Genome-wide identification and expression analyses of C2H2 zinc finger transcription factors in Pleurotus ostreatus

Introduction

Pleurotus ostreatus is a mushroom that is widely cultivated for its nutritional value and relatively simple cultivation techniques (Chang & Miles, 2004; Khan & Tania, 2012). The production of P. ostreatus relies on the precise control of fruiting body development. The formation of fruiting bodies starts when two hyphae with different mating types combine to form dikaryotic hyphae during a process called plasmogamy. If these dikaryotic hyphae aggregate, they will develop into primordia, which will then differentiate into fruiting bodies. Genome sequencing of model mushroom Schizophyllum commune indicated that many predicted transcription factors like zinc finger proteins (ZFPs), MYB, fungal specific transcription factor (fst), and so on are differentially expressed during sexual development (Ohm et al., 2010). The Pofst3 gene in P. ostreatus is a homolog of the fst3 gene in S. commune and was determined to play a role in primordia formation (Qi et al., 2019). These results indicated that certain transcription factors may mediate the development of P. ostreatus. However, only a few transcription factors have been identified in this commercial mushroom. The identification and characterization of more transcription factors in P. ostreatus could help researchers identify interesting proteins involved in various development processes or help breeders selectively breed for controlled mushroom development.

ZFPs are one of the largest transcription factor families in eukaryotic genomes (Laity, Lee & Wright, 2001). The term “zinc finger” refers to proteins harbor a conserved domain consisting of cysteine (C) and/or histidine (H) residues. This domain binds with a zinc ion and, structurally, consists of a two-stranded antiparallel beta-sheet and a helix (Takatsuji, 1998). ZFPs can be divided into the following categories based on the number and location of C and H residues in this conserved domain: C2H2, C2HC, C2HC5, C2C2, C3H, C3HC4, C4, C4HC3, C6, and C8 (Berg & Shi, 1996). Among these, C2H2-type zinc finger proteins (C2H2-ZFPs) are the most widely studied. The zinc finger domain in these proteins contains two C and two H residues, which are described as CX_(2-4)CX₁₂HX_(3-5)H (where X represents any amino acid) (Pabo, Peisach & Grant, 2001).

Functional analysis has shown that C2H2-ZFs participate in vegetative growth and reproductive development in plants (An et al., 2012; Lu et al., 2012; Sun et al., 2015). They also mediate growth and development (Tian et al., 2017), sexual development (Kim et al., 2009), oospores production (Wang et al., 2009), and so on in fungi and fungal hyphae. Moreover, while c2h2-overexpression strains did not affect normal development in Agaricus bisporus, the yield per day of the transgenic strains peaked 1 day earlier than the control strains did (Pelkmans et al., 2016). The effect of C2H2-ZFs on mushroom formation makes them a target for breeding of this commercial mushroom. However, no further studies have been conducted on the roles of C2H2-ZFs in P. ostreatus development thus far.

Apart from regulating various development processes, C2H2-ZFs have been found to play crucial roles in abiotic stress defense. In plants, they have been shown to respond to heat (Mittler et al., 2006), and functional analysis has shown they help temper the effects of drought (Yin et al., 2017), cold (Liu et al., 2017), and salt stress (Ciftci-Yilmaz et al., 2007). In China, traditional greenhouses are mainly used for cultivating P. ostreatus, but they often lack proper environmental control. Environmental stress, especially heat, consistently threatens the supply of greenhouse-grown mushrooms. Extremely and continuously high temperatures (>36 °C) disrupt the cell wall integrity of P. ostreatus and enhance the ability of Trichoderma asperellum to infect mycelia (Qiu et al., 2018). In addition, mycelia exposed to 40 °C for 3 h leads to the accumulation of lactate, which inhibits mycelial growth (Yan et al., 2020). On the other hand, treatment at 5 °C significantly decreases the activity of enzymes like laccase and Mn-peroxidase in mycelia (Snajdr & Baldrian, 2007). Therefore, identification of C2H2-ZFs in P. ostreatus could also be used to in breeding programs to improve environmental stress tolerance in mushrooms.

In this study, C2H2-ZFs in the P. ostreatus genome were identified and characterized using bioinformatic analysis. Then, the expression profiles of each transcription factor were measured in different tissues to better understand their roles in regulating P. ostreatus development and stress response. The results of this work provide useful information about the characterization of C2H2-ZFs in mushrooms and candidate genes for the control the development and abiotic stress tolerance in P. ostreatus.

Materials & Methods

Results

Identification, characterization, and phylogenetic analysis of C2H2-ZFPs in P. ostreatus

Using an HMM and manual correction, 18 C2H2-ZFPs were identified in the P. ostreatus genome. All these proteins contained one to four conserved C2H2 domains either in the N-terminus or the C-terminus (Fig. S1). Detailed information about the characteristics of these proteins like amino acid size, MW, isoelectric points, and so on were also analyzed (Table S1). The results showed that the PoC2H2-ZFPs had between 149 and 688 amino acids, molecular weights ranging from 16.4 (PleosPC15_2—1089905) to 74.8 kDa (PleosPC15_2—1054163), and isoelectric points ranging from 4.66 (PleosPC15_2—1079678) to 10.8 (PleosPC15_2—1089905). All the PoC2H2-ZFPs are predicted to be nuclear proteins based on subcellular localization analysis (Table S1).

To analyze the phylogenetic relationships of these C2H2-ZFPs in P. ostreatus, a phylogenetic tree was constructed with their full protein sequences. As a result, the 18 PoC2H2-ZFPs were clustered into four separate clades (Fig. 1A). PleosPC15_2—1104202 is the only gene in clade III, indicating that it originated independently of the other genes. Based on bootstrapping values, the other genes are distantly related to each other. Most PoC2H2-ZFPs in the same clade had low bootstrap values (<60%). Only PleosPC15_2—1111338 and PleosPC15_2—1095114, which are regarded as duplicate genes, had strong bootstrap values (100%). The results of sequence alignment indicates that the PoC2H2-ZFPs share low sequence homology (Fig. S1).

Conserved domain analysis of the PoC2H2-ZFPs

To better understand the characteristics of C2H2 domains in P. ostreatus, multiple sequence alignment was performed to identify conserved amino acids. The results revealed that the 29 predicted C2H2 domains consisted of 23-26 amino acids (Fig. 2A). The variation in sequence length was caused by amino acid changes in two regions: CX_(2-4)C and HX_(3-5)H. More specifically, 15 of the C2H2 domains contained two amino acids in the CX_(2-4)C region while the others contained four amino acids (Fig. 2A). In the HX_(3-5)H region, 24 of the C2H2 domains had three amino acids, three had four amino acids, and two had five amino acids (Fig. 2A). Two C and two H residues were conserved in every one of these domains. An F and an L residue were also highly conserved in these domains (Fig. 2A). A sequence similar to motif 1 (CX_(2-4)CX₃FX₅LX₂HX_(3-5)H) was also found (Fig. 2B), suggesting that motif is conserved in PoC2H2-ZFPs.

Expression analysis of PoC2H2-ZFs under auxin and cytokinin

To study the response of PoC2H2-ZFs to hormones, the primordia were treated with IAA and zeatin for 1 h and 3 h, respectively. Of the twelve genes studied, only the expression of PeosPC15_2—1046937 was not affected treatment with auxin or cytokinin (Fig. 4). The expression of PeosPC15_2—1091415 and PeosPC15_2—1102653 was down-regulated following 1 h of treatment with zeatin, whereas the expression of PeosPC15_2—1079678 and PeosPC15_2—1089905 increased (Fig. 4). In addition, four genes (PeosPC15_2—1079678, PeosPC15_2—1095114, PeosPC15_2—1051309, and PeosPC15_2—157229) were up-regulated following 3 h of treatment with zeatin (Fig. 4). On the other hand, the expression of PeosPC15_2—1079678 increased after 1 h of auxin treatment. The expression levels PeosPC15_2—1079678, PeosPC15_2—1089905, and PeosPC15_2—1095114 increased after 3 h of treatment with auxin, whereas PeosPC15_2—1102653 was down-regulated after 3 hours (Fig. 4). This shows that the PoC2H2-ZFs identified in this study are differentially regulated by auxin and cytokinin.

Expression analysis of PoC2H2-ZFs under different abiotic stresses

To investigate the potential roles of PoC2H2-ZFs in abiotic stress, their expression profiles were analyzed under heat and cold stress. The results showed that five genes (PeosPC15_2—1079678, PeosPC15_2—1089905, PeosPC15_2—1095114, PeosPC15_2—1051309, and PeosPC15_2—157229) were up-regulated after 1 h of heat stress (Fig. 5). After 3 h of heat treatment, PeosPC15_2—1102653 was down-regulated and PeosPC15_2—1102653 and PeosPC15_2—1051309 were up-regulated (Fig. 5). The expression of PeosPC15_2—1046937 and PeosPC15_2—1102653 was significantly suppressed by 3 h of cold stress, whereas PeosPC15_2—1111338, PeosPC15_2—1051309, PeosPC15_2—1089905, PeosPC15_2—157229, and PeosPC15_2—1104202 were up-regulated (Fig. 5). The expression levels of two PoC2H2-ZFs (PeosPC15_2—1091415 and PeosPC15_2—1053338) were not affected by cold and heat stress (Fig. 5). These data suggest that PoC2H2-ZFs are differentially regulated by abiotic stresses.

Discussion

C2H2-ZF proteins are one of the largest and most conserved transcription factor families in the eukaryotic kingdom. They have been reported to play important roles in mediating plant growth and responses to stress (An et al., 2012; Ciftci-Yilmaz et al., 2007; Liu et al., 2017; Lu et al., 2012; Sun et al., 2015; Yin et al., 2017). Moreover, it has been demonstrated that C2H2-ZFs participate in growth and development (Tian et al., 2017), microsclerotia formation (Tian et al., 2017), sexual development (Kim et al., 2009), and so on in fungi. For instance, it affected the yield of Agaricus bisporus (Pelkmans et al., 2016), one of the most widely cultivated commercial mushrooms. C2H2-ZFs could be candidate genes for edible mushrooms breeding. P. ostreatus is one of the widely cultivated mushrooms in China. Hence, it is useful to study C2H2-ZFs transcription factors in this species. The genome of P. ostreatus PC15 has been widely used since its release, but a systematic analysis of C2H2-ZFs has not yet been performed. The strain 3125 is the main cultivar for our laboratory work. However, the genome sequence of it was not obtaining. Thus, this study used the PC15 genome to identify phylogenetic relationships, gene structures, and conserved motifs among PoC2H2-ZFs. In addition, the effects of growth and development, various hormones, and abiotic stress treatments on the expression of PoC2H2-ZFs were analyzed.

Eighteen C2H2-ZFs were identified in the P. ostreatus genome using genome-wide analysis. The proteins were further divided into four subfamilies using phylogenetic relationship analysis. However, the C2H2-ZFPs in each subfamily had low bootstrap values (Fig. 1A), which may have been caused by the low sequence similarity of the non-C2H2 domain sequences (Fig. S1). The results show that the the molecular weight, pI values, protein length, exon and intron number, and motif composition of these PoC2H2-ZFs (Table S1, Figs. 1B and 1C) vary widely, suggesting that PoC2H2-ZFs have diverse structural and physicochemical properties, as well as distinct origins and functions.

The conserved “QALGGH” sequences located in the C2H2 domain was considered the plant-specific motif that animals and yeasts lacked (Takatsuji, 1999)). Like the rice and tomato, most of the C2H2-ZFPs that were detected had this sequence in their genomes (Cao et al., 2016; Xin et al. 2019). Such sequence was not detected in the C2H2-ZFPs of P. ostreatus (Fig. 2A). The sequence alignment indicated that the C2H2 domains in P. ostreatus have a CX_(2-4)CX₃FX₅LX₂HX_(3-5)H motif, and that the F and L residues are highly conserved (Fig. 2A). This signature sequence can also be written as CX₂CX₃FX₅LX₂HX₃H (Table S2). This motif, called, motif 1, was detected in all PoC2H2-ZFPs (Table S2, Fig. 1C). These results suggest that the CX₂CX₃FX₅LX₂HX₃H sequence was conserved in PoC2H2-ZFPs.

C2H2-ZFs have been shown to participate in multiple processes related to growth and development in fungi. An investigatioin of the expression profiles of PoC2H2-ZFs could provide information about their role in regulating the growth and development of P. ostreatus. In a strain of Verticillium dahlia with a C2H2 transcription factor loss-of-function mutation (VdMsn2), a significant reduction in hyphal growth was seen (Tian et al., 2017). It was hypothesized that C2H2-ZFPs could also influence hyphal growth in P. ostreatus. PeosPC15_2—1077016, PeosPC15_2—112393, and PeosPC15_2—1088285 were highly expressed in mycelia (Fig. 3B), suggesting they play an important role in mycelial growth. Previous studies have also shown that C2H2-ZFs regulate the formation of primordia. Inactivation of C2H2 in S. commune resulted in the formation of aggregates but not subsequent differentiation into primordia, for instance (Ohm et al., 2011). In this study, five genes (PeosPC15_2—1079678, PeosPC15_2—1089905, PeosPC15_2—1111338, PeosPC15_2—1095114, and PeosPC15_2—1095325) were expressed more in primordia than in mycelia (Fig. 3B), indicating they are involved in the formation of primordia in P. ostreatus. Moreover, it was shown that C2H2-ZFs are involved in the development of fruiting bodies. In Aspergillus nidulans, the deletion of nsdC (a gene that encoded one C2H2 transcription factor) resulted in the loss of fruiting body formation (Kim et al. 2009). Here, all the PoC2H2-ZFs was expressed more in the young fruiting bodies than in the primordia (Fig. 3B), suggesting that C2H2-ZFs play an important role in fruiting body development in P. ostreatus. Three genes (PeosPC15_2—1088285, PeosPC15_2—1089905, and PeosPC15_2—1102653) showed increased expression in the mature fruiting body (Fig. 3B), as well, implying that they are involved in the ripening process of P. ostreatus. The PsCZF1 gene (encoding a C2H2-ZFs in Phytophthora sojae) has been implicated in the production of oospores and swimming zoospores (Wang et al., 2009). Indeed, PeosPC15_2—1088285, PeosPC15_2—1089905, and PeosPC15_2—1102653 seem to participate in spore development in P. ostreatus (Fig. 3B).

In Arabidopsis thaliana, ZINC FINGER PROTEIN 5 (ZFP5) mediates the effects of cytokinin and ethylene on the formation and growth of root hairs (An et al., 2012). ZFP6 has been identified as essential regulator of trichome initiation by and is responsive to gibberellin and cytokinin (Zhou et al. 2013). In addition, GhWIP2 (encoded a C2H2-ZFP) mediates cell expansion during organ growth by modulating crosstalk between auxin, gibberellins, and abscisic acid in Gerbera hybrida (Ren et al., 2018). Thus, it was hypothesized that various hormones can regulate C2H2-ZFs which can, in turn, affect developmental processes. P. ostreatus produces auxin (Bose, Shah & Keharia, 2013), and exogenous auxin and cytokinin have been reported to affect mycelial growth (Ramachela & Sihlangu, 2016). In this study, expression levels of PoC2H2-ZFs in primordia changed significantly in the presence of auxin and cytokinin (Fig. 4). Therefore, auxin and cytokinin have the potential to affect PoC2H2-ZF-mediated growth and developmental processes.

Previous studies have revealed that C2H2-ZFs confer resistance to abiotic stress in plants. In Arabidopsis, root growth in transgenic plants constitutively expressing Zat10 were more tolerant to heat stress (Mittler et al., 2006)). Overexpression of ZAT18 in transgenic Arabidopsis plants also increased drought tolerance, whereas the mutation of this gene resulted in decreased drought tolerance (Yin et al., 2017). In soybean, the expression of the C2H2-ZF gene GmSCOF-1 was induced by low temperature, and the overexpression lines not only had increased cold tolerance, but also had increased expression levels of cold-responsive genes (Kim et al. 2001). During P. ostreatus cultivation, bad environmental conditions negatively impact the growth and development of the mushrooms by inhibiting mycelial growth and disrupting the integrity of the cell wall, thereby increasing the risk of fungal contamination and reducing yield (Qiu et al., 2018). In this study, PoC2H2-ZFs were induced by heat and cold stress (Fig. 5), meaning that this transcription factor may have a conserved function related to heat and cold tolerance in fungi. Notably, PeosPC15_2—1089905 and PeosPC15_2—1102653 were induced by heat and cold stress (Fig. 5), suggesting that these genes play a variety of roles in response to various stresses.

Conclusions

In this study, we identified 18 C2H2-ZFs in the P. ostreatus genome. Their phylogenetic relationship, gene structure, motif composition, and other structural factors were highly variable. The expression profiles of these PoC2H2-ZFs suggest they play diverse roles in tissue growth and development. In addition, hormones and abiotic stress treatments induced the expression of these PoC2H2-ZFs, meaning that they could also participate in hormone signaling and abiotic stress response pathways.

Supplemental Information