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Article

Function of Transcription Factors PoMYB12, PoMYB15, and PoMYB20 in Heat Stress and Growth of Pleurotus ostreatus

by
Hui Yuan
1,†,
Zongqi Liu
1,†,
Lifeng Guo
1,
Ludan Hou
1,
Junlong Meng
1,2 and
Mingchang Chang
1,2,*
1
College of Food Science and Engineering, Shanxi Agricultural University, Jinzhong 030801, China
2
Shanxi Engineering Research Center of Edible Fungi, Jinzhong 030801, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2023, 24(17), 13559; https://doi.org/10.3390/ijms241713559
Submission received: 15 July 2023 / Revised: 29 August 2023 / Accepted: 30 August 2023 / Published: 31 August 2023
(This article belongs to the Section Molecular Microbiology)
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Abstract

:
MYB transcription factors (TFs) have been extensively studied in plant abiotic stress responses and growth and development. However, the role of MYB TFs in the heat stress response and growth and development of Pleurotus ostreatus remains unclear. To investigate the function of PoMYB12, PoMYB15, and PoMYB20 TFs in P. ostreatus, mutant strains of PoMYB12, PoMYB15, and PoMYB20 were generated using RNA interference (RNAi) and overexpression (OE) techniques. The results indicated that the mycelia of OE-PoMYB12, OE-PoMYB20, and RNAi-PoMYB15 mutant strains exhibited positive effects under heat stress at 32 °C, 36 °C, and 40 °C. Compared to wild-type strains, the OE-PoMYB12, OE-PoMYB20, and RNAi-PoMYB15 mutant strains promoted the growth and development of P. ostreatus. These mutant strains also facilitated the recovery of growth and development of P. ostreatus after 24 h of 36 °C heat stress. In conclusion, the expression of PoMYB12 and PoMYB20 supports the mycelium’s response to heat stress and enhances the growth and development of P. ostreatus, whereas PoMYB15 produces the opposite effect.

1. Introduction

MYB transcription factors (TFs) are widely distributed in eukaryotes and feature highly conserved MYB binding domains that regulate diverse physiological processes in organisms [1]. In plants, the role of MYB TFs has been extensively studied, revealing their involvement in various abiotic stress responses and the growth and development of diverse species [2,3,4]. These abiotic stress responses primarily include drought stress, salt stress, cold stress, and heat stress. For instance, research on drought stress and salt stress has shown that rice’s OsFLP (MYB TF) plays a role in both drought stress and salt stress responses by regulating the expression of OsNAC1 and OsNAC6 [5]. The tobacco NtMYB102, analogous to Arabidopsis thaliana’s AtMYB70, shows increased tolerance to drought and salt stress upon overexpression (OE) of the coding gene [6]. In contrast, overexpressing FtMYB22 in Fagopyrum tataricum decreased transgenic A. thaliana’s tolerance to both drought and salt stress [7]. Studies on cold stress and heat stress have found that three MYB TFs (SaMYB098, SaMYB015, and SaMYB068) in Santalum album significantly participate in the cold stress response [8]. Within the tea plant, CsMYB45, CsMYB46, and CsMYB105 are involved in jasmonic acid signal transduction under cold stress [9]. Moreover, OE of OsMYB55 in rice enhances the amino acid metabolism of transgenic rice, thereby bolstering plant heat tolerance and mitigating the impact of high temperature on grain yield [10]. Meanwhile, Lilium LlMYB305 activates the LlHSC70 promoter activity under heat stress, contributing to plant heat tolerance [11].
MYB TFs also assume significant roles in growth and development, as evidenced by their participation in seed germination, seedling development, root growth, leaf development, and anther development. For example, the MYB protein RSM1 in A. thaliana interacts with HY5/HYH to regulate seed germination and seedling development [12], while AtMYB103 can regulate anther development [13]. Notably, the LcMYB2 in Leymus chinensis promotes seed germination and root growth during drought stress [14]. Similarly, NbPHAN (MYB TF) promotes leaf development in Nicotiana benthamiana [15]. These investigations collectively underscore the potential involvement of MYB TFs in stress responses and growth and development across diverse plant species. However, the exploration of the engagement of MYB TFs in stress responses and growth development in fungi has been less comprehensive, with investigations limited to Fusarium graminearum and Acremonium chrysogenum [16,17]. Among these, the role of MYB TFs in abiotic stress response and growth and development in macro-fungi (edible fungi) has been relatively overlooked.
Edible fungi possess significant edible and medicinal values. Currently, research into their abiotic stress response mechanism and growth and development has emerged as a research hotspot. For example, the transcription factor FvHmg1 has been identified as a negative regulator of fruiting body development in Flammulina velutiper [18]. Similarly, the transcription factor PDD1 influences the development and yield of F. velutiper [19]. Furthermore, genes such as hsp70, hsp90, and fes1 contribute to the regulation of heat stress during the initial stages of fruiting body development in F. velutiper [20]. Notably, within the phenol propane pathway, two pal genes were discovered to be implicated in fruiting body development and the mycelial heat stress response in P. ostreatus studies [21]. Additionally, the nitric-oxide-induced oxidase aox gene participates in regulating reactive oxygen species and enhancing resistance to heat stress in P. ostreatus [22]. In the realm of Lentinus edodes, research has revealed that the OE of the hsp20 can enhance mycelial heat resistance [23]. In summation, numerous genes, including TFs, play an important role in the abiotic stress response and growth and development of edible fungi. However, there are few reports about MYB TFs.
P. ostreatus is among the most widely cultivated edible mushrooms globally, valued for its nutritional and medicinal attributes. In China, the cultivation of P. ostreatus predominantly occurs within horticultural facilities, enabling year-round production. Among these practices, the price of P. ostreatus produced in summer is the highest [24]. This pricing is largely due to the elevated summer temperatures, which foster the potential for mycelial scalding during its growth phase, consequently retarding mycelial development and causing mycelial demise. Mycelia play a pivotal role in the growth and maturation of P. ostreatus’s fruiting body. The formation of the fruiting body depends on the interlacing of the mycelium, with the mycelium serving as a vital nutrient repository for P. ostreatus, facilitating its growth and development. As such, robust mycelial growth forms the bedrock for ensuring optimal yields. Moreover, P. ostreatus boasts a short production cycle and notable conversion efficiency [25]. Enhancing its growth and development rate could shorten the production cycle and heighten yields, especially during the summer, with its high commodity prices. Such advancements hold substantial potential to propel the industrial progression of P. ostreatus. Consequently, in-depth investigations into P. ostreatus’s responses to heat stress and the mechanisms underlying its growth and development remain of utmost significance.
Prior transcriptome studies regarding P. ostreatus’s response to heat stress and its growth and development have revealed that PoMYB12, PoMYB15, and PoMYB20 may be involved in heat stress response and growth and development [26]. Nevertheless, specific experiments to validate the functions of these TFs are lacking. In this study, RNA interference (RNAi) and OE techniques were used to verify the roles of these TFs in heat stress response and the growth and development of P. ostreatus. This work aims to provide a new theoretical basis for studying the heat stress response and growth and development mechanisms of P. ostreatus.

2. Results

2.1. PoMYB12, PoMYB15, and PoMYB20 May Be Involved in the Response of P. ostreatus Mycelium to Heat Stress

To investigate the alterations in gene expression of PoMYB12, PoMYB15, and PoMYB20 in response to heat stress in P. ostreatus hyphae, mycelia from the WT were collected following 24 h of heat stress at 32 °C, 36 °C, and 40 °C. Specific primers were devised based on the nucleotide sequences of these three MYB genes, and they were cloned for PoMYB12, PoMYB15, and PoMYB20, respectively. Their open reading frames had total lengths of 1272 bp, 1440 bp, and 1947 bp, respectively (Supplementary Figure S1). Subsequently, the gene expression was assessed. The findings revealed variable degrees of upregulation in the expression of PoMYB12, PoMYB15, and PoMYB20 under heat stress at 32 °C, 36 °C, and 40 °C. Notably, when the mycelium was subjected to heat stress at 32 °C to 40 °C, the expression level of PoMYB12 increased by 5–6 times compared with the control, and the expression level was the highest at 40 °C (Figure 1a). PoMYB15 also had the highest expression at 40 °C (Figure 1b). The highest expression level of PoMYB20 occurred at 36 °C (Figure 1c). These results suggest that PoMYB12, PoMYB15, and PoMYB20 may be involved in the heat stress response of P. ostreatus mycelia.

2.2. PoMYB12, PoMYB15, and PoMYB20 Are Involved in the Response of P. ostreatus Mycelium to Heat Stress

To verify the impacts of the PoMYB12, PoMYB15, and PoMYB20 in P. ostreatus hyphae’s heat stress response, OE and RNAi experiments were conducted on these three MYB genes. The structural diagrams of the OE and RNAi plasmids for PoMYB12, PoMYB15, and PoMYB20 are illustrated in Figure S2.
Using the research method of Ludan Hou [27], the amplification of the hygromycin (Hyg) gene in P. ostreatus, transformed with the OE-PoMYB12 plasmid, was conducted (Figure S3a). Subsequently, the relative expression level of the PoMYB12 in the transformed strains was detected. The results showed that the expression levels of the PoMYB12 in the OE-PoMYB12-8, OE-PoMYB12-14, and OE-PoMYB12-21 mutant strains were 4.74, 3.41, and 5.39 times that of the wild-type (WT) strain, respectively (Figure S3b). Similarly, the Hyg gene in P. ostreatus, transformed with the RNAi-PoMYB12 plasmid, was amplified using the same methodology (Figure S3c). The results demonstrated that the expression levels of the PoMYB12 in the RNAi-PoMYB12-11, RNAi-PoMYB12-16, and RNAi-PoMYB12-19 mutant strains were reduced by 70.1%, 61.7%, and 70.5%, respectively, compared with the WT strain (Figure 2b). Using the same method, OE mutant strains, namely OE-PoMYB15-12, OE-PoMYB15-19, and OE-PoMYB15-24 of PoMYB15, were obtained (Figure S3d,e). Concurrently, RNAi mutant strains, namely RNAi-PoMYB15-5, RNAi-PoMYB15-14, and RNAi-PoMYB15-17 of PoMYB15, were successfully generated (Figure S3e,f). Furthermore, OE mutant strains, namely OE-PoMYB20-7, OE-PoMYB20-16, and OE-PoMYB20-24 of PoMYB20 were obtained (Figure S3g,h). The RNAi mutant strains RNAi-PoMYB20-5, RNAi-PoMYB20-11, and RNAi-PoMYB20-18 of PoMYB20 were obtained (Figure S3h,i).
The WT strain, alongside OE and RNAi mutant strains of PoMYB12, PoMYB15, and PoMYB20, were cultured on potato dextrose agar (PDA) medium, cultured at 25 °C in the dark for 4 d, transferred to 32 °C, 36 °C, and 40 °C in the dark for 24 h, and then transferred to 25 °C in the dark to resume growth for 24 h. The WT strain was used as the control. The mycelia of OE strains of PoMYB12 and PoMYB20 had stronger recovery ability than the WT strain after 32 °C, 36 °C, and 40 °C heat stress, while RNAi strains were weaker than WT strains (Figure 2a,b,e,f). Interestingly, the mycelium recovery ability of OE strains of PoMYB15 were weaker than that of the WT strain after 32 °C, 36 °C, and 40 °C heat stress, whereas the research results of RNAi strains were the opposite (Figure 2c,d).

2.3. PoMYB12, PoMYB15, and PoMYB20 May Be Involved in the Growth and Development of P. ostreatus

In order to investigate the variations in gene expression of the PoMYB12, PoMYB15, and PoMYB20 within the WT strain during distinct stages of growth and development, samples were collected from the mycelium, primordium, young fruiting body, and mature fruiting body, respectively. These samples were subjected to gene expression analysis. The outcomes indicated a gradual increase in the expression level of PoMYB12 throughout the growth and development phases, with the highest expression observed in the mature fruiting body stage (Figure 3a). The expression level of PoMYB15 was highest in the primordium stage, and decreased in the young fruiting body and mature fruiting body stages (Figure 3b). PoMYB20 showed high gene expression in the primordium and mature fruiting body stage (Figure 3c).

2.4. PoMYB12, PoMYB15, and PoMYB20 Are Involved in the Growth and Development of P. ostreatus

In order to investigate the contribution of PoMYB12, PoMYB15, and PoMYB20 to the growth and development of P. ostreatus, the obtained OE and RNAi mutant strains were cultured and observed, with the WT strain serving as a reference. The results showed faster growth rates for mycelia, primordium, and fruiting bodies in OE strains of PoMYB12 and PoMYB20 compared to WT. In contrast, the RNAi strains displayed slower growth rates (Figure 4a,c–f). However, the mycelial growth, primordial growth, and fruiting body growth of the OE strains of PoMYB15 were slower than those of the WT strain, whereas the results of the RNAi strains showed the reverse trend (Figure 4b,d–f). These findings underscore that the expression of PoMYB12 and PoMYB20 serves to facilitate the growth and development of P. ostreatus, whereas the expression of PoMYB15 does not.

2.5. PoMYB12, PoMYB15, and PoMYB20 Are Involved in Growth and Development in Response to 36 °C Heat Stress of P. ostreatus

During the summer mycelial growth process, P. ostreatus frequently encounters brief episodes of high-temperature stress before resuming production. However, these brief instances of heat stress can lead to an elongation of the production cycle. To delve into the role of PoMYB12, PoMYB15, and PoMYB20 in addressing high-temperature stress and participating in growth during mycelial development, combined with the research results of the mycelial heat stress response in this study, 36 °C was selected for heat stress treatment for 24 h. The growth and development of the PoMYB12, PoMYB15, and PoMYB20 mutant strains were closely monitored. Results delineated that the mycelial recovery growth, primordial and fruiting body growth, and development of the OE strains of PoMYB12 and PoMYB20 were faster than those of the WT strain. Conversely, the RNAi strains displayed slower growth than those of the WT strain (Figure 5a,c–f). The mycelial recovery and primordial and fruiting body growth of the OE strains of PoMYB15 were slower than those of the WT strain, while the RNAi strain showed opposite results (Figure 5b,d–f). In conclusion, PoMYB12 and PoMYB20 demonstrate an ability to expedite the recovery of growth and development in P. ostreatus following exposure to 36 °C heat stress, whereas PoMYB15 does not.

3. Discussion

The involvement of MYB TFs in plant heat stress and growth, as well as development, has been extensively investigated [2,3,4]. Studies on heat stress and growth and development hold significant importance for the advancement of the P. ostreatus industry. This is because heat stress can disrupt the regular growth of P. ostreatus mycelia, subsequently affecting the pace of growth and development that, in turn, influences the overall growth cycle of P. ostreatus. Nevertheless, there have been limited inquiries into whether MYB TFs play a role in mycelial heat stress and growth and development in P. ostreatus. As such, the exploration of the involvement of MYB TFs in mycelial heat stress response and growth and development within P. ostreatus remains an especially crucial endeavor.
MYB TFs can participate in the heat stress response in Pennisetum glaucum [28], Morus alba [29], Vaccinium corymbosum [30], and rice [31], providing a foundation for exploring the potential role of MYB TFs in the heat stress response of P. ostreatus. In this study, PoMYB12, PoMYB15, and PoMYB20 were selected for functional exploration based on the results of the previous identification of the P. ostreatus MYB gene family [26]. The results from the identification of the MYB gene family in P. ostreatus showed that the expression patterns of PoMYB12, PoMYB15, and PoMYB20 under 40 °C heat stress varied somewhat from those in this study, which may be related to the duration of the heat stress. The predecessors treated their samples with heat stress for 48 h, whereas our samples were treated with heat stress for 24 h [26]. Despite these differences, both studies suggest that PoMYB12, PoMYB15, and PoMYB20 may be involved in the response of P. ostreatus hyphae to heat stress. Further OE and RNAi experiment results showed that the mycelial recovery growth rate of OE-PoMYB12, RANi-PoMYB15, and OE-PoMYB20 was faster than that of WT strain when subjected to heat stress at 32 °C, 36 °C, and 40 °C for 24 h. It is interesting that the results of the MYB gene family identification of P. ostreatus and the results of this study both found that PoMYB15 was highly expressed under heat stress, whereas strains that overexpress PoMYB15 showed delayed mycelial recovery and growth, suggesting that PoMYB15 might be a transcriptional suppressor, and the expression of PoMYB15 inhibited the expression of downstream target genes. It is not conducive to the heat stress response. The OE of PoMYB12 and PoMYB20 exhibits similar results to the RNAi of PoMYB15. This may be due to the fact that PoMYB12 and PoMYB20 may be transcriptional activators. In the study of the heat stress response in P. ostreatus, it was found that RNAi-pal1 strains could reduce the sensitivity of mycelia to ROS, whereas RNAi-pal2 strains responded to heat stress by reducing the sensitivity of mycelia to H2O2 [21]. The OE-aox strain can actively respond to 32 °C heat stress in mycelium [22]. After being subjected to heat stress, strain OE-Mnsod1 has a faster mycelial recovery rate than the WT strain and participates in the mycelial heat stress reaction of P. ostreatus [27]. Therefore, determining whether pal1, pal2, aox, and Mnsod1 are regulated by PoMYB12, PoMYB15, or PoMYB20 and their involvement in the heat stress regulatory pathway warrants further investigation.
The role of MYB TFs in fungal growth and development, particularly in macro-fungi, is becoming the focus of research. In fungi, AfMybA, MYT1, and MYT2 have been found to be involved in growth and development [32,33,34]. Among macro-fungi, F. velutiper and Ganoderma lucidum MYB TFs were found to be involved in growth and development [35,36]. In the context of P. ostreatus MYB gene family identification, there has been speculation that MYB might play a role in growth and development [26]. These findings provide a reference for us to explore the possible involvement of PoMYB12, PoMYB15, and PoMYB20 in the growth and development of P. ostreatus. The change trend of PoMYB12, PoMYB15, PoMYB20 expression during the growth and development of the WT strain explored in this study is similar to that in the identification of the MYB gene family of P. ostreatus [26]. Further phenotypic observations of the OE and RNAi strains revealed that the OE-PoMYB12, RNAi-PoMYB15, and OE-PoMYB20 strains promote the growth and development of P. ostreatus more than the WT strain. Interestingly, OE-PoMYB12 and OE-PoMYB20 exhibit similar effects in response to mycelial heat stress and growth and development. Whether there is possible interaction between PoMYB12 and PoMYB20, or whether they play a synergistic role in growth and development, needs to be further explored. Additionally, OE-PoMYB15 and OE-PoMYB12, and OE-PoMYB20, also showed opposite effects in the growth and development process, which promoted further speculation that PoMYB15 may be a transcription suppressor. A higher gene expression level corresponds to greater inhibition of downstream target gene expression, and inhibited expression of PoMYB15 results in accelerated mycelial growth and development. In the study of P. ostreatus growth and development, it was found that functional genes like Mnsod1 [27], LaeA-like [37], aco [38], Pofst3 [39], Pofst4 [40], pal1, and pal2 [21] can participate in the growth and development of P. ostreatus, but the transcriptional regulation of these functional genes is not clear. Whether they may be regulated by PoMYB12, PoMYB15, or PoMYB20 to participate in the growth and development of P. ostreatus is a worthwhile avenue for exploration.
In the production of P. ostreatus, when high temperatures are encountered during the mycelium growth process, producers disperse the hyphal rods of mushrooms to cool them. Subsequently, the mycelium continues to grow and produce mushrooms. Therefore, while examining the heat stress response and growth and development mechanism of P. ostreatus mycelia separately, this study integrated these two aspects to better simulate the actual production process, which is different than previous studies [21,22]. The research results indicate that the mycelium recovery and development rate of OE-PoMYB12, RNAi-PoMYB15, and OE-PoMYB20 strains is faster than that of WT strain after being subjected to 36 °C heat stress, which is consistent with the roles of PoMYB12, PoMYB15, and PoMYB20 in heat stress and growth and development in this study. The interesting results obtained in this study reveal that PoMYB12 and PoMYB20 play similar roles. It is worth further exploration to determine whether there is a synergistic effect between them and if an interaction relationship exists between them. In addition, future work should consider identifying the key genes under the control of MYB and explaining their roles in bringing the observed phenotype, enhancing our capacity to delve deeper into gene functions and improving our regulatory pathway information.
In this study, OE and RNAi experiments were performed on PoMYB12, PoMYB15, and PoMYB20. The results showed that OE of PoMYB12 and PoMYB20, along with RNAi of PoMYB15, improved recovery growth post mycelial heat stress, and could accelerate the growth and development of P. ostreatus. The results of this study are expected to provide an important reference basis for the breeding and technological improvement of new varieties of P. ostreatus, along with serving as a significant reference for in-depth research in the field of heat stress response and growth and development regulation of P. ostreatus and other macro-fungi.

4. Materials and Methods

4.1. Strains Tested

The P. ostreatus strain, “Da Ye 39”, was provided by the Shanxi Edible Fungi Germplasm Resource Collection Center of China. Escherichia coli DH5α was provided by Beijing Tsingke Biotechnology Co., Ltd. (Beijing, China). The Agrobacterium tumefaciens GV3101 strain was preserved in our laboratory.

4.2. Construction of OE and RNAi Plasmids of PoMYB12, PoMYB15, PoMYB20

The total RNA of mycelium was extracted using the TransZol Up kit (TransGen Biotech, Beijing, China). cDNA was synthesized by reverse transcription of total RNA using EasyScript® One-Step gDNA Removal and cDNA Synthesis SuperMix Kits (TransGen Biotech, Beijing, China). The coding sequences (CDS) of the PoMYB12, PoMYB15, and PoMYB20 genes were obtained from the NCBI database (GenBank: MH510323.1:55-1326; MH510318.1:1-1440; MH510311.1:1-1947). Primer Premier 5.0 was used to design PoMYB12-, PoMYB15-, and PoMYB20-specific primers (Table S1) to clone the PoMYB12, PoMYB15, and PoMYB20 genes. The original OE plasmids kept in the laboratory were double digested with SpeI and PspOMI, and the cloned OE-PoMYB12, OE-PoMYB15, and OE-PoMYB20 sequences (Table S1) were inserted into OE plasmids containing the Hyg resistance gene by homologous recombination to obtain OE-PoMYB12, OE-PoMYB15, and OE-PoMYB20 recombinant plasmids, respectively [25,41]. The original RNAi plasmids preserved in the laboratory were double digested with SpeI and BglII, and the cloned RNAi-PoMYB12-Sense, RNAi-PoMYB15-Sense, and RNAi-PoMYB20-Sense sequences (Table S1) were inserted into the RNAi plasmids containing Hyg resistance genes by homologous recombination to obtain recombinant plasmids. Subsequently, the obtained recombinant plasmids were double digested with SpeI and PspOMI, and the cloned RNAi-PoMYB12-Anti, RNAi-PoMYB15-Anti, and RNAi-PoMYB20-Anti sequences (Table S1) were inserted into the recombinant plasmids using homologous recombination to obtain the RNAi-PoMYB12, RNAi-PoMYB15, and RNAi-PoMYB20 recombinant plasmids [25,41].

4.3. Recombinant Plasmids Transformed into Competent Cells of A. tumefaciens

A. tumefaciens GV3101-competent cells were prepared according to the method in reference [42]. Recombinant plasmids of OE-PoMYB12, OE-PoMYB15, OE-PoMYB20, RNAi-PoMYB12, RNAi-PoMYB15, and RNAi-PoMYB20 were added to the competent cells, mixed, and incubated on ice for 5 min, in liquid nitrogen for 1 min, and in a water bath at 37 °C for 5 min, respectively. Subsequently, 600 μL of nonresistant lysogeny broth (LB) medium was added and expanded at 28 °C for 3 h. Finally, the cells were coated into culture dishes containing kanamycin (kan) 50 μg/mL and rifampicin (rif) 20 μg/mL resistance and incubated upside down at 28 °C for 2 d. After picking spots, colonies were PCR and expanded. Subsequently, 600 μL of nonresistant LB medium was added, shaken at 28 °C for 3 h, applied to kan (50 μg/mL) and rif (20 μg/mL) in a resistant petri dish, and cultured upside down at 28 °C for 2 d. A single colony was selected for colony PCR, and the culture was expanded for standby.

4.4. A. tumefaciens-Mediated Recombinant Plasmid Transfer to the Mycelium of P. ostreatus

The mycelium of P. ostreatus was inoculated into PDA medium, cultured in the dark at 25 °C for 5 d, and then inoculated into complete yeast medium (CYM) by punching 0.5 cm blocks with a hole puncher, which were left at 28 °C and cultured in the dark for 2 d. A. tumefaciens containing the OE-PoMYB12, OE-PoMYB15, OE-PoMYB20, RNAi-PoMYB12, RNAi-PoMYB15, and RNAi-PoMYB20 plasmids were expanded, centrifuged and collected, added to induction medium (IM) liquid medium, mixed, and induced for 5 h at 28 °C, 90 r/min and protected from light. The blocks in CYM were transferred into A. tumefaciens induced with OE-PoMYB12, OE-PoMYB15, OE-PoMYB20, RNAi-PoMYB12, RNAi-PoMYB15, and RNAi-PoMYB20 plasmids and incubated at 28 °C for 5 h in the dark. Then, they were transferred to IM solid medium, covered with cellophane, and cultured in the dark at 28 °C for 3 d. They were transferred to CYM containing Hyg and cefotaxime (Cef) resistance for 30 d. The surviving mycelia were transferred into PDA medium and collected. DNA was extracted, and Hyg gene fragments were amplified. RNA was extracted from the strain with the amplified Hyg fragment and reverse transcribed into cDNA, and the expression levels of PoMYB12, PoMYB15, and PoMYB20 were detected to obtain the transformed strain.

4.5. Detection of Gene Expression in WT Strain under Different Heat Stress Conditions

The mycelia of WT strain cultured at 25 °C in the dark for 4 d and subjected to 32 °C, 36 °C, and 40 °C heat stress for 24 h were collected. Then, RNA from mycelium was extracted and reverse transcribed into cDNA. The qPCR primers were designed using Primer Premier 5.0 for PoMYB12, PoMYB15, and PoMYB20 (Table S1). The gene expression of PoMYB12, PoMYB15, and PoMYB20 was detected in mycelium using ChamQ™ Uni-versal SYBR® qPCR Master Mix (Vazyme, Nanjing, China) kits. The Bio-Rad CFX Connect TM Real-Time PCR System was used. Reaction system: 0.4 μL 10 μM upstream and downstream primers, 10 μL ChamQ Universal SYBR qPCR Master Mix (2×), 4 μL cDNA template, and 5.2 μL ddH2O. The reaction procedure was as follows: predenaturation at 95 °C for 30 s, then 40 cycles of 10 s at 95 °C and 30 s at 60 °C, and finally, extension at 72 °C for 30 s. β-Actin and β-tubulin were used as internal reference genes [22], and the relative expression was calculated using the 2−ΔΔCt method with three biological replicates [43].

4.6. Detection of Heat Stress Resistance in PoMYB12, PoMYB15, and PoMYB20 Transformed Strains

To simulate mycelium scald encountered in the summer production of P. ostreatus, the obtained mutant strains were inoculated into PDA medium, cultured in the dark at 25 °C for 4 d, transferred to 32 °C, 36 °C, and 40 °C for 24 h, and then placed back in the dark at 25 °C for 24 h. The recovery of mycelia was observed. Mycelial recovery growth length was measured by the cross-cross method [44]. Measurement accuracy accurate to millimeters.

4.7. Analysis of Gene Expression Levels and Effects of Transformed Strains on the Growth and Development of P. ostreatus

To detect the gene expression levels of PoMYB12, PoMYB15, and PoMYB20 during the growth and development of P. ostreatus, samples of mycelium, primordium, young fruiting body, and mature fruiting body were collected. RNA from samples was extracted and reverse transcribed into cDNA. The procedure for gene expression measurement was the same as that described in the material method above.
The obtained transformants were cultured in PDA medium for 7 d at 25 °C in the dark, respectively. Then, the seed blocks were inoculated into the culture bottle (each bottle was inoculated with 5 evenly sized seeds 1 cm in diameter). The cultivar formulation was 78% cotton seed hulls, 2% lime, 18% bran, 1% glucose, 1% gypsum, and 60% water content and incubated at 25 °C for 16 d in the dark. With the WT strain as the control, the mycelial growth rates of transformed strains were observed. Additionally, mushroom emergence experiments were conducted. During the catalytic primordium stage, the temperature was alternated at 15 °C and 26 °C for alternating temperature difference stimulation, with a humidity of 85%, and scattered light alternated with dark conditions. During the growth stage of the fruiting body, the temperature was controlled alternately between 23 °C and 26 °C, with a humidity of 85%, and scattered light alternated with dark conditions. Mycelial growth length and cap length were measured by the cross-cross method [44]. Among them, the measurement of cap length selects the largest cap in each mushroom culture bottle as the measurement standard. Measurement accuracy accurate to millimeters.

4.8. Growth and Development of Transformed Strains after 24 h of Response to 36 °C Heat Stress

The transformed strains were inoculated into cultivation flasks, respectively. After the mycelia were cultured for 10 d, they were transferred to dark conditions at 36 °C for 24 h of heat stress. Following the heat stress, the mycelium was transferred to dark conditions at 25 °C for further cultivation. The subsequent conditions for catalytic primordium and growth of fruiting bodies remained consistent with the parameters described in the materials and methods section above. The mycelial growth length and cap length measurement was the same as that described in the material method above.

4.9. Statistical Analysis

All analyzed data were biologically replicated at least three times. Data represent the mean ± standard deviation. IBM SPSS Statistics 26 software was used to determine significant differences. Analysis of variance (ANOVA) and Duncan’s multiple range test were performed to assess differences between samples.

Supplementary Materials

The supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms241713559/s1.

Author Contributions

Conceptualization, H.Y., Z.L. and M.C.; methodology, H.Y., Z.L. and L.H.; software, H.Y.; formal analysis, H.Y., M.C., L.H. and J.M.; resources, H.Y., Z.L. and L.G.; writing—original draft preparation, H.Y.; writing—review and editing, H.Y. and Z.L.; project administration, M.C., J.M. and L.H.; funding acquisition M.C. and J.M. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by Key Project of Coal-based Science and Technology of Shanxi Province (FT2014-03-01) and Doctoral Research Initiation Program of Shanxi Agricultural University (2021BQ90).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All the research results and data in this work have been included in the manuscript and Supplementary Materials. The original data involved in this study can be obtained from the first author or corresponding author through email upon reasonable request.

Acknowledgments

We thank all the members of the research group for their cooperation in the laboratory.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Detection of gene expression changes in WT strain mycelium at 32 °C, 36 °C, and 40 °C after 24 h of heat stress. (a) PoMYB12 gene expression was detected in mycelia of WT strains. (b) PoMYB15. (c) PoMYB20. The expression levels of PoMYB12, PoMYB15, and PoMYB20 in the mycelium of the WT strain without stress (25 °C) were used as controls and were set to 1. Each value represents the mean ± SD (n = 3). ANOVA and Duncan’s multiple range test (p < 0.05).
Figure 1. Detection of gene expression changes in WT strain mycelium at 32 °C, 36 °C, and 40 °C after 24 h of heat stress. (a) PoMYB12 gene expression was detected in mycelia of WT strains. (b) PoMYB15. (c) PoMYB20. The expression levels of PoMYB12, PoMYB15, and PoMYB20 in the mycelium of the WT strain without stress (25 °C) were used as controls and were set to 1. Each value represents the mean ± SD (n = 3). ANOVA and Duncan’s multiple range test (p < 0.05).
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Figure 2. Observation of the recovery of colony morphology after 24 h of growth in mycelium of WT and mutant strains after 24 h of heat stress at 32 °C, 36 °C, and 40 °C. (a,b) Colony morphology and recovery of mycelial growth length of PoMYB12 mutant strains. (c,d) PoMYB15. (e,f) PoMYB20. The WT strain was used as a control. The red line indicates the length of mycelium recovery growth.
Figure 2. Observation of the recovery of colony morphology after 24 h of growth in mycelium of WT and mutant strains after 24 h of heat stress at 32 °C, 36 °C, and 40 °C. (a,b) Colony morphology and recovery of mycelial growth length of PoMYB12 mutant strains. (c,d) PoMYB15. (e,f) PoMYB20. The WT strain was used as a control. The red line indicates the length of mycelium recovery growth.
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Figure 3. Changes in gene expression levels of PoMYB12, PoMYB15, and PoMYB20 during the growth and development of WT strain. (a) PoMYB12. (b) PoMYB15. (c) PoMYB20. The expression levels of the PoMYB12, PoMYB15, and PoMYB20 in the mycelium of the WT strain were used as controls and were set to 1. My: mycelium, Pr: primordium, YF: young fruiting body, MF: mature fruiting body. Each value represents the mean ± SD (n = 3). ANOVA and Duncan’s multiple range test (p < 0.05).
Figure 3. Changes in gene expression levels of PoMYB12, PoMYB15, and PoMYB20 during the growth and development of WT strain. (a) PoMYB12. (b) PoMYB15. (c) PoMYB20. The expression levels of the PoMYB12, PoMYB15, and PoMYB20 in the mycelium of the WT strain were used as controls and were set to 1. My: mycelium, Pr: primordium, YF: young fruiting body, MF: mature fruiting body. Each value represents the mean ± SD (n = 3). ANOVA and Duncan’s multiple range test (p < 0.05).
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Figure 4. Growth and development of mutant strains. (a) Growth and development of PoMYB12 mutant strains. (b) PoMYB15 mutant strains. (c) PoMYB15 mutant strains. (d) Mycelial growth length of mutant strains cultured for 12 d. (e) Cap length of mutant strains cultured for 22 d. (f) Cap length of mutant strains cultured for 26 d. WT strains were used as controls.
Figure 4. Growth and development of mutant strains. (a) Growth and development of PoMYB12 mutant strains. (b) PoMYB15 mutant strains. (c) PoMYB15 mutant strains. (d) Mycelial growth length of mutant strains cultured for 12 d. (e) Cap length of mutant strains cultured for 22 d. (f) Cap length of mutant strains cultured for 26 d. WT strains were used as controls.
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Figure 5. Observation of the recovery of mycelium growth and development of each mutant strain after 24 h of heat stress at 36 °C. (a) Growth and development results of PoMYB12 mutant strains. (b) PoMYB15 mutant strains. (c) PoMYB20 mutant strains. (d) Mycelial growth length of mutant strains cultured for 15 d. (e) Cap length of mutant strains cultured for 25 d. (f) Cap length of mutant strains cultured for 29 d. The growth and development results of WT strain recovered after 24 h of 36 °C heat stress were taken as the control.
Figure 5. Observation of the recovery of mycelium growth and development of each mutant strain after 24 h of heat stress at 36 °C. (a) Growth and development results of PoMYB12 mutant strains. (b) PoMYB15 mutant strains. (c) PoMYB20 mutant strains. (d) Mycelial growth length of mutant strains cultured for 15 d. (e) Cap length of mutant strains cultured for 25 d. (f) Cap length of mutant strains cultured for 29 d. The growth and development results of WT strain recovered after 24 h of 36 °C heat stress were taken as the control.
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Yuan, H.; Liu, Z.; Guo, L.; Hou, L.; Meng, J.; Chang, M. Function of Transcription Factors PoMYB12, PoMYB15, and PoMYB20 in Heat Stress and Growth of Pleurotus ostreatus. Int. J. Mol. Sci. 2023, 24, 13559. https://doi.org/10.3390/ijms241713559

AMA Style

Yuan H, Liu Z, Guo L, Hou L, Meng J, Chang M. Function of Transcription Factors PoMYB12, PoMYB15, and PoMYB20 in Heat Stress and Growth of Pleurotus ostreatus. International Journal of Molecular Sciences. 2023; 24(17):13559. https://doi.org/10.3390/ijms241713559

Chicago/Turabian Style

Yuan, Hui, Zongqi Liu, Lifeng Guo, Ludan Hou, Junlong Meng, and Mingchang Chang. 2023. "Function of Transcription Factors PoMYB12, PoMYB15, and PoMYB20 in Heat Stress and Growth of Pleurotus ostreatus" International Journal of Molecular Sciences 24, no. 17: 13559. https://doi.org/10.3390/ijms241713559

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