Investigation of Mating Pheromone–Pheromone Receptor Specificity in Lentinula edodes

The B mating-type locus of Lentinula edodes, a representative edible mushroom, is highly complex because of allelic variations in the mating pheromone receptors (RCBs) and the mating pheromones (PHBs) in both the Bα and Bβ subloci. The complexity of the B mating-type locus, five Bα subloci with five alleles of RCB1 and nine PHBs and three Bβ subloci with 3 alleles of RCB2 and five PHBs, has led us to investigate the specificity of the PHB–RCB interaction because the interaction plays a key role in non-self-recognition. In this study, the specificities of PHBs to RCB1-2 and RCB1-4 from the Bα sublocus and RCB2-1 from the Bb sublocus were investigated using recombinant yeast strains generated by replacing STE2, an endogenous yeast mating pheromone receptor, with the L. edodes RCBs. Fourteen synthetic PHBs with C-terminal carboxymethylation but without farnesylation were added to the recombinant yeast cells and the PHB–RCB interaction was monitored by the expression of the FUS1 gene—a downstream gene of the yeast mating signal pathway. RCB1-2 (Bα2) was activated by PHB1 (4.3-fold) and PHB2 (2.1-fold) from the Bα1 sublocus and RCB1-4 (Bα4) was activated by PHB5 (3.0-fold) and PHB6 (2.7-fold) from the Bα2 sublocus and PHB13 (3.0-fold) from the Bα5 sublocus. In particular, PHB3 from Bβ2 and PHB9 from Bβ3 showed strong activation of RCB2-1 of the Bβ1 sublocus by 59-fold. The RCB–PHB interactions were confirmed in the monokaryotic S1–10 strain of L. edodes by showing increased expression of clp1, a downstream gene of the mating signal pathway and the occurrence of clamp connections after the treatment of PHBs. These results indicate that a single PHB can interact with a non-self RCB in a sublocus-specific manner for the activation of the mating pheromone signal pathways in L. edodes.


Introduction
The mating process of mushrooms initiates when the mycelial hyphae of compatible monokaryotic strains fuse together to generate new dikaryotic hypha. Compatibility of the mating is determined by genes lying at the chromosomal A and B mating-type loci, which are involved in various steps of the mating process, including hyphal fusion, nuclear pairing, clamp cell formation and nuclear division, clamp cell fusion and nuclear migration to establish dikaryons throughout the connected mycelial cells [1][2][3]. The B mating-type locus has been of particular interest because it is composed of mating pheromone genes (PHBs) and pheromone receptor genes (RCBs) that play a key role in non-self-recognition at the first stage of the whole mating process. RCBs encode G-protein-coupled receptors (GPCRs), which consist of seven transmembrane domains, an extracellular mating pheromone-interacting domain an intracellular domain that interacts with the α subunit of heterotrimeric G protein [3]. PHBs encode pre-pheromone polypeptides of 53-72 amino acids as shown in Coprinopsis cinerea and Schizophyllum commune [4][5][6]. The pre-pheromones undergo a maturation process to form a mature pheromone which includes proteolytic cleavage of the N-terminal pre-peptide, the strain RCY1420 (Table 1, Figure S1 and Table S1). Second, the C-terminal amino acids, K-I-G-G-I, of yeast Gpa1 were replaced with E-A-G-L-L of L. edodes Gpa1 homolog. For this, the strain RCY1432 was generated by transformation with the modification module (C-GPA1-EAGLL (250 bp)-TRP1-Terminator of GPA1) to the strain RCY1420 and subsequent selection against SD agar without histidine and tryptophan (Table 1, Figure S2). Transformation of the yeast cell was performed by the PEG-lithium acetate method using salmon sperm DNA (Invitrogen, Carlsbad, CA, USA) as a carrier DNA. The HIS and TRP markers were from the yeast 2-micron vectors pRS423 and pRS424, respectively.

Total RNA Extraction and PCR Conditions
RCB genes in L. edodes were obtained by reverse transcriptase-PCR (RT-PCR) using L. edodes total RNA as a template. For this, total RNA was extracted from mycelial powder of L. edodes grown on PDA for 14 days using RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). The extracted total RNA was subjected to RT-PCR to obtain cDNA using a HiPi RT-Prime Kit (ELPIS, Daejeon, Korea). PCR was performed with the cDNA and the primer sets specific to RCB genes (Table S2) under the following conditions: hold for 10 min at 95 • C; 25 cycles of 95 • C for 30 s and 60 • C for 30 s and 72 • C for 30-120 s; and 72 • C for 5 min. PCR products were analyzed by 1% agarose gel electrophoresis and recovered using a Gel Elution Kit (Geneall, Seoul, Korea).

Pheromone Response Assay with Synthetic PHBs
Pheromone peptides containing methyl ester at C-terminal cysteine residue were synthesized through a commercial service (GenScript, Piscataway, NJ, USA) ( Table 2). The synthetic PHBs (20 µg/mL) were applied to the recombinant yeast culture grown in fresh YPD (OD600 = 0.6). After incubation for 6 h at 25 • C, cells were harvested by centrifugation (3500 rpm, 10 min) and were washed twice with distilled water. The recovered cells were subjected to total RNA extraction using TRI-RNA reagent (Invitrogen). The expression of FUS1 as a marker of PHB-RCB interaction in yeast was monitored through real-time qRT-PCR using TOPreal™ qPCR 2X PreMix (Enzynomics, Daejeon, Korea). Real-time qPCR was performed under the following conditions: Hold for 10 min at 95 • C; 45 cycles of 95 • C for 20 s, 57 • C for 30 s and 72 • C for 20 s. Relative expression of the target gene was calculated based on the Cq value of β-tubulin gene. All data were obtained in triplicates from three independent experiments. Statistical significance of the mean difference was examined by one-way ANOVA using Microsoft Excel program with Real Statistics Resource Pack software (www.real-statistics.com).
EHDTSDSTDIGYC-OME * OME indicates the carboxymethylation at the C-terminal cysteine residue.
For the flow cytometry analysis, the yeast cells grown at the exponential growth phase (OD600 = 0.2) were harvested. The collected cells were suspended in fresh YPD medium containing 20 µg/mL of PHB and then incubated for 3 h at 25 • C. After the incubation, the yeast cells were collected and then washed twice with phosphate-buffered saline (PBS). The washed cells (2 × 10 6 cells) were resuspended in 1 mL of cold PBS. A volume of 9 mL of 70% ethanol was added to the cell suspension with gentle shaking. The suspension was incubated at −20 • C for 12 h. For the propidium iodide (PI, Sigma-Aldrich) staining, the supernatant was removed after centrifugation and the collected cells were washed with PBS. The PBS-suspended cells were treated with RNase A (100 µg/mL) for 2 h at 37 • C. PI stock solution was added to achieve the final concentration of 20 µg/mL. The PI-treated cells were subjected to flow cytometry analysis using BD FACSVerse TM (Becton Dickinson).

Effect of Synthetic PHBs on Lentinula Edodes S1-10
The monokaryotic S1-10 strain of Lentinula edodes was grown in PDB (100 mL) for a week at 25 • C. The mycelia were harvested by centrifugation. The collected mycelia were suspended in fresh PDB and were fragmented by shaking (150 rpm) in the presence of 5 pieces of glass bead (d = 1 cm, Sigmund Linder GmbH, Germany) for 12 h. The culture broth was incubated additional 2 days after removal of the glass beads. The mycelia were washed twice with fresh PDB after centrifugation. The mycelia were resuspended in 100 mL PDB. Synthetic PHB (final 20 µg/mL) was added to the mycelial suspension at 25 • C. Samples (10 mL) were taken at given time intervals and were subjected to total RNA extraction. The real-time qRT-PCR analysis was performed using the same procedure described above with primer sets specific to target genes (Table S2).
For the microscopic analysis, the PHB-treated mycelia were stained with equivalent mixture of 10% potassium hydroxide and calcofluor white (Sigma-Aldrich), which binds to the chitin component of the fungal cell wall. The stained mycelia were examined under fluorescence microscope (DP-70, Olympus, Japan) at 1000X magnification with excitation at 355 nm.

Construction of Yeast Model System for the Investigation of Lentinula edodes Mating Pheromone Receptor-Pheromone Interactions
The yeast strain RCY1419 carrying L. edodes RCB1-4 instead of yeast STE2 did not respond to the treatment of L. edodes mating pheromones, PHB5 and PHB6, which had been effective in the activation of RCB1-4 in L. edodes [12] (Figure 1a). To sensitize the L. edodes RCB to the mating pheromone in yeast, SST2 in the host yeast cell was deleted. Sst2 is a negative regulator of the mating pheromone signal pathway [29] and its deletion is known to sensitize the mating pheromone signal in yeast [30]. However, RCB1-4 in sst2∆ background (RCY1421) was still unresponsive to PHB5 and PHB6 (Figure 1a). We next replaced the C-terminal five amino acids (KIGGI) of the yeast Gpa1 with the corresponding C-terminal five amino acids (EAGLL) of L. edodes Gpa1. The C-terminal region of Gpa1 is important for the receptor-Gpa1 interaction and the C-terminal modification can increase the interaction between heterogenous receptors in S. cerevisiae [31]. These modifications resulted in the stable response of RCB1-4 to PHB treatment by showing a 5.3-fold increase in FUS1 expression (Figure 1a), even though the RCB1-4-PHB5/PHB6 interaction was not fully comparable to the Ste2-α-factor interaction, which showed a 24-fold increase in the FUS1 expression and complete change in the yeast morphology ( Figure  S4). The optimal PHB treatment time was 6 h, and the FUS1 level decreased beyond 6 h ( Figure 1b). The FUS1 expression increased along with the increase in PHB concentration. However, it gradually decreased after the peak at 20 µg/mL (Figure 1c).
Genes 2020, 11, x FOR PEER REVIEW 5 of 12 Gpa1 is important for the receptor-Gpa1 interaction and the C-terminal modification can increase the interaction between heterogenous receptors in S. cerevisiae [31]. These modifications resulted in the stable response of RCB1-4 to PHB treatment by showing a 5.3-fold increase in FUS1 expression ( Figure 1a), even though the RCB1-4-PHB5/PHB6 interaction was not fully comparable to the Ste2α-factor interaction, which showed a 24-fold increase in the FUS1 expression and complete change in the yeast morphology ( Figure S4). The optimal PHB treatment time was 6 h, and the FUS1 level decreased beyond 6 h ( Figure 1b). The FUS1 expression increased along with the increase in PHB concentration. However, it gradually decreased after the peak at 20 μg/mL ( Figure 1c). The FUS1 expression relative to the β-tubulin gene expression was normalized using triplicated data.
Error bars indicate standard deviations of means.

Interaction between Lentinula edodes RCBs and PHBs in the Yeast Model System
Yeast strains carrying eight RCBs were constructed using RCY1432 as a background strain. Significant activation of the yeast mating pheromone signal pathway was observed from the yeast strains carrying RCB1-2, RCB1-4 and RCB2-1 after the treatment of each of the 14 PHBs at the concentration of 20 μg/mL whereas other RCBs were not responsive to the PHBs for unknown reason ( Figure S5). We focused on these three RCBs for further investigation.

Interaction between Lentinula edodes RCBs and PHBs in the Yeast Model System
Yeast strains carrying eight RCBs were constructed using RCY1432 as a background strain. Significant activation of the yeast mating pheromone signal pathway was observed from the yeast strains carrying RCB1-2, RCB1-4 and RCB2-1 after the treatment of each of the 14 PHBs at the concentration of 20 µg/mL whereas other RCBs were not responsive to the PHBs for unknown reason ( Figure S5). We focused on these three RCBs for further investigation.
The mating pheromone receptor RCB1-2 is one of the alleles of RCB1 found from the Bα sublocus of the B mating-type locus in the commercial strains of L. edodes, including SJ701, SJ707, KFRI619, SMR1 and SMR2 [27]. It has two associated mating pheromone genes, PHB5 and PHB6 [27]. To investigate the interaction between RCB1-2 with 14 PHBs found from different alleles of the 5 Bα and 3 Bβ subloci (Figure 2a), the synthetic PHBs were added with the yeast RCY1433 strain, which was created by incorporating RCB1-2 into the RCY1432 strain. In the single PHB treatment, only PHB1 and PHB2 showed a 4.3-fold and 2.1-fold increase in the FUS1 expression, respectively, while there was no response by other PHBs, including the self PHBs (PHB5 and PHB6) (Figure 2b). Treatment of PHBs in the pair showed a 2.7-fold increase in FUS1 expression only from the PHB1/PHB2 pair, which constitutes the Bα1 sublocus with the mating pheromone receptor RCB1-1 (Figure 2c). This indicates that a single PHB is enough and that PHBs in a pair are not essential for activation of the mating pathway. It also suggests that RCB1-2 in Bα2 interacts with PHBs from the Bα1 sublocus to activate the mating pathway in L. edodes.
Genes 2020, 11, x FOR PEER REVIEW 6 of 12 The mating pheromone receptor RCB1-2 is one of the alleles of RCB1 found from the Bα sublocus of the B mating-type locus in the commercial strains of L. edodes, including SJ701, SJ707, KFRI619, SMR1 and SMR2 [27]. It has two associated mating pheromone genes, PHB5 and PHB6 [27]. To investigate the interaction between RCB1-2 with 14 PHBs found from different alleles of the 5 Bα and 3 Bβ subloci (Figure 2a), the synthetic PHBs were added with the yeast RCY1433 strain, which was created by incorporating RCB1-2 into the RCY1432 strain. In the single PHB treatment, only PHB1 and PHB2 showed a 4.3-fold and 2.1-fold increase in the FUS1 expression, respectively, while there was no response by other PHBs, including the self PHBs (PHB5 and PHB6) (Figure 2b). Treatment of PHBs in the pair showed a 2.7-fold increase in FUS1 expression only from the PHB1/PHB2 pair, which constitutes the Bα1 sublocus with the mating pheromone receptor RCB1-1 (Figure 2c). This indicates that a single PHB is enough and that PHBs in a pair are not essential for activation of the mating pathway. It also suggests that RCB1-2 in Bα2 interacts with PHBs from the Bα1 sublocus to activate the mating pathway in L. edodes. The picture is reproduced from our previous study [27]. (b) Interaction of RCB1-2 with different mating pheromones. P1-P14 represent the mating pheromones, PHB1-PHB14. Individual PHB at a concentration of 20 μg/mL was added to the yeast RCY1433 strain at 30 °C for 6 h. (c) Treatment of PHB pairs representing each sublocus shown in (a). PHBs in total 20 μg/mL (10 μg/mL of each PHB) were added to RCY1433 at 30 °C for 6 h. PHB8, sole PHB representing the Bα3 sublocus, was added as 20 μg/mL. Basal level of the expressed FUS1 in RCY1433 without PHB treatment was used as an experimental control. The FUS1 expression relative to the β-tubulin gene expression was normalized using triplicated data. Error bars indicate standard deviations of means. The statistical significance of the mean difference between PHB-treated sample and the untreated sample (control) is indicated on the bar with asterisks (**** for p ≤ 0.0001, in this case).
We next examined the effects of PHB on RCB1-4 from the Bα sublocus of L. edodes and RCB2-1 from the Bβ sublocus using the yeast RCY1434 and RCY1440 strains, respectively. The enhanced FUS1 The picture is reproduced from our previous study [27]. (b) Interaction of RCB1-2 with different mating pheromones. P1-P14 represent the mating pheromones, PHB1-PHB14. Individual PHB at a concentration of 20 µg/mL was added to the yeast RCY1433 strain at 30 • C for 6 h. (c) Treatment of PHB pairs representing each sublocus shown in (a). PHBs in total 20 µg/mL (10 µg/mL of each PHB) were added to RCY1433 at 30 • C for 6 h. PHB8, sole PHB representing the Bα3 sublocus, was added as 20 µg/mL. Basal level of the expressed FUS1 in RCY1433 without PHB treatment was used as an experimental control. The FUS1 expression relative to the β-tubulin gene expression was normalized using triplicated data. Error bars indicate standard deviations of means. The statistical significance of the mean difference between PHB-treated sample and the untreated sample (control) is indicated on the bar with asterisks (**** for p ≤ 0.0001, in this case). We next examined the effects of PHB on RCB1-4 from the Bα sublocus of L. edodes and RCB2-1 from the Bβ sublocus using the yeast RCY1434 and RCY1440 strains, respectively. The enhanced FUS1 expression through the interaction with RCB1-4 from PHB5, PHB6 and PBH13 was found to be 3.0-, 2.7-and 3.0-fold, respectively (Figure 3a). The interaction of PHB5 and PHB6 from the B2 sublocus with RCB1-4 is already known in L. edodes [12]; however, the activation by PHB13 from the Bα5 sublocus with RCB1-4 is a new finding. Treatment of PHBs in a pair did not show synergistic effect by PHB13/PHB14 from the Bα5 sublocus, however, PHB5/PHB6 from the Bα2 sublocus showed additive effect in the interaction with RCB1-4 ( Figure 3b). RCB2-1 from the Bβ1 sublocus was highly activated by PHB3 and PHB9 from Bβ2 and Bβ3 subloci, respectively (Figure 3c). PHB3-or PHB9-RCB2-1 interaction resulted in a 59-fold increase in the FUS1 expression, which was even higher than the activation by the Ste2-α-factor interaction (24-fold). However, the treatment of both pheromones from Bβ2 (PHB3 and PHB4) or Bβ3 (PHB9 and PHB10) was rather inferior to the individual treatment of PHB3 or PHB9 (Figure 3d), suggesting that the presence of nonspecific pheromone inhibits the specific PHB-RCB interaction.
Genes 2020, 11, x FOR PEER REVIEW 7 of 12 expression through the interaction with RCB1-4 from PHB5, PHB6 and PBH13 was found to be 3.0-, 2.7-and 3.0-fold, respectively (Figure 3a). The interaction of PHB5 and PHB6 from the B2 sublocus with RCB1-4 is already known in L. edodes [12]; however, the activation by PHB13 from the Bα5 sublocus with RCB1-4 is a new finding. Treatment of PHBs in a pair did not show synergistic effect by PHB13/PHB14 from the Bα5 sublocus, however, PHB5/PHB6 from the Bα2 sublocus showed additive effect in the interaction with RCB1-4 ( Figure 3b). RCB2-1 from the Bβ1 sublocus was highly activated by PHB3 and PHB9 from Bβ2 and Bβ3 subloci, respectively (Figure 3c). PHB3-or PHB9-RCB2-1 interaction resulted in a 59-fold increase in the FUS1 expression, which was even higher than the activation by the Ste2-α-factor interaction (24-fold). However, the treatment of both pheromones from Bβ2 (PHB3 and PHB4) or Bβ3 (PHB9 and PHB10) was rather inferior to the individual treatment of PHB3 or PHB9 (Figure 3d), suggesting that the presence of nonspecific pheromone inhibits the specific PHB-RCB interaction. In yeast, the mating pathway activation by Ste2-α-factor interaction results in cell cycle arrest at the G1 phase accompanied by a morphologic change to the "Shmoo" form ( Figure 4). We analyzed the effect of the RCB-PHB interaction on the yeast cell cycle and cell morphology. The RCB2-1-PHB9 interaction, which showed strong activation of the FUS1 expression, resulted in complete morphologic change and cell cycle arrest (Figure 4a, b), whereas, regarding the effects of RCB1-2-PHB1 or RCB1-4-PHB5, PHB6 interactions were less severe. In these cases, although the interactions Basal level of the expressed FUS1 in RCY1434 or RCY1440 without PHB treatment was used as an experimental control. The FUS1 expression relative to the β-tubulin gene expression was normalized using triplicated data. Error bars indicate standard deviations of means. The statistical significance of the mean difference between PHB-treated sample and the untreated sample (control) is indicated on the bar with asterisks (** for p ≤ 0.01, *** for p ≤ 0.01 and **** for p ≤ 0.0001).
In yeast, the mating pathway activation by Ste2-α-factor interaction results in cell cycle arrest at the G1 phase accompanied by a morphologic change to the "Shmoo" form ( Figure 4). We analyzed the effect of the RCB-PHB interaction on the yeast cell cycle and cell morphology. The RCB2-1-PHB9 interaction, which showed strong activation of the FUS1 expression, resulted in complete morphologic change and cell cycle arrest (Figure 4a,b), whereas, regarding the effects of RCB1-2-PHB1 or RCB1-4-PHB5, PHB6 interactions were less severe. In these cases, although the interactions appeared to fail to arrest the cell division cycle, both interactions resulted in swollen cell morphologies, but not the "Shmoo" form ( Figure 4b).
Genes 2020, 11, x FOR PEER REVIEW 8 of 12 appeared to fail to arrest the cell division cycle, both interactions resulted in swollen cell morphologies, but not the "Shmoo" form ( Figure 4b).

Effects of PHBs on the Activation of the Lentinula edodes Mating Pathway
The effect of PHB on the mating pathway activation in L. edodes was investigated by the treatment of PHB1 or PHB9 to the monokaryotic L. edodes S1-10 strain, whose B mating-type locus consists of Bα2 (RCB1-2) and Bβ1 (RCB2-1) subloci. Although it would be ideal to use knock-out strain that only carries single RCB, our investigation was carried out using the S1-10 strain without modification due to lack of available transformation method for L. edodes. It is notable that the sublocus specific PHB-RCB interaction seen in the yeast model study may exclude cross-activation by PHB1-RCB2-1 or PHB9-RCB1-2 interaction.
Upon treatment of PHB1 or PHB9, there were small changes in the expression of HD1, HD2 and znf2 ( Figure 5). A slight decrease in HD1 and HD2 expression was observed 12 h after the PHB1 treatment whereas the expression of znf2 was gradually decreased ( Figure 5). On the other hand, the transcript levels of HD1 and HD2 were peaked after 6-12 h after the PHB9 treatment while the level of znf2 was unchanged. However, clp1, a downstream gene of HD1 and HD2, showed a strong gradual increase in gene expression during 24 h of incubation ( Figure 5). This trend was also true for the PHB9 with more clp1 expression. The elevated expression of clp1 has been considered as a hallmark of successful mating in mushrooms [24,25]. The expression of priA, which may play a role during the beginning of fruiting body formation, increased shortly after the treatment of PHB1 or PHB9. However, it decreased gradually 3-6 h later ( Figure 5). The effect of PHBs was further confirmed by the occurrence of clamp connection, as evidence of the mating pathway activation in the monokaryotic strain ( Figure 5).

Effects of PHBs on the Activation of the Lentinula edodes Mating Pathway
The effect of PHB on the mating pathway activation in L. edodes was investigated by the treatment of PHB1 or PHB9 to the monokaryotic L. edodes S1-10 strain, whose B mating-type locus consists of Bα2 (RCB1-2) and Bβ1 (RCB2-1) subloci. Although it would be ideal to use knock-out strain that only carries single RCB, our investigation was carried out using the S1-10 strain without modification due to lack of available transformation method for L. edodes. It is notable that the sublocus specific PHB-RCB interaction seen in the yeast model study may exclude cross-activation by PHB1-RCB2-1 or PHB9-RCB1-2 interaction.
Upon treatment of PHB1 or PHB9, there were small changes in the expression of HD1, HD2 and znf2 ( Figure 5). A slight decrease in HD1 and HD2 expression was observed 12 h after the PHB1 treatment whereas the expression of znf2 was gradually decreased ( Figure 5). On the other hand, the transcript levels of HD1 and HD2 were peaked after 6-12 h after the PHB9 treatment while the level of znf2 was unchanged. However, clp1, a downstream gene of HD1 and HD2, showed a strong gradual increase in gene expression during 24 h of incubation ( Figure 5). This trend was also true for the PHB9 with more clp1 expression. The elevated expression of clp1 has been considered as a hallmark of successful mating in mushrooms [24,25]. The expression of priA, which may play a role during the beginning of fruiting body formation, increased shortly after the treatment of PHB1 or PHB9. However, it decreased gradually 3-6 h later ( Figure 5). The effect of PHBs was further confirmed by the occurrence of clamp connection, as evidence of the mating pathway activation in the monokaryotic strain ( Figure 5).

Figure 5.
Effect of PHBs on the monokaryotic S1-10 strain of Lentinula edodes. Expression of the downstream genes in the mating pathway was monitored time-dependently. PHB1 or PHB9 (20 μg/mL) was added to actively growing mycelia of L. edodes S1-10 strain at 25 °C. Inset pictures show the microscopic image of the mushroom mycelia after the treatment of PHB1 or PHB9. The mycelia were stained with calcofluor. The circles indicate clamp connections. Error bars indicate standard deviations of means. The statistical significance of the mean difference is indicated on the bar with asterisks (** for p ≤ 0.01, *** for p ≤ 0.01 and **** for p ≤ 0.0001).

Discussion
Mating of fungi belonging to Basidiomycota is initiated by the interaction between pheromone (PHB) and pheromone receptor (RCB). PHBs and RCBs are, necessarily, multiallelic to increase mating opportunities in the wild environment since the specificity of the PHB-RCB interaction is an important factor in self/non-self-recognition in the mating process [1,4,11,31,32]. In accordance with this, C. cinerea [1,4,23] and S. commune [26] are known to have more than 79 and 81 B mating types, respectively. The PHB-RCB interaction in C. cinerea occurs selectively between non-self PHB and RCB [10,14,23]. Studies using S. cerevisiae as a model system have shown that the fungal RCBs expressed in the yeast cells interact selectively with synthetic PHBs or plasmid-expressed PHBs from C. cinerea [18,33] and S. commune [26,34]. The B mating-type of L. edodes is known to consist of five Bα subloci by the combination of five alleles of RCB1 and nine PHBs and three Bβ subloci by combination of three alleles of RCB2 and five PHBs [27,35]. Due to the complexity of PHBs and RCBs, it was necessary to investigate the interaction of PHB and RCB in L. edodes. As in the study in C. cinerea [18,33] and S. commune [26,34], we have used synthetic PHBs and the RCB-expressing yeast strain. In our yeast model, the C-terminal modification of Gpa1 and the deletion of SST2 were necessary to sensitize the RCB interaction with Gpa1 ( Figure 1)-a mediator of pheromone signal to the mating pheromone signal pathway-as previously reported [30,31]. In the treatment of synthetic PHBs of the RCB-expressing yeast strain with modified GPA1 and SST2 deletion, RCB1-2 from the Bα2 sublocus showed a moderate interaction with PHB1 and PHB2 from the Bα1 sublocus ( Figure 2). RCB1-4 from the Bα4 sublocus also showed moderate interactions with PHBs (PHB5 and PHB6) from the Bα2 sublocus and PHB13 from the Bα5 sublocus ( Figure 3). RCB2-1 from the Bβ1 sublocus showed a strong interaction with PHB3 from the Bβ2 sublocus and PHB9 from the Bβ3 sublocus, which was even higher than that of the Ste2-α-factor interaction in S. cerevisiae (Figure 3). The specific PHB-RCB interaction revealed from the yeast study was further confirmed by the expression of clp1 and the formation of a clamp connection in the monokaryotic L. edodes S1-10 strain, as indications of the mating pheromone pathway activation.
It appears that RCBs in L. edodes have a specificity toward a certain group of PHBs. In our previous study, we categorized the L. edodes PHBs into three different groups based on the amino acid composition: PHB1, PHB2, PHB8 and PHB14 to group I; PHB5, PHB6, PHB11 and PHB13 to group II; and PHB3, PHB9, PHB7 and PHB10 to group III [27]. RCB1-2 in this study shows interaction with the group I PHBs (PHB1 and PHB2) whereas RCB1-4 and RCB2-1 interact with the group II Figure 5. Effect of PHBs on the monokaryotic S1-10 strain of Lentinula edodes. Expression of the downstream genes in the mating pathway was monitored time-dependently. PHB1 or PHB9 (20 µg/mL) was added to actively growing mycelia of L. edodes S1-10 strain at 25 • C. Inset pictures show the microscopic image of the mushroom mycelia after the treatment of PHB1 or PHB9. The mycelia were stained with calcofluor. The circles indicate clamp connections. Error bars indicate standard deviations of means. The statistical significance of the mean difference is indicated on the bar with asterisks (** for p ≤ 0.01, and **** for p ≤ 0.0001).

Discussion
Mating of fungi belonging to Basidiomycota is initiated by the interaction between pheromone (PHB) and pheromone receptor (RCB). PHBs and RCBs are, necessarily, multiallelic to increase mating opportunities in the wild environment since the specificity of the PHB-RCB interaction is an important factor in self/non-self-recognition in the mating process [1,4,11,31,32]. In accordance with this, C. cinerea [1,4,23] and S. commune [26] are known to have more than 79 and 81 B mating types, respectively. The PHB-RCB interaction in C. cinerea occurs selectively between non-self PHB and RCB [10,14,23]. Studies using S. cerevisiae as a model system have shown that the fungal RCBs expressed in the yeast cells interact selectively with synthetic PHBs or plasmid-expressed PHBs from C. cinerea [18,33] and S. commune [26,34]. The B mating-type of L. edodes is known to consist of five Bα subloci by the combination of five alleles of RCB1 and nine PHBs and three Bβ subloci by combination of three alleles of RCB2 and five PHBs [27,35]. Due to the complexity of PHBs and RCBs, it was necessary to investigate the interaction of PHB and RCB in L. edodes. As in the study in C. cinerea [18,33] and S. commune [26,34], we have used synthetic PHBs and the RCB-expressing yeast strain. In our yeast model, the C-terminal modification of Gpa1 and the deletion of SST2 were necessary to sensitize the RCB interaction with Gpa1 ( Figure 1)-a mediator of pheromone signal to the mating pheromone signal pathway-as previously reported [30,31]. In the treatment of synthetic PHBs of the RCB-expressing yeast strain with modified GPA1 and SST2 deletion, RCB1-2 from the Bα2 sublocus showed a moderate interaction with PHB1 and PHB2 from the Bα1 sublocus ( Figure 2). RCB1-4 from the Bα4 sublocus also showed moderate interactions with PHBs (PHB5 and PHB6) from the Bα2 sublocus and PHB13 from the Bα5 sublocus ( Figure 3). RCB2-1 from the Bβ1 sublocus showed a strong interaction with PHB3 from the Bβ2 sublocus and PHB9 from the Bβ3 sublocus, which was even higher than that of the Ste2-α-factor interaction in S. cerevisiae (Figure 3). The specific PHB-RCB interaction revealed from the yeast study was further confirmed by the expression of clp1 and the formation of a clamp connection in the monokaryotic L. edodes S1-10 strain, as indications of the mating pheromone pathway activation.
It appears that RCBs in L. edodes have a specificity toward a certain group of PHBs. In our previous study, we categorized the L. edodes PHBs into three different groups based on the amino acid composition: PHB1, PHB2, PHB8 and PHB14 to group I; PHB5, PHB6, PHB11 and PHB13 to group II; and PHB3, PHB9, PHB7 and PHB10 to group III [27]. RCB1-2 in this study shows interaction with the group I PHBs (PHB1 and PHB2) whereas RCB1-4 and RCB2-1 interact with the group II PHBs (PHB5, PHB6 and PHB13) and group III PHBs (PHB3 and PHB9), respectively. The results imply that the membrane-embedded RCBs have group-specific recognition motifs in their loop domains, potentially in the L3, L5 and L7 domains described in our previous report [27]. It also implies that the non-self recognition in the mushroom mating can occur though the PHB group-specific interaction with RCBs.
C-terminal farnesylation in fungal PHBs is known to be important for the function of pheromone [36][37][38][39][40]. Little is known about the maturation of mating pheromones in L. edodes. However, the presence of CAAX motif suggests possible farnesylation at the C-terminus. The present study demonstrates that some synthetic PHBs can activate the mating pheromone signal pathway without the C-terminal farnesylation. However, the C-terminal farnesylation appears to be requisite for the full function of PHB since the synthetic PHBs without farnesylation failed to interact with other L. edodes RCBs in the yeast model ( Figure S5).

Conclusions
Through the yeast model study, we discovered that a single PHB, despite found as a pair to constitute a sublocus, is enough to activate a certain RCB. We also found that RCB is only activated by PHB of the same sublocus; that is, the Bα receptors are specific to Bα PHBs and Bβ receptors are specific to Bβ PHBs.
Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4425/11/5/506/s1, Figure S1: Deletion of SST2 using integration module; Figure S2: Modification of GPA1 using integration module, Figure S3: Replacement of yeast STE2 with RCB from Lentinula edodes, Figure S4: Effects of α-factor on the morphology and expression of FUS1 in yeast, Figure S5: Effects of PHBs on the activation of RCBs in yeast model, Table S1: Primers used for cloning, Table S2