Overexpression of the Capebp2 Gene Encoding the PEBP-like Protein Promotes the Cap Redifferentiation in Cyclocybe aegerita

Phosphatidylethanolamine-binding protein (PEBP) is widely involved in various physiological behaviors, such as the transition from vegetative growth to reproductive growth in plants, tumorigenesis in the human, etc. However, few functional studies have examined pebp genes affecting the development of fungi. In this study, Capebp2 was cloned from Cyclocybe aegerita AC0007 strains based on the genome sequence and gene prediction, and the sequence alignment of CaPEBP2 with other PEBP proteins from other biological sources including plant, animal, fungi, and bacteria indicated that PEBP had low sequence similarity in fungi, whereas all protein sequences had some conserved motifs such as DPDAP and HRY. Expression analysis showed the transcription level of Capebp2 increased approximately 20-fold in fruiting bodies compared with mycelia. To uncover the function of Capebp2 in C. aegetita development, Capebp2 was cloned into a pATH vector driven by the actin promoter for obtaining overexpression transformant lines. Fruiting experiments showed the transformed strains overexpressing Capebp2 exhibited redifferentiation of the cap on their surface, including intact fruiting bodies or partial lamella during fruiting development stage, and the longitudinal section indicated that all regenerated bodies or lamella sprouted from the flesh and shared the epidermis with the mother fruiting bodies. In summary, the sequence characterization of Capebp2, expression level during different development stages, and function on fruiting body development were documented in this study, and these findings provided a reference to study the role of pebp in the development process of basidiomycetes. Importantly, gene mining of pebp, function characterization, and the regulating pathways involved need to be uncovered in further studies.


Introduction
Phosphatidylethanolamine-binding protein (PEBP) is a class of regulatory proteins that widely exist in bacteria, fungi, plants, and animals. It was originally isolated from the bovine brain [1] and named PEBP1 due to its preferred binding to phosphatidylethanolamine [2]. In mammals, the PEBP2 and PEBP4 proteins have been successively discovered based on their functional characteristics, although the transcript of pebp3 is not found in the tested species and is presumed to be a pseudogene [3].
PEBP1 is widely involved in various cellular physiological behaviors, including cell growth and differentiation [4], cell cycle [5], and genome stability [6]. In plants, the functions and molecular regulatory pathways of the PEBP protein family have been thoroughly remains unknown. As an important commercial mushroom, C. aegerita is a delicious species with high nutrient value. It is a typical basidiomycete species with a short life cycle, which renders this species important in fruiting body development research. In this study, a gene encoding PEBP named Capebp2 of C. aegerita 0007 was cloned based on genome annotation, and the sequence characterization was performed compared with the homology genes in other species. Functional analysis of pepb2 was conducted by the overexpression of Capebp2 in the C. aegerita 0007 strain, and the morphologies of transformants were compared with those of wild strains during various developmental stages, providing new insights to understand the functions of PEBP in fungi.

Strains and Culture Conditions
C. aegerita dikaryotic strain AC0007 was employed for the experiment in this study. The mycelia were cultivated on a YPD medium plate (0.2% yeast extract, 0.2% peptone, 2% dextrose, and 1.5% agar) at 25 • C. Three agar disks (1 cm diameter each) were inoculated in a sawdust medium (64% hardwood sawdust, 15% wheat bran, 1% plaster, and 20% cottonseed shells) to produce fruiting bodies. Two batches of fruiting tests for transformed strains were separately conducted in a greenhouse or with a thermostat with constant humidity. E. coli DH5α strain used for plasmid amplification was cultured in Luria-Bertani (LB) medium containing 100 µg/mL ampicillin.

Capebp2 Cloning and Sequence Analysis
The genomic DNA of AC0007 was extracted from mycelia using the fungal gDNA isolation kit (Biomiga, San Diego, CA, USA). Total RNA was prepared from the tissues using the RNAiso reagent (TaKaRa, Dalian, China), and single-stranded cDNA was synthesized from 0.75 µg of total RNA using the PrimeScript TM RT reagent kit (TaKaRa, Kyoto, Japan). The complete DNA and cDNA products of Capebp2 were obtained using the primer pairs capebp2F/R (Table 1) based on the genome information of AC0007. These products were then cloned into the PMD19T vector for sequencing. For phylogenetic analyses, multiple protein sequence alignments were performed by the program MUSCLE in the software MEGA6 v6.06 [40] using default parameters, and the phylogenetic trees for the PEBP sequences were inferred using the neighbor-joining (NJ) method. PEBP sequences from species of plants, animals, fungi, and bacteria were obtained by blasting CaPEBP sequences against the NCBI GenBank database.

Vector Construction and Transformation
The overexpression vector was constructed and transformed using the method described by a previous study [41]. In brief, The pATH vector contains the CaMV35S terminator, the hygromycin B phosphotransferase gene (HygR), the gpd promoter from Ustilago maydis (Pum-gpd), and C. aegerita actin promoter (Ca-actin) for driving the overexpression of HygR and the targeted gene, respectively. The amplified products of Capebp2 cDNA using the primer pairs capebp2F/R (Table 1) were ligated into the EcoRV enzymedigested site between the pCa-actin and T35s elements of the pATH vector. The constructed vector pATH-pebp2 was transferred to DH5α for plasmid amplification. The genetic transformation was conducted using the PEG-mediated method, as described previously [28]. The mycelia were digested using a lysing enzyme solution (1% cellulase R-10 (Yakult Pharmaceutical Industry Co., Ltd, Tokyo, Japan), 1% Lywallzyme (Biktak Bio Tech, Beijing, China), and 1% lysing enzyme (Sigma-Aldrich, St. Louis, MI, USA) in a 0.6 M mannitol buffer), following which the protoplasts were washed in STC solution (1.2 M sorbitol, 10 mM CaCl 2 , and 50 mM Tris-HCl, pH 7.0) to mix them with plasmid DNA for transformation. The reaction mixture was added to a YPD regeneration medium (20.5% sucrose, 0.2% tryptone, 0.2% yeast extract, 1% agar), following which a YPD medium containing 150 µg/mL hygromycin B was poured over the plates to screen transformants after five days.

Transformant Verification and Expression Analysis
The regenerated strains were transferred to a YPD medium containing 120 µg/mL hygromycin B to further verify strain resistance. Primer pairs 19 ha3/19 ha4 anchored to the flanking region of the inserted fragment were used for PCR validation ( Table 1). The positive transformants were deposited in a YPD medium containing 50 µg/mL hygromycin B as the selection pressure. All strains and plasmids were deposited in our laboratory.
The qPCR reaction was performed in a CFX96 real-time PCR detection system (Bio-Rad). The gpd gene was used as the internal reference gene for qPCR. The primer pairs gpd qF/R and pebp qF/R were used for the amplification of gpd and Capebp2, respectively ( Table 1). The reaction system included 10 µL of iTaqTM universal SYBR green SuperMix (Bio-Rad Laboratories, Hercules, CA, USA), 1 µL of upstream primer, 1 µL of downstream primer, and 8 µL of ultrapure water, in a total volume of 20 µL. Each sample was analyzed in triplicate. The reaction conditions were as follows: initial polymerase activation at 95 • C for 20 s, followed by 40 cycles of 95 • C for 5 s and 60 • C for 30 s. Each sample was performed in triplicate. Glyceraldehyde-3-phosphate dehydrogenase (GPD) was used as the reference gene for the normalization of the qPCR data. The gene expression levels were calculated using the 2 −∆∆CT method.

Data Analysis
The compilation and mapping of experimental data were performed using Graphpad prism5 software. One-way analysis of variance (ANOVA) and the least significant difference (LSD) test were used to examine the significant differences among samples. All experiments were conducted in triplicate for data precision.

Sequence Analysis of Capebp2
Based on the genome sequence and gene prediction, the primer pairs capebp2F/R were used to amplify the complete DNA and cDNA sequences. Sequence analysis revealed that Capebp2 was 1182 bp in length and contained five introns and six exons encoding 299 aa ( Figure 1A). The sequence alignment of CaPEBP2 with other PEBP proteins from other biological sources indicated that PEBP had low sequence similarity in fungi, whereas all protein sequences had three conserved motifs (C1-C3) in the D1 and D2 regions ( Figure 1B). The phylogenetic analysis of the PEBP protein sequences indicated that species from plants, animals, fungi, and bacteria were divided into independent branches. Among them, proteins from plants and animals were clustered together and further clustered with those of fungi ( Figure 1C). In the clade of fungi, the sequence of CaPEBP2 was closest to that of C. aegerita (CAA7265478), indicating that both shared high similarity ( Figure 1C). The DNA and protein sequences of Capebp2 are shown in Supplementary File S1. species from plants, animals, fungi, and bacteria were divided into independent branches. Among them, proteins from plants and animals were clustered together and further clustered with those of fungi ( Figure 1C). In the clade of fungi, the sequence of CaPEBP2 was closest to that of C. aegerita (CAA7265478), indicating that both shared high similarity (Figure 1C). The DNA and protein sequences of Capebp2 are shown in Supplementary File S1.

Characterization of the Expression of Capebp2 during the Development of C. aegerita
To characterize the expression of Capebp2 in different developmental stages, mycelia and fruiting bodies were collected to prepare for RNA extraction. The primer pairs Pebp qF/R were used to determine the expression of Capebp2, and Gpd qF/R was used as the reference gene for the normalization of the qPCR data. The results revealed that the expression level increased approximately 20-fold in fruiting bodies ( Figure 2).

Characterization of the Expression of Capebp2 during the Development of C. aegerita
To characterize the expression of Capebp2 in different developmental stages, mycelia and fruiting bodies were collected to prepare for RNA extraction. The primer pairs Pebp qF/R were used to determine the expression of Capebp2, and Gpd qF/R was used as the reference gene for the normalization of the qPCR data. The results revealed that the expression level increased approximately 20-fold in fruiting bodies ( Figure 2). species from plants, animals, fungi, and bacteria were divided into independent branches. Among them, proteins from plants and animals were clustered together and further clustered with those of fungi ( Figure 1C). In the clade of fungi, the sequence of CaPEBP2 was closest to that of C. aegerita (CAA7265478), indicating that both shared high similarity (Figure 1C). The DNA and protein sequences of Capebp2 are shown in Supplementary File S1.

Characterization of the Expression of Capebp2 during the Development of C. aegerita
To characterize the expression of Capebp2 in different developmental stages, mycelia and fruiting bodies were collected to prepare for RNA extraction. The primer pairs Pebp qF/R were used to determine the expression of Capebp2, and Gpd qF/R was used as the reference gene for the normalization of the qPCR data. The results revealed that the expression level increased approximately 20-fold in fruiting bodies ( Figure 2).

Validation of Transformants and Expression of Capebp2
The overexpression transformants were obtained by constructing a pATH-pebp2 vector driven by the Ca-actin promoter ( Figure 3A,B). The transformants were determined through hygromycin screening and PCR verification. The regeneration strains were transferred to a medium containing hygromycin B for analysis of resistance. Four strains, namely, T2-1, T2-7, T2-10, and T2-11, showed significant resistance to hygromycin B, whereas the growth of wild-type strains was completely inhibited ( Figure 3C).
The overexpression transformants were obtained by constructing a pATH-pebp2 vector driven by the Ca-actin promoter ( Figure 3A,B). The transformants were determined through hygromycin screening and PCR verification. The regeneration strains were transferred to a medium containing hygromycin B for analysis of resistance. Four strains, namely, T2-1, T2-7, T2-10, and T2-11, showed significant resistance to hygromycin B, whereas the growth of wild-type strains was completely inhibited ( Figure 3C).
The resistance transformants were further verified by PCR amplification using the specific primer pair adhF and adhR targeting the actin promoter and T35s regions, and the sequencing results revealed that they were all positive transformants ( Figure 3D). The mycelia of four transformants were collected for extraction of total RNA, and an equal amount of RNA was used for cDNA synthesis. The expression of Capebp2 in all four transformants increased to a high level of approximately 9.60-fold, 12.53-fold, 22.69-fold, and 27.74-fold in T2-1, T2-7, T2-10, and T2-11, respectively, compared to that in the WT strain ( Figure 3E).  The resistance transformants were further verified by PCR amplification using the specific primer pair adhF and adhR targeting the actin promoter and T35s regions, and the sequencing results revealed that they were all positive transformants ( Figure 3D). The mycelia of four transformants were collected for extraction of total RNA, and an equal amount of RNA was used for cDNA synthesis. The expression of Capebp2 in all four transformants increased to a high level of approximately 9.60-fold, 12.53-fold, 22.69-fold, and 27.74-fold in T2-1, T2-7, T2-10, and T2-11, respectively, compared to that in the WT strain ( Figure 3E).

The Effect of Overexpression of Capebp2 on the Fruiting Bodies
The mycelia of WT strains and the four transformants were inoculated in a sawdust medium at 25 • C for 40 days and then transferred to a greenhouse or thermostat with constant humidity for fruiting. The plasmid detection was conducted in fruiting bodies of four transformants using the specific primer pair adhF and adhR targeting the actin promoter and T35s regions for PCR verification, and the result showed the plasmids were all present in tested samples (Supplementary File S2).
All fruiting bodies of Capebp2-overexpression strains exhibited redifferentiation of the cap on their surface ( Figure 4A-C). These fruiting bodies were of two types: (1) complete fruiting bodies in T2-1, T2-10, and T2-11, among which the regeneration fruiting bodies sprouted from the flesh of fungi with a stalk or cap; and (2) partial lamella without stalk in T2-1 and T2-7, among which the cap surface sprouted partial lamella and the longitudinal section indicated that all regenerated bodies or lamella sprouted from the flesh and shared the epidermis with the mother fruiting bodies. cate data that differed significantly based on a p-value of < 0.0001 (t-test) as the significance threshold.

The Effect of Overexpression of Capebp2 on the Fruiting Bodies
The mycelia of WT strains and the four transformants were inoculated in a sawdust medium at 25 °C for 40 days and then transferred to a greenhouse or thermostat with constant humidity for fruiting. The plasmid detection was conducted in fruiting bodies of four transformants using the specific primer pair adhF and adhR targeting the actin promoter and T35s regions for PCR verification, and the result showed the plasmids were all present in tested samples (Supplementary File S2).
All fruiting bodies of Capebp2-overexpression strains exhibited redifferentiation of the cap on their surface ( Figure 4A-C). These fruiting bodies were of two types: (1) complete fruiting bodies in T2-1, T2-10, and T2-11, among which the regeneration fruiting bodies sprouted from the flesh of fungi with a stalk or cap; and (2) partial lamella without stalk in T2-1 and T2-7, among which the cap surface sprouted partial lamella and the longitudinal section indicated that all regenerated bodies or lamella sprouted from the flesh and shared the epidermis with the mother fruiting bodies.
To examine the expression level of Capebp2 in fruiting bodies, the tissues in the regeneration site were sampled for cDNA synthesis. Capebp2 in fruiting bodies also showed a high expression level in four transformants, approximately 22.94-fold, 5.07-fold, 45.08fold, and 8.12-fold in T2-1, T2-7, T2-10, and T2-11, respectively, compared to the level in the WT strain ( Figure 5).  To examine the expression level of Capebp2 in fruiting bodies, the tissues in the regeneration site were sampled for cDNA synthesis. Capebp2 in fruiting bodies also showed a high expression level in four transformants, approximately 22.94-fold, 5.07-fold, 45.08-fold, and 8.12-fold in T2-1, T2-7, T2-10, and T2-11, respectively, compared to the level in the WT strain ( Figure 5).

Discussion
The function of the PEBP protein family has been thoroughly studied in plants and is known to finely regulate flower formation under the regulation of light signals [14,31,42]. It also plays a regulatory role in endosperm development [43], tuber and bulb formation, and bud development [44,45]. However, the function of this gene family has not been studied in fungi. C. aegerita is a delicious species widely cultivated in eastern Asia, with high nutrient value. Importantly, its short life cycle renders this species as an ideal species for studying the development of fruiting bodies. In the previous study, we screened some genes highly expressed at the fruiting body stage through transcriptome analysis, one of which was annotated as pebp gene. However, the protein sequence of it was quite different from that of PEPB gene in other species such as plants; we further analyzed the sequence characteristics and function of this gene in C. aegerita.
The PEBP family protein sequence is highly conserved in plants and animals [9,12,17,26], and is easily found through omics analysis. However, it has not been explored in fungi for a long time, mainly due to the significant differences in sequence characteristics between animals and plants, which affects the finding of PEBP sequences in fungi. In this study, systematic analysis revealed that the PEBP protein had high similarity in plants, animals, fungi, and bacteria, and was divided into four clades in the phylogenetic tree. Sequence alignment indicated that all PEBP protein sequences had several conserved motifs, including DPDAP and HRY, thus providing a reference for the identification of PEBP sequences in other species. In plants, PEBP gene numbers ranges from 6-8 to nearly 24 [9]. In Zea mays, 24 genes that encode PEBP-like proteins are identified, which is divided into three major subfamilies including TFL1-like (six members), mother of ft and TFL1like (three members), and FT-like based on the protein phylogeny analysis (fifteen members). Protein sequence analysis shows several motifs DPD, PS, HR, and HW are all conserved in the PEBP family [9]. However, due to the limited number of sequences of pebp genes in fungi, it is difficult to classify them into groups based on the sequence characteristics and infer their functions.
The overexpression of Capebp2 in transformant strains during the fruiting stage led to cap redifferentiation, including lamella and intact fruiting bodies. This phenomenon has not been reported before, and more interestingly, both regenerated tissues and intact

Discussion
The function of the PEBP protein family has been thoroughly studied in plants and is known to finely regulate flower formation under the regulation of light signals [14,31,42]. It also plays a regulatory role in endosperm development [43], tuber and bulb formation, and bud development [44,45]. However, the function of this gene family has not been studied in fungi. C. aegerita is a delicious species widely cultivated in eastern Asia, with high nutrient value. Importantly, its short life cycle renders this species as an ideal species for studying the development of fruiting bodies. In the previous study, we screened some genes highly expressed at the fruiting body stage through transcriptome analysis, one of which was annotated as pebp gene. However, the protein sequence of it was quite different from that of PEPB gene in other species such as plants; we further analyzed the sequence characteristics and function of this gene in C. aegerita.
The PEBP family protein sequence is highly conserved in plants and animals [9,12,17,26], and is easily found through omics analysis. However, it has not been explored in fungi for a long time, mainly due to the significant differences in sequence characteristics between animals and plants, which affects the finding of PEBP sequences in fungi. In this study, systematic analysis revealed that the PEBP protein had high similarity in plants, animals, fungi, and bacteria, and was divided into four clades in the phylogenetic tree. Sequence alignment indicated that all PEBP protein sequences had several conserved motifs, including DPDAP and HRY, thus providing a reference for the identification of PEBP sequences in other species. In plants, PEBP gene numbers ranges from 6-8 to nearly 24 [9]. In Zea mays, 24 genes that encode PEBP-like proteins are identified, which is divided into three major subfamilies including TFL1-like (six members), mother of ft and TFL1-like (three members), and FT-like based on the protein phylogeny analysis (fifteen members). Protein sequence analysis shows several motifs DPD, PS, HR, and HW are all conserved in the PEBP family [9]. However, due to the limited number of sequences of pebp genes in fungi, it is difficult to classify them into groups based on the sequence characteristics and infer their functions.
The overexpression of Capebp2 in transformant strains during the fruiting stage led to cap redifferentiation, including lamella and intact fruiting bodies. This phenomenon has not been reported before, and more interestingly, both regenerated tissues and intact fruiting bodies were regenerated from the flesh of the fungus. This indicates that the trigger point for regeneration lies in the flesh of the fungus, and further studies are needed to determine whether it is related to the aggregation of the PEBP protein in the flesh. In plants, the transporting path of florigens from the leaf to shoot apical meristem has been well studied. The FT interacting protein 1 (FTIP1) is accompanied by FT proteins moving from companion cells to sieve elements by passive diffusion [46], and other components involved in FT long-distance movement were also uncovered [47,48], which provides a research idea for the study of intracellular production and metastasis of PEBP in fungi.
The phenomenon of cap redifferentiation has been rarely observed in fungi. Environmental signals, oxygen, CO 2 , and light can exert a profound influence on fruiting body development. Moreover, temperature, humidity, volatiles, pH, salinity, and availability of nutrients may play an important role [49][50][51][52]. Light as a signal induces fruiting body formation in many fungi, regulating the primordia formation [53,54] and maturation [55,56]. A red-light receptor FphA represses sexual development [57], while the blue-light LreA/LreB receptor complex stimulates this process [58]. However, the light response genes revealed no correlation with fruiting body structure except stipe bend, and the studies on the cap development are also rarely reported. In Coprinopsis cinerea, the exp1 gene encoding an HMG1/2-like protein is strongly induced in the pileus 3 h before pileus expansion, and the mature fruiting bodies of exp1 mutant strains normally elongated stipes but with unexpanded pilei remained intact for a long time [59]. In addition, the phenotype of fruiting bodies directly affects the commercial traits of edible mushrooms. To control the phenotype of fruiting bodies through environmental condition changes, it is necessary to clarify the key genes and their molecular regulatory pathways that control the phenotype. Although numerous gene-phenotype combinations have been found in previous studies, there have been no relevant reports on the trait of fruiting body redifferentiation. The regenerative feature requires further clarification of the functional characteristics of the pebp genes in basidiomycetes during fruiting body development, including its action and regulation mechanisms.
As important regulation factors, PEBP family proteins are involved in multiple molecular pathways. In Arabidopsis, rice, and potato, it was confirmed that the FT and TFL1 proteins bind to the bZIP-type transcription factor FD through the 14-3-3 protein as a coactivator or inhibitor [29,[60][61][62]. TFL1-FD inhibits the key regulators CO and GIGANTEA (GI) that promote flowering and repress the expression of FRUITFULL (FUL), LEAFY (LFY), and APETALA1 (AP1), which causes the switch from the branching to flowering state in axillary meristems [28,44,63,64]. These results indicated that the PEBP protein targeting the upstream genes in the molecular pathway regulates the transition from vegetative growth to reproductive stages in plants. In future studies, the environmental factors regulating the PEBP protein and the molecular signaling pathways involved need clarification to explore the function of the PEBP protein family in fungi.

Conclusions
PEBP family proteins exist widely in biological sources; they regulate various physiological behaviors. However, due to differences in sequence composition, the function of PEBP family proteins has not been revealed in fungi. In this study, a pebp-like gene, Capebp2, was cloned and identified in the basidiomycetes C. aegerita. The sequence analyses showed PEBP were highly conserved in plants and animals, but had low sequence similarity in fungi. However, some conserved motifs, such as DPDAP and HRY, provided references for pebp gene discovery and identification in fungi. The transcription investigation showed Capebp2 may be related to fruiting development reaching a high level in fruiting bodies. The overexpression of Capebp2 led to cap redifferentiation including intact fruiting bodies and lamella from the flesh of the fungus. These results should provide an important insight for further investigations into the role of the PEBP protein in fruiting body development in C. aegerita. However, the production and transportation pathways of PEBP protein, as well as its target and regulatory pathways for fruiting body development, still require further research. In future research, pepb gene mining, response to environmental factors, function features, and the regulatory pathways involved need to be elucidated in fungi.

Data Availability Statement:
The data presented in the study are deposited in the Supplementary File S1.

Conflicts of Interest:
The authors declare no conflict of interest.