Molecular Characterization and Functional Analysis of GPCR Gene Bx-srh-1 in Pinewood Nematode (Bursaphelenchus xylophilus)

Abstract

Bursaphelenchus xylophilus, also known as the pine wood nematode (PWD), which causes pine wilt disease (PWD), is one of the most devastating diseases affecting pine forests globally. G protein-coupled receptors (GPCRs) are crucial in many biological processes and serve as privileged points of communication between cells and the surrounding environment. α-pinene has been found to play a crucial role in combating the infection, colonization, and early stages of pathogenesis caused by B. xylophilus. In this study, we investigated the molecular characteristics and biological functions of the GPCR gene Bx-srh-1 in B. xylophilus. Fluorescence in situ hybridization (FISH) was performed to determine the spatial expression patterns of Bx-srh-1 in B. xylophilus. The results indicated that Bx-srh-1 is expressed in the intestine and subcutaneous tissues of J2 and J3 juveniles and in the spicules of adult males and vulvae of adult females. RNA interference (RNAi) was used to analyze Bx-srh-1 gene function, and we examined the expression patterns of Bx-srh-1 in B. xylophilus under α-pinene stress. The RNA interference indicated that Bx-srh-1 was involved in the reproductive ability and pathogenicity of B. xylophilus; the expression levels of Bx-srh-1 significantly increased after the exposure to α-pinene for 12 h, and they peaked at 48 h. Silencing Bx-srh-1 may therefore lead to a reduction in B. xylophilus reproduction and pathogenicity. These results demonstrate that Bx-srh-1 is related to the feeding behavior, reproduction, pathogenicity, and resistance to α-pinene process of B. xylophilus.

1. Introduction

Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle, commonly referred to as the pinewood nematode (PWN), is a highly menacing endoparasitic plant nematode. This nematode, which is indigenous to North America, is responsible for the onset of pine wilt disease (PWD), leading to significant economic and ecological damages in pine forests. [1,2]. Under field conditions, PWNs are transmitted by the Monochamus beetle, which emerges from dead pine trees to eat young, succulent, and healthy pine tree branches. When Monochamus beetles feed, they cause wounds and provide entry portals (infection courts) for the PWN. Upon penetration of the tree, the PWN proceeds to consume the plant epithelial cells located in the cortex and xylem tissues, thereby disseminating throughout the tree via tissue migration during the phytophagous phase. This ultimately culminates in the manifestation of wilting symptoms, ultimately resulting in the demise of the pine tree. [3]. B. xylophilus poses a significant threat to pine forests worldwide, resulting in substantial damage to these forests in North America, Europe, and East Asia [4,5,6,7,8,9]. In China, this disease was first identified in Nanjing City, Jiangsu Province, in 1982 and has since spread rapidly to 19 provinces, encompassing 731 county administrative regions and covering an area of 650,000 ha [10,11,12]. Despite extensive research on the pathogenicity, biological properties, infection cycle, and vector insects [13,14,15,16,17], the high mortality rate of host trees and the rapid spread of the disease continue to pose a significant challenge.

G protein-coupled receptors (GPCRs) constitute the largest family of transmembrane ™ receptors, playing a crucial role in various physiological processes [18]. Prior research has demonstrated the involvement of GPCRs in innate/adaptive immunity, environmental signal detection, and pathogen defense in the model nematode Caenorhabditis elegans [19,20,21]. The prefix “sr” designates over 1300 potential chemoreceptor genes identified in C. elegans, which belong to the receptor superfamilies Sra (Sra, Srb, Srab, Sre) and Str (Srh, Str, Sri, Srd, Srj, Srm, Srn), and Srg (Srx, Srt, Srg, Sru, Srv, Srxa) [22]. Some researchers have identified ~1059 C. elegans neuropeptide G-protein coupled receptor (Ce-NP-GPCR), a genome-wide prediction of homologous genes in 10 key parasitic nematodes covering a wide range of phylogenetic clades and lifestyle types [23]. However, there are still only a few studies on B. xylophilus with GPCRs. Previously, it was demonstrated that B. xylophilus responds to low temperatures through GPCRs; thus, GPCR genes play a crucial role in abiotic stress adaption [24]. Other researchers have found that GPCRs are extremely important for the locomotion, early ontogenesis, and mating behavior in B. xylophilus [25]. Therefore, GPCRs are extremely important in B. xylophilus. However, it is unknown whether they are involved in the host defense and B. xylophilus invasion.

Monoterpenes are important part of terpenoids produced by pine trees, which can provide resistance to external pathogen invasion [26]. The monoterpene secreted by pine trees are largely α-pinene [27], and α-pinene has low toxicity to B. xylophilus [28]. Previous studies have proved that low concentration of α-pinene could reduce the reproduction of B. xylophilus. The detoxification-related and resistance-related genes of B. xylophilus are upregulated when B. xylophilus is exposed to α-pinene [29,30]. However, it is unknown whether GPCRs are involved under the stress of α-pinene.

In order to delineate the expression pattern and biological functions of GPCRs in B. xylophilus, we cloned the GPCR gene Bx-srh-1. Subsequently, we conducted an investigation into the molecular characteristics and biological functions of Bx-srh-1. To determine the spatial expression patterns of Bx-srh-1 in B. xylophilus, we utilized fluorescence in situ hybridization (FISH). The identification of Bx-srh-1 with altered expressions induced by α-pinene was accomplished through the utilization of real-time quantitative reverse-transcription PCR. The objective of this study is to examine the role of Bx-srh-1 in the reproduction, pathogenicity, and defense against α-pinene stress of B. xylophilus.

2. Materials and Methods

2.1. Biological Materials

The highly virulent isolate QH-1 of B. xylophilus [31], obtained from infected P. koraiensis in Liaoning, China, was cultured on Botrytis cinerea Pers. (1794) fungus grown on barley grains at 25 °C for two weeks [32]. The Baermann funnel technique was employed at room temperature to extract mixed stages of nematodes [33], which were subsequently washed using 1 × M9 buffer for the experiments. The counting methods of B. xylophilus were based on Wang’s methods [34]. After the nematode suspension was collected from cultured B. cinerea, the volume of nematode suspension was adjusted to 10 mL with 1 × M9 buffer and mixed. Next, the nematode suspension was retrieved to the counting dish by gentle suction. The number of nematodes in the suspension was counted using stereomicroscope (Olympus, Tokyo, Japan). Four-year-old P. koraiensis seedlings were grown in a nursery situated as previous methods [31].

2.2. RNA Extraction and cDNA Synthesis

The nematodes were subjected to RNA extraction using the Tissue/Cell Total RNA Mini Kit (GeneBetter, Beijing, China) in accordance with the manufacturer’s guidelines. The total RNA concentration was determined at 260/280 nm using an ultraviolet spectrophotometer (NanoDrop ND-2000, Thermo Fisher, Waltham, MA, USA), and its quality was assessed via electrophoresis on a 1% agarose gel. Subsequently, the RNA was reverse-transcribed to cDNA using the Scripit III RT Kit with gDNA Eraser (GeneBetter, Beijing, China).

2.3. Bx-srh-1 Gene Cloning

Utilizing transcriptome data from WormBase ParaSite (http://parasite.wormbase.org/index.html, (accessed on 10 October 2022)), the Bx-srh-1 sequence was obtained for the B. xylophilus genome. Subsequently, a specific primer pair (Bx-srh-1-F/R) was designed to target the complete Bx-srh-1 via polymerase chain reaction (PCR) from cDNA reverse-transcribed from B. xylophilus mRNA (Table S1). The PCR protocol begins with 94 °C pre-denaturation for 5 min, then 35 cycles of 28 °C denaturation for 30 s each, 56 °C annealing for 30 s, 72 °C extension for 2 min, and 72 °C extension for 10 s each. The amplified PCR products were subjected to electrophoresis on 1% agarose gels for confirmation and subsequently purified using the Fast-pure Gel DNA Extraction Mini Kit protocol (Vazyme, Nanjing, China). The purified PCR products were then cloned into the pUC57 Vector (TsingKe, Beijing, China) and transformed into competent cells of Escherichia coli Trans1-T1 (TsingKe, Beijing, China). We incubated a transformed E. coli culture overnight at 37 °C on Luria-Bertani (LB) plates containing ampicillin. Positive transformants were identified using the M13F/R primers by PCR (Table S1).

2.4. Sequence Analysis of Bx-srh-1

In this study, NCBI tools were employed for amino acid prediction and homology analysis, while MEGA7 with the N-J method was utilized to construct the phylogenetic tree for the protein encoded by Bx-srh-1. To ensure the reliability of the phylogenetic tree, a self-expansion test was conducted by repeating the sampling test 1000 times. Furthermore, the molecular weight and other molecular function of each protein were predicted through the bioinformatics website ExPASy (SIB Swiss Institute of Bioinformatics, Zurich, Switzerland, https://www.expasy.org/, (accessed on 15 October 2022)). To analyze the signal peptide, transmembrane structure, and tertiary structure of the proteins, the ExPASy, SMART (http://smart.embl-heidelberg.de/, (accessed on 15 October 2022)), TMHMM (Center for Biological Sequence Analysis, Technical University of Denmark, Lyngby Denmark, https://services.healthtech.dtu.dk/services/TMHMM-2.0/, (accessed on 15 October 2022)), and SWISS-MODEL programs (Swiss Institute of Bioinformatics Biozentrum, University of Basel Spitalstrasse, Switzerland, https://swissmodel.expasy.org/, (accessed on 15 October 2022)) were utilized.

2.5. Fluorescence In Situ (FISH)

To determine the spatiotemporal expression patterns of Bx-srh-1, mRNA in situ hybridization was performed. A red fluorescence-labeled probe was synthesized from the cloned sequence of Bx-srh-1, as outlined in Table S1. The FISH in situ hybridization kit C007 (Gefan, Shanghai, China) was utilized following the manufacturer’s instructions, with the sense probe (Table S1) serving as the negative control. Visualization and capture of the samples were conducted using a Zeiss Axio Image M2 microscope (Zeiss, Oberkochen, Germany).

2.6. RNA Interference

By using RNA interference technology, we investigated Bx-srh-1’s function in vitro, and green fluorescent protein (GFP) was performed using the T7 RNAi Transcription Kit (Vazyme, Nanjing, China) with the following primers: Bx-srh-1T7-F/R, Bx-srh-1T7-R/F, and Bxgfp-F/R (Table S1). The soaking method was used to analyze the nematodes at mixed developmental stages, as per previous studies [34]. For dsRNA treatment, nematodes were soaked in ddH₂O containing 800 ng/L of dsRNA with intermittent stirring (25 °C, 180 rpm) for 48 h. The control group nematodes were soaked in ddH₂O. After soaking, nematodes were collected by centrifuging and were then thoroughly washed with ddH₂O for subsequent experiments.

2.7. Quantitative Real-Time PCR (qRT-PCR)

The qRT-PCR (20 μL) was performed to detect the gene expression levels using the Taq Pro Universal SYBR qPCR Master Mix for qPCR (Vazyme, Nanjing, China) and CFX96TM Real-Time system (Bio-Rad, Hercules, CA, USA). Primers were designed with Primer Premier 5 software (Premier Biosoft International, San Francisco, CA, USA) for the target gene Bx-srh-1 and reference gene Actin (Table S1).

The three-step reaction procedure for qRT-PCR was executed with the following parameters: denaturing stage at 95 °C for 30 s, PCR stage consisting of 40 cycles of 95 °C for 10 s and 60 °C for 30 s, melting stage at 95 °C for 15 s, 60 °C for 60 s, and 95 °C for 10 s, and cooling stage at 50 °C for 30 s. The experiment was conducted with three biological and technical replicates for each sample. The relative expression levels were calculated using the 2^−ΔΔCt method [35].

2.8. Assay for Feeding, Reproduction, and Pathogenicity after RNAi

In order to assess the impact of RNA interference (RNAi) on the reproductive capacity of B. xylophilus, approximately 100 nematodes subjected to RNAi treatment were cultivated on potato dextrose agar (PDA) that had been overgrown with Botrytis cinerea at a temperature of 25 °C. Daily observations and photographic documentation were made of the nematodes’ feeding behavior. After a period of nine days, the nematodes were removed from the cultures, counted, and used to calculate the rate of reproduction. Additionally, to examine the effects of RNAi on the pathogenicity of B. xylophilus, 2000 nematodes that had undergone dsBx-srh-1 treatment were inoculated into four-year-old P. koraiensis seedlings. A control group consisting of nematodes soaked in ddH₂O was utilized, while negative controls were treated with dsgfp. The infective ability was periodically monitored, and the severity of disease on P. koraiensis was classified into five stages (0, I, II, III, and IV) using Xiang’s methodology [36]. The experiment was conducted with ten biological replicates. The mortality rate was calculated using the following formula:

$$\mathrm{Motality} \mathrm{rate}=\frac{\Sigma \mathrm{Number} \mathrm{of} \mathrm{dead} \mathrm{plants}}{\mathrm{Total} \mathrm{number} \mathrm{of} \mathrm{plants}}\times 100\%$$

2.9. Analysis of Expression Levels of Bx-srh-1 in B. xylophilus under Abiotic Stress

Li’s method was employed to administer treatment for a brief duration [29]. Specifically, 500 nematodes were subjected to soaking in a 200 μL solution of α-pinene (56.33 mg/mL) dissolved in 0.5% (w/w) Triton X-100 and ddH₂O for 6, 12, 24, and 48 h, while Triton X-100 and ddH₂O were utilized as controls. Each treatment was replicated thrice biologically. Following the washing of nematodes from the solution, RNA extraction was carried out, and cDNA was synthesized. Subsequently, gene expression levels of Bx-srh-1 were determined by quantitative real-time PCR using specific primers (Table S1).

2.10. Data Analysis and Processing

The mean and standard deviation (SD) of triplicate measurements for each group were calculated using Microsoft Excel software (Office excel 2016, Microsoft Corp., Redmond, WA, USA). Statistical analysis was conducted using SPSS17.0 (SPSS Inc., Chicago, IL, USA) and Origin8.0 (OriginLab, Northampton, MA, USA). Significance was determined at p < 0.05 using one-way ANOVA with appropriate t-tests.

3. Results

3.1. Alignment and Phylogenetic Analysis of Bx-srh-1

PCR amplification of the Bx-srh-1 sequence was accomplished with success, as depicted in Figure S1. Subsequent sequence analysis revealed that the PCR product encompasses 1080 base pairs, encompassing a full open reading frame (ORF) that encodes 359 amino acids. The coding sequence for Bx-srh-1 was deposited in the NCBI GenBank, with an assigned accession number of OQ383310. Phylogenetic analysis revealed that the protein Bx-SRH-1 is relatively conservative in its evolution between plant parasitic nematodes (Figure 1B). Bx-SRH-1 shares a 99% homology with SRH1 from Bursaphelenchus okinawaensis. We found that Bx-SRH-1 is not closely related to free-living nematode species (Caenorhabditis elegans and C. briggsae). The results of the conserved domain analysis by SMART indicated that Bx-SRH-1 belongs to the srh family of chemoreceptors of G protein-coupled receptors (GPCRs) (Figure 1C). We analyzed the physicochemical properties of the Bx-SRH-1 protein on the ExPASy, and the results showed that the molecular weight of Bx-SRH-1 is 40.819 kDa, with isoelectric points (pI) of 9.25. Based on the prediction of the signal peptides and transmembrane and tertiary structures, the grand average of the hydropathicity (GRAVY) of Bx-SRH-1 was 0.502, it had no signal peptides, and it had seven transmembrane regions (Figure 1D,E), indicating that Bx-SRH-1 is a typical transmembrane protein.

3.2. Gene Expression Pattern of Bx-srh-1 in Bursaphelenchus xylophilus

The spatiotemporal expression of Bx-srh-1 was identified using FISH. Hybridization signals were observed in both juvenile (J2 and J3) and adult nematodes. In the adult stage, the hybridization signals were predominantly detected in the intestine and subcutaneous tissues, with a notable increase in expression near the spicules of adult males and vulvae of adult females. Specifically, the hybridization signals were observed in close proximity to the spicules of adult males and vulvae of adult females (as depicted in Figure 2A,C). The hybridization signals were also detected in the intestine and subcutaneous tissues of J2 and J3 juveniles, as illustrated in Figure 2B,D. Conversely, no hybridization signals were observed in the negative controls, as depicted in Figure 2E. It is speculated that that Bx-srh-1 could play important roles in the sex specificity in adult nematodes and in the feeding in juvenile nematodes.

3.3. RNA Interference Effect on B. xylophilus Feeding, Reproduction, and Pathogenicity

In comparison to the non-dsRNA-treated B. xylophilus, a significant reduction of 74.9% (p < 0.0001) in the expression of Bx-srh-1 was observed in the dsBx-srh-1-treated B. xylophilus (Figure 3A). Additionally, the Bx-srh-1 expression in the dsgfp-treated B. xylophilus was similar to that of the non-dsRNA-treated group. The feeding rate of the ds Bx-srh-1 treatment groups was notably slower than that of the other treated group (Figure 3C). Within 9 days, almost all the hyphae were consumed by B. xylophilus in the water treatment and dsgfp treatment. In the group treated with dsBx-srh-1, consumption of hyphae was observed in only one- to two-thirds of the sample. The reproductive rate of B. xylophilus, when soaked in dsBx-srh-1, was significantly lower compared to the water treated and dsgfp groups (Figure 3B). These findings suggest that Bx-srh-1 may have significant implications in feeding and reproduction.

Within 10 days after inoculation, seedlings inoculated with B. xylophilus treated with sterile water displayed initial wilt symptoms, as depicted in Figure 3D. Within 20 days after inoculation, P. koraiensis inoculated with dsBx-srh-1-treated B. xylophilus exhibited wilt symptoms, with mortality rates of 60% for the sterile-water treatment and 30% for dsgfp treatment seedlings, respectively (Figure 3E). By the 30th day after inoculation, all P. koraiensis seedlings inoculated with sterile-water-treated B. xylophilus had perished, while the mortality rate for the dsBx-srh-1 treatment seedlings was 10%.

3.4. Expression Levels of Bx-srh-1 in B. xylophilus under α-Pinene Stress

The expression levels of Bx-srh-1 were compared between the α-pinene treatment and TritonX-100 treatment (Figure 4). The expressions of Bx-srh-1 gradually increased with the B. xylophilus stress with α-pinene for 12 h, with 1.7-fold changes from the TritonX-100 treatment. After treatment for 48 h, the expression levels of Bx-srh-1 from the α-pinene treatment were upregulated to 4.37-fold changes from the TritonX-100 treatment. These results indicate that the α-pinene exposure directly upregulated the expression levels of Bx-srh-1. Therefore, Bx-srh-1 may play an important role in the nematode resistance to α-pinene stress.

4. Discussion

GPCRs constitute a large protein family with diverse functions, such as autocrine, paracrine, and endocrine functions [37]. GPCR genes transduce extracellular signals in C. elegans [38]. The srh family is part of the str superfamily of chemoreceptors of GPCRs in C. elegans, and it is very large, containing 214 genes and 90 pseudogenes [39]. Previous studies have found that the srh family genes srh-34 and srh-234 are regulated by the integration of the sensory and internal feeding state signals in C. elegans. When nematodes are under starvation, srh-34 and srh-234 are regulated by multiple pathways [40]. Previous studies have reported that GPCR genes are involved with abiotic stress adaption, locomotion, early ontogenesis, and mating in B. xylophilus [24,25]. However, the other roles of GPCRs in B. xylophilus are still unclear. In this study, we investigated the phylogenetics of SRH-1 between B. xylophilus and six other species of nematodes. The results showed that the SRH-1 protein of B. xylophilus had the closest relationship with that of B. okinawaensis. It formed a founded cluster with the SRH-1 proteins of other plant parasitic nematodes, and it further clustered with the SRH-1 protein of C. elegans and C. briggsae as one branch. These results indicate that SRH-1 is evolutionarily conserved across the species of different plant parasitic and free-living nematodes. In free-living nematodes, GPCRs have a larger number of genes; however, the number of GPCRs is reduced in parasitic nematodes. Nearly 50% of the srh family genes were lost in B. xylophilus compared with C. elegans [41]. The mechanism of the gene loss in the srh family needs further investigation.

Previous research investigated the expression pattern of the GPCR gene Bxy-octr-1 in B. xylophilus using whole-mount in situ hybridization, and Bxy-octr-1 is expressed throughout all developmental stages of B. xylophilus. Specifically, during the larval stage, Bxy-octr-1 is expressed in the entire body, while in the sexually mature adult stage, it is expressed in the gonads and other reproductive organs [25]. These findings suggest that Bxy-octr-1 may play a role in the locomotion and feeding behavior during the larval stage, as well as influence mating behavior at adult stage. Our study utilized FISH analysis to determine that Bx-srh-1 is expressed in the J2 juvenile, J3 juvenile, and adult stages of B. xylophilus. The gene was specifically expressed in the intestine and hypodermis of the J2, J3, and adult stages, with particularly high expression near the spicules of adult males and vulvae of adult females. Our study shows that Bx-srh-1 has various functions in B. xylophilus, which is consistent with the findings of Wang’s [25]. However, the regulatory mechanism of Bx-srh-1 in B. xylophilus remains unclear. Further investigation is needed to determine the precise tissue or cells where Bx-srh-1 is expressed in B. xylophilus.

The first instance of RNAi-mediated transcriptional gene silencing in C. elegans was reported in 1998 [42], and since then, RNAi has been extensively used in gene-silencing research on many multicellular organisms, including plant parasitic nematodes [43,44,45]. Several studies have been conducted on B. xylophilus using RNAi mediation, revealing that the efficacy and reproducibility of RNAi mediation vary significantly depending on the target gene [46,47]. The study found that Bx-srh-1 RNAi efficiency in B. xylophilus was 74.9%. The knockdown of Bx-srh-1 through RNAi showed that it plays a crucial role in feeding, reproduction, and pathogenicity of the nematode. The results suggest that RNAi treatment could potentially reduce the population growth and pathogenicity of B. xylophilus. However, the mechanism of Bx-srh-1 remains unknown, and further investigation is required to determine if it regulates the expression of other srh family genes.

In C. elegans, toxicants can trigger a response by inhibiting or activating GPCRs located on the cytoplasmic membrane. Several GPCRs that control the stress response to toxicants have been identified in studies [48,49]. Previous research on B. xylophilus found that GPCRs regulate hormone levels during the invasion of pine trees, resulting in the upregulation of genes associated with food digestion, consumption, and xenobiotic metabolism. Thus, genes involved in xenobiotic metabolism are essential for detoxification and transport of toxicants [41]. α-pinene, a critical volatile monoterpene, is responsible for a significant proportion of the monoterpene production in pine trees [27] and exhibits low toxicity to B. xylophilus [28]. Studies have demonstrated that α-pinene effectively inhibits the reproduction of B. xylophilus at low concentration and upregulates t detoxification-related genes [29]. Our study revealed a gradual increase in the expression levels of Bx-srh-1 in response to α-pinene-induced stress in B. xylophilus, reaching their peak at 48 h. This finding suggests that Bx-srh-1 may play a crucial role in the detoxification process. Further investigation is required to determine if other GPCR genes respond to α-pinene.

5. Conclusions

This study mainly focuses on molecular characterization and functional determination of the GPCR gene Bx-srh-1 in B. xylophilus. The results revealed that Bx-srh-1 plays a crucial role in regulating feeding behavior, reproduction, pathogenicity, and resistance to α-pinene in B. xylophilus. The findings suggest that Bx-srh-1 is involved in the pathogenicity of B. xylophilus, which could enhance our understanding of the physiological mechanisms of PWD.