Phylogeny of Regulators of G-Protein Signaling Genes in Leptographium qinlingensis and Expression Levels of Three RGSs in Response to Different Terpenoids

Leptographium qinlingensis is a bark beetle-vectored pine pathogen in the Chinese white pine beetle (Dendroctonus armandi) epidemic in Northwest China. L. qinlingensis colonizes pines despite the trees’ massive oleoresin terpenoid defenses. Regulators of G-protein signaling (RGS) proteins modulate heterotrimeric G-protein signaling negatively and play multiple roles in the growth, asexual development, and pathogenicity of fungi. In this study, we have identified three L. qinlingensis RGS genes, and the phylogenetic analysis shows the highest homology with the regulators of G-protein signaling proteins sequence from Ophiostoma piceae and Grosmannia clavigera. The expression profiles of three RGSs in the mycelium of L. qinlingensis treated with six different terpenoids were detected, as well as their growth rates. Under six terpenoid treatments, the growth and reproduction in L. qinlingensis were significantly inhibited, and the growth inflection day was delayed from 8 days to 12–13 days. By analyzing the expression level of three RGS genes of L. qinlingensis with different treatments, results indicate that LqFlbA plays a crucial role in controlling fungal growth, and both LqRax1 and LqRgsA are involved in overcoming the host chemical resistances and successful colonization.


Introduction
Phytophagous insects with fungal pathogens that attack coniferous trees have drawn a lot of attention due to the enormous amount of damage they cause. The damage caused by these fungal pathogens has important implications for climate change through the obvious impact of tree mortality on forest carbon dynamics [1]. The ascomycete Leptographium qinlingensis (Lq) is a fungal pathogen of Chinese white pine trees (Pinus armandii) and is vectored by the Chinese white pine beetle (Dendroctonus armandi, CWPB) [2]. CWPB and Lq form an interactive biological complex that has caused a rapid, large-scale decline in the Chinese white pine (P. armandii) in the Qinling and Bashan Mountains of China [3][4][5]. L. qinlingensis can stain the sapwood blue when the CWPB-Lq complex is inoculated manually into healthy host trees; such discoloration reduces the commercial value of lumber [6][7][8]. Like all conifers, the pine hosts of the CWPB-Lq complex have complex oleoresin-based chemical defences that protect these trees against most potential pests and pathogens [9,10]. The highly specialized CWPB-Lq complex, which colonizes the monoterpene-rich environment of pine phloem and sapwood, requires mechanisms to overcome the host defence chemicals [11,12].
The heterotrimeric G-protein (G-protein) signaling pathway is one of the most important signaling pathways that transmits external signals into the inside of the cell [13]. G-proteins are composed of α (Gα), β (Gβ), and γ (Gγ) subunits. The β and γ subunits are tightly associated and can be regarded as one functional unit [14,15]. In the presence

Terpenoid Treatments
Experimental group: Monoterpenes (±)-α-pinene((+)-α-pinene:(−)-α-pinene = 1:1), (−)-β-pinene, (+)-3-carene, (+)-limonene, turpentine, mix-monoterpene ((+)-limonene: (+)-3-carene: (±)-α-pinene: (−)-β-pinene = 5:3:1:1) were diluted with DMSO to three concentrations of 5%, 10% and 20% [31][32][33]. The main steps of terpenoid treatment are shown in Supplementary Figure S1. According to the purity of the terpenoid reagent, the volume (V1) of DMSO to be added to each 100 mL stock solution when preparing a 20% concentration terpenoid dilution was calculated (Table 1). Add 100 mL of terpenoid stock solution and V1 volume of DMSO to the burette, and after mixing, it is a 20% concentration of terpenoid Microorganisms 2022, 10, 1698 3 of 14 dilution (solution A). Take 100 mL of solution A into a new burette, add 100 mL of DMSO, and mix to obtain a 10% concentration of terpenoid dilution (solution B). Take 100 mL of solution B into a new burette, add 100 mL of DMSO, and mix to obtain a 5% terpenoid dilution (solution C). Pipette 200 mL of each concentration test chemical onto the centre of each MEA medium overlaid with cellophane and gently swirl over the agar surface before inoculation. Control group: 200 mL DMSO was added to the MEA medium overlaid with cellophane. A 1 cm diameter L. qinlingensis mycelial plug was inserted into the center of the above medium, incubated at 28 • C in the dark, and the growth (colony area measured by its diameter in cm) was measured every 3 days in four directions and averaged until the strain brought the fungus to the edge of the plate [32,34]. For the eight different media, the growth condition was obtained by calculating the area of the colony. Each treatment was repeated five times.

RNA Isolation and cDNA Synthesis
Total RNA was isolated from L. qinlingensis by the UNIQ-10 Column Trizol Total RNA Isolation Kit (Sangon Biotech, Shanghai, China) following the manufacturer's protocol. Its integrity was checked on 1% agarose gels and quantified using NANO DROP 2000 spectrophotometry (Thermo Scientific, Pittsburgh, PA, USA). The purity was calculated by the mean of relation A260/A280 ratio (µg/mL = A260 × dilution factor × 40). The synthesized cDNA obtained from the sample was used as the template using the HisScript ® III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme Biotech, Nanjing, China).

Gene Amplification and Cloning
The cDNA synthesized from the sample was used as a template for the PCR reaction. Degenerate primers (Table 2) were designed in Primer Premier 5.0, based on the regulator of G-protein signaling (RGS) protein sequences of Grosmannia clavigera and Ophiostoma piceae from NCBI (http://www.ncbi.nlm.nih.gov/, accessed on 18 July 2022). PCR amplifications were performed in a C1000 Touch Thermal Cycler (Bio-Rad, Hercules, CA, USA), and the cDNA amplification was performed in a 50 µL reaction volume: 5 µL cDNA, 10 µM each primer, 25 µL Green Taq Mix (Vazyme Biotech, Nanjing, China), with ddH 2 O added to 50 µL. The reaction conditions were as follows: 95 • C for 5 min, 30 cycles of 95 • C for 30 s, TM of each pair of primers for 30 s and 72 • C for 1 min, with a final extension for 10 min at 72 • C. The PCR products were visualized on 1% agarose gels stained with Ultra GelRed (10,000×) (Vazyme Biotech, Nanjing, China) and compared with a DL2000 Plus DNA Marker (Vazyme Biotech, Nanjing, China). Single-stranded 5' and 3' RACE-ready cDNA was synthesized from RNA using a SMARTer™ RACE cDNA Amplification Kit (Clontech Laboratories Inc., Mountain, CA, USA) according to the manufacturer's protocol. Partial sequences were used in the primer design (Table 2), and the PCR was performed as described in the SMARTer™ RACE cDNA Amplification Kit (Clontech Laboratories Inc., Mountain, CA, USA). The amplicons were purified, cloned, and sequenced. Sequences were manually edited with DNAMAN 6.0 software (Lynnon BioSoft, Vaudreuil, Quebec, Canada) to obtain inserts sequences, which were then BLASTed against the NCBI database.

Analysis of Full-Length cDNA Sequences
Full-length cDNA sequences were assembled in DNAMAN 6.0, using sequence fragments and RACE results. To avoid chimera sequences, specific primers (Table 2) from initiation to terminator codon were designed based on complete sequences. Open reading frames (ORFs) of full-length cDNA were obtained via ORF Finder (https:// www.ncbi.nlm.nih.gov/orffinder/, accessed on 18 July 2022), and cDNA was then translated to amino acid sequences using the ExPASy Translate Tool (http://www.expasy. org/tools/dna.html, accessed on 18 July 2022), and colored in DNAMAN 6.0. Molecular mass (kDa) and isoelectric points were determined in the ProtParam tool. RGSs of L. qinlingensis were checked for likely subcellular localization using Target P 2.0 software (http://www.cbs.dtu.dk/services/TargetP/, accessed on 18 July 2022) with the default parameters. RGSs of L. qinlingensis homologs were identified with the NCBI-BlastP network server (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 18 July 2022). Amino acid identity was analyzed through the construction of a homology tree in DNA-MAN6.0. A neighbor-joining phylogenetic tree was built in MEGA-X, employing ClustalW with default parameters, p-distance model, pairwise gap deletion, and 1000 bootstrap replicates. The putative N-terminal signal peptide was predicted in SignalP 5.0 Server (https://services.healthtech.dtu.dk/service.php?SignalP-5.0, accessed on 18 July 2022).

Effects of Terpenoids on Expression Levels of three L. qinlingensis RGSs (Real Time-qPCR)
In the Ultra-clean workbench, the mycelium cultured for 15 days on the solid medium was gently scraped with tweezers into the RNase-free microfuge tubes (1.5 mL) and stored at −80 • C until use. RNA isolation and cDNA synthesis followed previous descriptions ("RNA Isolation and cDNA Synthesis"). Five repetitions per treatment were prepared.
The CFX96TM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) was used for qRT-PCR, with L. qinlingensis EF1 (accession number: AHZ56579.1) as the reference gene. Specific qRT-PCR primers were designed in PRIMER 3Plus (https://www.primer3 plus.com/, accessed on 18 July 2022), based on nucleotide sequences (Table 2), and their amplification efficiencies were calculated using relative standard curves with a five-fold cDNA dilution series; the efficiency values for the primers were 100 ± 5%. The sizes of the amplicons were 128 bp (EF1), 135 bp (FlbA), 127 bp (Rax1), and 136 bp (RgsA). Amplicons were confirmed as the correct size after the qRT-PCR assay via gel electrophoresis and were then sequenced by a biotechnology company (TSINGKE Biotechnology, Beijing, China) to make sure the correct amplification products were obtained.
The reaction mixture (20 µL) contained 10 µL of ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech, Nanjing, China), 2 µL of cDNA (diluted 4 times), 0.4 µL of each primer, and 7.2 µL of nuclease-free water. Template-free negative controls were included in every reaction. Thermocycling conditions were as follows: 95 • C for 2 min, followed by 39 cycles of 95 • C for 10 s, and 60 • C for 30 s, melting curve analysis at 95 • C for 5 s, 65 • C for 5 s. At the end of each reaction, a melting curve analysis was performed to detect single gene-specific peaks and check for primer dimers. Three biological and three technical replicates were included to ensure reproducibility.

Statistical Analysis
Relative expression values of L. qinlingensis RGSs were determined using the Ct (∆∆Ct) method (Livak and Schmittgen 2008) and analyzed with Excel 2019 (Microsoft Office). To evaluate significant differences in the expression for L. qinlingensis RGSs, 2 −∆∆Ct values transformed at log 2 were subjected to a one-way analysis of variance (ANOVA) and Tukey's honest significant difference test (HSD) to determine whether the gene expression differed among the treatments. The 2 −∆∆Ct values and standard error (SE) were transformed at log 2 to generate graphs. All of the statistical analyses were performed with SPSS 22.0 (IBM SPSS Statistics, Chicago, IL, USA) and plotted with OriginPro 2021 software (OriginLab, Northampton, MA, USA).

Identification of L. qinlingensis RGS Genes
In L. qinlingensis, three regulators of G-protein signaling (RGS) genes were identified, all of which possess the conserved RGS domain and were termed LqFlbA, LqRax1, and LqRgsA. The full-length sequence of RGSs in L. qinlingensis showed a relatively high amino acid identity to the sequences predicted from the genomes of the bark beetle-associated fungi O. piceae UAMH 11346 and G. clavigera kw1407, respectively, LqFlbA (87.98% and 77.18%), LqRax1 (92.35% and 83.74%), LqRgsA (79.38% and 65.87%). RGSs in L. qinlingensis also shared a strong identity with other RGSs genes from the genus Sporothrix (S. brasiliensis, S. insectorum, and S. schenckii) ( Table 3).
Phylogenetic analysis revealed that these three L. qinlingensis RGSs amino acid sequences had the highest identity to O. piceae UAMH 11346 according to a neighbor-joining method analysis of the putative full-length amino acid sequences ( Figure 1).

Physicochemical Properties and Bioinformation Analysis
The

Physicochemical Properties and Bioinformation Analysis
The full-length open reading frames (ORFs) of RGSs in L. qinlingensis were 2349 bp (LqFlbA), 1101 bp (LqRax1), and 1296 bp (LqRgsA) encoding 782 (LqFlbA), 366 (LqRax1), and 431 (LqRgsA) amino acids. The predicted molecular masses were 85.09 kDa (LqFlbA), 41.23 (LqRax1), and 48.46 kDa (LqRgsA). The lowest isoelectric point of RGSs was 5.91 (LqRax1), and the isoelectric point of LqFlbA and LqRgsA were 9.04 and 9.60, respectively. The predicted subcellular location of LqFlbA and LqRax1 by the Target P1.1 program suggests a cytoplasmic location, but LqRgsA suggests a nuclear location (Table 4). The deduced amino acid sequences and domain structures of RGSs in L. qinlingensis were analyzed (Figures 2 and S2). LqFlbA contains a C-terminal RGS domain and two DEP domains at the N-terminus (Figure 2A), which is the same as the Sst2 in S. cerevisiae [35]. LqRax1 contains an N-terminal RGS domain and three DEP domains transmembrane domains at the C-terminus ( Figure 2B), which is the same as the Rax1 in S. cerevisiae [36]. The structure of LqRgsA is similar to that of LqRax1 ( Figure 2C).
The logistic curve fits the growth curve of L. qinlingensis in different terpenoid treatments (R 2 > 0.97). According to the logistic curve fitting of the growth curve, the growth inflection day of L. qinlingensis growth on MEA media occurred after approximately 9 days and the growth inflection day on different terpenoid treatments occurred after approximately 12-13 days (Table 5). However, the growth inflection day occurred after over 15 days for the medium with 5% concentration (+)-limonene treatment (Table 5).
domains transmembrane domains at the C-terminus ( Figure 2B), which is the same as the Rax1 in S. cerevisiae [36]. The structure of LqRgsA is similar to that of LqRax1 ( Figure 2C). The logistic curve fits the growth curve of L. qinlingensis in different terpenoid treatments (R 2 > 0.97). According to the logistic curve fitting of the growth curve, the growth inflection day of L. qinlingensis growth on MEA media occurred after approximately 9  Logistic equation: Y = K/(1 + ae −bt ), Y means size of the colony (cm 2 ), t means culture time, K is maximum size of the colony (cm 2 ), a is parameter and b is maximum of relative growth rate (cm 2 /day).   (C) Relative expression of LqRgsA. RGSs expressions were normalized with EF1 gene. The 2 −∆∆Ct and SE values were transformed at log 2 for plotting. Different letters indicate significant differences at p < 0.05 (Tukey test, no letter means no significant difference) among different concentrations of the same stimulus. Mycelial growth on MEA medium with 200 mL DMSO was set as the control group (CK). Based on the value of CK (X-axis), the expression levels of other treatments are higher than CK as a positive value (expression up-regulation), and vice versa for a negative value (expression down-regulation). Table 6. Statistics significant of RGSs expression from L. qinlingensis in different terpenoids.  Figure 4 with different letters.

Discussion
As the primary upstream components of the G-protein signaling pathway, regulators of G-protein signaling (RGSs) are the key negative regulators of the G-proteins to control the activities of GTPase in Gα subunits [17,26,37]. It has been known that RGSs are highly conserved in most filamentous fungi and play diverse roles in the growth, reproduction, and pathogenicity of fungi [38,39]. In this study, three RGS genes in the bark beetle-associated fungi L. qinlingensis were identified, all of which possess the conserved RGS domain, and were termed FlbA, Rax1, and RgsA, respectively.
The FlbA gene from L. qinlingensis, homologous to Sst2 from S. cerevisiae and FlbA from the model filamentous fungus A. nidulans, has the same domains including one RGS and two DEP [24,40]. The DEP domain is responsible for specific GPCRs recognition and targets RGS proteins to the Golgi and plasma membranes [35,41]. In this study, both mycelial growth and FlbA expression of L. qinlingensis were inhibited after exposure to the terpenoids ( Figure 4A), which is consistent with studies on the positive regulation of asexual development by FlbA in other fungi such as A. nidulans and A. flavus [26,42].
As a fungal-specific RGS protein, Rax1 has no mammalian counterparts [24]. The Rax1 from L. qinlingensis-which contains one RGS domain and three putative transmembrane domains at the C-terminus-is similar to the Rax1 protein in S. cerevisiae [43], which plays a key role in yeast bipolar budding reproduction [36,44,45]. In the opportunistic human pathogenic fungus A. fumigatus, Rax1 has been reported to play a positive controlling role in the growth, development, and oxidative stress response [46], as well as helping to regulate the normal growth in fungi under ER stress conditions [47]. The transcription level in LqRax1 was significantly overexpressed after treatment with (±)-α-pinene and turpentine but was downregulated after treatment with mix-monoterpene at 10% ( Figure 4B). The differences in the functional expression in Rax1 between plant pathogenic fungi and mammalian pathogenic fungi require further comparative study.
Except for the isoelectric point, the properties and functional domains of LqRgsA are similar to that of LqRax1. The bark beetle-associated fungi O. piceae UAMH 11346 and G. clavigera kw1407 also contain two RGS genes of similar size and structure [33,48], which belong to different groups in the phylogenetic tree ( Figure 1). LqRgsA was expressed more significantly than LqRax1 in response to terpenoid stress. The expression in LqRgsA was upregulated at low concentrations (5%) and downregulated at high concentrations (10% and 20%) in almost all terpenoid treatments except that (+)-3-carene caused downregulation at all concentrations ( Figure 4C). LqRgsA might contribute to the fungus' ability to overcome host defense chemicals and survive in an unfavorable environment at the beginning of the invasion of the insect-fungal complex in the host trees [8].
The beetle-fungi complex requires mechanisms to overcome host defences when colonizing healthy host trees [8,48]. Beetle-symbiotic fungi can not only help metabolize phenolics and terpenoids [49,50], but also provide nutritional support for bark beetles [34,51]. In this study, the growth rate in L. qinlingensis was slowed down after exposure to terpenoids, and the logistic curve showed that its growth inflection point was delayed by 4-5 days, which is consistent with the results of previous studies [31,32].
Overall, our results demonstrate that there are three RGS genes in L. qinlingensis, containing conserved RGS domains. The physicochemical properties and phylogenetic analysis showed that the three RGS genes in L. qinlingensis had high homology with the RGS genes in the beetle-symbiotic fungi O. piceae and G. clavigera. To explore the role of RGS genes in beetle-fungal complexes against host resistance, we diluted six terpenoids into three concentrations to treat L. qinlingensis separately. By monitoring the growth rate in hyphae exposed to host-volatile terpenoids and measuring the expression in three LqRGS genes, it was shown that LqRGS genes play an important role in both growth and overcoming host resistance.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/microorganisms10091698/s1, Figure S1. The main steps of terpenoid treatment. Figure S2. Domain structure analysis of three putative RGS proteins in L. qinlingensis.