUpFw GGCCTAATAGGCCTGGTCAAGGCATCCCGCAG 220

dGATAlUpRev GGCCTCGCAGGCCCTTCTTCTCAAGCCCAAATG 221

dGATAlDwFw GGCCTGCGAGGCCTACTCTCATGCGAGACCCAC 222

dGATAlDwRev GGCCTATTAGGCCCGTGGGTTGTTGAACTTACC 223 Gene Deletion construct (pROxxxx) with primers used to SEQ ID NO:

(ProteinID) amplify flanking sequences fst4 (66861) pR0191

dFst4UpFw GGCCTAATAGGCCACAAGCAGCAGAGGCTTGG 224 dFst4UpRev GGCCTCGCAGGCCGATTCGGACAGTCGAG 225 dFst4DwFw GGCCTGCGAGGCCGACTATAGGATGGTGAGCG 226 dFst4DwRev GGCCTATTAGGCCCAAACGGTGTCGGGAACGC 227 fst3 (257422) pRO097

dFst3UpFw GGCCTAATAGGCCCGTTTCCTAGTACACCTGTC 228 dFst3UpRev GGCCTCGCAGGCCGGAGAACGGGGTCCAGCAGG 229 dFst3DwFw GGCCTGCGAGGCCAGACCACCGAAGGATAGTTG 230 dFst3DwRev GGCCTATTAGGCCTCGTTGCTATCAGGAGCGGC 231 wc2 (13988) pR0192

dWC2UpFw2 GGCCTAATAGGCCACCGTCACGTCCATGTTCG 232 dWC2UpRev2 GGCCTCGCAGGCCCGAAACAACAATGATTG 233 dWC2DwFw GGCCTGCGAGGCCCTAGATGTTCGGTAATTGCC 234 dWC2DwRev GGCCTATTAGGCCCAGCCACCCATCTCGACTTG 235

Hom2 (257987) pR0189

dHom2UpFw GGCCTAATAGGCCTTGAGATGTTGCCTTGTCG 236 dHom2UpRev GGCCTCGCAGGCCCAAGAGCAAGCGTTGAG 237 dHom2DwFw GGCCTGCGAGGCCCACGATCTACCCAAACAG 238 dHom2DwRev GGCCTATTAGGCCAGATCCAACGTGAGAGCCAG 239

Homl (257652) pRO093

dHomlUpFw2 GGCCTAATAGGCCAGTGCTGGTGAGACTCACG 240 dHomlUpRev2 GGCCTCGCAGGCCCGATTGGTACGAGCTGGATG 241 dHomlDwFw GGCCTGCGAGGCCCATTCTCATATGCCTCAAAC 242 dHomlDwRev GGCCTATTAGGCCTCGTCTCTATTCACAACCGC 243 bril (255701) pDelcas-BRIGHT

dBrightUpFw GGCCGAATGGGCCGTATGAAGGAAG 244 dBrighUpRev GGCCCCGCTGGCCCTGCAAACGAAC 245 dBrightDwFw GGCCAGCGAGGCCAGGTCCGTGATCCTTTGTG 246 dBrightDwRev GGCCTATTAGGCCTGAAGGGCGGTAATGCTG 247 D/ Proof of principle: knockouts of other putative transcription factors

In addition to fst3 and fst4, 6 other putative transcription factor genes have been inactivated. Inactivation followed the procedures described above using primers indicated in Table 2. These deletions also affected mushroom formation (see Table 3)

Table 3 Transcription factors of S. commune that have been inactivated.

These results clearly show that with the above described method we have identified transcription factors that are involved in fruiting body development in S. commune.

Example 2: identification of heat shock promoters in mushrooms

Material and methods Strains

S. commune strain H4-8 (FGSC #9210) and the compatible co-isogenic strain H4- 8.3 (L.G. Lugones, Utrecht University) were used.

Expression profile of putative heat shock protein genes

In order to determine the expression profiles of putative heat shock protein genes, we used the technique MPSS (massive parallel signature sequencing). Total RNA was isolated from the monokaryotic strain 4-40 and from the dikaryon resulting from a cross between 4-40 and 4-39. A 7-day-old colony grown on solid MM at 30°C in the dark was homogenized in 200 ml MM using a Waring blender for 1 min at low speed. 2 ml of the homogenized mycelium was spread out over a polycarbonate membrane that was placed on top of solidified MM. Vegetative monokaryotic mycelium was grown for 4 days in the light. The dikaryon was grown for 2 and 4 days in the light to isolate mycelium with stage I aggregates and stage II primordia, respectively. Mature mushrooms of 3 days old were picked from dikaryotic cultures that had grown for 8 days in the light. R A was isolated as described (van Peer et al, 2009). MPSS was performed essentially as described (Brenner et al, 2000) except that after DpnII digestion Mmel was used to generate 20 bp tags. Tags were sequenced using the Clonal Single Molecule Array technique (Illumina, Hayward, CA, US). Programs were developed in the programming language Python to analyze the data. Tag counts were normalized to transcripts per million (TPM). Tags with a maximum of < 4 TPM were removed from the data set. TPM values of tags originating from the same transcript were summed to assess their expression levels. MPSS expression analysis agreed with expression studies that have been performed in the past (for a review see Wosten, H.A.B. & Wessels, 2006).

Promoter analysis

The MEME Motif Discovery Tool (Bailey et al. 1994) was used to identify motifs in promoter sequences (defined here as the 1000 bp region upstream of the translation start site, containing the putative core promoter and upstream regulatory elements). The TOMTOM Motif Comparison Tool (Gupta et al. 2007) was used to compare the identified motifs to known motifs in the TRANSFAC database (Wingender et al. 2000).

Subcloning of dTomato

The coding sequence of the gene encoding the red fluorescent protein dTomato (Shaner et al. 2004) was mutagenised in order to remove the internal Ncol site from the coding sequence by changing nucleotide 423 from a cytosine into a guanine without changing the amino acid sequence, using the procedure described previously (Ko et al, 2005). Furthermore, an Ncol site at the 5' end of the gene and a BamHI site at the 3 ' end of the gene were incorporated. The primers are listed in Table 4. The resulting dTomato gene was cloned into pGEM®-T Easy Vector (Promega), resulting in plasmid pRO020. The gene was excised using the restriction enzymes Ncol and BamHI and ligated into plasmid pGDSi3, which had been opened with the same enzymes. This resulted in plasmid pR0151, which is a pUC vector containing the gpd promoter, the dTomato coding sequence, an intron, the sc3 terminator and the phleomycin resistance cassette.

Table 4. Primers used to amplify coding sequences of dTomato, wc2 and fst4

Subcloning of hsp promoters

The regions upstream of the predicted translation start site of three heat shock genes were amplified from S. commune chromosomal DNA using Phusion polymerase (Finnzymes, Finland). Primer pairs, product sizes and introduced restriction sizes are given in Table 5. The amplified fragment is cloned into the Smal site of pUC20, resulting in the plasmid pR0195, pR0196, pR0197 and pR0198 for hspl, hsp2, and hsp3, respectively. The promoters were excised using the respective restriction enzymes in Table 5 and ligated into pR0151 which had been opened with Hindlll and Ncol. The resulting plasmids were pR0199, pRO200, and pRO201 for hspl, hsp2, and hsp3, respectively. The resulting plasmids consist of a pUC vector containing the promoter of the respective heat shock gene, the coding sequence of dTomato, an intron, the sc3 terminator and the phleomycin resistance gene. Table 5. Primers used to amplify the upstream regions of three heat shock genes.

a: restriction with this enzyme results in sticky ends compatible with Hindlll. b: restriction with this enzyme results in sticky ends compatible wit

Construction of a hsp3_vrom - wc2 and a hsp3_vrom - fst4 construct

The coding sequence of the putative blue light receptor WC2 (proteinID 13988) was amplified from S. commune chromosomal DNA using Phusion polymerase (Finnzymes, Finland) and the primers WC2CodingFw and WC2CodingRev (Table 4), which introduce an Ncol and a BamHI site, respectively. The amplified fragment was 1760 bp in length and was cloned into the Smal site of pUC20, resulting in the plasmid pRO203. The coding sequence of wc2 was excised using the restriction enzymes Ncol and BamHI and was cloned into the plasmid pRO201 , which was opened with the same enzymes, resulting in plasmid pRO205.

The coding sequence of the putative transcription factor FST4 (proteinID 66861) was amplified from S. commune chromosomal DNA using Phusion polymerase (Finnzymes, Finland) and the primers Fst4CodingFw and Fst4CodingRev (Table 4), which introduce an Eco31I and a BamHI site, respectively. The amplified fragment was 3854 bp in length and was cloned into the Smal site of pUC20, resulting in the plasmid pRO204. The coding sequence of fst4 was excised using the restriction enzymes Eco31I (resulting in a sticky overhang that is compatible with Ncol) and BamHI and was cloned into the plasmid pRO201 which was opened with the restriction enzymes Ncol and BamHI, resulting in plasmid pRO207. Transformation of S. commune

Transformation of S. commune strain 4.8 was done according to Van Peer et al. (2009). Regeneration medium and selection plates contained 5 and 25 μg ml^"1 phleomycin, respectively. Plates were incubated at 30 °C for 2 days and the resulting colonies were grown on a second selection plate containing 25 μg ml^"1 phleomycin. Per construct at least 50 colonies were screened.

Fluorescence microscopy

Fluorescence of dTomato was visualised using the Leica MZ16F stereomicroscope with dsRED3 filters (Leica Microsystems, Germany).

Results

Heat shock genes in the genome of S. commune The genome of S. commune contains 7 genes encoding putative small heat shock proteins (Table 6). These genes have been annotated as "Molecular chaperones (small heat-shock protein Hsp26/Hsp42) (KOG0710)" based on sequence similarity (Koonin et al. 2004). Promoter analysis showed that the promoters of these 7 genes all contain at least one sequence containing 3 inverted repeats of the sequence nGAAn, varying between 100 bp and 350 bp upstream of the predicted translation start site (see Figure 1). The consensus sequence showed significant similarity with a heat shock factor binding site (TRANSFAC motif M00169, p-value 7.8e-l l), indicating that these promoters are likely to be regulated by this transcription factor.

Table 6. Putative heat shock genes in the genome of S. commune and their expression in four developmental stages as determined by Massively Parallel Signature Sequencing (MPSS). Samples were grown at 25 °C. n.d.: non-detectable (expression < 4 tpm)

Gene expression analysis using MPSS showed that hspl-3 were not expressed in any of the developmental stages when grown at 25 °C (Table 6). In contrast, hsp4-7 were expressed under these conditions in at least 1 developmental stage. Only hsp7 was found to be highly expressed in one of the developmental stages when grown at 25°C. The 1000 bp promoter fragments of hspl-3 were cloned in front of the coding sequence of the red fluorescent protein dTomato and introduced in the monokaryotic strain 4-8 of S. commune. 50 transformants of each construct were screened. The transformants were not fluorescent when grown at 25 °C. This was expected on basis of the MPSS analysis. After an incubation of 1 hour at 42 °C, about 75 % of these colonies did show fluorescence of dTomato. This was irrespective of the promoter that was used (data not shown). Absence of fluorescence at 42 °C in 25% of the transformants can be explained by a single cross-over event in the hsp promoter or dTomato gene. This part makes up about 25% of the expression construct.

Expression of the hsp3 promoter was studied in more detail in strain scRO034. 5 day old colonies that were grown at 25 °C were incubated for 1 hour at either 25 °C, 30 °C, 37 °C or 42 °C, after which they were placed back at 25 °C. After 1 hour, no fluorescence was observed in colonies incubated at 25 °C or 30 °C (Figure 2). However, weak fluorescence was observed after incubation at 37 °C and strong fluorescence at 42 °C. Monokaryotic and dikaryotic colonies gave an identical pattern of fluorescence. Wild type colonies showed no fluorescence in any of the conditions (data not shown).

A hot needle (1 mm in diameter; heated to 100 °C in boiling water) was used to locally heat the mycelium for 10 seconds. After 5 hours, fluorescence was only observed in a ring around the heated area (Figure 3). The wild type showed no fluorescence. This shows that the hsp3 promoter can be used to locally induce gene expression.

Importantly, the local or systemic heat shocks that were used in this study showed no effect on colony diameter, biomass and, in the case of a dikaryon, on number and shape of the mushrooms.

Inducible mushroom formation in a wc2 deletion mutant

The promoter of hsp3 was used to induce expression of the transcriptional regulator wc2 (see Table 3 for details) in a Awc2Awc2 dikaryon and thus complement the deletion. Colonies were pre-grown for 4 days at 25 °C and then, once a day, transferred to a 42 °C stove for one hour. The negative controls were kept at 25 °C. In these negative controls, there was no difference between the Awc2Awc2 strain and the Awc2Awc2 strain containing the hsp3_prom-wc2 construct, confirming that the hsp3 promoter is not active at 25 °C (Figure 4). The wild type and Awc2Awc2 strain that were incubated at 42 °C showed no changed phenotype compared to the strains grown at 25 °C, showing that a heat shock treatment has no apparent effect on these colonies. On the other hand, after 2 days the Awc2Awc2 strain containing the hsp3_prom-wc2 construct that was incubated at 42 °C showed irregular growth at the periphery of the colony, similar to the wild type (data not shown). After 4 days, primordia were visible, which formed mature fruiting bodies (Figure 4).

Formation of fruiting bodies could also be induced by a heat treatment after introduction of a hsp3_prom-fst4 construct in a Afst4Afst4 dikaryon (for details about fst4 see Table 3).

These results show that the promoter of hsp3 can be used to induce mushroom formation, for example by applying a daily heat pulse of 1 hour at 42 °C.

Table 7. Domains of S. commune transcription factors that are conserved in C. cinerea and L. bicolor. (SEQ ID NO: 208-215)

Transcription Domain Domain Domain sequence

factor start stop

Bril 283 358 RRKIEYVPFAREVDTFGGRDLAALEKYAE

EARRRPIRDFNDWGNIDVDHLIMSLRSRVA

TELSYALTTLSMLSAMR

C2h2 192 247 KKHVCTTCNKRFNRPS SLRIHLNTHTGATP

FRCPWPHCGREFNVNSNMRRHLRNHT

Fst3 593 716 IAMHFAKHSAATALIDGWKSVELCQAYIL

MSIYAVPARRWEEDRSWLYTGLAIRIATDL NLHQVSTAKPS SERHEREILNRTRVWLICF NLDRSTATQFGKPSTIKEDYIVQHAKDWY KKSKYN

Fst4 329 463 LSPRRLALLLMVLSIGSLVDLKRPLGYLSA

EAYHHLARASVCEIPLMEEPDFDTVHALFF MI WYHLIF SDNRKALGYAWNLLGF VAKL VQGVHRETSGSSKLIPEESERRRNIFWELLN LDYRMSLTLGRPPSIS

Gatl 221 291 VQHTDDAASKETQYLRRRCFNCHTTEPPS

WRRSTLNPGKIVCNKCGLYERTHLRPRPLR

FDELRAGSKTRK

Homl 222 228 KKKRKRADANQLRVLNDVYMRTAFPSTE

ERHQLAKQLDMSPRSVQIWFQNKRQAMR STNRQ

Hom2 67 139 DYRTFFPYQPNEVKHRRRTTAVQLKVLEGI

FKTETKPNAALRNKLAVQLEMTARGVQV WFQNRRAKEKLKASK

Wc2 27 83 FTKRKRWADLLVTELADAIILVLGVPNPKI

LYCGAAVEELLGWRDTDVIDLDLTELM

Table 8. Relationship Protein ID (see http://jgi.doe.gov/Scommiine; table 1) and Protein Domain (see Table 7) and SEQ ID NO's as used herein.

SEQ ID SEQ ID SEQ ID

ProteinID NO: ProteinID NO: ProteinID NO:

84275 8 86194 35 257652 62

112067 9 66861 36 114363 63

75142 10 255327 37 103949 64

255701 11 84267 38 250177 65

17463 12 84657 39 258543 66

80413 13 102836 40 85474 67

269940 14 102971 41 11542 68

68168 15 103145 42 110354 69

80935 16 105299 43 230646 70

65208 17 108072 44 110178 71

269941 18 269950 45 237000 72

81364 19 110010 46 110445 73

236086 20 110310 47 269975 74

258244 21 110595 48 256910 75

233954 22 269932 49 84085 76

81115 23 111234 50 104304 77

50846 24 269957 51 111555 78

269944 25 113625 52 258217 79

11907 26 114874 53 257931 80

13988 27 230844 54 269938 81

255004 28 233513 55 110229 82

85539 29 236631 56 112017 83

83110 30 255185 57 110458 84

16376 31 255490 58 255386 85

53446 32 255941 59 14572 86

67562 33 256135 60 81262 87

54452 34 257422 61 269939 88 SEQ ID SEQ ID SEQ ID

ProteinID NO: ProteinID NO: ProteinID NO:

48318 89 233370 123 81806 157

257380 90 256320 124 84684 158

112634 91 109936 125 269945 159

255183 92 114988 126 254988 160

80526 93 111683 127 255385 161

107138 94 230584 128 104000 162

236743 95 232060 129 269948 163

105290 96 66326 130 269949 164

84273 97 78316 131 256993 165

254923 98 86018 132 108591 166

71685 99 257247 133 109190 167

237374 100 232448 134 269952 168

255836 101 232514 135 110136 169

255161 102 63699 136 110416 170

62967 103 111405 137 250298 171

79748 104 231700 138 111623 172

82694 105 256693 139 269956 173

112825 106 257455 140 231698 174

257915 107 258832 141 234557 175

103232 108 52392 142 234560 176

81412 109 257495 143 269958 177

269943 110 57298 144 255656 178

102719 111 57817 145 255852 179

255207 112 83015 146 256706 180

17379 113 269928 147 257056 181

73063 114 63410 148 257622 182

104375 115 66095 149 257926 183

85886 116 66586 150 257987 184

232127 117 73210 151 269960 185

257445 118 74309 152 257265 186

104344 119 74719 153 269961 187

81107 120 77191 154 248401 188

255863 121 78089 155 82883 189

232771 122 81726 156 84749 190 SEQ ID

ProteinID NO:

110478 191

254870 192

258883 193

108216 194

108605 195

102516 196

256746 197

258642 198

112405 199

83895 200

109596 201

269979 202

77161 203

233354 204

233946 205

12349 206

250247 207

Protein domain SEQ ID

NO:

Bril domain 208

C2h2 domain 209

Fst3 domain 210

Fst4 domain 211

Gatl domain 212

Homl domain 213

Hom2 domain 214

Wc2 domain 215 References

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Claims

1. A method for the production of a mushroom, mycelium, fungal cell, a fruiting body, a spore or a gene product of interest comprising the steps of:

a) culturing a fungal cell, fruiting body, spore, mycelium or mushroom comprising a nucleic acid molecule represented by a nucleic acid sequence coding for a gene product of interest wherein said nucleic acid sequence is operably linked to a heat inducible promoter functional in said mushroom, mycelium, fungal cell, fruiting body and/or spore;

b) treating the mushroom, mycelium, fungal cell, fruiting body, or spore with heat during part of the culturing under a); and

c) optional recovery of the mushroom, mycelium, fungal cell, fruiting body, spore or gene product of interest.

2. A method according to claim 1, wherein the method comprises the step of transforming a mushroom, mycelium or a fungal cell with an expression vector comprising the nucleic acid sequence coding for a gene product of interest operably linked to the heat inducible promoter.

3. A method according to claim 1 or 2, wherein the heat treatment is selected from the group consisting of:

i) 2 minutes to 2 hour placement in an incubator of 37-95 °C;

iii) 0.01 sec to 16 hour exposure to light, preferably laser light and

4. A method according to any one of the preceding claims, wherein the heat inducible promoter is from a basidiomycete, more preferably an Agaricales, more preferably a schizophyllaceae, even more preferably a Schizophyllum, most preferably Schizophyllum commune.

5. A method according to any one of the preceding claims, wherein the heat inducible promoter is represented by:

i) any of SEQ ID NO:3, 1 , 2, 4-7 or has at least 60% identity with any of SEQ ID NO:3, 1, 2, 4-7;

ii) a nucleic acid sequence of 100-1000 bp upstream of the start codon of a heat shock protein, said heat shock protein having at least 60% identity or similarity with any of SEQ ID NO: 263-269;

and/or

iii) the following nucleic acid sequence comprising:

GAAX1X2X3TCX4X5GX6X7 (SEQ ID NO:262), wherein X_u X₂ are A, G, C, T; X₃ is T or G; X₄ is C, T, or G, X₅ is A, G or T, X₆ is A or T and X₇ is A or C.

6. A method according to any one of the preceding claims, wherein the gene of interest is a gene which upon expression of the gene product of interest induces mushroom formation.

7. A method according to any one of claims 1 - 5, wherein the gene of interest is a gene which upon expression of the gene inhibits mushroom formation.

8. A method according to any one of claims 1 - 5, wherein the gene of interest is represented by a nucleotide sequence encoding a polypeptide that comprises an amino acid sequence:

(a) that has at least 40 % amino acid identity or similarity with a sequence selected from SEQ ID NO: 36, 8-35, 37-207; and/or,

(b) that has at least 50% amino acid identity or similarity with a sequence selected from SEQ ID NO: 21 1, 208-210, 212-215.

9. A heat inducible promoter having a length of 100 - 1000 base pairs and comprising a nucleic acid sequence represented by any of SEQ ID NO: 1-7 or having at least 60% identity with any of SEQ ID NO: 1-7.

10. A heat inducible promoter having a length of 100 - 1000 base pairs, comprising the following nucleic acid sequence: GAAX1X2X3TCX4X5GX6X7 (SEQ ID NO:262), wherein X_{l s} X₂ are A, G, C, T; X₃ is T or G; X₄ is C, T, or G, X₅ is A, G or T, X6 is A or T and X₇ is A or C.

11. A heat inducible promoter having a length of 100 - 1000 bp and which is present upstream of the start codon of a nucleic acid sequence encoding a heat shock protein having at least 60% identity or similarity with any of SEQ ID NO: 263-269.

12. A nucleic acid construct comprising the promoter as defined in any one of claim 9 to 11.

13. A nucleic acid construct according to claim 12, wherein the nucleic acid construct further comprises a gene of interest operably linked to the heat inducible promoter.

14. An expression vector comprising a nucleic acid construct of claim 12 or claim 13.

15. A fungal cell, a mycelium a spore, a fruiting body or a mushroom, comprising a nucleic acid construct as defined in claim 12 or claim 13 or an expression vector according to claim 14.