Characterization of a G protein α subunit encoded gene from the dimorphic fungus-Tremella fuciformis

Tremella fuciformis is a dimorphic fungus which can undertake the reversible transition between yeast and pseudohypha forms. G protein α subunit (Gα) carries different signals to regulate a variety of biological processes in eukaryotes, including fungal dimorphism. In this study, a novel Gα subunit encoded gene, TrGpa1, was firstly cloned from T. fuciformis. The TrGpa1 open reading frame has 1059 nucleotides, and encodes a protein which belongs to the group I of Gαi superfamily. Furthermore, the role of TrGpa1 in the T. fuciformis dimorphism was analysed by gene overexpression and knockdown. Stable integration of the target gene into the genome was confirmed by PCR and Southern blot hybridization. Transformants with the highest and lowest TrGpa1 expression levels were selected via quantitative real-time PCR analysis and Western blot. Each transformant was compared with the wild-type strain about the morphological change under different environmental factors, including pH values, temperature, cultivation time, inoculum size, and quorum-sensing molecules (farnesol and tyrosol). Comparing with the wild-type strain, the overexpression transformant always had higher ratios of pseudohyphae, while the knockdown transformant had less proportions of pseudohyphae. Therefore, the TrGpa1 is involved in the dimorphism of T. fuciformis and plays a positive role in promoting pseudohyphal growth.


Introduction
The heterotrimeric guanine nucleotide-binding proteins (G proteins), universal signaling proteins in eukaryotes, carry different signals from the receptors to various effectors, then regulate a variety of biological processes (Kang et al. 2011). The G proteins are highly conserved, and consist of Ga, Gb and Gc subunits. In the inactive state, these three subunits are tightly associated together. Once the Ga subunit was activated by G protein-coupled receptors (GPCRs), G protein dissociates to form Ga and Gb-Gc dimeric subunits. Each of them can interact with downstream effectors, which subsequently trigger a series of intracellular responses (Perez-Sanchez et al. 2010;Valle-Maldonado et al. 2015).
Fungal dimorphism is an intriguing morphological transition, which undertakes a morphological interconversion between the yeast form and the mycelial/ pseudohypha form (Nickerson and Atkin 2017). This switch promotes the disease progression in the pathogenic filamentous species, which is necessary for the invasion to hosts and the expression of virulence factors (Wilson et al. 2010;Boyce and Andrianopoulos 2015). Lots of researches showed Ga subunits had linkage with morphological transition in dimorphic fungi. For Ustilago maydis, the yeast colonies of cap1-defective cells failed to form filamentous colonies, which resulted in a significantly decreased pathogenicity (Takach and Gold 2010). In Sporothrix schenckii, Ga subunits SSG-1 and SSG-2 are involved in the dimorphism and pathogenicity (Pérez-Sánchez et al. 2010;Yemelin et al. 2017). The DMgGpa3 mutant of Mycosphaerella graminicola showed more pronounced yeast-like growth accompanied with hampered filamentation, which suppressed the transition from yeast-like form to filamentous form (Orton et al. 2011). As to Mucor circinelloides, the gpa3 expression levels was decreased during the dimorphic transition from mycelium to yeast cell (Valle-Maldonado et al. 2015). In Candida albicans, the Gpa2 played an important role in the yeast-hypha dimorphic transition during the response of C. albicans to some environmental inducers (Wilson et al. 2010). The Dgpa2 mutant strains of Saccharomyces cerevisiae had a defect in pseudohyphal growth, while constitutive overexpression of gpa2 stimulated filamentation of the mutant (Kayikci and Magwene 2018).
Tremella fuciformis, or white jelly mushroom, is a typical dimorphic fungus having the yeast-hypha and yeast-pseudohypha transition triggered by environmental cues (Hou et al. 2011). Previous studies about the T. fuciformis dimorphism mainly focused on the environmental factors which affected its dimorphism and cell wall polysaccharides changes during dimorphic transition (Zhu et al. 2016). Little is known about the functions of signaling proteins during the dimorphic change of T. fuciformis. In the present study, a Ga subunit gene (TrGpa1) which was cloned and characterized the contributions to the T. fuciformis dimorphism. The gene overexpression and knockdown vectors were constructed to evaluate its roles in the dimorphic transition in response to the environmental inducers.

Full-length gene cloning and bioinformatical analysis
Ga subunit gene, named as TrGpa1, and the DNA (GenBank accession no. MH091706) and cDNA (GenBank accession no. MH101517) were acquired in our previous work (data not published). The qRT-PCR analysis of TrGpa1 was performed in the yeast form and mycelial form of T. fuciformis, and results showed TrGpa1 was differentially expressed, which indicates this gene may be involved in the dimorphic changes (data not shown). Total RNA and DNA were extracted from Y32 strain using the RNaiso TM plus (Takara, Dalian, China) and the cetyltrimethylammonium bromide (CTAB) method (Yin et al. 2015), respectively. The TrGpa1 was cloned by PCR with the specific primers listed on Table 1. The amplification procedures were carried out as follows: an initial denaturation at 94°C for 5 min; 35 cycles of 94°C denaturation for 30 s, 60°C annealing for 30 s, 72°C elongation for 90 s; and a final extension at 72°C for 10 min.
Multi-sequence alignment was generated using ClustalW (http://www. clustal.org/). The phylogenetic tree was constructed using neighbor joining method implemented in Molecular Evolutionary Genetics Analysis (MEGA) version 6 program.
Vector construction and Agrobacterium-mediated transformation The overexpression and knockdown vectors of TrG-pa1 were constructed according to the vector pGEH-GH (Zhu et al. 2017) based on pCAMBIA 1302 backbone (Cambia, Brisbane, Australia). The TrGpa1 amplified using the primers with MluI and AsuII restriction sites were digested and introduced into pGEH-GH to generate the overexpression vector pTrGpa1-OE (Fig. 1a). The knockdown vector (pTrG-pa1-hp) was generated by the ligation of a 439 bp fragment (a 325 bp fragment and a 124 bp spacer fragment, flanking MluI and BlnI restriction sites) and a 325 bp (flanking BlnI and AsuII restriction sites) reverse complementary fragments. The plasmid was expected to encode a hairpin RNA included two 325 bp complementary regions separated by a 124 bp spacer fragment (Fig. 1b). The overexpression and knockdown elements were under control of T. fuciformis endogenous constitutive promoter-glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter.
All vectors were transformed into the A. tumefaciens strain EHA105 component cells. Agrobacterium-mediated transformation of T. fuciformis Y32 Data were analysed by one-way analysis of variance (ANOVA), followed by Ducan's multiple range tests using SPSS 26.0 software. Transformants selected based on the qRT-PCR assays and Y32 were analysed by Western blot. Cells were lysed in buffer containing protease and phosphatase inhibitor cocktails (Sigma, St. Louis, MO, USA). Then total protein content was measured with BCA Protein Assay Kit according to the manufacturer's protocol, and 40 lg of proteins were separated on 10% SDS polyacrylamide gels and transferred to PVDF membrane (Thermo Fisher Scientific, USA) using a Semi Dry Blotter (Thermo Fisher Scientific, USA) for 1 h at 20 V. After blocking with TBST buffer (5% dry milk powder in tris-buffered saline and 1% v/v Tween 20) for 1 h, the membrane was incubated with the primary rabbit antibodies against TrGpa1 at 4°C overnight. After 5 washing steps with TBST buffer, blots were incubated with the secondary antibodies: horseradish peroxidase labeled anti-rabbit IgG in the dilution of 1:2000 in 5% milk/TBST at room temperature for 2 h. The membranes were performed using the SuperSignal ECL Solution for Western blot (Willget Biotech, Shanghai, China). Densitometric evaluation was performed with ImageJ software (National Institute of Health, New York, NY).

Phenotypic analysis
The sub-cultured cells in LM medium were aseptically collected by centrifugation at 5000 9 g for 5 min, washed three times and distributed in 50 mL of medium to obtain final concentration of 10 5 Fig. 1 Map of the TrGpa1 over-expression vector (a) and knockdown vector (b). a The TrGpa1 was ligated with the endogenous gpd promoter to generate the overexpression vector pTrGpa1-OE. b The knockdown vector pTrGpa1-hp was generated by the ligation of a 325 bp fragment, a 124 bp spacer fragment, and a 325 bp reverse complementary fragments cellsÁmL -1 . Except the given situations, the 50 mL of LM medium containing cells were incubated at 25°C on an orbital shaker at pH 7 for 3 to 5 d. The pH of the medium was adjusted by the addition of dibasic phosphate-citric acid buffer to the desired pH value.
Transformants and Y32 were cultured under different conditions to test the morphological changes in transformants and Y32. 20°C, 25°C, 28°C, 30°C, 37°C were chosen as the temperature parameters and 3, 4, 5, 6, 7, 8 as the pH parameters. The cells were incubated for 2 to 9 d for the culture time parameters. Each LM medium containing different concentrations of cells (10 3 , 10 4 , 10 5 , 10 6 , and 10 7 cellsÁmL -1 ) was cultured respectively. Quorum sensing molecules (QSMs) including farnesol and tyrosol (Sigma, USA) in different concentrations (5, 25, 50, 100, 200 lmolÁL -1 ) were also prepared to observe their effects on morphological changes. Strains supplemented with 1% methanol were the controls for each assay. All experiments were performed in triplicate of each treatment. Data were analysed by ANOVA, followed by Ducan's multiple range tests.
Samples were observed at 20 9 objective by an optical microscope (Leica, Germany). Three or more cells connected at the end of the long axes or in a definite direction or an elongated cell with a daughter cell and an ellipsoidal cell having two branched daughter cells were counted as a pseudohypha. Only differentiated cells were quantified and normalized to 100% (yeast/pseudohypha cells). For each repetition, at least 300 cells were counted under the microscopy.

The bioinformation analysis of TrGpa1
The DNA sequence of TrGpa1 is 1436 bp and contains eight introns of 53, 61, 41, 40, 45, 47, 46, and 44 nucleotides, respectively (data not shown). The 5' and 3' borders of the eight introns showed the same splicing sites (GT-AG) which are the common sequences for introns of filamentous fungi (Yin et al. 2015). The CDs encoded a protein of 352 amino acid residues. The calculated theoretical isoelectric point of TrGpa1 was 5.55, and the molecular weight was 40.18 kDa. The conserved domain analysis of the amino acid sequence revealed that TrGpa1 contained the GTPase domain (G1-G5), an ATP/GTP binding regions (G/AXXXXGKT/S), and the bc complex interaction site (data not shown).
A phylogenetic tree was constructed based on the multiple sequence alignments with the Ga subunits from other species (Fig. 3). The Ga proteins were divided into four groups according to their evolutionary relationships. It revealed that TrGpa1 belongs to the group I of mammalian Ga i superfamily. The amino acid sequence of TrGpa1 contained the consensus myristoylation site, indicated as MGXXXS at the N terminus, but did not present a consensus CXXX sequence (pertussis toxin-catalyzed ADP-ribosylation site) at the C terminus (Fig. 2).

Stability test of transformants
Single colonies of T. fuciformis transformants were selected randomly and sub-cultured in PDSA for five rounds. To confirm the integration of the overexpression and knockdown fragments, the DNA was extracted from 12 randomly selected transformants, and Y32. The positive amplification of a 500 bp DNA product, suggesting that the egfp-hph fusion gene had been transferred into these transformants (Fig. 4a). Southern blot analysis performed in 10 PCR positive transformants showed that all transformants except one appeared to have copies of the hph gene at random sites, but Y32 showed no hybridization (Fig. 4b).
Fluorescence microscopy showed eGFP expressed successfully in the individual transformants (Fig. 5).

Gene expression analysis of transformants
The qRT-PCR assays were used to test the mRNA accumulation of TrGpa1. For the overexpression transformants (Fig. 6a), the expression level increased from 1.5 to 2.5 folds, and the transformant with the highest relative mRNA level was selected and subcultured. In addition, the gene suppression ratio ranged from 52.10 to 68.26% among the knockdown transformants (Fig. 6a). Thus, the transformant having the highest gene suppression ratio was chosen. Then Western blot was subsequently undertaken in Y32 and transformants with the maximum and minimum expression level. As shown in Fig. 6b, TrGpa1 in the overexpression transformants was expressed at a higher level than Y32, whereas at a lower level in the knockdown transformants, which is in accordance with the qRT-PCR analysis.
TrGpa1 contributes to T. fuciformis dimorphism The dimorphic-related functions of TrGpa1 were characterized. It was shown that the TrGpa1 was b Fig. 2 Multiple amino acid sequence alignment of TrGpa1 with homologues from other fungi: K. heveanensis (OCF36875.1), C. gattii (XP_003191999.1), C. neoformans (XP_566528.1), Pisolithus sp. (AAK15759.1), S. commune (XP_003029155.1), L. bicolor (XP_001888946.1), L. edodes (AAP13579.1), and H. marmoreus (KYQ31721.1). Conserved residues are shown in dark blue boxes, identical residues in light blue boxes, and unrelated residues in a white bachground. Dots indicates gaps or the lack of a matching sequence of the protein sequences. Amino acid numbers are shown on the right. Black Boxes in the N-terminal and N-terminal indicate the MGXXXS and CXXX sequence Fig. 3 Phylogenetic tree of TrGpa1 was generated by the neighbor-joining (NJ) method using MEGA 6.0, based on the amino acid sequences of Ga proteins from 22 species. One thousand bootstrap replicates were calculated, and bootstrap values are shown at each node. The scale bar indicates an evolutionary distance of amino acid substitutions per position required for pseudohyphal differentiation of T. fuciformis. When the environmental conditions change, the transition from yeast to pseudohypha in Y32, the overexpression and knockdown transformants were influenced by the environmental factors, including pH, temperature, inoculum size, culture time, farnesol and tyrosol concentration (Fig. 7). Comparing with Y32, the overexpression transformant always had higher ratios of pseudohyphae, and the knockdown transformant always had less proportions of pseudohyphae (Fig. 7).

Discussion
The dimorphism is a reversible transition and depends upon the environment to which the fungi are exposed (Wang et al. 2020). T. fuciformis, an edible jelly mushroom, has the capacity to perform this type of morphogenesis (Zhu et al. 2016). It has been demonstrated that G proteins are key regulators of this morphological transition in many dimorphic fungi (Park et al. 2020). However, little is known about the G proteins of T. fuciformis and their functions.
In this work, we have cloned the TrGpa1, a gene encoding a Ga subunit from T. fuciformis. According to the phylogenetic tree, TrGpa1, encoded by TrGpa1, belongs to the group I of Ga i superfamily containing the characteristic sequence sites (Fig. 3). The site for Fig. 4 Stability test of the overexpression and knockdown transformants. a PCR assays of the egfp-hph in the transformants, plasmid pPEH-PH, and Y32. Lane 1, DNA marker. Lane 2-7, PCR products of overexpression transformants. Lane 8-13, PCR products of knockdown transformants. Lane 14, PCR product of pPEH-PH. Lane 15, PCR product of Y32. b Analysis of the integration of hph in T. fuciformis transformants by Southern blot. Genomic DNA digested with XhoI was probed using * 500 bp DIG-labeled egfp-hph. Lane 1, DNA marker. Lane 2-6, TrGpa1 overexpression transformants. Lane 7-11, TrGpa1 knockdown transformants. Lane 12, Y32. Lane 13, pPEH-PH. The molecular weight of DNA marker (bp) is shown on the left myristoylation at the N terminus is important to attachment to the GPCRs in membrane (Li et al. 2019). The amino acid sequence of TrGpa1 did not contain the conserved pertussis toxin site at the C terminus. ADP ribosylation of the Ga i subunits locks the activity of G proteins and prevents the activation by GPCRs (Appleton et al. 2014).
Furthermore, the overexpression and knockdown vectors were constructed for identifying the function of TrGpa1. Since the gene knockout methods are lacking in T. fuciformis, RNA interference (RNAi) was performed for identifying the function of TrGpa1 . Though it causes only partial gene silencing, RNAi technology provides variable rates of gene suppression transformants. Therefore, it makes it possible to investigate the effects of genes on the phenotypes of interest and the minimum effective inhibition rate. For example, in Magnaporthe oryzae, only a slight decrease in the expression of some calcium signaling related genes caused a complete loss of infection-related morphogenesis and pathogenicity (Lange and Peiter 2020). Nevertheless, strong knockdown of hydrophobin gene Mpg1 did not severely  affect its pathogenicity, despite the fact that a knockout of this gene presented a drastic reduction in pathogenicity (Han 2018). Thus, the impacts on the phenotypes would differ among different genes. Here we only chose the transformant with the highest or lowest mRNA accumulation level. The relationship of gene suppression rate and the dimorphic phenotypes is worthy to be discussed in our future work.
The characterization of dimorphic related functions indicated that TrGpa1 played a positive role in the promotion of pseudohyphal growth. The overexpression of TrGpa1 enhances the response to different conditions and promotes the pseudohyphal formation. Cells lacking TrGpa1 have a defect in pseudohyphal development in response to specific environmental cues. For dimorphic fungi, cell morphology is depending on the inoculation size. There is a general  (Wedge et al. 2016), that is when inoculation at C 10 6 cells mL -1 , budding yeasts are produced, while pseudohyphae and mycelia are produced following inoculation at \ 10 6 cells mL -1 . In the present study, interestingly, the overexpression transformant had high ratios of pseudohyphae, even when the inoculum size was larger than 10 6 cells mL -1 . In addition to the inoculation size, extracellular QSMs contribute to T. fuciformis dimorphism. In C. albicans, farnesol is a characterized QSM, which suppresses filamentous formation. Tyrosol, another QSM produced by C. albicans, stimulates the yeast-to-hypha conversion (Han et al. 2011;Monteiro et al. 2017). In Y32 and the transformants, farnesol and tyrosol played the same roles. Farnesol effectively blocked the transition from yeast to pseudohypha at the concentration of 50 lmolÁL -1 , while tyrosol stimulated this transition. The pseudohyphae ratios in knockdown transformant were largely decreased with the addition of farnesol, while the pseudohyphae ratios in the overexpression transformant were slightly decreased, which shows the overexpression transformant was less sensitive to the QSMs (Fig. 7).
However, it is still unclear in which signal pathways TrGpa1 is involved. Former studies showed many Ga subunits are involved in the cAMP-protein kinase A (PKA) and the mitogen-activated protein kinase (MAPK) pathways (Nogueira et al. 2015;Shwab et al. 2017;Martínez-Soto et al. 2020). Here the exogenous addition of cAMP (10 mmolÁL -1 ) had no apparent influences on the yeast-pseudohypha transition, which indicates TrGpa1 may not be involved in the cAMP/PKA pathway (data not shown). Other reagent including dibutyryl cAMP and MAPK inhibitors should be further performed in investigating the TrGpa1 related pathways.
In conclusion, TrGpa1, a gene encoding a G protein a subunit was cloned from the dimorphic fungus T. fuciformis. The TrGpa1, encoded by TrGpa1, belongs to the group I of Ga i superfamily. The function of TrGpa1 was characterized by gene overexpression and knockdown. The results have demonstrated that TrG-pa1 is involved in T. fuciformis dimorphism and supports a positive role in the transition from yeast to pseudohyphal growth under different environmental conditions. In our future study, the pathways wherein TrGpa1 is involved and the relationship of TrGpa1 suppression rate and dimorphic phenotypes need to be further investigated.
Author contributions HZ and AM designed the study; LZ and LC collected the samples; HZ and DL performed the laboratory work; HZ performed the data analysis and wrote the manuscript; AM, LZ, and LC reviewed and revised the writing.
Funding This work was supported by grants from the National Natural Science Foundation of China (NSFC) (No. 30972072 and No. 31572182) to Aimin Ma and from the General Project of Hunan Provincial Education Department (No. 19C0289) to Hanyu Zhu.
Availability of data and materials The DNA and cDNA sequences of TrGpa1 can be downloaded from the National Center for Biotechnology Information (NCBI), and the GenBank accession numbers are MH091706 and MH101517.

Declarations
Conflict of interest The authors declare no conflicts of interest.
Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.
Consent for publication All authors read and approved the final version of the manuscript.