Background

Coprinopsis cinerea is a well-known model fungus for studying biological processes in Agaricomycetes. As early as in 1987 and for one of the first fungi of all, protoplast transformation of C. cinerea was successfully established by Binninger et al. [1]. For DNA transformation, protoplasts are usually generated from easy to regenerate single-celled haploid aerial mitotic spores (oidia) and are commonly treated in PEG 4000/CaCl2-mediated cold-shock transformation with ca. 1 µg plasmid DNA. The protocol is highly efficient with in best cases up to several hundreds of transformants per µg DNA [1,2,3,4]. Up till today, the protoplasting and transformation protocol of Binninger et al. [1] has not much been changed in the principles. However, the method was later more simplified and specified in details as compared to the original description [2, 3]. Comprehensive troubleshooting tips have been provided to identify and correct possible subconscious while crucial small handling errors in order to ensure reliable transformation [4].

One reason for the very high transformation rates of C. cinerea is that mostly homologous selection markers are used for the complementation of auxotrophies. The bifunctional tryptophan synthase gene trp1+ cloned in the pUC9-based 9.8 kb-sized plasmid pCc1001 [1] is so far most often applied in transformation. More recently, the shorter pBluescript KS−-based trp1+-plasmid pBD5 (7 kb) with higher copy number in Escherichia coli and the trp1+ yeast-shuttle vector pYtrp1 (9.9 kb) have been established [5]. The two gene halves of trp1+, i.e. trpA+ for the Trp1 A domain responsible for the aldo-cleavage of indole-3-glycerol-phosphate (IGP) into indole and trpB+ for the Trp1 B domain for the subsequent pyridoxal phosphate cofactor-dependent conversion of indole with serine to tryptophan [5], have been functionally separated into individual yeast-shuttle vectors pYAdom (8.3 kb) and pYBdom (8.7 kb) to allow successive rounds of transformation into C. cinerea trp1.1,1.6 double mutant strains with first trp1.6 (trpB) and then trp1.1 (trpA) complementation [6].

Two other genes from the tryptophan biosynthesis pathway cloned in vectors for transformation of suitable C. cinerea mutant strains are trp2+ [2] for a trifunctional enzyme with glutamine amidotransferase (GATase; anthranilate synthase component II which releases ammonia from glutamine), phosphoribosylanthranilate isomerase (PRAI) and indol-3-glycerol-phosphate synthase (IGPS) activities [5], and the gene trp3+ [7, 8] for anthranilate synthase component I which uses ammonia and chorismate to produce anthranilate, 2-aminobenzoic acid [5]. Cloned is also a positively selectable mutant gene trp3iar for a dominant 5-fluoroindole-resistant anthranilate synthase component I mutant [9]. pab1+ vectors [3, 10] have been provided for complementation of auxotrophies in para-aminobenzoic acid (PABA) synthesis caused by defects in the bifunctional enzyme Pab1. Conventionally, this fungal enzyme is known as PABA synthase but more precisely, it is a 4-amino-4-deoxychorismate (ADC) synthase. The enzyme consists of an N-terminal PabA domain (37% identity, 53% similarity to E. coli PabA; Fig. 1a) and a C-terminal PabB domain (30% identity, 49% similarity to E. coli PabB; Fig. 1a). PabA presents PABA synthase component II (or better called ADC synthase component II) and has a PabB-dependent GATase function. The PabB domain as PABA synthase component I (or more precisely ADC synthase component I) will aminate chorismate in order to yield ADC as the direct precursor of PABA to be formed by an ADC lyase (PabC) [11, 12]. Regarding further functional C. cinerea selection markers, a cosmid is mentioned in a conference proceeding that could complement an uncharacterized ade8 defect of C. cinerea in transformation [13].

Fig. 1
figure 1figure 1

Alignment of A. wt Pab1 from C. cinerea monokaryon OK130 (CcPab1) with PabA (EcPabA, underlaid in yellow) and PabB of E. coli (EcPabB, underlaid in dusky pink) and B. wt Ade8 from C. cinerea strain AmutBmut (CcAde8) with PurD (EcPurD, underlaid in yellow) and PurM of E. coli (EcPurM, underlaid in dusky pink), respectively. a The catalytic triad, glutamine binding residues and residues involved in ammonia tunnel formation in PabA are marked with red, green and blue symbols *, respectively. Other residues affecting enzymatic activities and bonding to PabB are marked with grey squares. The position of a stabilizing residue stretch called oxyanion hole is underlaid in light blue, a sequence stretch for chorismate signal transfer in olive [29, 30, 75]. Red letters in PabB mark helical regions, blue letters β-sheets. The conserved PIKGT motif, sequences for interaction with PabA, for signal transfer of chorismate binding, and of a binding pocket for tryptophan implicated in structural stabilization are underlaid in olive, bright yellow, grey and light blue, respectively. The residue K in the PIKGT motif which is mutated in C. cinerea AmutBmut (K546E) is marked in red. Symbols * in red and black mark (predicted) active site residues and Mg2+-binding residues in two chorismate-interacting helices, respectively. Triangles in black indicate residues that contact the bound tryptophan and grey squares further residues where mutations affect functionality [28,29,30,31, 76]. b Red, blue, green and magenta letters mark the N, B, A, and C domains of PurD. The positions of the P-loop and the flexible A and B loops in PurD [56] are underlaid in light blue, olive and orange, respectively. Symbols * in black, red, and blue mark residues that recognize the adenine base, ribose and phosphate of the nucleotide, whereas grey squares indicate residues interacting with the ligand PRA [56, 57]. The residue N in the A loop which is mutated in C. cinerea OK130 (N231D) is marked in red. In PurM, symbols * mark (predicted) nucleotide binding residues and triangles (in grey predicted) binding sites of the substrate N-formylglycinamidine ribonucleotide (FGAM) [58]

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Selection for dominant resistances is another strategy to obtain transformants. A carboxin resistance selection marker (sdi1R) has been generated by site-specific mutation of the native C. cinerea sdi1 gene for the iron-sulphur protein subunit (subunit SdhB) of the mitochondrial succinate dehydrogenase (SDH) complex [14]. Flutolanil and carboxin resistance is moreover mediated through a spontaneous point mutation by an allele of the sdhC gene for the SdhC cytochrome b560 subunit of the SDH complex [15]. The sdi1R allele has been cloned behind the heterologous constitutive gpdII promoter of Agaricus bisporus [14] which is highly active in C. cinerea [16]. Transformation rates of such optimized sdi1R vectors were then high with > 100 transformants/µg plasmid DNA [14]. Transformation rates with the sdhC mutant allele under natural regulatory sequences in contrast were low with 1.0 to 4.8 transformants/105 viable protoplasts [15].

As functional bacterial resistance genes in C. cinerea, vectors with the E. coli hygromycin B phospotransferase gene hph [14, 17] and the Streptoalloteichus hindustanus gene ble for a phleomycin binding protein are available [14]. Insertion of a functional intron after the second codon of the ble gene was essential for successful expression of the gene in C. cinerea behind the A. bisporus gpdII promoter [14]. Regarding expression of hph, presence of an intron was not crucial. However, the entire coding region of hph is required to be inserted behind an active promoter in C. cinerea (native tub1 promoter or heterologous A. bisporus gpdII promoter) [14, 17]. The best-known hph-vector pAN7-1 from transformation in filamentous ascomycetes for example lacks the first two codons for two lysine residues and by this reason did not function in C. cinerea transformation [14] unlike, although at low frequency (1 to 5 transformants/µg plasmid DNA), in the basidiomycetes Hebeloma cylindrosporium [18] and Crinipellis perniciosa [19].

The obvious advantage of usage of dominant resistance markers for selection is that transformation becomes independent of any auxotrophies that are needed to be generated. Though, using dominant resistance markers for C. cinerea somewhat complicates the transformation procedure. Protoplasts are spread onto regeneration agar but for suppression of unwanted background growth, it requires an extra regeneration agar overlay with antibiotics for selection for positive transformants to grow through this overlay [14, 16]. Handling of complementation of auxotrophies in transformation in contrast is much easier by just plating and then incubating protoplasts on regeneration agar [2,3,4]. However, through complementation of available auxotrophies and selections for dominant resistance markers, extra rounds of successive transformations in a same background become possible. Such makes strains more versatile for repeated genetic manipulations.

So far, the genomes of two distinct C. cinerea strains, the monokaryon Okayama 7/#130 (short OK130) and the self-fertile homokaryon AmutBmut, have been sequenced by the Broad Institute (Boston, MA) and the JGI (Joint Genome Institute, Walnut Creek, CA), respectively [20, 21]. AmutBmut carries a pab1-1 mutation and is easily be transformed by pab1+ vectors, a feature which is very useful in studying dikaryon-specific growth behavior and fruiting body development in this self-fertile strain, independently of a second genome [22,23,24]. On the other hand, to the best of our knowledge, strain OK130 with the first C. cinerea reference genome established had not yet been transformed before. This reference monokaryon carries an ade8-1 mutation [8] which we used here in transformation for selection by complementation. Missense mutations in the defective alleles pab1-1 and ade8-1 were identified in this study. In addition, transformants of OK130 were obtained with the dominant bacterial hygromycin resistance selection marker hph.

Results and discussion

Genes pab1 and ade8 in C. cinerea

Classical map** of C. cinerea localized gene pab1 0.5 cM upstream and gene ade8 1.3 cM downstream to the bipartite A mating type locus (consisting of Aα and Aβ) on linkage group I [25, 26]. The ca. 20 kb-long A43 mating type allele with all its homeodomain transcription factor genes locates at position Chr_1:2,666,138–2,647,809 in the sequenced OK130 genome [20, 27]. pab1 [11] is found at location Chr_1:2,699,078–2,701,362, 32.94 kb apart from the 3′ end of the closest A43α gene a1-1 [20, 27]. pab1+ in OK130 (Broad model CC1G_01849T0) distinguishes from the pab1-1 allele in AmutBmut (JGI ID 414607) by a point mutation in codon 546, with a change from AAG to GAG. This missense mutation resulted in a K546E exchange in the PabB domain within the highly conserved ADC synthase component I motif PIKGT. Lysine in the wildtype (wt) covalently binds to the C2 of chorismate to initiate with the ammonia-group of glutamine the enzymatic formation of ADC ([28,29,30,31], Fig. 1a).

The recombination rate between pab1 and Aα calculates as ≥ 66 kb/cM (≥ 70-75 kb/cM with the whole pab1 gene sequence included [8, 32]). Other studies estimated the average recombination frequency over the C. cinerea genome higher as 27.9 kb/cM [33] and 33 kb/cM [20], respectively. With the same kb/map unit relations, ade8 should then locate about 40 to 100 kb downstream of Aβ. A gene for a bifunctional purine biosynthetic protein (CC1G_01782T0; Table 1) was found in the OK130 genome at location Chr_1:2,548,109–2,550,858, 97 kb downstream to the closest A43β gene d1-1 [20, 27], with a possible recombination rate of 74.6 kb/cM using 1.3 cM for calculation.

Table 1 Identification of gene functions in de novo purine biosynthesis, formation of folates and THF-mediated one-carbon metabolism in C. cinerea OK130
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Many mutations leading to adenine-auxotrophies belong directly to the de novo purine biosynthesis pathway [34,35,36]. Other indirect mutations include defects in tetrahydrofolate (THF) cofactor formation, further folate metabolism and THF-mediated C1-metabolism, as well as defects in cross-pathway regulation of de novo purine biosynthesis and syntheses of amino acids (histidine, methionine) mediated by feedback control of certain metabolites [5´-phosphoribosyl-5-monophosphate (AICAR)] or shared transcriptional regulators [35, 37,38,39,40,41,42,43,44,45,46,47,48]. We screened the OK130 genome for such genes, using known E. coli and Saccharomyces cerevisiae proteins in tblastn searches. Spread over 7 chromosomes, genes for all enzymatic functions for de novo purine biosynthesis and for other mentioned functions were found (Table 1). Previously, twelve different ade complementation groups have been described in C. cinerea, two more mutants that react to adenine and histidine (ad/his1 and ad/his2) and another that reacts alternatively to adenine or methionine (ad/met) [49, 50]. Ten of these genes have been mapped onto 7 linkage groups [50,51,52]. Though, in our analysis only four to possibly seven genes (ade2, ade8, ade1, ade5, and possibly ade4, ade9, and ade12) from only four linkage groups could be assigned to specific positions on sequenced chromosomes (Table 1), using as additional information their clearly defined biochemical reactions (cases ade1, ade5 [49]) or approximate positions in the de novo purine biosynthesis pathway (ade2, ade3, ade4 and ade8 all act prior to imidazole ring closure [49]) and/or their linkages (ade2, ade3, ade5, ade8, ade9 and ade12) to other unquestionably identifiable gene functions on the classical C. cinerea map ( [33, 50,51,52]; see footnote of Table 1). However, no other convincing candidate for gene ade8 were found in appropriate distance to the A locus on chromosome 1 (Table 1).

The protein encoded by the gene at Chr_1:2,548,109–2,550,858 has been annotated in GenBank (EAU92737.2) as ADE1 [Coprinopsis cinerea Okayama 7/#130] which conflicts the traditional C. cinerea gene nomenclature. C. cinerea gene ade1 resides on linkage group IV of the fungus [51, 52] which corresponds to chromosome 5 in the OK130 genome assorted by chromosome sequence length ( [20], Table 1). Moreover, Ade1 of C. cinerea had been shown in the de novo purine biosynthesis to function in the 6th step directly after 5-aminoimidazole ribonucleotide (AIR) ring closure as phosphoribosylaminoimidazole carboxylase in the formation of 5-amino-4-imidazolecarboxamide ribonucleotide (CAIR) ( [49], Table 1).

The gene at location Chr_1:2,548,109–2,550,858 has homologs in other fungi that, by historical naming of adenine-auxotrophic mutants, are variably known as ade1 such as in Phanerochaete chrysosporium, ade5 in Schizophyllum commune, ade2 in Neurospora crassa, ade5,7 in S. cerevisiae and pur2, pur2,5 and pur2,7 in Yarrowia lipolytica, Ogataea angusta and Scheffersomyces stipitis, respectively (Fig. 2). Gene ade5+ of S. commune can complement ade1 defects of P. chrysosporium like the homologous native ade1+ gene and it can complement ade2 defects of the ascomycete N. crassa [53, 54]. All mentioned fungal genes encode bifunctional enzymes for the de novo biosynthesis of purines, with an N-terminal glycinamide ribonucleotide synthase (GARS) domain and a C-terminal aminoimidazole ribonucleotide synthase (AIRS) domain (Fig. 1b; Table 1) which act in the 2nd and the ring-closing 5th step in de novo purine biosynthesis, respectively [34,35,36]. ade5 of S. commune and ade8 of C. cinerea are conserved in chromosomal location relative to the position of Aβ, similar as their pab1 genes are relative to Aα [8, 32, 55]. The gene for a bifunctional GARS-AIRS enzyme identified here on C. cinerea chromosome I with good likelihood thus presents its ade8 gene.

Fig. 2
figure 2

Neighbor-joining phylogenetic tree of bifunctional fungal GARS-AIRS enzymes clustering according to fungal clades. Note that corrections in exon/intron splicing sites have been done for the OK130 Ade8 model (GenBank EAU92737.2 = Broad model CC1G_ 01782T0 = JGI ID 1589), following the RNAseq-supported model for the ade8+ gene of strain AmutBmut (JGI ID 414375). The Drosophila melanogaster Ade3 protein used as outgroup is trifunctional with GARS, AIRS and GART domains, the latter of which was excluded from the analysis

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The N-terminal halves of the fungal bifunctional GARS-AIRS enzymes correspond to bacterial PurD enzymes (49% identity, 67% similarity between the C. cinerea enzyme and E. coli PurD; Fig. 1b) which are glycinamide ribonucleotide (GAR) synthases represented in structure e.g. by the crystalized E. coli PurD protein (1GSO_A). PurD catalyzes the 2nd step of the de novo purine biosynthetic pathway, the conversion of phosphoribosylamine (PRA), glycine, and ATP to GAR, ADP (adenosine diphosphate), and phosphate (Pi) ( [35, 56, 57], Table 1). The C-terminal halves of the fungal bifunctional GARS-AIRS enzymes are homologous to bacterial PurM enzymes (55% identity, 67% similarity of the C. cinerea enzyme to E. coli PurM; Fig. 1b). PurM represented in structure by E. coli 1CLI_A is a phosphoribosylformylglycinamidine cyclo-ligase that catalyzes the conversion of formylglycinamide ribonucleotide (FGAM) and ATP to AIR, ADP, and Pi, in the 5th step in de novo purine biosynthesis ( [35, 58], Table 1).

The folded bacterial GARSs consist of the three domains N, A, and C forming the central core of the enzyme and, connected to them by flexible hinges, the outward-extended domain B [56]. Substrate PRA is recognized by specific amino acids in the N, A, and C domains. The A domain further confers the binding site for the ligand glycine ( [56, 57], Fig. 1b). GARSs are members of the ATP-grasp superfamily of enzymes with an atypical ATP-binding site (ATP-grasp fold) comprised by the two domains A and B that catch an ATP between them [59]. Accordingly, the A and B domains primarily define the ATP/ADP binding site of GARSs, with distinct residues in domains A and B and also in N contacting the adenine base, ribose and phosphate, respectively ( [56, 57], please see Fig. 1b for details). Further, the A domain possesses a flexible specific A loop with a highly conserved unique sequence (DHKRVGDKDTGPNTGGMG in E. coli, see Fig. 1b) which distinguishes GARSs well from all other members of the ATP-grasp superfamily [56, 57, 59]. Structural analyses of bacterial enzymes revealed N226 in the E. coli A loop to recognize ribose [57]. The E. coli A loop shares 83–89% sequence identity and 94% sequence similarity with the loops in the fungal enzymes analyzed in Fig. 2, with amino acid N231 of wt C. cinerea Ade8 = N226 in PurD of E. coli (Fig. 1b). Sequence comparison between the functional ade8+ copy from AmutBmut and the defective ade8-1 allele in OK130 revealed a point mutation that altered codon 231 from AAT into GAT and then, within the flexible A loop in the GARS A domain, the highly conserved amino acid N231 into D231 (Fig. 1b). The D231 mutation in the N-terminal GARS half explains then the former observation that Ade8 acts prior to imidazole ring formation [49] and, more specifically, assigns the loss of the Ade8 function in OK130 to the 2nd step of de novo purine biosynthesis.

The pCcAde8 vector in fungal transformations

The wt genomic sequence with the ade8+ coding region (with 9 exons and 8 introns) and 483 and 569 bp upstream and downstream, respectively were PCR-amplified with chimeric primers Ade8f and Ade8r in order to construct vector pCcAde8 (Fig. 3) by in vivo recombination in yeast with plasmid pRS426 [60]. pCcAde8 was transformed into monokaryon OK130, alone and, using protoplasts from same batches, in parallel co-transformations with other vectors (Table 2). Adenine prototrophic transformants were selected by growth on adenine-free regeneration agar. Diagnosis PCR with amplicon sequencing verified for 25 transformants randomly chosen from group pCcAde8 + pYSK-lcc5 (experiment 1 in Table 2, 1st to 4th day of collection) in all cases the presence and function of the ade8+ allele.

Fig. 3
figure 3

Physical map of the yeast-E. coli shuttle vector pCcAde8 with the cloned C. cinerea gene ade8+

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Table 2 Transformations of C. cinerea OK130 (ade8-1) with ade8+-vector pCcAde8 alone or, using same batches of protoplasts, in combination with various pYSK7 laccase gene derivatives
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Transformation rates of OK130 to ade8+ prototrophy in single-plasmid and two-plasmid transformations were in ranges of about 40 to 60 clones each (Table 2). Gene ade8+ therefore might not confer any significant feedback inhibition on the de novo purine biosynthesis pathway in C. cinerea. On the contrary, the trp1+ selection marker of C. cinerea can cause suicidal feedback inhibition on tryptophan biosynthesis with loss of affected clones by a sudden overflow of the amino acid from more expressed trp1+ copies [5, 6]. This adverse effect on clone viabilities is greater with the single-plasmid transformation than when using mixtures of two plasmids, because singular plasmids in transformation without competition are likely to integrate into twice as many spontaneous DNA breaks per nucleus [5, 6]. As in our previous work with trp1.1,1.6 monokaryons [5, 6], reduced amounts of tryptophan prototrophs were obtained in only trp1+-vector pDB5 transformations of strains FA2222 and PG78 as compared to any co-transformations (Tables 3 and 4). pCcAde8 was newly tested in such co-transformations. Numbers of total transformants under trp1+ selection were about 1.5–2.5 times higher in the co-transformations with pCcAde8 than in the single-vector transformation, similar to results of co-transformations with other plasmids (Tables 3 and 4). In co-transformations of monokaryon PG78 with pab1+-vector pPAB1-2 for selection for PABA-prototrophy, total transformation rates were slightly higher with pCcAde8 (1.9 × and 1.3x) as compared to other plasmids and in single-plasmid transformation (Table 4). PABA is an intermediate in the biosynthesis of folate [61] which in turn is required in steps of de novo purine biosynthesis for the cofactor THF (Table 1). Co-transforming pab1+-vector pPAB1-2 with pCcAde8 might have an initial promoting effect on protoplast regeneration and clone numbers. Typically in transformations of C. cinerea with selection schemes other than adenine, we add adenine sulfate as optional supplement to regeneration agar (50 or 100 mg/l) [3, 4] because this can stimulate protoplast regeneration [advice by late L.A. Casselton kindly given to UK].

Table 3 Transformations of C. cinerea FA2222 (trp1.1,1.6) with plasmid pBD5 alone or, using same batches of protoplasts, in combination with other non-directly selectable vectors
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Table 4 Transformations of C. cinerea PG78 (trp1.1,1.6, pab1-1) with either trp1+ plasmid pBD5 or pab1+ vector pPAB1-2 alone or, using same batches of protoplasts, in combination with other non-directly selectable vectors
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Co-transformation of a selectable vector together with one or more other plasmids is an efficient means to introduce and find non-selectable genes in transformed C. cinerea clones [62]. Because we have a deeper interest in laccase functions and applications [16, 63,64,65,5. The fused sequences were then digested with EcoRV and ApaI and inserted into the EcoRV and ApaI polylinker sites of pCRII-hph.

Sequence analyses

The published genomes of monokaryon Okayama 7/#130 (https://mycocosm.jgi.doe.gov/Copci1/Copci1.home.html) and homokaryon AmutBmut (https://mycocosm.jgi.doe.gov/Copci_AmutBmut1/Copci_AmutBmut1.home.html) on the JGI Mycocosm side were used for defining chromosomal loci of genes of interest and obtaining relevant DNA and protein sequences. Protein sequences from E. coli and S. cerevisiae (Table 1) were used in tblastn searches. Homologous protein sequences retrieved from the JGI homepages and from NCBI were aligned by ClustalX 2.0 [73] and the MEGA 6.0 software was used with 1000 bootstrap values for constructing a neighbor-joining tree [74].