- Email: [email protected]
Molecular analysis of B mating type diversity in Lentinula edodes
Scientia Horticulturae 243 (2019) 55–63
Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Molecular analysis of B mating type diversity in Lentinula edodes a
a
a
a
a
T b
Byeongsuk Ha , Yoon Jung Moon , Yelin Song , Sinil Kim , Minseek Kim , Cheol-Won Yoon , ⁎ Hyeon-Su Roa, a b
Division of Applied Life Science and Research Institute of Life Sciences, Gyeongsang National University, Jinju 52828, Republic of Korea School of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea
A R T I C LE I N FO
A B S T R A C T
Keywords: B mating type Diversity Lentinula edodes Mating Pheromone Receptor
Pheromone and pheromone receptor genes in B mating type locus constitute B mating type of fungi belonging to Basidiomycota. In Lentinula edodes, multiple B mating types have been suggested based on genome sequence analysis and mating assay. Here we report finding of new alleles of pheromones (phbs) and pheromone receptors (rcbs) to constitute five alleles of rcb1 (rcb1-1∼rcb1-5) with nine associated phbs in Bα sublocus and three alleles of rcb2 (rcb2-1∼rcb2-3) with five associated phbs in Bβ sublocus. Each rcb was primarily associated with two phbs. Each phb-rcb pair was recurrently discovered as a distinct unit in various monokarytotic and dikaryotic strains, regardless of subloci. This allowed us to propose 15 B mating types through combinations of five Bα and three Bβ subloci. PCR analyses with primer sets probing alleles of rcb1 and rcb2 demonstrated that all monokaryotic strains contained one of the five rcb1s and one of the three rcb2s representing Bα and Bβ subloci, respectively, thereby enabling designation of their B mating types via combinations of Bα and Bβ. PCR analyses also revealed the presence of one or two alleles of rcb1 and rcb2 representing B mating type of each nuclei in dikaryotic cytoplasm. Further investigation of 111 dikaryotic strains, including 83 cultivated and 28 wild strains collected from East Asia, revealed that B8 and B11 mating types constructed by rcb1-3 + rcb2-2 and rcb1-4 + rcb2-2, respectively, were more prevalent in cultivated strains. In B mating type pairs representing each nucleus in dikaryons, B2B12, B4B8, and B8B11 were frequently occurring pairs in cultivated strains while none of them was found in wild strains. Such prevalence indicates that certain nuclei have been preferentially selected in the generation of cultivated strains. Our findings shed new light on the construction of B mating type in L. edodes and provide practical tools in the breeding of new cultivars.
1. Introduction Filamentous fungi belonging to Basidiomycota spend most of their life cycle as mycelia with one (monokaryon) or two nuclei (dikaryon) in the cytoplasm. For sexual reproduction, monokaryotic mycelia will mate with compatible partners to form fused mycelia which maintain two compatible nuclei inside cells. In fused mycelial cells, these two nuclei undergo mitotic division. Divided nuclei then move to next cells through clamp connections to establish dikaryons throughout connected mycelial cells (Kües, 2000). Dikaryotic mycelia can occasionally form fruiting bodies with basidia as sexual organs upon physico-chemical signals. In the basidium of heterothallic basidiomycetes, nuclear fusion (karyogamy) followed by meiotic division produces four sexual spores (basidiospores), resulting in monokaryotic mycelia upon germination (Kües, 2000). Self or non-self recognition in sexual reproduction is enabled by two unlinked mating type loci, A and B (Bakkeren et al., 2008; Casselton ⁎
and Olesnicky, 1998). The A mating type locus encodes two homeodomain proteins (HDs). Each HD proteins forms heterodimeric transcription factor with HD protein from compatible partner to control mating events, including nuclear pairing, clamp cell formation, and coordination of nuclear division (Casselton and Olesnicky, 1998). The B mating type locus is consisted of pheromone and pheromone receptor genes (Kües, 2015). Mating pheromones in basidiomycetes contain CaaX motif, implying their localizations to cell membrane through acylation at the cysteine residue (Huyer et al., 2006; Michaelis and Barrowman, 2012), similar to a-factor of ascomycete fungi (Caldwell et al., 1995). Pheromone receptors in basidiomycetes are homologous to Ste3p (a-factor receptor) of yeasts (Kües, 2015). Pheromone and receptor interaction is thought to activate the mating pathway involving MAP kinase-dependent signal transduction (Casselton and Olesnicky, 1998; Raudaskoski et al., 2012). Both mating types can be multiple to increase chances of inter-strain mating in nature. Multiplicity of mating types arises from allelic
Corresponding author. E-mail address: [email protected] (H.-S. Ro).
https://doi.org/10.1016/j.scienta.2018.08.009 Received 26 June 2018; Received in revised form 27 July 2018; Accepted 7 August 2018 0304-4238/ © 2018 Elsevier B.V. All rights reserved.
Scientia Horticulturae 243 (2019) 55–63
B. Ha et al.
(CCDBM, University of Incheon, Korea), Korea Forest Research Institute (KFRI, Korea), National Institute of Agricultural Sciences (NAAS, Korea), and Forest Mushroom Research Center (FMRC, Korea) (Supplementary Table S1). Of these 111 strains, 83 were cultivated strains collected from East Asian countries, including 18, 20, and 45 strains from China, Japan, and Korea, respectively, and 28 were wild strains collected from mountainous areas in southern part of Korean peninsula. Mycelia of L. edodes were grown on potato dextrose agar (PDA, Oxoid, UK) or in potato dextrose broth (PDB, Difco, USA) at 25 °C. Mating analysis and genomic DNA extraction were performed as described previously (Ha et al., 2018).
variations of genes residing in multiple subloci constituting both A and B mating type loci. Coprinopsis cinerea has a, b, and d subloci in the archetypal A mating type locus and Groups 1, 2, and 3 subloci in the B mating type locus consisted of HD1-HD2 gene pairs and pheromonereceptor pairs, respectively (Riquelme et al., 2005; Kües, 2015). Allelic variations in genes at each sublocus together with combinations of subloci have generated more than 240 A and 70 or 79 B mating types (Riquelme et al., 2005; O’Shea et al., 1998). Similarly, it has been reported that Schizophyllum commune contains 288 A and 81 B mating types (Raper et al., 1958; Kües, 2015). Mating behavior of Lentinula edodes has been of our interest because of its practical importance in the mushroom industry (Ha et al., 2015). Similar to C. cinerea and S. commune, L. edodes carries multiallelic A and B mating types. Through mating analyses of wild populations, Tokimoto et al. (1973) have suggested 40-65 As and 63–100 Bs from 33 Japanese wild strains. Later, Lin et al. (2003) have reported 66 As and 72 Bs (predicted to be 121 A and 151 B) from 53 Chinese wild strains. However, these numbers have inherent uncertainty because they have been mainly estimated by mating analyses that rely on empirical observations such as the presence of clamp connections and the formation of barrage lines as measures of successful mating. To establish more reliable method for estimating mating types, we have focused on allelic variations in pheromone and receptor to see if we can employ them to verify B mating types. Using similar approach, we have recently reported variable sequence regions in A mating type loci of L. edodes that represent different mating type alleles (Ha et al., 2018). Genome analyses of three monokaryotic strains (939 P26, 939 P42, and SUP2) of L. edodes have revealed that the genetic structure of B mating type locus is more complex than that of A mating type locus (Wu et al., 2013). Each B mating type locus consists of two mating typespecific receptors (rcbs), one or two pheromone genes (phbs), and two non-mating type rcbs. The whole gene-set in this locus lies in a single chromosome with an approximate length of 37 Kbp. Mating type-specific receptors rcb1 and rcb2 are found together with their associated phbs whereas non-mating type receptors (rcb3 and rcb4) are devoid of associated phb gene. rcb1 and rcb2 with their coupled phbs reside in subloci Bα and Bβ, respectively, to constitute the B mating type locus. Recent genome analysis of L. edodes strain W1-26 has revealed the same Bα and Bβ subloci as strain 939 P42, with an additional sublocus at approximately 30 Kbp downstream from the Bα sublocus that contains two phbs (le_pp4 and le_pp5) and an rcb (le_pr5) (Chen et al., 2016). rcb1 and rcb2 contain significant amounts of allelic variations, suggesting a multiallelic nature of the B mating type. In strain 939 P26, the Bα sublocus is comprised of phb5, phb6, and rcb1-939 P26 (rcb1-2), while the Bβ sublocus contains phb7, phb4, and rcb2-939 P26 (rcb2-1). Similarly, strain SUP2 carries phb1, phb2, and rcb1-SUP2 (rcb1-1) in the Bα sublocus and phb3, phb4, and rcb2-SUP2 (rcb2-2) in the Bβ sublocus. Each mating type-specific receptor pairs with two pheromone genes except rcb1-939 P42 (rcb1-3) of strain 939 P42 which has only one pheromone gene phb8 in the Bα sublocus whereas rcb2-939 P42 (rcb2-2) retains phb3 and phb4 in the Bβ sublocus (Wu et al., 2013). The pheromone gene names published as “Lephbx” (Wu et al., 2013) are simplified to phbx in this study. The pheromone receptor genes published as “Lercb1 or 2-strain name” are also renamed to rcb1-x or rcb2-x to better represent the allelic variations. Based on these previous findings, we explored the diversity of L. edodes B mating type at molecular level using sequence information of rcbs and phbs from 111 strains, including 83 cultivated and 28 wild strains, collected from East Asia.
2.2. PCR analysis For sequence determination of pheromone and receptor genes, primer sets specific for phbs and rcbs were designed using consensus sequence regions (Supplementary Table S2). PCR was performed with the following conditions: 94 °C for 5 min; 30 cycles of amplification reaction at 94 °C for 45 s 45 s, 60 °C for 30 s, and 72 °C for 30∼120 s; and 72 °C for 5 min. PCR products were analyzed by agarose gel electrophoresis. Target DNA was extracted from agarose gel and then cloned into a TA vector as described previously (Ha et al., 2018). Sequences of phbs and rcbs were determined by sequencing 1st phb and 2nd phb-rcb1 for Bα sublocus and rcb2-3rd phb and 4th phb for Bβ sublocus. Results are summarized in Supplementary Fig. S1. All sequences discovered in this study were deposited at Genbank (Supplementary Table S3). 2.3. Generation of monokaryon by de-dikaryotization When necessary, monokaryotic strain was generated directly from a dikaryotic strain without making fruiting body using modified protocol of Miyazaki et al. (2000). In brief, mycelia cultured in mushroom complete medium (MCM, MB cell, USA) for 10 days were isolated using a filter paper (Grade 41, Whatman, UK) and subsequently washed twice with distilled water. Cell walls were disrupted in 0.6 M sucrose solution by treatment with a 2.5% a lysing enzyme solution (Sigma, USA) for 3 h at 25℃. Resulting protoplasts were filtered through Miracloth (Calbiochem, USA). The filtrate was then centrifuged at 1000 rpm for 1 min. Protoplasts were washed twice with 0.6 M sucrose solution and suspended in washing solution followed by spreading onto MCM agar containing 0.6 M sucrose. Agar plate was incubated for one week. Monokaryotic mycelia devoid of clamp connections were isolated under a light microscope. 2.4. Gene and protein analyses Exon structures of rcb genes were analyzed using FGENESH with gene-finding parameters specific for Moniliophthora (Salamov and Solovyev, 2000). Transmembrane domain in RCB was predicted with Phobius software (http://phobius.sbc.su.se/, Käll et al., 2004). Phylogenetic trees for RCBs and PHBs were constructed by Maximum Likelihood method with 1000 repeats of bootstrapping using MEGA7 (Kumar et al., 2016). 3. Results 3.1. Identification of alleles of phb and rcb in B mating type locus Analysis of recently published genome sequence of CHAM-B17 strain (Shim et al., 2016), a monokaryotic strain derived from basidiospore of L. edodes CHAM strain (Ha et al., 2015), revealed that the B mating type locus consisted of phb11, phb12, and rcb1-4 in the Bα sublocus and phb9, phb10, and rcb2-3 in the Bβ sublocus (Fig. 1A). Comparison of this new B mating type with other three known B mating types (Wu et al., 2013) showed that each mating type-specific rcb paired
2. Materials and methods 2.1. Strains, culture conditions, and mating assay A total of 111 strains of L. edodes were obtained from various collections, including Culture Collection and DNA Bank of Mushroom 56
Scientia Horticulturae 243 (2019) 55–63
B. Ha et al.
Fig. 1. Structure of the B mating type locus in L. edodes. (A) Arrangement of phbs and rcbs in CHAM-B17 and KFRI976-M2 strains. Arrow boxes represent transcriptional direction. Numbers under genes and intergenic regions indicate lengths in bp. (B) Structure of the B mating type locus in sixteen monokaryotic strains. For comparison, three published B mating types (939 P26, 939 P42, and SUP2) are shown at the bottom (Wu et al., 2013).
CHAM-B17 for M2 strains (Fig. 1B). B mating type loci of all remaining strains essentially consisted of one of phbs-rcb1 pairs in the Bα sublocus and one of phbs-rcb2 pairs in the Bβ sublocus (Fig. 1B, Supplementary Table S3).
with at least one phb and that phb and rcb were arranged in the order of 1st phb-2nd phb-rcb1 or rcb1-2nd phb in the Bα sublocus and rcb2-3rd phb-4th phb in the Bβ sublocus. Based on this finding, we analyzed sequences of phbs and rcbs in Bα and Bβ subloci of 16 monokaryotic strains (Fig. 1B and Supplementary Table S3). Sequence of each rcb-phb pair was nearly identical regardless of its origin. For example, rcb1-1 and phb1-phb2 pairs in the Bα sublocus were identical for strains SUP2, KFRI1478, SJ111, and KFRI3008 except that there were two single nucleotide polymorphisms (SNPs) in rcb1-1 (Supplementary Data S1). In the B mating type locus of monokaryotic CHAM-M1 strains, a compatible partner strain of CHAM-B17 strain, phb11, phb12, and rcb14 in Bα sublocus and phb3, phb4, and rcb2-2 in Bβ sublocus were found (Fig. 1A). This new B mating type was composed of the same Bα sublocus as CHAM-B17 strain and the same Bβ sublocus as SUP2 strain. KFRI976-M1 strain, a monokaryotic strain that can generate dikaryotic KFRI976 strain through mating with KFRI976-M2 strain, carried the same B mating type as 939 P42 strain, consisting of rcb1-3 and phb8 in the Bα sublocus and phb3, phb4, and rcb2-2 in the Bβ sublocus. However, the Bα sublocus in KFRI976-M2 strain was composed of new phbs (phb13 and phb14) and rcb1 (rcb1-5) whereas the Bβ sublocus contained known phbs (phb4 and phb7) and rcb2 (rcb2-1) found in 939 P26 strain. KFRI1478-M1 strain carried the same B mating type discovered in SUP2 whereas the B mating type of KFRI1478-M2 strain was composed of the same Bα sublocus as KFRI976-M1 and 939 P42 strains (rcb1-3 and phb8) and the same Bβ sublocus as CHAM-B17 strain (phb9, phb10, and rcb23). The B mating type locus of FMRC1315-M1 consisted of the same Bα sublocus (phb5, phb6, and rcb1-2) found in strain 939 P26 and the same Bβ sublocus (phb9, phb10, and rcb2-3) found in CHAM-B17 and KFRI1478-M2. Dikaryotic strains SJ701, SJ707, and KFRI619 carried the same B mating type as strain 939 P26 for M1 strains and strain
3.2. Analysis of pheromones and pheromone receptors in B mating type We next performed comparative analyses on protein sequences of RCBs and PHBs. The five and three alleles of RCB1 and RCB2, respectively, were homologous to Ste3 of S. cerevisiae (Nakayama et al., 1985) regardless of mating type subloci where they belonged to (Supplementary Fig. S2 A). Despite having high homology, RCB1s and RCB2s were clustered in different homology groups as reported previously (Wu et al., 2013). Transmembrane topology analysis predicted that all RCBs were membrane proteins consisting of seven transmembrane domains and eight loop domains (Fig. 2A), similar to Ste3 of S. cerevisiae (Nakayama et al., 1985). Among the three loop domains (L3, L5 and L7) predicted to locate outside cell membrane (Fig. 2A), L3 and L5 domains were homologous for all eight RCBs whereas L7 loop domains in RCB1s and RCB2s showed significant variations (Fig. 2B). Particularly, the second β-strand region in L7 was hypervariable in both RCB1s and RCB2s. RCB2s contained additional variable region at the starting nine amino acid residues in L7. Fourteen phb genes encoded polypeptides with 49∼66 amino acid residues. Sequence alignment showed that PHBs at Bα and Bβ subloci were separated by two distinct groups (Fig. 2C and Supplementary Fig. S2B). Bα pheromones commonly contained EH (ER for PHB11) signature for proteolytic cleavage, C-terminal CV(I)V(I)A as CaaX motif for acylation, and GY motif for RCB function (Casselton and Olesnicky, 1998; Brown and Casselton, 2001) whereas Bβ pheromones contained 57
Scientia Horticulturae 243 (2019) 55–63
B. Ha et al.
Fig. 2. Primary structures of RCBs and PHBs. (A) Predicted topology of RCB proteins. Transmembrane domains and loop domains are numbered after TM and L prefixes, respectively. Position of the β-sheet in the loop domain is indicated by arrow box. (B) Sequence comparison of loop domains in RCBs predicted to locate outside membrane. Polymorphic positions are marked in red. Variable regions in loop domains are boxed. (C) Sequence comparison of PHB polypeptides. Predicted proteolytic cleavage site and the C-terminal CaaX box are highlighted in red. Predicted mature pheromones are boxed. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
phbs units from the Bβ sublocus (Table 1).Validity of these suggested B mating types was assessed using eight randomly collected monokaryotic strains. We analyzed genes in B mating type loci of eight monokaryotic strains using primer sets specific to each allele of phb, rcb1, and rcb2 genes. PCR with these specific primer sets yielded distinct DNA bands for phbs and rcbs of each strain (Fig. 4A). The B mating type of each strain was allocated using phb and rcb information according to the scheme described in Table 1. For example, the B mating type locus of FMRC1315-1 strain carried rcb1-2 with its associated pheromones phb5 and phb6 to constitute the Bα sublocus and rcb2-3 associated with phb9 and phb10 for the Bβ sublocus. Thus, the B mating type of this strain was assigned to B6. Similarly, the B mating type of FMRC1315-2 was B8 because it contained phb8-rcb1-3 in the Bα sublocus and phb3-phb4-rcb2-2 in the Bβ sublocus. By such simple PCR analysis, we could determine the identity of allelic genes in the B mating type locus, thereby identifying the B mating type of L. edodes strain within the context of 15 B mating types. We further validated our B mating type classification system through mating assay using eight monokaryotic strains with different B mating types. A mating types of these strains were determined using the method described in our previous paper (Ha et al., 2018). As shown in Fig. 4B, all monokaryotic strains with different B mating types were compatible for mating unless their A mating types were identical as shown in mating pairs FMRC1315-1 x FMRC1315-2, KFRI1478-2 x FMRC1315-1, and KFRI1478-2 x FMRC1315-2, which carried mating type pairs A1B6 X A1B8, A1B6 X A1B9, and A1B8 X A1B9, respectively.
EA for cleavage, CVIS(L) as CaaX motif, and GF or AF motif just before acylation site. Since both Bα and Bβ pheromones are predicted to be acylated through CaaX motif, they might be incorporated into cell membrane, similar to a-factor in yeast in which they interact with RCBs (Michaelis and Herskowitz, 1988). It is known that a-factor interacts with Ste3 receptor to turn on the mating pathway in α-cell of yeast (Caldwell et al., 1995; Lin et al., 2010). No diffusible pheromone was found in L. edodes. Primary structures of pre-PHBs varied. However, predicted mature structures after proteolytic cleavage showed high similarity (Fig. 2C). Overall, mature PHBs could be categorized into three groups. Mature PHBs from the Bα sublocus belonged to Group I (13 amino acids) or Group II (15 amino acids) whereas those from the Bβ sublocus fell into Group III (12 amino acids). Most notably, mature PHB3 and PHB9 in the different Bβ subloci were identical while PHB12 and PHB14 in the Bα subloci differed only in a single amino acid (S/T).
3.3. Determination of B mating types Our sequence analysis on B mating type locus revealed that B mating types of L. edodes were constructed by combinations of Bα and Bβ subloci that could be represented by alleles of rcb1 and rcb2, respectively, with their associated phbs (Fig. 1). It was also revealed that the same phb(s)-rcb pair was recurrently discovered as a unit independently of mushroom strains (Fig. 1). As summarized in Fig. 3, a total of five phb(s)-rcb units, including phb1-phb2-rcb1-1, phb5-phb6rcb1-2, rcb1-3-phb8, phb11-phb12-rcb1-4, and phb13-phb14-rcb1-5, were found from the Bα sublocus whereas three phb(s)-rcb units, including rcb2-1-phb7-phb4, rcb2-2-phb3-phb4, and rbc2-3-phb9-phb10, were discovered from the Bβ sublocus. No additional unit was discovered from the strains used in this study. Based on these findings, we formulated fifteen B mating types of L. edodes through combinations of the five allelic phbs-rcb1 units from the Bα sublocus and the three allelic rcb2-
3.4. Rcb pairs can represent B mating type One important implication derived from a series of our analyses was that a certain rcb always paired with specific phbs such as phb5-phb6rcb1-2 and phb11-phb12-rcb1-4 in the Bα sublocus and phb4-phb7-rcb2-1 and phb9-phb10-rcb2-3 in the Bβ sublocus, regardless of strains (Fig. 3). 58
Scientia Horticulturae 243 (2019) 55–63
B. Ha et al.
Fig. 3. Pheromones (phbs) and pheromone receptor (rcb) pairs recurrently found in different strains of L. edodes. Alleles of rcb1 and rcb2 are numbered as rcb1-x and rcb2-x representing pheromone receptors for Bα and Bβ subloci, respectively. Pheromone genes (phbs) pair with corresponding rcbs are shown in different color. Arrow boxes indicate direction of transcription. Strains in which phb-rcb unit is found are described at the right side of each unit.
Table 1 Proposed B mating types and the selected monokaryotic strains carrying corresponding B mating types. Mating type B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 B13 B14 B15
Bα sublocus
Bβ sublocus
Bα1
rcb1-1, phb1, phb2
Bα2
rcb1-2, phb5, phb6
Bα3
rcb1-3, phb8
Bα4
rcb1-4, phb11, phb12
Bα5
rcb1-5, phb12, phb14
Strains
Bβ1 Bβ2 Bβ3 Bβ1 Bβ2 Bβ3 Bβ1 Bβ2 Bβ3 Bβ1 Bβ2 Bβ3 Bβ1 Bβ2 Bβ3
rcb2-1, rcb2-2, rcb2-3, rcb2-1, rcb2-2, rcb2-3, rcb2-1, rcb2-2, rcb2-3, rcb2-1, rcb2-2, rcb2-3, rcb2-1, rcb2-2, rcb2-3,
This suggests that one can determine B mating type locus of any monokaryotic strain by verifying allelic variants at sites of rcb1 and rcb2 as markers of Bα and Bβ subloci, respectively, instead of determining
phb7, phb3, phb9, phb7, phb3, phb9, phb7, phb3, phb9, phb7, phb3, phb9, phb7, phb3, phb9,
phb4 phb4 phb10 phb4 phb4 phb10 phb4 phb4 phb10 phb4 phb4 phb10 phb4 phb4 phb10
Songo-M1 KFRI1478-M1, SUP2 SJ109-M1 SJ701-M1, SJ707-M1, KFRI619-M1, 939 P26 SMR1, SMR2 FMRC1315-M1 SJ709-M2 KFRI976-M1, FMRC1315-M2, 939 P42 KFRI1478-M2 Songo-M2 CHAM-M1 CHAM-B17, SJ701-M2, SJ707-M2, KFRI619-M2 KFRI976-M2, FMRC8120-M2 IUM4848-M1 IUM4848-M2
whole sets of 14 phbs and 8 rcbs. One such example is shown in Fig. 5. We knew that dikaryotic CHAM strain contained two nuclei with mating types of A1 and A5 at A mating type locus (Ha et al., 2018) and Fig. 4. Detection of mating type genes by PCR. (A) Detection of phbs and rcbs in different monokaryotic strains. phbs and rcbs in monokaryotic strains were detected by PCR with specific primer sets. B mating types assigned by compositions of phbs and rcbs are described at the right side. (B) Confirmation of mating type by mating analysis. ‘O’ indicates a successful mating whereas ‘X’ means failure in mating due to incompatibility in A mating types in this occasion.
59
Scientia Horticulturae 243 (2019) 55–63
B. Ha et al.
Fig. 5. Determination of complete mating types by PCR analysis. PCR with primer sets targeting variable regions in A locus and rcbs in B locus yielded distinct PCR bands. Each column represents A and B mating types of monokaryotic strains of CHAM, SJ701, and SJ707. Name of monokaryotic strains are provided on the top of each column. Mating types of each strain are written under the gel image.
could identify the exact B mating type of each nucleus if we could obtain monokaryons through de-dikaryotization of dikaryons (SMR1, SJ108, SJ705, etc.) or from basidiospores (KFRI1478, SJ701, FMRC1315, etc.) (Table 2, Category 3).
B11 and B12 at the B mating type locus. Monokaryotic strains derived from basidiospores can have tetrapolar combinations of mating type loci, including A5B12, A5B11, A1B11, and A1B12. To confirm this, we analyzed the B locus with primer sets targeting variants of rcb1 and rcb2 and the A locus with A1 and A5 specific primer sets (Ha et al., 2018). PCR yielded two different combinations of rcbs. rcb1-4 and rcb2-2 were found in monokaryotic strains CH2 and CH3 while rcb1-4 and rcb2-3 were found in CH1 and CH15 strains. This indicated that the former two had B11 whereas the latter two had B12 in the B locus. Combining this with information on A locus determined by specific PCR, we could determine tetrapolar mating types of all monokaryotic strains. The same approach also verified tetrapolar mating types of monokaryotic strains derived from basidiospores of SJ701 and SJ707 (Fig. 5). In dikaryotic strains, PCR detected three or four rcbs depending on strains (Table 2). Alleles of rcb1 or rcb2 could be single when these two different mating types representing two nuclei, M1 and M2, in a dikaryotic cytoplasm were constituted by the same rcb1 with two different rcb2s or by two different rcb1s with the same rcb2. For example, ‘Songo’ strain had rcb1-1 and rcb1-4 in Bα and rcb2-1 in Bβ. In this case, the B mating type of one nucleus (M1) contained rcb1-1 + rcb2-1 and that of another nucleus (M2) carries rcb1-4 + rcb2-1 (Table 2, Category 1). Similarly, ‘CHAM’ strain contained rcb1-4 in Bα and rcb2-2 and rcb23 in Bβ. Combinations of these rcbs generated a pair of B mating types that carried rcb1-4 + rcb2-2 in one nucleus (M1) and rcb1-4 + rcb2-3 in another nucleus (M2). When a dikaryotic strain contains two alleles of rcb1 and two of rcb2, there are four possible combinations of rcb pairs. In this case, the exact B mating type of individual nucleus is uncertain. Strains KFRI673, 20,701, SJ103, and some others were representatives of such cases (Table 2, Category 2). For example, rcb analysis on strain 20,701 revealed the presence of rcb1-2 and rcb1-3 in Bα and rcb2-2 and rcb2-3 in Bβ that could have four combinations of rcbs, including rcb1-2 + rcb22, rcb1-3 + rcb2-3, rcb1-2 + rcb2-3, and rcb1-3 + rcb2-2. When B5 (rcb1-2 + rcb2-2) is identified as one B mating type, another nucleus should have B9 (rcb1-3 + rcb2-3) while if B6 (rcb1-2 + rcb2-3) is one B mating type, B8 (rcb1-3 + rcb2-2) should be another type. Therefore, we can only conclude that there is a possibility of two different combinations of B mating types (B5B9 or B6B8 for this strain) for dikaryotic strains belonging to this category. However, even for these strains, we
3.5. Diversity of B mating types To further understand B mating types of L. edodes in terms of their diversity, we analyzed alleles of rcb1 and rcb2 and determined B mating types of 111 dikaryotic strains, including 83 cultivated strains and 28 wild strains collected from East Asian countries (Supplementary Table S4). Among alleles of rcbs, rcb1-3 and rcb1-4 in rcb1s and rcb2-2 and rcb2-3 in rcb2s were more prevalent as shown in Table 3. rcb1-4 was mostly discovered in cultivated strains. Fifty-three out of 58 rcb1-4 were found from cultivated strains while the remaining five were from wild strains. On the contrary, the number of rcb1-5, the rarest rcb, had higher frequency (13/21) in wild strains than that (8/21) in cultivated strains. After comprehensive analysis of rcb compositions, we found seven new combinations of alleles of rcb1 and rcb2, including rcb1-1 + rcb2-1 (B1), rcb1-1 + rcb2-3 (B3), and rcb1-2 + rcb2-2 (B5) (Table 2). These comprised all possible combinations of rcb1s and rcb2s when they were combined with eight different combinations found in CHAM (B11, B12), 939 P (B4, B8), SUP2 (B2), KFRI1478 (B2, B9), and KFRI976 (B8, B13). Together, these combinations of rcb1 and rcb2 represented 15 different B mating types. Ten cultivated strains and thirteen wild strains carried two alleles of rcb1s and two alleles of rcb2s which belonged to ‘Category 2′ in Table 2. Alternative mating pairs of these strains are provided in parentheses (Supplementary Table S4). Different from the diversification of A mating type in wild strains (Ha et al., 2018), distributions of B mating types were relatively random in both cultivated and wild strains (Supplementary Table S4). Mating types B8 and B12 were most frequent B mating types, occurring 37 and 34 times, respectively (Fig. 6A). The least selected B type was B14 which only occurred in a single strain. Mating types B3, B14, and B15 were present only in wild strains when we excluded B mating types uncertain in 23 strains in ‘Category 2′. B mating type pairs, including B2B12, B8B11, and B4B8, occurred most prevalently in cultivated strains (Fig. 6B). 60
Scientia Horticulturae 243 (2019) 55–63
B. Ha et al.
Table 2 Identification of B mating type through combination of rcb1s and rcb2s. Strain
PCR detection
Nucleus M1
rcb1 1
rcb2 2
3
4
5
1
Category 1. One rcb1 + Two rcb2 or Two rcb1 + One rcb2 KFRI57 O Songo O O SJ111 O O IUM1405 O O KFRI1520 O O NAAS4255 O ASI3220 O SL8 O O SJ109 O KFRI2521 O KFRI955 O O SJ110 O SL1 O O Mori250 O CHAM O KFRI2290 O IUM4848 O Category 2. Two rcb1 + Two rcb2a KFRI673 O O 20,701
O
O
KFRI58
O
IUM1851
O O
O O O
O
O O O O O
O O O O
O O O
O
O O
O
O O O O O
O O
O
O
O
O
O
O
Category 3. Two rcb1 + Two rcb2 confirmed by mating or de-dikaryotization KFRI1478 O O O SJ704 O O O IUM3178 O O O SJ701 O O O SMR1 O O O FMRC1315 O O O Yujiro O O O SJ108 O O O O SJ705 O O O KFRI976 O O O O
rcb Combination
B mating type
rcb Combination
3
O O
O
O
2
O O
O
SJ103
B mating type
Nucleus M2
O O O O O O O O
B1 B1 B2 B2 B3 B4 B4 B6 B7 B7 B7 B8 B8 B10 B11 B13 B14
rcb1-1, rcb1-1, rcb1-1, rcb1-1, rcb1-1, rcb1-2, rcb1-2, rcb1-2, rcb1-3, rcb1-3, rcb1-3, rcb1-3, rcb1-3, rcb1-4, rcb1-4, rcb1-5, rcb1-5,
rcb2-1 rcb2-1 rcb2-2 rcb2-2 rcb2-3 rcb2-1 rcb2-1 rcb2-3 rcb2-1 rcb2-1 rcb2-1 rcb2-2 rcb2-2 rcb2-1 rcb2-2 rcb2-1 rcb2-2
B3 B10 B8 B11 B6 B5 B6 B9 B8 B9 B13 B9 B11 B12 B12 B15 B15
rcb1-1, rcb1-4, rcb1-3, rcb1-4, rcb1-2, rcb1-2, rcb1-2, rcb1-3, rcb1-3, rcb1-3, rcb1-5, rcb1-3, rcb1-4, rcb1-4, rcb1-4, rcb1-5, rcb1-5,
rcb2-3 rcb2-1 rcb2-2 rcb2-2 rcb2-3 rcb2-2 rcb2-3 rcb2-3 rcb2-2 rcb2-3 rcb2-1 rcb2-3 rcb2-2 rcb2-3 rcb2-3 rcb2-3 rcb2-3
B1 B2 B5 B6 B8 B9 B8 B9 B10 B11
rcb1-1, rcb1-1, rcb1-2, rcb1-2, rcb1-3, rcb1-3, rcb1-3, rcb1-3, rcb1-4, rcb1-4,
rcb2-1 rcb2-2 rcb2-2 rcb2-3 rcb2-2 rcb2-3 rcb2-2 rcb2-3 rcb2-1 rcb2-2
B8 B7 B9 B8 B12 B11 B14 B13 B14 B13
rcb1-3, rcb1-3, rcb1-3, rcb1-3, rcb1-4, rcb1-4, rcb1-5, rcb1-5, rcb1-5, rcb1-5,
rcb2-2 rcb2-1 rcb2-3 rcb2-2 rcb2-3 rcb2-2 rcb2-2 rcb2-1 rcb2-2 rcb2-1
B2 B2 B3 B4 B5 B6 B6 B8 B8 B8
rcb1-1, rcb1-1, rcb1-1, rcb1-2, rcb1-2, rcb1-2, rcb1-2, rcb1-3, rcb1-3, rcb1-3,
rcb2-2 rcb2-2 rcb2-3 rcb2-1 rcb2-2 rcb2-3 rcb2-3 rcb2-2 rcb2-2 rcb2-2
B9 B12 B11 B12 B12 B8 B8 B10 B12 B13
rcb1-3, rcb1-4, rcb1-4, rcb1-4, rcb1-4, rcb1-3, rcb1-3, rcb1-4, rcb1-4, rcb1-5,
rcb2-3 rcb2-3 rcb2-2 rcb2-3 rcb2-3 rcb2-2 rcb2-2 rcb2-1 rcb2-3 rcb2-1
a B mating type pair in this category could not be designated. Dikaryotic strains can have one of two possible pairs. For example, KFRI673 can have either B1B8 or B2B7 pair.
pheromone-receptor interactions (Casselton and Olesnicky, 1998; Raudaskoski et al., 2012). In the present study, we showed recurrent discovery of five alleles of rcb1 with nine associated phbs and three alleles of rcb2 with five associated phbs to represent Bα sublocus and Bβ sublocus, respectively (Fig. 3). All alleles of RCB1 and RCB2 proteins were predicted to contain seven transmembrane helical domains with three loop domains positioning outside the cell membrane (Fig. 2A). Loop domain L7 was particularly heterologous between RCB1s and RCB2 (Fig. 2B). Variations within RCB1s or RCB2s were also concentrated in this loop domain, suggesting its role in specificity of pheromones. In yeast Ste3p, two extracellular loop domains corresponding to L5 and L7 of RCBs can bind mating pheromone as some mutations in these loops can block pheromone signaling (Gastaldi et al., 2016). Therefore, it is highly conceivable that RCBs in L. edodes can distinguish different PHBs through variations in this loop. Unlike diffusible α-factor in yeast (Kurjan, 1985), all L. edodes PHBs are conceivably a-factor-like membrane-bound pheromones because of the presence of acylation motif (CaaX) at the C-termini (Michaelis and Herskowitz, 1988). PHBs in Bα and Bβ subloci are distinguishable by their proteolytic cleavage motifs (EH for Bα and EA for Bβ) and primary structures of mature pheromones. Mature Bα pheromones are further
Table 3 Occurrence of alleles of pheromone receptors in cultivated and wild strains. Alleles
Bα sublocus
Bβ sublocus
a b
rcb1-1 rcb1-2 rcb1-3 rcb1-4 rcb1-5 rcb2-1 rcb2-2 rcb2-3
Cultivated (C)a
Wild (W)b
27 23 42 53 8 32 66 51
11 3 14 5 13 18 15 19
Total (C + W) 38 26 56 58 21 50 81 70
Ratio (W/ C) 0.41 0.13 0.33 0.09 1.63 0.56 0.23 0.37
Number of occurrence in 83 cultivated strains. Number of occurrence in 28 wild strains.
4. Discussion The B mating type of L. edodes can be determined by mating specific genes residing in the B mating type locus that consists of two subloci, Bα and Bβ (Kües, 2000). Both subloci encode distinct PHBs and RCBs to enable reciprocal activation of the mating pathway through 61
Scientia Horticulturae 243 (2019) 55–63
B. Ha et al.
Fig. 6. Diversity of B mating types. (A) Prevalence of B mating types of 176 nuclei in 88 dikaryotic strains of L. edodes. ‘Frequency’ means the number of occurrence of a certain type of B mating type. (B) Occurrence of B mating type pairs in 88 dikaryotic strains. Twenty-three dikaryotic strains carrying uncertain B mating type pairs described in Supplementary Table S4 are excluded.
divided into two groups (Group1 and Group 2 in Fig. 2C). Mature polypeptides in these two groups are completely different in size and sequence. This may function in the discrimination of RCBs. However, the specificity of RCB to PHB is largely elusive for now. Another notably feature in the primary structure of PHB is that some mature pheromones in the same group share high sequence similarity. PHB12 and PHB14 in Bα sublocus differed only in a single amino acid while PHB3 and PHB9 in Bβ sublocus were even identical (Fig. 2C), meaning that PHB3 and PHB9 are recognized as self pheromone for RCB2-2 and RCB2-3 in Bβ2 and Bβ3 units, respectively (Fig. 3). For a mated strain having Bβ2 and Bβ3 in the Bβ subloci, activation of pheromone response pathway can only occur through cross interactions between PHB4 (Bβ2) and RCB2-3 (Bβ3), and PHB10 (Bβ3) and RCB2-2 (Bβ2). This may explain the reason why the majority of rcbs are in pair with two phb genes. Further biochemical and genetic studies on the pairing of PHB and RCB in the activation of pheromone signal pathway are needed. Among mating specific rcbs, rcb1-3 and rcb1-4 in the Bα sublocus and rcb2-3 and rcb2-3 in the Bβ sublocus are more prevalent. rcb1-4 is particularly frequent in cultivated strains whereas rcb1-5, the rarest allele, has higher frequency in wild strains (Table 3). Prevalence in rcbs reflect biased selection of the nucleus with a certain B mating type. B8 and B12 are prevalent B mating types that contain rcb1-3 and rcb1-4, respectively. It is currently unclear how the nuclei with these B mating types become more competent in cultivated strains. Our previous studies have hinted that dikaryotic strains generated by mating with a certain monokaryotic strain (with certain nucleus) show better fruiting rate and fruiting body production (Ha et al., 2015) and that laccase genes are preferentially expressed by one of the two nuclei in the dikaryotic cytoplasm (Ha et al., 2017). Therefore, prevalent B mating types might have advantages in the expression of desirable characteristics. Findings of the present study lead us to propose 15 B mating types through combinations of five Bα subloci and three Bβ subloci. Multiallelism of B mating type locus has also been observed in Flammulina velutipes (Wang et al., 2016), C. cinerea (Casselton and Olesnicky, 1998; Stajich et al., 2010; Riquelme et al., 2005), and S. commune (Fowler et al., 2001, 2004). Similar to L. edodes, S. commune carries Bα and Bβ subloci with nine alleles per each sublocus. Combinations of them constitute 81 B mating types (Parag and Koltin, 1971; Fowler et al., 2001). The B mating type locus of C. cinerea is more complex. It consists of Group 1, Group 2, and Group 3 subloci with 2, 5, and 7 alleles, respectively, thereby having 70 B mating types (Riquelme et al., 2005). The actual numbers of B mating types for these species can be fewer than predicted because of the suppression of recombination between some subloci (Stamberg and Koltin, 1971). However, our
results suggest that the B mating type locus of L. edodes in the context of 5 Bα and 3 Bβ subloci is freely recombinable since all 15 predicted B mating types are present in our collection of strains. For L. edodes, two independent papers based on mating analyses of wild strains have reported the presence of 40-65 A and 63–100 B mating types from 33 Japanese strains (Tokimoto et al., 1973) and 66 A and 72 B mating types from 53 Chinese strains (Lin et al., 2003). Our previous study on A mating type of 126 strains of L. edodes also provided similar number of A mating types (63 As) (Ha et al., 2018). Therefore, these fifteen B mating types proposed here seem to be too little to cover all B mating types present in nature. However, the proposed number can nicely represent B mating types of all cultivated and wild strains within our strain collection which covers most of cultivated strains in Korea and some Chinese and Japanese strains. If we consider most cultivated strains in East Asia share common origins, the proposed number can serve as a good guide for further study on mating system and breeding of new cultivars. Competing interests statement No potential conflict of interest was reported by the authors. Acknowledgments This work was carriedout with the support of “Cooperation Research Program for Agriculture Science and Technology Development (Project No. PJ01368102)” Rural Development Administration, Republic of Korea. YS, SK, and MSK were supported by a scholarship from the BK 21 Plus Program, the Ministry of Education, Korea. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.scienta.2018.08.009. References Bakkeren, G., Kämper, J., Schirawski, J., 2008. Sex in smut fungi: structure, function and evolution of mating-type complexes. Fungal Genet. Biol. 45, S15–S21. Brown, A.J., Casselton, L.A., 2001. Mating in mushrooms: increasing the chances but prolonging the affair. Trends Genet. 17, 393–400. Caldwell, G.A., Naider, F., Becker, J.M., 1995. Fungal lipopeptide mating pheromones: a model system for the study of protein prenylation. Microbiol. Rev. 59, 406–422. Casselton, L.A., Olesnicky, N.S., 1998. Molecular genetics of mating recognition in basidiomycete fungi. Microbiol. Mol. Biol. Rev. 62, 55–70. Chen, L., Gong, Y., Cai, Y., Liu, W., Zhou, Y., Xiao, Y., Xu, Z., Liu, Y., Lei, X., Wang, G., Guo, M., Ma, X., Bian, Y., 2016. Genome sequence of the edible cultivated mushroom
62
Scientia Horticulturae 243 (2019) 55–63
B. Ha et al.
heterozygous DNA markers in shiitake (Lentinula edodes) using de-dikaryotization via preparation of protoplasts and isolation of four meiotic monokaryons from one basidium. J. Wood Sci. 46, 395–400. Nakayama, N., Miyajima, A., Arai, K., 1985. Nucleotide sequences of STE2 and STE3, cell type-specific sterile genes from Saccharomyces cerevisiae. EMBO J. 4, 2643–2648. O’Shea, S.F., Chaure, P.T., Halsall, J.R., Olesnicky, N.S., Leibbrandt, A., Connerton, I.F., Casselton, L.A., 1998. A large pheromone and receptor gene complex determines multiple B mating type specificities in Coprinus cinereus. Genetics 148, 1081–1090. Parag, Y., Koltin, Y., 1971. The structure of the incompatibility factors of Schizophyllum commune: constitution of the three classes of B factors. Mol. Gen. Genet. 112, 43–48. Raper, J.R., Krongelb, G.S., Baxter, M.G., 1958. The number and distribution of incompatibility factors in Schizophyllum commune. Am. Nat. 92, 221–232. Raudaskoski, M., Kothe, E., Fowler, T.J., Jung, E.M., Horton, J.S., 2012. Ras and Rho small G proteins: Insights from the Schizophyllum commune genome sequence and comparisons to other fungi. Biotechnol. Genet. Eng. Rev. 28, 61–100. Riquelme, M., Challen, M.P., Casselton, L.A., Brown, A.J., 2005. The origin of multiple B mating specificities in Coprinus cinereus. Genetics 170, 1105–1119. Salamov, A.A., Solovyev, V.V., 2000. Ab initio gene finding in Drosophila genomic DNA. Genome Res. 10, 516–522. Shim, D., Park, S.G., Kim, K., Bae, W., Lee, G.W., Ha, B.S., Ro, H.S., Kim, M., Ryoo, R., Rhee, S.K., Nou, I.S., Koo, C.D., Hong, C.P., Ryu, H., 2016. Whole genome de novo sequencing and genome annotation of the world popular cultivated edible mushroom, Lentinula edodes. J. Biotechnol. 223, 24–25. Stajich, J.E., Wilke, S.K., Ahrén, D., Au, C.H., Birren, B.W., Borodovsky, M., Burns, C., Canbäck, B., Casselton, L.A., Cheng, C.K., Deng, J., Dietrich, F.S., Fargo, D.C., Farman, M.L., Gathman, A.C., Goldberg, J., Guigó, R., Hoegger, P.J., Hooker, J.B., Huggins, A., James, T.Y., Kamada, T., Kilaru, S., Kodira, C., Kües, U., Kupfer, D., Kwan, H.S., Lomsadze, A., Li, w., Lilly, W.W., Ma, L.J., Mackey, A.J., Manning, G., Martin, F., Muraguchi, H., Natvig, D.O., Palmerini, H., Ramesh, M.A., Rehmeyer, C.J., Roe, B.A., Shenoy, N., Stanke, M., Ter-Hovhannisyan, V., Tunlid, A., Velagapudi, R., Vision, T.J., Zeng, Q., Zolan, M.E., Pukkila, P.J., 2010. Insights into evolution of multicellular fungi from the assembled chromosomes of the mushroom Coprinopsis cinerea (Coprinus cinereus). Proc. Nat. Acad. Sci. USA 107, 11889–11894. Stamberg, J., Koltin, Y., 1971. Selectively recombining B incompatibility factors of Schizophyllum commune. Mol. Gen. Genet. 113, 157–165. Tokimoto, K., Komatsu, M., Takemaru, T., 1973. Incompatibility factors in the natural population of Lentinus edodes in Japan. Rep. Tottori Mycol. Inst. Jpn. 10, 371–376. Wang, W., Lian, L., Xu, P., Chou, T., Mukhtar, I., Osakina, A., Waqas, M., Chen, B., Liu, X., Liu, F., Xie, B., van Peer, A.F., 2016. Advances in understanding mating type gene organization in the mushroom-forming fungus Flammulina velutipes. G3 6, 3635–3645. Wu, L., van Peer, A., Song, W., Wang, H., Chen, M., Tan, Q., Song, C., Zhang, M., Bao, D., 2013. Cloning of the Lentinula edodes B mating-type locus and identification of the genetic structure controlling B mating. Gene 531, 270–278.
Lentinula edodes (shiitake) reveals insights into lignocellulose degradation. PLoS ONE 11, e0160336. Fowler, T.J., Mitton, M.F., Vaillancourt, L.J., Raper, C.A., 2001. Changes in mate recognition through alterations of pheromones and receptors in the multisexual mushroom fungus Schizophyllum commune. Genetics 158, 1491–1503. Fowler, T.J., Mitton, M.F., Rees, E.I., Raper, C.A., 2004. Crossing the boundary between the Bα and Bβ mating-type loci in Schizophyllum commune. Fungal Genet. Biol. 41, 89–101. Gastaldi, S., Zamboni, M., Bolasco, G., Segni, G.D., Tocchini‐Valentini, G.P., 2016. Analysis of random PCR‐originated mutants of the yeast Ste2 and Ste3 receptors. MicrobiologyOpen 5, 670–686. Ha, B.S., Kim, S., Ro, H.S., 2015. Isolation and characterization of monokaryotic strains of Lentinula edodes showing higher fruiting rate and better fruiting body production. Mycobiology 43, 24–30. Ha, B., Lee, S., Kim, S., Kim, M., Moon, Y.J., Song, Y., Ro, H.S., 2017. Nucleus-selective expression of laccase genes in the dikaryotic dtrain of Lentinula edodes. Mycobiology 45, 379–384. Ha, B., Kim, S., Kim, M., Moon, Y.J., Song, Y., Ryu, J.S., Ryu, H., Ro, H.S., 2018. Diversity of A mating type in Lentinula edodes and mating type preference in domesticated strains. J. Microbiol. 56, 416–425. Huyer, G., Kistler, A., Nouvet, F.J., George, C.M., Boyle, M.L., Michaelis, S., 2006. Saccharomyces cerevisiae a-factor mutants reveal residues critical for processing, activity, and export. Eukaryot. Cell 5, 1560–1570. Käll, L., Krogh, A., Sonnhammer, E.L.L., 2004. A combined transmembrane topology and signal peptide prediction method. J. Mol. Biol. 338, 1027–1036. Kües, U., 2000. Life history and developmental processes in the basidiomycete Coprinus cinereus. Microbiol. Mol. Biol. Rev. 64, 316–353. Kües, U., 2015. From two to many: multiple mating types in Basidiomycetes. Fungal Biol. Rev. 29, 126–166. Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874. Kurjan, J., 1985. Alpha-factor structural gene mutations in Saccharomyces cerevisiae: effects on alpha-factor production and mating. Mol. Cell. Biol. 5, 787–796. Lin, F.C., Wang, Z.W., Sun, Y., Cai, Y.J., 2003. Analysis of the mating type factors in natural population of Lentinula edodes in china. Mycosystema 22, 235–240. Lin, X., Jackson, J.C., Feretzaki, M., Xue, C., Heitman, J., 2010. Transcription factors Mat2 and Znf2 operate cellular circuits orchestrating opposite-and same-sex mating in Cryptococcus neoformans. PLoS Genet 6, e1000953. Michaelis, S., Barrowman, J., 2012. Biogenesis of the Saccharomyces cerevisiae pheromone a-factor, from yeast mating to human disease. Microbiol. Mol. Biol. Rev. 76, 626–651. https://doi.org/10.1128/MMBR.00010-12. Michaelis, S., Herskowitz, I., 1988. The a-factor pheromone of Saccharomyces cerevisiae is essential for mating. Mol. Cell. Biol. 8, 1309–1318. Miyazaki, K., Maeda, H., Sunagawa, M., Tamai, Y., Shiraishi, S., 2000. Screening of
63
Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Molecular analysis of B mating type diversity in Lentinula edodes a
a
a
a
a
T b
Byeongsuk Ha , Yoon Jung Moon , Yelin Song , Sinil Kim , Minseek Kim , Cheol-Won Yoon , ⁎ Hyeon-Su Roa, a b
Division of Applied Life Science and Research Institute of Life Sciences, Gyeongsang National University, Jinju 52828, Republic of Korea School of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea
A R T I C LE I N FO
A B S T R A C T
Keywords: B mating type Diversity Lentinula edodes Mating Pheromone Receptor
Pheromone and pheromone receptor genes in B mating type locus constitute B mating type of fungi belonging to Basidiomycota. In Lentinula edodes, multiple B mating types have been suggested based on genome sequence analysis and mating assay. Here we report finding of new alleles of pheromones (phbs) and pheromone receptors (rcbs) to constitute five alleles of rcb1 (rcb1-1∼rcb1-5) with nine associated phbs in Bα sublocus and three alleles of rcb2 (rcb2-1∼rcb2-3) with five associated phbs in Bβ sublocus. Each rcb was primarily associated with two phbs. Each phb-rcb pair was recurrently discovered as a distinct unit in various monokarytotic and dikaryotic strains, regardless of subloci. This allowed us to propose 15 B mating types through combinations of five Bα and three Bβ subloci. PCR analyses with primer sets probing alleles of rcb1 and rcb2 demonstrated that all monokaryotic strains contained one of the five rcb1s and one of the three rcb2s representing Bα and Bβ subloci, respectively, thereby enabling designation of their B mating types via combinations of Bα and Bβ. PCR analyses also revealed the presence of one or two alleles of rcb1 and rcb2 representing B mating type of each nuclei in dikaryotic cytoplasm. Further investigation of 111 dikaryotic strains, including 83 cultivated and 28 wild strains collected from East Asia, revealed that B8 and B11 mating types constructed by rcb1-3 + rcb2-2 and rcb1-4 + rcb2-2, respectively, were more prevalent in cultivated strains. In B mating type pairs representing each nucleus in dikaryons, B2B12, B4B8, and B8B11 were frequently occurring pairs in cultivated strains while none of them was found in wild strains. Such prevalence indicates that certain nuclei have been preferentially selected in the generation of cultivated strains. Our findings shed new light on the construction of B mating type in L. edodes and provide practical tools in the breeding of new cultivars.
1. Introduction Filamentous fungi belonging to Basidiomycota spend most of their life cycle as mycelia with one (monokaryon) or two nuclei (dikaryon) in the cytoplasm. For sexual reproduction, monokaryotic mycelia will mate with compatible partners to form fused mycelia which maintain two compatible nuclei inside cells. In fused mycelial cells, these two nuclei undergo mitotic division. Divided nuclei then move to next cells through clamp connections to establish dikaryons throughout connected mycelial cells (Kües, 2000). Dikaryotic mycelia can occasionally form fruiting bodies with basidia as sexual organs upon physico-chemical signals. In the basidium of heterothallic basidiomycetes, nuclear fusion (karyogamy) followed by meiotic division produces four sexual spores (basidiospores), resulting in monokaryotic mycelia upon germination (Kües, 2000). Self or non-self recognition in sexual reproduction is enabled by two unlinked mating type loci, A and B (Bakkeren et al., 2008; Casselton ⁎
and Olesnicky, 1998). The A mating type locus encodes two homeodomain proteins (HDs). Each HD proteins forms heterodimeric transcription factor with HD protein from compatible partner to control mating events, including nuclear pairing, clamp cell formation, and coordination of nuclear division (Casselton and Olesnicky, 1998). The B mating type locus is consisted of pheromone and pheromone receptor genes (Kües, 2015). Mating pheromones in basidiomycetes contain CaaX motif, implying their localizations to cell membrane through acylation at the cysteine residue (Huyer et al., 2006; Michaelis and Barrowman, 2012), similar to a-factor of ascomycete fungi (Caldwell et al., 1995). Pheromone receptors in basidiomycetes are homologous to Ste3p (a-factor receptor) of yeasts (Kües, 2015). Pheromone and receptor interaction is thought to activate the mating pathway involving MAP kinase-dependent signal transduction (Casselton and Olesnicky, 1998; Raudaskoski et al., 2012). Both mating types can be multiple to increase chances of inter-strain mating in nature. Multiplicity of mating types arises from allelic
Corresponding author. E-mail address: [email protected] (H.-S. Ro).
https://doi.org/10.1016/j.scienta.2018.08.009 Received 26 June 2018; Received in revised form 27 July 2018; Accepted 7 August 2018 0304-4238/ © 2018 Elsevier B.V. All rights reserved.
Scientia Horticulturae 243 (2019) 55–63
B. Ha et al.
(CCDBM, University of Incheon, Korea), Korea Forest Research Institute (KFRI, Korea), National Institute of Agricultural Sciences (NAAS, Korea), and Forest Mushroom Research Center (FMRC, Korea) (Supplementary Table S1). Of these 111 strains, 83 were cultivated strains collected from East Asian countries, including 18, 20, and 45 strains from China, Japan, and Korea, respectively, and 28 were wild strains collected from mountainous areas in southern part of Korean peninsula. Mycelia of L. edodes were grown on potato dextrose agar (PDA, Oxoid, UK) or in potato dextrose broth (PDB, Difco, USA) at 25 °C. Mating analysis and genomic DNA extraction were performed as described previously (Ha et al., 2018).
variations of genes residing in multiple subloci constituting both A and B mating type loci. Coprinopsis cinerea has a, b, and d subloci in the archetypal A mating type locus and Groups 1, 2, and 3 subloci in the B mating type locus consisted of HD1-HD2 gene pairs and pheromonereceptor pairs, respectively (Riquelme et al., 2005; Kües, 2015). Allelic variations in genes at each sublocus together with combinations of subloci have generated more than 240 A and 70 or 79 B mating types (Riquelme et al., 2005; O’Shea et al., 1998). Similarly, it has been reported that Schizophyllum commune contains 288 A and 81 B mating types (Raper et al., 1958; Kües, 2015). Mating behavior of Lentinula edodes has been of our interest because of its practical importance in the mushroom industry (Ha et al., 2015). Similar to C. cinerea and S. commune, L. edodes carries multiallelic A and B mating types. Through mating analyses of wild populations, Tokimoto et al. (1973) have suggested 40-65 As and 63–100 Bs from 33 Japanese wild strains. Later, Lin et al. (2003) have reported 66 As and 72 Bs (predicted to be 121 A and 151 B) from 53 Chinese wild strains. However, these numbers have inherent uncertainty because they have been mainly estimated by mating analyses that rely on empirical observations such as the presence of clamp connections and the formation of barrage lines as measures of successful mating. To establish more reliable method for estimating mating types, we have focused on allelic variations in pheromone and receptor to see if we can employ them to verify B mating types. Using similar approach, we have recently reported variable sequence regions in A mating type loci of L. edodes that represent different mating type alleles (Ha et al., 2018). Genome analyses of three monokaryotic strains (939 P26, 939 P42, and SUP2) of L. edodes have revealed that the genetic structure of B mating type locus is more complex than that of A mating type locus (Wu et al., 2013). Each B mating type locus consists of two mating typespecific receptors (rcbs), one or two pheromone genes (phbs), and two non-mating type rcbs. The whole gene-set in this locus lies in a single chromosome with an approximate length of 37 Kbp. Mating type-specific receptors rcb1 and rcb2 are found together with their associated phbs whereas non-mating type receptors (rcb3 and rcb4) are devoid of associated phb gene. rcb1 and rcb2 with their coupled phbs reside in subloci Bα and Bβ, respectively, to constitute the B mating type locus. Recent genome analysis of L. edodes strain W1-26 has revealed the same Bα and Bβ subloci as strain 939 P42, with an additional sublocus at approximately 30 Kbp downstream from the Bα sublocus that contains two phbs (le_pp4 and le_pp5) and an rcb (le_pr5) (Chen et al., 2016). rcb1 and rcb2 contain significant amounts of allelic variations, suggesting a multiallelic nature of the B mating type. In strain 939 P26, the Bα sublocus is comprised of phb5, phb6, and rcb1-939 P26 (rcb1-2), while the Bβ sublocus contains phb7, phb4, and rcb2-939 P26 (rcb2-1). Similarly, strain SUP2 carries phb1, phb2, and rcb1-SUP2 (rcb1-1) in the Bα sublocus and phb3, phb4, and rcb2-SUP2 (rcb2-2) in the Bβ sublocus. Each mating type-specific receptor pairs with two pheromone genes except rcb1-939 P42 (rcb1-3) of strain 939 P42 which has only one pheromone gene phb8 in the Bα sublocus whereas rcb2-939 P42 (rcb2-2) retains phb3 and phb4 in the Bβ sublocus (Wu et al., 2013). The pheromone gene names published as “Lephbx” (Wu et al., 2013) are simplified to phbx in this study. The pheromone receptor genes published as “Lercb1 or 2-strain name” are also renamed to rcb1-x or rcb2-x to better represent the allelic variations. Based on these previous findings, we explored the diversity of L. edodes B mating type at molecular level using sequence information of rcbs and phbs from 111 strains, including 83 cultivated and 28 wild strains, collected from East Asia.
2.2. PCR analysis For sequence determination of pheromone and receptor genes, primer sets specific for phbs and rcbs were designed using consensus sequence regions (Supplementary Table S2). PCR was performed with the following conditions: 94 °C for 5 min; 30 cycles of amplification reaction at 94 °C for 45 s 45 s, 60 °C for 30 s, and 72 °C for 30∼120 s; and 72 °C for 5 min. PCR products were analyzed by agarose gel electrophoresis. Target DNA was extracted from agarose gel and then cloned into a TA vector as described previously (Ha et al., 2018). Sequences of phbs and rcbs were determined by sequencing 1st phb and 2nd phb-rcb1 for Bα sublocus and rcb2-3rd phb and 4th phb for Bβ sublocus. Results are summarized in Supplementary Fig. S1. All sequences discovered in this study were deposited at Genbank (Supplementary Table S3). 2.3. Generation of monokaryon by de-dikaryotization When necessary, monokaryotic strain was generated directly from a dikaryotic strain without making fruiting body using modified protocol of Miyazaki et al. (2000). In brief, mycelia cultured in mushroom complete medium (MCM, MB cell, USA) for 10 days were isolated using a filter paper (Grade 41, Whatman, UK) and subsequently washed twice with distilled water. Cell walls were disrupted in 0.6 M sucrose solution by treatment with a 2.5% a lysing enzyme solution (Sigma, USA) for 3 h at 25℃. Resulting protoplasts were filtered through Miracloth (Calbiochem, USA). The filtrate was then centrifuged at 1000 rpm for 1 min. Protoplasts were washed twice with 0.6 M sucrose solution and suspended in washing solution followed by spreading onto MCM agar containing 0.6 M sucrose. Agar plate was incubated for one week. Monokaryotic mycelia devoid of clamp connections were isolated under a light microscope. 2.4. Gene and protein analyses Exon structures of rcb genes were analyzed using FGENESH with gene-finding parameters specific for Moniliophthora (Salamov and Solovyev, 2000). Transmembrane domain in RCB was predicted with Phobius software (http://phobius.sbc.su.se/, Käll et al., 2004). Phylogenetic trees for RCBs and PHBs were constructed by Maximum Likelihood method with 1000 repeats of bootstrapping using MEGA7 (Kumar et al., 2016). 3. Results 3.1. Identification of alleles of phb and rcb in B mating type locus Analysis of recently published genome sequence of CHAM-B17 strain (Shim et al., 2016), a monokaryotic strain derived from basidiospore of L. edodes CHAM strain (Ha et al., 2015), revealed that the B mating type locus consisted of phb11, phb12, and rcb1-4 in the Bα sublocus and phb9, phb10, and rcb2-3 in the Bβ sublocus (Fig. 1A). Comparison of this new B mating type with other three known B mating types (Wu et al., 2013) showed that each mating type-specific rcb paired
2. Materials and methods 2.1. Strains, culture conditions, and mating assay A total of 111 strains of L. edodes were obtained from various collections, including Culture Collection and DNA Bank of Mushroom 56
Scientia Horticulturae 243 (2019) 55–63
B. Ha et al.
Fig. 1. Structure of the B mating type locus in L. edodes. (A) Arrangement of phbs and rcbs in CHAM-B17 and KFRI976-M2 strains. Arrow boxes represent transcriptional direction. Numbers under genes and intergenic regions indicate lengths in bp. (B) Structure of the B mating type locus in sixteen monokaryotic strains. For comparison, three published B mating types (939 P26, 939 P42, and SUP2) are shown at the bottom (Wu et al., 2013).
CHAM-B17 for M2 strains (Fig. 1B). B mating type loci of all remaining strains essentially consisted of one of phbs-rcb1 pairs in the Bα sublocus and one of phbs-rcb2 pairs in the Bβ sublocus (Fig. 1B, Supplementary Table S3).
with at least one phb and that phb and rcb were arranged in the order of 1st phb-2nd phb-rcb1 or rcb1-2nd phb in the Bα sublocus and rcb2-3rd phb-4th phb in the Bβ sublocus. Based on this finding, we analyzed sequences of phbs and rcbs in Bα and Bβ subloci of 16 monokaryotic strains (Fig. 1B and Supplementary Table S3). Sequence of each rcb-phb pair was nearly identical regardless of its origin. For example, rcb1-1 and phb1-phb2 pairs in the Bα sublocus were identical for strains SUP2, KFRI1478, SJ111, and KFRI3008 except that there were two single nucleotide polymorphisms (SNPs) in rcb1-1 (Supplementary Data S1). In the B mating type locus of monokaryotic CHAM-M1 strains, a compatible partner strain of CHAM-B17 strain, phb11, phb12, and rcb14 in Bα sublocus and phb3, phb4, and rcb2-2 in Bβ sublocus were found (Fig. 1A). This new B mating type was composed of the same Bα sublocus as CHAM-B17 strain and the same Bβ sublocus as SUP2 strain. KFRI976-M1 strain, a monokaryotic strain that can generate dikaryotic KFRI976 strain through mating with KFRI976-M2 strain, carried the same B mating type as 939 P42 strain, consisting of rcb1-3 and phb8 in the Bα sublocus and phb3, phb4, and rcb2-2 in the Bβ sublocus. However, the Bα sublocus in KFRI976-M2 strain was composed of new phbs (phb13 and phb14) and rcb1 (rcb1-5) whereas the Bβ sublocus contained known phbs (phb4 and phb7) and rcb2 (rcb2-1) found in 939 P26 strain. KFRI1478-M1 strain carried the same B mating type discovered in SUP2 whereas the B mating type of KFRI1478-M2 strain was composed of the same Bα sublocus as KFRI976-M1 and 939 P42 strains (rcb1-3 and phb8) and the same Bβ sublocus as CHAM-B17 strain (phb9, phb10, and rcb23). The B mating type locus of FMRC1315-M1 consisted of the same Bα sublocus (phb5, phb6, and rcb1-2) found in strain 939 P26 and the same Bβ sublocus (phb9, phb10, and rcb2-3) found in CHAM-B17 and KFRI1478-M2. Dikaryotic strains SJ701, SJ707, and KFRI619 carried the same B mating type as strain 939 P26 for M1 strains and strain
3.2. Analysis of pheromones and pheromone receptors in B mating type We next performed comparative analyses on protein sequences of RCBs and PHBs. The five and three alleles of RCB1 and RCB2, respectively, were homologous to Ste3 of S. cerevisiae (Nakayama et al., 1985) regardless of mating type subloci where they belonged to (Supplementary Fig. S2 A). Despite having high homology, RCB1s and RCB2s were clustered in different homology groups as reported previously (Wu et al., 2013). Transmembrane topology analysis predicted that all RCBs were membrane proteins consisting of seven transmembrane domains and eight loop domains (Fig. 2A), similar to Ste3 of S. cerevisiae (Nakayama et al., 1985). Among the three loop domains (L3, L5 and L7) predicted to locate outside cell membrane (Fig. 2A), L3 and L5 domains were homologous for all eight RCBs whereas L7 loop domains in RCB1s and RCB2s showed significant variations (Fig. 2B). Particularly, the second β-strand region in L7 was hypervariable in both RCB1s and RCB2s. RCB2s contained additional variable region at the starting nine amino acid residues in L7. Fourteen phb genes encoded polypeptides with 49∼66 amino acid residues. Sequence alignment showed that PHBs at Bα and Bβ subloci were separated by two distinct groups (Fig. 2C and Supplementary Fig. S2B). Bα pheromones commonly contained EH (ER for PHB11) signature for proteolytic cleavage, C-terminal CV(I)V(I)A as CaaX motif for acylation, and GY motif for RCB function (Casselton and Olesnicky, 1998; Brown and Casselton, 2001) whereas Bβ pheromones contained 57
Scientia Horticulturae 243 (2019) 55–63
B. Ha et al.
Fig. 2. Primary structures of RCBs and PHBs. (A) Predicted topology of RCB proteins. Transmembrane domains and loop domains are numbered after TM and L prefixes, respectively. Position of the β-sheet in the loop domain is indicated by arrow box. (B) Sequence comparison of loop domains in RCBs predicted to locate outside membrane. Polymorphic positions are marked in red. Variable regions in loop domains are boxed. (C) Sequence comparison of PHB polypeptides. Predicted proteolytic cleavage site and the C-terminal CaaX box are highlighted in red. Predicted mature pheromones are boxed. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
phbs units from the Bβ sublocus (Table 1).Validity of these suggested B mating types was assessed using eight randomly collected monokaryotic strains. We analyzed genes in B mating type loci of eight monokaryotic strains using primer sets specific to each allele of phb, rcb1, and rcb2 genes. PCR with these specific primer sets yielded distinct DNA bands for phbs and rcbs of each strain (Fig. 4A). The B mating type of each strain was allocated using phb and rcb information according to the scheme described in Table 1. For example, the B mating type locus of FMRC1315-1 strain carried rcb1-2 with its associated pheromones phb5 and phb6 to constitute the Bα sublocus and rcb2-3 associated with phb9 and phb10 for the Bβ sublocus. Thus, the B mating type of this strain was assigned to B6. Similarly, the B mating type of FMRC1315-2 was B8 because it contained phb8-rcb1-3 in the Bα sublocus and phb3-phb4-rcb2-2 in the Bβ sublocus. By such simple PCR analysis, we could determine the identity of allelic genes in the B mating type locus, thereby identifying the B mating type of L. edodes strain within the context of 15 B mating types. We further validated our B mating type classification system through mating assay using eight monokaryotic strains with different B mating types. A mating types of these strains were determined using the method described in our previous paper (Ha et al., 2018). As shown in Fig. 4B, all monokaryotic strains with different B mating types were compatible for mating unless their A mating types were identical as shown in mating pairs FMRC1315-1 x FMRC1315-2, KFRI1478-2 x FMRC1315-1, and KFRI1478-2 x FMRC1315-2, which carried mating type pairs A1B6 X A1B8, A1B6 X A1B9, and A1B8 X A1B9, respectively.
EA for cleavage, CVIS(L) as CaaX motif, and GF or AF motif just before acylation site. Since both Bα and Bβ pheromones are predicted to be acylated through CaaX motif, they might be incorporated into cell membrane, similar to a-factor in yeast in which they interact with RCBs (Michaelis and Herskowitz, 1988). It is known that a-factor interacts with Ste3 receptor to turn on the mating pathway in α-cell of yeast (Caldwell et al., 1995; Lin et al., 2010). No diffusible pheromone was found in L. edodes. Primary structures of pre-PHBs varied. However, predicted mature structures after proteolytic cleavage showed high similarity (Fig. 2C). Overall, mature PHBs could be categorized into three groups. Mature PHBs from the Bα sublocus belonged to Group I (13 amino acids) or Group II (15 amino acids) whereas those from the Bβ sublocus fell into Group III (12 amino acids). Most notably, mature PHB3 and PHB9 in the different Bβ subloci were identical while PHB12 and PHB14 in the Bα subloci differed only in a single amino acid (S/T).
3.3. Determination of B mating types Our sequence analysis on B mating type locus revealed that B mating types of L. edodes were constructed by combinations of Bα and Bβ subloci that could be represented by alleles of rcb1 and rcb2, respectively, with their associated phbs (Fig. 1). It was also revealed that the same phb(s)-rcb pair was recurrently discovered as a unit independently of mushroom strains (Fig. 1). As summarized in Fig. 3, a total of five phb(s)-rcb units, including phb1-phb2-rcb1-1, phb5-phb6rcb1-2, rcb1-3-phb8, phb11-phb12-rcb1-4, and phb13-phb14-rcb1-5, were found from the Bα sublocus whereas three phb(s)-rcb units, including rcb2-1-phb7-phb4, rcb2-2-phb3-phb4, and rbc2-3-phb9-phb10, were discovered from the Bβ sublocus. No additional unit was discovered from the strains used in this study. Based on these findings, we formulated fifteen B mating types of L. edodes through combinations of the five allelic phbs-rcb1 units from the Bα sublocus and the three allelic rcb2-
3.4. Rcb pairs can represent B mating type One important implication derived from a series of our analyses was that a certain rcb always paired with specific phbs such as phb5-phb6rcb1-2 and phb11-phb12-rcb1-4 in the Bα sublocus and phb4-phb7-rcb2-1 and phb9-phb10-rcb2-3 in the Bβ sublocus, regardless of strains (Fig. 3). 58
Scientia Horticulturae 243 (2019) 55–63
B. Ha et al.
Fig. 3. Pheromones (phbs) and pheromone receptor (rcb) pairs recurrently found in different strains of L. edodes. Alleles of rcb1 and rcb2 are numbered as rcb1-x and rcb2-x representing pheromone receptors for Bα and Bβ subloci, respectively. Pheromone genes (phbs) pair with corresponding rcbs are shown in different color. Arrow boxes indicate direction of transcription. Strains in which phb-rcb unit is found are described at the right side of each unit.
Table 1 Proposed B mating types and the selected monokaryotic strains carrying corresponding B mating types. Mating type B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 B13 B14 B15
Bα sublocus
Bβ sublocus
Bα1
rcb1-1, phb1, phb2
Bα2
rcb1-2, phb5, phb6
Bα3
rcb1-3, phb8
Bα4
rcb1-4, phb11, phb12
Bα5
rcb1-5, phb12, phb14
Strains
Bβ1 Bβ2 Bβ3 Bβ1 Bβ2 Bβ3 Bβ1 Bβ2 Bβ3 Bβ1 Bβ2 Bβ3 Bβ1 Bβ2 Bβ3
rcb2-1, rcb2-2, rcb2-3, rcb2-1, rcb2-2, rcb2-3, rcb2-1, rcb2-2, rcb2-3, rcb2-1, rcb2-2, rcb2-3, rcb2-1, rcb2-2, rcb2-3,
This suggests that one can determine B mating type locus of any monokaryotic strain by verifying allelic variants at sites of rcb1 and rcb2 as markers of Bα and Bβ subloci, respectively, instead of determining
phb7, phb3, phb9, phb7, phb3, phb9, phb7, phb3, phb9, phb7, phb3, phb9, phb7, phb3, phb9,
phb4 phb4 phb10 phb4 phb4 phb10 phb4 phb4 phb10 phb4 phb4 phb10 phb4 phb4 phb10
Songo-M1 KFRI1478-M1, SUP2 SJ109-M1 SJ701-M1, SJ707-M1, KFRI619-M1, 939 P26 SMR1, SMR2 FMRC1315-M1 SJ709-M2 KFRI976-M1, FMRC1315-M2, 939 P42 KFRI1478-M2 Songo-M2 CHAM-M1 CHAM-B17, SJ701-M2, SJ707-M2, KFRI619-M2 KFRI976-M2, FMRC8120-M2 IUM4848-M1 IUM4848-M2
whole sets of 14 phbs and 8 rcbs. One such example is shown in Fig. 5. We knew that dikaryotic CHAM strain contained two nuclei with mating types of A1 and A5 at A mating type locus (Ha et al., 2018) and Fig. 4. Detection of mating type genes by PCR. (A) Detection of phbs and rcbs in different monokaryotic strains. phbs and rcbs in monokaryotic strains were detected by PCR with specific primer sets. B mating types assigned by compositions of phbs and rcbs are described at the right side. (B) Confirmation of mating type by mating analysis. ‘O’ indicates a successful mating whereas ‘X’ means failure in mating due to incompatibility in A mating types in this occasion.
59
Scientia Horticulturae 243 (2019) 55–63
B. Ha et al.
Fig. 5. Determination of complete mating types by PCR analysis. PCR with primer sets targeting variable regions in A locus and rcbs in B locus yielded distinct PCR bands. Each column represents A and B mating types of monokaryotic strains of CHAM, SJ701, and SJ707. Name of monokaryotic strains are provided on the top of each column. Mating types of each strain are written under the gel image.
could identify the exact B mating type of each nucleus if we could obtain monokaryons through de-dikaryotization of dikaryons (SMR1, SJ108, SJ705, etc.) or from basidiospores (KFRI1478, SJ701, FMRC1315, etc.) (Table 2, Category 3).
B11 and B12 at the B mating type locus. Monokaryotic strains derived from basidiospores can have tetrapolar combinations of mating type loci, including A5B12, A5B11, A1B11, and A1B12. To confirm this, we analyzed the B locus with primer sets targeting variants of rcb1 and rcb2 and the A locus with A1 and A5 specific primer sets (Ha et al., 2018). PCR yielded two different combinations of rcbs. rcb1-4 and rcb2-2 were found in monokaryotic strains CH2 and CH3 while rcb1-4 and rcb2-3 were found in CH1 and CH15 strains. This indicated that the former two had B11 whereas the latter two had B12 in the B locus. Combining this with information on A locus determined by specific PCR, we could determine tetrapolar mating types of all monokaryotic strains. The same approach also verified tetrapolar mating types of monokaryotic strains derived from basidiospores of SJ701 and SJ707 (Fig. 5). In dikaryotic strains, PCR detected three or four rcbs depending on strains (Table 2). Alleles of rcb1 or rcb2 could be single when these two different mating types representing two nuclei, M1 and M2, in a dikaryotic cytoplasm were constituted by the same rcb1 with two different rcb2s or by two different rcb1s with the same rcb2. For example, ‘Songo’ strain had rcb1-1 and rcb1-4 in Bα and rcb2-1 in Bβ. In this case, the B mating type of one nucleus (M1) contained rcb1-1 + rcb2-1 and that of another nucleus (M2) carries rcb1-4 + rcb2-1 (Table 2, Category 1). Similarly, ‘CHAM’ strain contained rcb1-4 in Bα and rcb2-2 and rcb23 in Bβ. Combinations of these rcbs generated a pair of B mating types that carried rcb1-4 + rcb2-2 in one nucleus (M1) and rcb1-4 + rcb2-3 in another nucleus (M2). When a dikaryotic strain contains two alleles of rcb1 and two of rcb2, there are four possible combinations of rcb pairs. In this case, the exact B mating type of individual nucleus is uncertain. Strains KFRI673, 20,701, SJ103, and some others were representatives of such cases (Table 2, Category 2). For example, rcb analysis on strain 20,701 revealed the presence of rcb1-2 and rcb1-3 in Bα and rcb2-2 and rcb2-3 in Bβ that could have four combinations of rcbs, including rcb1-2 + rcb22, rcb1-3 + rcb2-3, rcb1-2 + rcb2-3, and rcb1-3 + rcb2-2. When B5 (rcb1-2 + rcb2-2) is identified as one B mating type, another nucleus should have B9 (rcb1-3 + rcb2-3) while if B6 (rcb1-2 + rcb2-3) is one B mating type, B8 (rcb1-3 + rcb2-2) should be another type. Therefore, we can only conclude that there is a possibility of two different combinations of B mating types (B5B9 or B6B8 for this strain) for dikaryotic strains belonging to this category. However, even for these strains, we
3.5. Diversity of B mating types To further understand B mating types of L. edodes in terms of their diversity, we analyzed alleles of rcb1 and rcb2 and determined B mating types of 111 dikaryotic strains, including 83 cultivated strains and 28 wild strains collected from East Asian countries (Supplementary Table S4). Among alleles of rcbs, rcb1-3 and rcb1-4 in rcb1s and rcb2-2 and rcb2-3 in rcb2s were more prevalent as shown in Table 3. rcb1-4 was mostly discovered in cultivated strains. Fifty-three out of 58 rcb1-4 were found from cultivated strains while the remaining five were from wild strains. On the contrary, the number of rcb1-5, the rarest rcb, had higher frequency (13/21) in wild strains than that (8/21) in cultivated strains. After comprehensive analysis of rcb compositions, we found seven new combinations of alleles of rcb1 and rcb2, including rcb1-1 + rcb2-1 (B1), rcb1-1 + rcb2-3 (B3), and rcb1-2 + rcb2-2 (B5) (Table 2). These comprised all possible combinations of rcb1s and rcb2s when they were combined with eight different combinations found in CHAM (B11, B12), 939 P (B4, B8), SUP2 (B2), KFRI1478 (B2, B9), and KFRI976 (B8, B13). Together, these combinations of rcb1 and rcb2 represented 15 different B mating types. Ten cultivated strains and thirteen wild strains carried two alleles of rcb1s and two alleles of rcb2s which belonged to ‘Category 2′ in Table 2. Alternative mating pairs of these strains are provided in parentheses (Supplementary Table S4). Different from the diversification of A mating type in wild strains (Ha et al., 2018), distributions of B mating types were relatively random in both cultivated and wild strains (Supplementary Table S4). Mating types B8 and B12 were most frequent B mating types, occurring 37 and 34 times, respectively (Fig. 6A). The least selected B type was B14 which only occurred in a single strain. Mating types B3, B14, and B15 were present only in wild strains when we excluded B mating types uncertain in 23 strains in ‘Category 2′. B mating type pairs, including B2B12, B8B11, and B4B8, occurred most prevalently in cultivated strains (Fig. 6B). 60
Scientia Horticulturae 243 (2019) 55–63
B. Ha et al.
Table 2 Identification of B mating type through combination of rcb1s and rcb2s. Strain
PCR detection
Nucleus M1
rcb1 1
rcb2 2
3
4
5
1
Category 1. One rcb1 + Two rcb2 or Two rcb1 + One rcb2 KFRI57 O Songo O O SJ111 O O IUM1405 O O KFRI1520 O O NAAS4255 O ASI3220 O SL8 O O SJ109 O KFRI2521 O KFRI955 O O SJ110 O SL1 O O Mori250 O CHAM O KFRI2290 O IUM4848 O Category 2. Two rcb1 + Two rcb2a KFRI673 O O 20,701
O
O
KFRI58
O
IUM1851
O O
O O O
O
O O O O O
O O O O
O O O
O
O O
O
O O O O O
O O
O
O
O
O
O
O
Category 3. Two rcb1 + Two rcb2 confirmed by mating or de-dikaryotization KFRI1478 O O O SJ704 O O O IUM3178 O O O SJ701 O O O SMR1 O O O FMRC1315 O O O Yujiro O O O SJ108 O O O O SJ705 O O O KFRI976 O O O O
rcb Combination
B mating type
rcb Combination
3
O O
O
O
2
O O
O
SJ103
B mating type
Nucleus M2
O O O O O O O O
B1 B1 B2 B2 B3 B4 B4 B6 B7 B7 B7 B8 B8 B10 B11 B13 B14
rcb1-1, rcb1-1, rcb1-1, rcb1-1, rcb1-1, rcb1-2, rcb1-2, rcb1-2, rcb1-3, rcb1-3, rcb1-3, rcb1-3, rcb1-3, rcb1-4, rcb1-4, rcb1-5, rcb1-5,
rcb2-1 rcb2-1 rcb2-2 rcb2-2 rcb2-3 rcb2-1 rcb2-1 rcb2-3 rcb2-1 rcb2-1 rcb2-1 rcb2-2 rcb2-2 rcb2-1 rcb2-2 rcb2-1 rcb2-2
B3 B10 B8 B11 B6 B5 B6 B9 B8 B9 B13 B9 B11 B12 B12 B15 B15
rcb1-1, rcb1-4, rcb1-3, rcb1-4, rcb1-2, rcb1-2, rcb1-2, rcb1-3, rcb1-3, rcb1-3, rcb1-5, rcb1-3, rcb1-4, rcb1-4, rcb1-4, rcb1-5, rcb1-5,
rcb2-3 rcb2-1 rcb2-2 rcb2-2 rcb2-3 rcb2-2 rcb2-3 rcb2-3 rcb2-2 rcb2-3 rcb2-1 rcb2-3 rcb2-2 rcb2-3 rcb2-3 rcb2-3 rcb2-3
B1 B2 B5 B6 B8 B9 B8 B9 B10 B11
rcb1-1, rcb1-1, rcb1-2, rcb1-2, rcb1-3, rcb1-3, rcb1-3, rcb1-3, rcb1-4, rcb1-4,
rcb2-1 rcb2-2 rcb2-2 rcb2-3 rcb2-2 rcb2-3 rcb2-2 rcb2-3 rcb2-1 rcb2-2
B8 B7 B9 B8 B12 B11 B14 B13 B14 B13
rcb1-3, rcb1-3, rcb1-3, rcb1-3, rcb1-4, rcb1-4, rcb1-5, rcb1-5, rcb1-5, rcb1-5,
rcb2-2 rcb2-1 rcb2-3 rcb2-2 rcb2-3 rcb2-2 rcb2-2 rcb2-1 rcb2-2 rcb2-1
B2 B2 B3 B4 B5 B6 B6 B8 B8 B8
rcb1-1, rcb1-1, rcb1-1, rcb1-2, rcb1-2, rcb1-2, rcb1-2, rcb1-3, rcb1-3, rcb1-3,
rcb2-2 rcb2-2 rcb2-3 rcb2-1 rcb2-2 rcb2-3 rcb2-3 rcb2-2 rcb2-2 rcb2-2
B9 B12 B11 B12 B12 B8 B8 B10 B12 B13
rcb1-3, rcb1-4, rcb1-4, rcb1-4, rcb1-4, rcb1-3, rcb1-3, rcb1-4, rcb1-4, rcb1-5,
rcb2-3 rcb2-3 rcb2-2 rcb2-3 rcb2-3 rcb2-2 rcb2-2 rcb2-1 rcb2-3 rcb2-1
a B mating type pair in this category could not be designated. Dikaryotic strains can have one of two possible pairs. For example, KFRI673 can have either B1B8 or B2B7 pair.
pheromone-receptor interactions (Casselton and Olesnicky, 1998; Raudaskoski et al., 2012). In the present study, we showed recurrent discovery of five alleles of rcb1 with nine associated phbs and three alleles of rcb2 with five associated phbs to represent Bα sublocus and Bβ sublocus, respectively (Fig. 3). All alleles of RCB1 and RCB2 proteins were predicted to contain seven transmembrane helical domains with three loop domains positioning outside the cell membrane (Fig. 2A). Loop domain L7 was particularly heterologous between RCB1s and RCB2 (Fig. 2B). Variations within RCB1s or RCB2s were also concentrated in this loop domain, suggesting its role in specificity of pheromones. In yeast Ste3p, two extracellular loop domains corresponding to L5 and L7 of RCBs can bind mating pheromone as some mutations in these loops can block pheromone signaling (Gastaldi et al., 2016). Therefore, it is highly conceivable that RCBs in L. edodes can distinguish different PHBs through variations in this loop. Unlike diffusible α-factor in yeast (Kurjan, 1985), all L. edodes PHBs are conceivably a-factor-like membrane-bound pheromones because of the presence of acylation motif (CaaX) at the C-termini (Michaelis and Herskowitz, 1988). PHBs in Bα and Bβ subloci are distinguishable by their proteolytic cleavage motifs (EH for Bα and EA for Bβ) and primary structures of mature pheromones. Mature Bα pheromones are further
Table 3 Occurrence of alleles of pheromone receptors in cultivated and wild strains. Alleles
Bα sublocus
Bβ sublocus
a b
rcb1-1 rcb1-2 rcb1-3 rcb1-4 rcb1-5 rcb2-1 rcb2-2 rcb2-3
Cultivated (C)a
Wild (W)b
27 23 42 53 8 32 66 51
11 3 14 5 13 18 15 19
Total (C + W) 38 26 56 58 21 50 81 70
Ratio (W/ C) 0.41 0.13 0.33 0.09 1.63 0.56 0.23 0.37
Number of occurrence in 83 cultivated strains. Number of occurrence in 28 wild strains.
4. Discussion The B mating type of L. edodes can be determined by mating specific genes residing in the B mating type locus that consists of two subloci, Bα and Bβ (Kües, 2000). Both subloci encode distinct PHBs and RCBs to enable reciprocal activation of the mating pathway through 61
Scientia Horticulturae 243 (2019) 55–63
B. Ha et al.
Fig. 6. Diversity of B mating types. (A) Prevalence of B mating types of 176 nuclei in 88 dikaryotic strains of L. edodes. ‘Frequency’ means the number of occurrence of a certain type of B mating type. (B) Occurrence of B mating type pairs in 88 dikaryotic strains. Twenty-three dikaryotic strains carrying uncertain B mating type pairs described in Supplementary Table S4 are excluded.
divided into two groups (Group1 and Group 2 in Fig. 2C). Mature polypeptides in these two groups are completely different in size and sequence. This may function in the discrimination of RCBs. However, the specificity of RCB to PHB is largely elusive for now. Another notably feature in the primary structure of PHB is that some mature pheromones in the same group share high sequence similarity. PHB12 and PHB14 in Bα sublocus differed only in a single amino acid while PHB3 and PHB9 in Bβ sublocus were even identical (Fig. 2C), meaning that PHB3 and PHB9 are recognized as self pheromone for RCB2-2 and RCB2-3 in Bβ2 and Bβ3 units, respectively (Fig. 3). For a mated strain having Bβ2 and Bβ3 in the Bβ subloci, activation of pheromone response pathway can only occur through cross interactions between PHB4 (Bβ2) and RCB2-3 (Bβ3), and PHB10 (Bβ3) and RCB2-2 (Bβ2). This may explain the reason why the majority of rcbs are in pair with two phb genes. Further biochemical and genetic studies on the pairing of PHB and RCB in the activation of pheromone signal pathway are needed. Among mating specific rcbs, rcb1-3 and rcb1-4 in the Bα sublocus and rcb2-3 and rcb2-3 in the Bβ sublocus are more prevalent. rcb1-4 is particularly frequent in cultivated strains whereas rcb1-5, the rarest allele, has higher frequency in wild strains (Table 3). Prevalence in rcbs reflect biased selection of the nucleus with a certain B mating type. B8 and B12 are prevalent B mating types that contain rcb1-3 and rcb1-4, respectively. It is currently unclear how the nuclei with these B mating types become more competent in cultivated strains. Our previous studies have hinted that dikaryotic strains generated by mating with a certain monokaryotic strain (with certain nucleus) show better fruiting rate and fruiting body production (Ha et al., 2015) and that laccase genes are preferentially expressed by one of the two nuclei in the dikaryotic cytoplasm (Ha et al., 2017). Therefore, prevalent B mating types might have advantages in the expression of desirable characteristics. Findings of the present study lead us to propose 15 B mating types through combinations of five Bα subloci and three Bβ subloci. Multiallelism of B mating type locus has also been observed in Flammulina velutipes (Wang et al., 2016), C. cinerea (Casselton and Olesnicky, 1998; Stajich et al., 2010; Riquelme et al., 2005), and S. commune (Fowler et al., 2001, 2004). Similar to L. edodes, S. commune carries Bα and Bβ subloci with nine alleles per each sublocus. Combinations of them constitute 81 B mating types (Parag and Koltin, 1971; Fowler et al., 2001). The B mating type locus of C. cinerea is more complex. It consists of Group 1, Group 2, and Group 3 subloci with 2, 5, and 7 alleles, respectively, thereby having 70 B mating types (Riquelme et al., 2005). The actual numbers of B mating types for these species can be fewer than predicted because of the suppression of recombination between some subloci (Stamberg and Koltin, 1971). However, our
results suggest that the B mating type locus of L. edodes in the context of 5 Bα and 3 Bβ subloci is freely recombinable since all 15 predicted B mating types are present in our collection of strains. For L. edodes, two independent papers based on mating analyses of wild strains have reported the presence of 40-65 A and 63–100 B mating types from 33 Japanese strains (Tokimoto et al., 1973) and 66 A and 72 B mating types from 53 Chinese strains (Lin et al., 2003). Our previous study on A mating type of 126 strains of L. edodes also provided similar number of A mating types (63 As) (Ha et al., 2018). Therefore, these fifteen B mating types proposed here seem to be too little to cover all B mating types present in nature. However, the proposed number can nicely represent B mating types of all cultivated and wild strains within our strain collection which covers most of cultivated strains in Korea and some Chinese and Japanese strains. If we consider most cultivated strains in East Asia share common origins, the proposed number can serve as a good guide for further study on mating system and breeding of new cultivars. Competing interests statement No potential conflict of interest was reported by the authors. Acknowledgments This work was carriedout with the support of “Cooperation Research Program for Agriculture Science and Technology Development (Project No. PJ01368102)” Rural Development Administration, Republic of Korea. YS, SK, and MSK were supported by a scholarship from the BK 21 Plus Program, the Ministry of Education, Korea. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.scienta.2018.08.009. References Bakkeren, G., Kämper, J., Schirawski, J., 2008. Sex in smut fungi: structure, function and evolution of mating-type complexes. Fungal Genet. Biol. 45, S15–S21. Brown, A.J., Casselton, L.A., 2001. Mating in mushrooms: increasing the chances but prolonging the affair. Trends Genet. 17, 393–400. Caldwell, G.A., Naider, F., Becker, J.M., 1995. Fungal lipopeptide mating pheromones: a model system for the study of protein prenylation. Microbiol. Rev. 59, 406–422. Casselton, L.A., Olesnicky, N.S., 1998. Molecular genetics of mating recognition in basidiomycete fungi. Microbiol. Mol. Biol. Rev. 62, 55–70. Chen, L., Gong, Y., Cai, Y., Liu, W., Zhou, Y., Xiao, Y., Xu, Z., Liu, Y., Lei, X., Wang, G., Guo, M., Ma, X., Bian, Y., 2016. Genome sequence of the edible cultivated mushroom
62
Scientia Horticulturae 243 (2019) 55–63
B. Ha et al.
heterozygous DNA markers in shiitake (Lentinula edodes) using de-dikaryotization via preparation of protoplasts and isolation of four meiotic monokaryons from one basidium. J. Wood Sci. 46, 395–400. Nakayama, N., Miyajima, A., Arai, K., 1985. Nucleotide sequences of STE2 and STE3, cell type-specific sterile genes from Saccharomyces cerevisiae. EMBO J. 4, 2643–2648. O’Shea, S.F., Chaure, P.T., Halsall, J.R., Olesnicky, N.S., Leibbrandt, A., Connerton, I.F., Casselton, L.A., 1998. A large pheromone and receptor gene complex determines multiple B mating type specificities in Coprinus cinereus. Genetics 148, 1081–1090. Parag, Y., Koltin, Y., 1971. The structure of the incompatibility factors of Schizophyllum commune: constitution of the three classes of B factors. Mol. Gen. Genet. 112, 43–48. Raper, J.R., Krongelb, G.S., Baxter, M.G., 1958. The number and distribution of incompatibility factors in Schizophyllum commune. Am. Nat. 92, 221–232. Raudaskoski, M., Kothe, E., Fowler, T.J., Jung, E.M., Horton, J.S., 2012. Ras and Rho small G proteins: Insights from the Schizophyllum commune genome sequence and comparisons to other fungi. Biotechnol. Genet. Eng. Rev. 28, 61–100. Riquelme, M., Challen, M.P., Casselton, L.A., Brown, A.J., 2005. The origin of multiple B mating specificities in Coprinus cinereus. Genetics 170, 1105–1119. Salamov, A.A., Solovyev, V.V., 2000. Ab initio gene finding in Drosophila genomic DNA. Genome Res. 10, 516–522. Shim, D., Park, S.G., Kim, K., Bae, W., Lee, G.W., Ha, B.S., Ro, H.S., Kim, M., Ryoo, R., Rhee, S.K., Nou, I.S., Koo, C.D., Hong, C.P., Ryu, H., 2016. Whole genome de novo sequencing and genome annotation of the world popular cultivated edible mushroom, Lentinula edodes. J. Biotechnol. 223, 24–25. Stajich, J.E., Wilke, S.K., Ahrén, D., Au, C.H., Birren, B.W., Borodovsky, M., Burns, C., Canbäck, B., Casselton, L.A., Cheng, C.K., Deng, J., Dietrich, F.S., Fargo, D.C., Farman, M.L., Gathman, A.C., Goldberg, J., Guigó, R., Hoegger, P.J., Hooker, J.B., Huggins, A., James, T.Y., Kamada, T., Kilaru, S., Kodira, C., Kües, U., Kupfer, D., Kwan, H.S., Lomsadze, A., Li, w., Lilly, W.W., Ma, L.J., Mackey, A.J., Manning, G., Martin, F., Muraguchi, H., Natvig, D.O., Palmerini, H., Ramesh, M.A., Rehmeyer, C.J., Roe, B.A., Shenoy, N., Stanke, M., Ter-Hovhannisyan, V., Tunlid, A., Velagapudi, R., Vision, T.J., Zeng, Q., Zolan, M.E., Pukkila, P.J., 2010. Insights into evolution of multicellular fungi from the assembled chromosomes of the mushroom Coprinopsis cinerea (Coprinus cinereus). Proc. Nat. Acad. Sci. USA 107, 11889–11894. Stamberg, J., Koltin, Y., 1971. Selectively recombining B incompatibility factors of Schizophyllum commune. Mol. Gen. Genet. 113, 157–165. Tokimoto, K., Komatsu, M., Takemaru, T., 1973. Incompatibility factors in the natural population of Lentinus edodes in Japan. Rep. Tottori Mycol. Inst. Jpn. 10, 371–376. Wang, W., Lian, L., Xu, P., Chou, T., Mukhtar, I., Osakina, A., Waqas, M., Chen, B., Liu, X., Liu, F., Xie, B., van Peer, A.F., 2016. Advances in understanding mating type gene organization in the mushroom-forming fungus Flammulina velutipes. G3 6, 3635–3645. Wu, L., van Peer, A., Song, W., Wang, H., Chen, M., Tan, Q., Song, C., Zhang, M., Bao, D., 2013. Cloning of the Lentinula edodes B mating-type locus and identification of the genetic structure controlling B mating. Gene 531, 270–278.
Lentinula edodes (shiitake) reveals insights into lignocellulose degradation. PLoS ONE 11, e0160336. Fowler, T.J., Mitton, M.F., Vaillancourt, L.J., Raper, C.A., 2001. Changes in mate recognition through alterations of pheromones and receptors in the multisexual mushroom fungus Schizophyllum commune. Genetics 158, 1491–1503. Fowler, T.J., Mitton, M.F., Rees, E.I., Raper, C.A., 2004. Crossing the boundary between the Bα and Bβ mating-type loci in Schizophyllum commune. Fungal Genet. Biol. 41, 89–101. Gastaldi, S., Zamboni, M., Bolasco, G., Segni, G.D., Tocchini‐Valentini, G.P., 2016. Analysis of random PCR‐originated mutants of the yeast Ste2 and Ste3 receptors. MicrobiologyOpen 5, 670–686. Ha, B.S., Kim, S., Ro, H.S., 2015. Isolation and characterization of monokaryotic strains of Lentinula edodes showing higher fruiting rate and better fruiting body production. Mycobiology 43, 24–30. Ha, B., Lee, S., Kim, S., Kim, M., Moon, Y.J., Song, Y., Ro, H.S., 2017. Nucleus-selective expression of laccase genes in the dikaryotic dtrain of Lentinula edodes. Mycobiology 45, 379–384. Ha, B., Kim, S., Kim, M., Moon, Y.J., Song, Y., Ryu, J.S., Ryu, H., Ro, H.S., 2018. Diversity of A mating type in Lentinula edodes and mating type preference in domesticated strains. J. Microbiol. 56, 416–425. Huyer, G., Kistler, A., Nouvet, F.J., George, C.M., Boyle, M.L., Michaelis, S., 2006. Saccharomyces cerevisiae a-factor mutants reveal residues critical for processing, activity, and export. Eukaryot. Cell 5, 1560–1570. Käll, L., Krogh, A., Sonnhammer, E.L.L., 2004. A combined transmembrane topology and signal peptide prediction method. J. Mol. Biol. 338, 1027–1036. Kües, U., 2000. Life history and developmental processes in the basidiomycete Coprinus cinereus. Microbiol. Mol. Biol. Rev. 64, 316–353. Kües, U., 2015. From two to many: multiple mating types in Basidiomycetes. Fungal Biol. Rev. 29, 126–166. Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874. Kurjan, J., 1985. Alpha-factor structural gene mutations in Saccharomyces cerevisiae: effects on alpha-factor production and mating. Mol. Cell. Biol. 5, 787–796. Lin, F.C., Wang, Z.W., Sun, Y., Cai, Y.J., 2003. Analysis of the mating type factors in natural population of Lentinula edodes in china. Mycosystema 22, 235–240. Lin, X., Jackson, J.C., Feretzaki, M., Xue, C., Heitman, J., 2010. Transcription factors Mat2 and Znf2 operate cellular circuits orchestrating opposite-and same-sex mating in Cryptococcus neoformans. PLoS Genet 6, e1000953. Michaelis, S., Barrowman, J., 2012. Biogenesis of the Saccharomyces cerevisiae pheromone a-factor, from yeast mating to human disease. Microbiol. Mol. Biol. Rev. 76, 626–651. https://doi.org/10.1128/MMBR.00010-12. Michaelis, S., Herskowitz, I., 1988. The a-factor pheromone of Saccharomyces cerevisiae is essential for mating. Mol. Cell. Biol. 8, 1309–1318. Miyazaki, K., Maeda, H., Sunagawa, M., Tamai, Y., Shiraishi, S., 2000. Screening of
63