https://orcid.org/0000-0001-7115-542X
Figures
Abstract
The pine wood nematode (PWN) Bursaphelenchus xylophilus is a highly destructive invasive pest that has spread from North America to Eurasia, demonstrating remarkable adaptability across environments. However, the molecular mechanisms underlying environmental adaptation of B. xylophilus remain poorly understood. In this study, we integrated genomic and transcriptomic analyses of B. xylophilus and its native sibling species B. mucronatus, which is native in China. Functional validation of key genes was conducted using RNA interference, BAX, and inoculation assays. Our research focused on three rapidly expanding gene families in B. xylophilus, including the BolA-like superfamily, diacylglycerol acyltransferase (DGAT), and papain-like cysteine peptidase (PLCP). Key genes were functionally validated to elucidate their roles in environmental adaptability. The BolA-like genes were identified as critical stress-response elements, enabling B. xylophilus to survive under harsh conditions. The DGAT genes are essential for lipid biosynthesis and play pivotal roles in resisting starvation and cold. Regarding pathogenicity, the PLCP gene family has been identified as a critical virulence determinant facilitating B. xylophilus infection on host pine trees. These expanded gene families collectively enhance stress tolerance and virulence in B. xylophilus. The findings of this study not only reveal the genetic basis of PWN’s invasive success, but also provide a foundation for managing climate-driven disease spread.
Author summary
The PWN is an invasive species that causes pine wilt disease (PWD). As a major globally recognized forest pest, it is listed as an A1 quarantine pest by the European and Mediterranean Plant Protection Organization. PWN has adapted to environments across North America, Asia, and Europe, leading to severe infestations worldwide. However, the mechanisms underlying its environmental adaptability remain unclear. Our study reveals that the genetic advantage contributing to PWN’s destructiveness stems from the expansion of three gene families, which together enable survival under environmental stress and drive PWD. First, BolA-like genes act as a universal cellular repair system, allowing the nematode to withstand abiotic stresses. Second, DGAT genes enhance lipid storage, generating energy reserves that support survival during starvation and cold exposure. Third, the PLCP gene family has been identified as a critical virulence determinant facilitating B. xylophilus infection on host pine trees. Collectively, these findings indicate that the invasive success of PWN arises from the co-expansion and functional integration of the BolA-like, DGAT, and PLCP gene families. Their coordinated activity forms an integrated system that enhances both environmental adaptability and pathogenicity, thereby driving global population expansion.
Citation: Shao H, Chen J, Hu W, Zou Y, Liu H, Zhang Y, et al. (2026) Decoding the Pine Wood Nematode’s survival mystery: Gene family expansion drives adaptation revealed by dual-omics. PLoS Pathog 22(3): e1014033. https://doi.org/10.1371/journal.ppat.1014033
Editor: Shahid Siddique, University of California Davis, UNITED STATES OF AMERICA
Received: August 23, 2025; Accepted: February 23, 2026; Published: March 6, 2026
Copyright: © 2026 Shao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This work was supported by the National Natural Science Foundation of China (32371892, 31670652 and 31870633) to JH and (32401591) to HS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
The pine wood nematode B. xylophilus is distributed across both North America and Eurasia, whereas its close relative B. mucronatus is restricted to Eurasia [1]. This geographic disparity may be attributed to the comparatively greater environmental adaptability of B. xylophilus. Notably, B. xylophilus has caused severe ecological damage in Eurasia after being spread from North America to Eurasia [2,3]. In contrast, B. mucronatus has not been associated with similar pathogenic effects. This indicates that B. xylophilus has a significantly greater capacity for environmental adaptation than B. mucronatus, enabling its rapid establishment and proliferation in new ecosystems. Limited research addresses the molecular basis of B. xylophilus’s environmental adaptability. However, elucidating underlying molecular mechanisms is essential for advancing understanding of PWD epidemiology, predicting its spread, and developing effective control strategies.
Bursaphelenchus xylophilus exists in two distinct developmental forms, i.e., reproductive and dispersal form, and relies on insect vectors for host transfer. At an optimal temperature of 25°C with abundant food, B. xylophilus remains in the reproductive form and can complete its life cycle in four days [4]. Faced with environmental stressors such as low temperature or nutrient limitation, it executes a key survival strategy by transitioning from propagative form to dispersal form [4]. Dispersal-form nematodes can sense and aggregate toward their vector beetle’s pupal chambers, colonize the pupae. And subsequently are transmitted to new host trees by the emerging adults [5]. After being transferred to a new host, B. xylophilus switches back to the reproductive form, feeds on the resin duct epithelial cell (i.e., phytophagous phase), and initiates mass reproduction of progeny. With the rapid proliferation of nematode, the host pine tree begins to wilt, facilitating extensive fungal growth inside. B. xylophilus then shift to feeding on the fungal hyphae, entering the mycophagous phase [6].The closed life cycle of B. xylophilus, involving transitions between propagative and dispersal forms, represents an active evolutionary adaptation to environmental challenges. Temperature acts as a key environmental signal, directly regulating this developmental plasticity and influencing subsequent pathogenicity [7,8].
Although B. xylophilus and B. mucronatus have highly similar ecological niches, vector insects, and biological characteristics, they exhibit a significant divergence in their pathogenicity to pine hosts [1]. B. xylophilus acts as the pathogen responsible for severe pine wilt, in contrast B. mucronatus typically provokes only mild symptoms [1]. Traditional pathological observations reveal that this disparity correlates with its rate of migration within the host and capacity for cellular destruction [9]. B. xylophilus displays rapid dispersal via resin canals post-inoculation, causing extensive parenchyma and epithelial cell necrosis. Conversely, B. mucronatus exhibits slow migration, confining lesion development primarily to the inoculation site with markedly reduced cell death. With advancements in molecular biology, comparative proteomic analyses have identified distinct protein expression profiles between two nematodes; [10] however, functional validation of these differential proteins and analysis of their links to pathogenicity remain largely unexplored. Given the complexity of the pathogenic mechanisms of two nematodes, the multidimensional mechanistic differentiation remains to be elucidated. This is also the core scientific question concerning the pathogenic of B. xylophilus.
Comparative genomics and transcriptomics offer new perspectives for investigating the stress resistance and pathogenicity in closely related species. For instance, multi-omics analyses of Taenia saginata and T. asiatica have highlighted gene families involved in homeostasis, epidermal development, and lipid uptake, which are crucial for adaptation to new hosts [11]. Similarly, comparative studies of Trichinella pseudospiralis and Trichinella spiralis have revealed key excretory secretory genes important for immune evasion [12]. These examples demonstrate how comparative genomic and transcriptomic analyses can uncover the molecular basis of differences in environmental adaptation between B. xylophilus and B. mucronatus. It has been previously reported that in Caenorhabditis elegans, BolA-like proteins and diacylglycerol acyltransferase (DGAT) function as critical regulators of stress resistance [13,14]. BolA-like superfamily proteins confer stress resistance by maintaining cellular homeostasis, while diacylglycerol acyltransferase (DGAT) ensures energy availability during stress through lipid storage. In addition, papain-like cysteine peptidases (PLCPs) have been identified as key pathogenicity factors in plant-parasitic nematodes [15,16]. Nevertheless, research on B. xylophilus and B. mucronatus in this respect is limited.
In this work, the genomes of B. xylophilus and B. mucronatus were assembled, and multi-transcriptome analyses were conducted, including B. xylophilus and B. mucronatus in different developmental stages, and B. xylophilus isolated from pine after different inoculating times. The functional validation of key genes was performed by qPCR, in situ hybridization, RNAi, BAX, and inoculation assays. This study provides direct evidence of understanding the environmental adaptation mechanisms of B. xylophilus and suggests targeted strategies for controlling PWD.
Results
Two high-quality de novo genome assemblies
De novo genome assembly of B. xylophilus was based on 152-fold-coverage single-molecule real-time (SMRT) sequencing reads (S1 Table). The final assembly, BxV1.0, spans 76.32 Mb with a contig N50 of 4.10 Mb (S1 Fig and S2 Table). The genome contains 76.5% complete benchmarking universal single copy orthologs (BUSCO genes) (S3 Table), and over 29% consists of repetitive elements (S2 Fig and S4 Table). To assess assembly quality and structural consistency, BxV1.0 was aligned to the previous assembly BXYJv5 using MUMmer. Alignment rates reached 100% for both reference and query sequences, with 97.58% and 98.31% base alignment for reference and query sequences, respectively. Average nucleotide identity was 97.75% for 1-to-1 alignments (n = 12,642) and 97.32% for many-to-many alignments (n = 39,833), indicating high sequence similarity. Structural analysis revealed 79,528–79,645 breakpoints, comprising 2,104 shifts, 1,610 translocations, 262–369 inversions, and >23,000 insertions, along with >1 million single-nucleotide polymorphisms and ~515,000 indels (S3 Fig). These findings indicated that there are differences between genomic structure of B. xylophilus from different geographical region. RNA-seq datasets from different developmental stages of B. xylophilus in this study and all expressed sequence tags of Bursaphelenchus from the National Center for Biotechnology Information (NCBI) were used to predict genes and annotate the genome. A total of 16,072 high-confidence protein-coding genes and 1,183 non-coding RNAs were identified, with functional annotations derived from eight publicly available databases (S5 and S6 Tables). More than 85% of these genes were successfully annotated using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases (S5 Table).
Similarly, de novo genome assembly of B. mucronatus was performed using a set of 124-fold coverage SMRT reads (S7 Table). The resulting assembly of B. mucronatus (BmV1.0) had a size of 80.39 Mb and a contig N50 of1.27 Mb (S4 Fig and S8 Table). BUSCO completeness was estimated at 77.39% as estimated (S9 Table). Repetitive sequences spanned 18 Mb, accounting for 22% of the assembly (S5 Fig and S10 Table). Detailed annotation of 17,248 protein-coding genes and 1,241 non-coding RNAs was conducted based on the RNA-Seq datasets from eight different stages of B. mucronatus (S11 and S12 Tables).
The predicted number of genes was lower in B. xylophilus (N = 16,072) than in B. mucronatus (N = 17,248). However, gene density was similar between the two genomes (210 per Mb in B. xylophilus vs. 214 per Mb in B. mucronatus). A total of 11,118 homologous gene gene pairs were identified based on reciprocal best BLAST hits. The average intron size in B. xylophilus was longer than that in B. mucronatus (254 bp vs. 236 bp), whereas the average exon size was comparable (239 bp vs. 235 bp). In addition, B. xylophilus has more tandem repeats (3.77 Mb vs. 1.71 Mb) and long terminal repeats (1.58 Mb vs. 0.079 Mb) than B. mucronatus (S4 and S10 Tables).
Gene family evolution
Gene family expansion and contraction refer to increases or decreases in gene copy numbers within a family due to duplication or loss events. To investigate the dynamic patterns of gene family gain and loss across nematode lineages during evolution, we constructed a phylogenomic tree based on 674 single-copy orthologs from nine nematode species, with Drosophila melanogaster as the outgroup (Fig 1 and S13 Table). This time-calibrated tree was then used as input for computational analysis of gene family evolution (CAFE) to infer lineage-specific rates of gene family gain and loss, treating OrthoFinder orthogroups as gene families.
(A) Analysis of gene gain or loss during nematode evolution. Numbers indicate the number of orthogroups under expansion (+ and red) or contraction (- and blue) and rapidly evolving orthogroups (* and green). The proteome of D. melanogaster (UniProt ID: UP000000803) was used as an outgroup. (B) Venn diagram of the orthogroups of four nematodes (B. okinawaensis, B. mucronatus, C. elegans, and C. briggsae).
At the nematode superfamily level, 12 gene families from Sphaerularioidea and Tylenchoidea exhibited significant rapid expansion, primarily involving epidermal growth factor-like domains and concanavalin A-like lectins (S14 Table). In contrast, 19 rapidly expanding gene families were identified in Aphelenchoidea (S15 Table), enriched in metabolic enzymes (e.g., cytochrome P450s and carboxylesterases), signal transduction components (tyrosine phosphatases, adenylyl/guanylyl cyclases, and protein kinases), and membrane transporters (major facilitator superfamily proteins and proton-dependent oligopeptide transporters). Collectively, these results reveal a clear divergence in the functional categories of rapidly expanding gene families among major nematode superfamilies.
Within the Aphelenchoidea superfamily, most observed gene family expansions and contractions were due to B. xylophilus and B. mucronatus (Fig 1 and S15–S23 Tables). A total of 152 gene families showed expansion across the two Bursaphelenchus species based on evolutionary dynamics analysis using CAFE. Among these, 61 were classified as rapidly expanding (family-wise P < 0.05 in CAFE) (S18 Table). To explore the biological functions of these gene families, GO enrichment analysis was performed on the 61 rapidly expanding families combined with species-specific orthogroups (S16–S22 Tables). GO analysis revealed that rapidly expanding families in B. xylophilus and B. mucronatus were predominantly involved in metabolism (e.g., serine carboxypeptidases and lipases), detoxification (cytochrome P450s), membrane transport (phosphate transporters, major facilitator superfamily members, and P-type ATPases), and signal transduction (7TM G-protein-coupled receptors and serpentine receptor class H proteins) (S16 Table). Specifically, rapidly expanding families in B. mucronatus were enriched in homeodomain/homeobox transcription factors, glutathione S-transferases, and epidermal growth factor-like proteins (S20 Table). Notably, 14 gene families in B. xylophilus were identified as rapidly expanding, with the BolA-like superfamily, PLCPs, and DGATs showing the greatest degree of expansion (S18 Table).
Differential expression of the BolA-like superfamily
The BolA-like superfamily, present in prokaryotes and eukaryotes, plays essential roles in regulating cell morphology, stress responses, biofilm formation, and developmental processes [17]. Therefore, the number and evolutionary relationships (S6 Fig) of BolA-like proteins across genomes from algae to mammals were analyzed. The B. xylophilus genome encodes 24 BolA-like superfamily members, whereas other 95 species possess at most five, with an average of three. (Fig 2A). This result aligns with the rapid expansion of BolA-like orthologs in B. xylophilus (S18 Table). Phylogenetic analysis results categorized the BolA-like superfamily into BolA1, BolA2, BolA3, and BolA4 families. BolA1 is widely distributed, with at least one member in both prokaryotes and eukaryotes, and the BolA1 family of nematode BolA1 clade shows closest affinity to bacterial BolA1. BolA2 family is present in all eukaryotes, including algae, fungi, insects, nematodes, and land plants, but is restricted to Rhabditida among nematodes. BolA3 family is exclusive to non-photosynthetic eukaryotes; In nematodes, one copies present in all species, except cyst nematodes. BolA4 occurs in Archaea, bacteria, cyanobacteria, and photosynthetic plants but is absent in nematodes. All BolA-like proteins from eight nematode species were included in phylogenetic analysis, revealing nematode-specific subfamilies: BolA1 L1, BolA1 L2, BolA2, and BolA3. Notably, all members of the BolA1 L1 subfamily are unique to B. xylophilus, indicating a lineage specific expansion of this gene family in B. xylophilus (S7 Fig).
Bars show means ± standard deviation (SD). n denotes the number of species in each group (n = 1 for B. xylophilus; n = 95 for the other species representing algae, bacteria, cyanobacteria, fungi, land plants, insects, mammals and nematodes shown in S6 Fig). Summary statistics are provided in the Sheet C in S1 Data. (B) Simplified phylogenetic maximum likelihood (ML) tree of BolA-like superfamily proteins from algae, bacteria, cyanobacteria, fungi, land plants, insects, mammals, and eight nematodes (B. xylophilus, B. mucronatus, B. okinawaensis, Ditylenchus destructor, Globodera pallida, Meloidogyne hapla, C. briggsae, and C. elegans). Genes from B. xylophilus are marked in dark red, while clades from other species are collapsed. (C) BolA-like superfamily expression in different stages of B. xylophilus and B. mucronatus development. Fragments Per Kilobase of transcript per Million mapped reads (FPKM) values of genes in different stages are scaled by rows to emphasize the stage with the highest expression. Source data are provided in the Sheet D in S1 Data. (D) Total expression levels of B. xylophilus and B. mucronatus BolA-like superfamily at different stages. It represents the cumulative transcriptional abundance of the entire gene family. (E) SCBN-normalized expression of the orthologous pair Bxy004826 and Bmu006654 at the J2 (P = 3.88e-56) and J3 (P = 7.96e-22) stages. P-values were calculated using the SCBN method. (F) Expression levels of a pair of genes, Bxy002668 and Bxy015400, at the J2 and J3 stages. (G) Survival rate of B. xylophilus under different treatment conditions at 40°C. (H) Survival rate of B. mucronatus under different treatment conditions at 40°C. (I) Survival rate of B. xylophilus under different treatment conditions at -10°C. (J) Survival rate of B. mucronatus under different treatment conditions at -10°C. (K) Survival rate of B. xylophilus under different treatment conditions at 15mM H2O2 (L) Survival rate of B. mucronatus under different treatment conditions at 15mM H2O2. (M) Phenotypic characteristics of B. xylophilus and B. mucronatus in different treatments groups under 3% NaCl stress (N, O) Abnormal percentage of B. xylophilus and B. mucronatus under 3% NaCl solution. Different letters above the bars indicate statistically significant groups at P < 0.05 (one-way analysis of variance followed by Fisher’s least significant difference multiple comparison test). Exact P values are provided in in the Sheet K in S1 Data. All experiments were performed three times with similar results. ***P < 0.001 as determined by dsRNA. (P) Percentage statistics of B. xylophilus developmental progress in different treatments. (Q) Percentage statistics of B. mucronatus developmental progress in different treatments.
Based on the reconstructed phylogenetic tree, the BolA-like superfamily in B. xylophilus and B. mucronatus primarily clusters within the BolA1 and BolA3 subfamilies (Figs 2B and S6), with higher expression observed at the J2 and J3 stages, respectively. (Figs 2C, 3D, and S8). Notably, the total expression of the BolA-like superfamily in B. xylophilus was higher than that in B. mucronatus across all developmental stages (Figs 2D and S9). Bxy004826 and Bmu006654 are the most highly expressed genes in the BolA-like superfamily that are homologous genes in B. xylophilus and B. mucronatus, respectively. Both genes were expressed at significantly higher levels at J2 and J3 than at other stages. Using SCBN-normalized cross-species RNA-seq data analysis, Bxy004826 showed significantly higher expression than its ortholog Bmu006654 at both J2 and J3 stages (Fig 2E; P < 0.001, SCBN). Bxy002668 and Bxy015400 are the two most highly expressed genes in the BolA1 L1 family in B. xylophilus (Figs 2F and S9). The protein structures of BXY004826, BMU006654, BXY002668, and BXY015400 were predicted. The overall topology of BXY004826 and BMU006654 adopt an αβββα topology, where β1 and β2 are antiparallel, and β3 is parallel to β2 (S8A, S8B, and S8F Fig). This fold resembles the class II KH domain but lacks conserved GXXG loop. In contrast, BXY002668 and BXY015400, which are specifically expanded in B. xylophilus, exhibit an αββα topology, with antiparallel β1 and β2 strands. Compared with BXY004826, both BXY002668 and BXY015400 lack the β3. (S8C, S8D, and S8E Fig).
(A) Number of DGAT genes in Bursaphelenchus SP and in other species. Bars show means ± SD. n denotes the number of species in each group (n = 3 for Bursaphelenchus species; n = 22 for the other species shown in S11 Fig). Species names are labelled in S11 Fig., and summary statistics are provided in in the Sheet M in S1 Data. (B) Simplified phylogenetic ML tree of DGAT proteins across taxa, with B. xylophilus genes marked in dark red, while clades from other species are collapsed. (C) DGAT expression of B. xylophilus and B. mucronatus at different developmental stages, FPKM values are scaled by rows to highlight highest expression. (D) Total DGAT expression levels at different stages of B. xylophilus and B. mucronatus. It represents the cumulative transcriptional abundance of the entire gene family. (E) Comparison of DGAT expression in the J3 and J4 stages of propagative and dispersal forms of B. xylophilus, with significant differentially expressed genes (DEGs; log2 fold change>1) colored red.(F) Expression profiles of DGAT genes in J2, virgin female, and mated female stages, highlighting the dispersal phenotype of B. xylophilus and homologous genes in B. mucronatus; Significant genes are colored red. (G) SCBN-normalized expression of the orthologous pair Bxy007358 and Bmu009603 at the J2 (P = 9.49e-121), V-F (P = 2.34e-90), and M-F (P = 2.67e-35) stages. P-values were calculated using the SCBN method. (H) SCBN-normalized expression of the orthologous pair Bxy011316 and Bmu010594 at the J2 (P = 2.64e-26), V-F (P = 2.00e-30), and M-F (P = 7.91e-13) stages. P-values were calculated using the SCBN method. (I) Predicted structure of BXY007358 (red) and BMU009603 (blue), with TM-score values reflecting structural similarity. (J) Comparison of the expression of Bxy011316 in the J3 and J4 stages of propagative and dispersal forms of B. xylophilus. ***P < 0.001, as determined by two-tailed Student’s t t-test. (K) Expression levels of Bxy011316 and Bmu010594, in J2, virgin female and mated female stages. ns, not significant, ***P < 0.001, as determined by two-tailed Student’s t test. (L) Predicted structure of BXY011316 (red) and BMU010594 (blue).
Based on the phylogenetic analysis of the BolA-like superfamily (Figs 2B and S7), The most highly expressed homologous pair in B. xylophilus and B. mucronatus, Bxy004826 and Bmu006654, are assigned to the BolA2 clade. From the BolA1 L1 clade, which is uniquely expanded in B. xylophilus, the two most highly expressed genes, Bxy015400 and Bxy002668, were selected. The BolA3 clade comprises two members in both species: Bmu008858 in B. mucronatus and Bxy014563 in B. xylophilus. To validate the function of BolA genes, RNAi was performed using six representative BolA-like genes (Bxy004826, Bxy002668, Bxy015400, Bxy014563, Bmu008858 and Bmu006654). Both Bmu008858 and Bxy014563were cloned and subjected to RNAi. However, no stable or reproducible phenotypic changes were observed, likely due to their low transcriptional levels across developmental stages and poor interference efficiency (Sheet D in S1 Data). The qRT‐PCR confirmed that other BolA‐targeting dsRNA achieved robust, specific silencing of its respective transcript with minimal impact on non‐target BolA paralogs (S10 Fig). Survival rates in RNAi-treated groups were significantly lower than controls under heat stress at 40°C. Control survival rates were 82.67% for B. xylophilus and 83.81% for B. mucronatus, whereas RNAi-treated groups dropped to 30.03% (Bxy004826), 30.01% (Bxy002668), and 22.67% (Bmu006654), underscoring the crucial role of BolA-like genes in thermal stress resistance. (Fig 2G and 2H).
A similar trend was observed under cold stress at -10°C. Control groups maintained high survival rates (B. xylophilus: 63.50%, B. mucronatus: 59.86%), whereas RNAi-treated groups showed significantlyreduced survival: 4.67% (Bxy004826), 8.84% (Bxy002668), and 10.71% (Bmu006654) (Fig 2I and 2J). This confirms the essential role of BolA-like genes in low-temperature resistance. Oxygen stress yielded consistent results: control survival rates were 89.72% (B. xylophilus) and 89.94% (B. mucronatus), whereas RNAi-treated groups showed markedly reduced survival rate, withBxy004826 RNAi-treated group exhibited the lowest survival rate of 21.75%, whereas other BolA RNAi-treated groups also showed decreased survival (Fig 2K and 2L).
Under saline stress (3% NaCl), RNAi-treated nematodes displayed higher abnormality rates, characterized by dehydration and shrinkage. The Bxy004826 RNAi-treated group showed the highest abnormality rate (38%), while other RNAi-treated groups ranged from 11%-24% (Fig 2M, 2N, and 2O).
In addition, knockdown of the BolA-like gene affects the development of nematodes, preventing some larvae from developing into adults. In the Bxy004826 RNAi-treated group, 3.16% of nematodes remained at J2, 8.99% at J3, and 19.73% at J4, resulting in a total of 31.90% failure to reach adulthood (Fig 2P). In the Bmu006654 RNAi-treated group, 0.42% remained at J2, 14.10% at J3, and 22.83% at J4, for a total of 37.36% failing to develop into adults (Fig 2Q). These findings indicate that BolA-like is essential for both stress resistance and normal development in B. xylophilus and B. mucronatus, with a particularly pronounced developmental arrest in B. mucronatus.
DGAT is involved in lipid droplet synthesis
DGAT enzymes DGAT1 and DGAT2 catalyze the final step of triglyceride biosynthesis by converting diacylglycerol and acyl-CoA into triglycerides, which are crucial for energy storage and lipid metabolism in animals, plants, and microorganisms [13,18]. Phylogenetic analysis showed that the DGAT gene family is divided into DGAT1 and DGAT2, with DGAT2 present in algae, fungi, plants, nematodes, fish, amphibians, and mammals. The DGAT gene family of the Bursaphelenchus genus is classified into the DGAT2 group, possessing significantly more DGAT genes (9) than other species (3) (P < 0.001; Fig 3A). The DGAT1 clade had only one cluster (DGAT1), but the DGAT2 clade consisted of several clusters, including DGAT1, DGAT2 L1, DGAT2 L2, DGAT2 L3, DGAT2 L4, DGAT2 L5, DGAT2 L6, and DGAT2 L7 (S11 Fig). In addition, DGAT1 from animals clustered separately from DGAT1 from plants.
The reconstructed phylogenetic tree showed that all DGAT members in B. xylophilus and B. mucronatus belong to the DGAT2 group (Figs 3B and S9), with peak expression at the J2 and female stage. (Figs 3C, 3D, 3E, and S12). Comparative transcriptome analysis revealed that DGAT genes (Bxy00019, Bxy007358, Bxy012589, Bxy011316, and Bxy002397) were significantly upregulated during the dispersal stage compared with the reproductive stage (Log 2 Fold Change > 1) (Fig 3F, 3J, and 3K). SCBN-normalized cross-species analysis results showed that Bxy007358 was expressed at significantly higher levels than its ortholog Bmu009603 at J2, virgin female and mated female stages (Fig 3G; P < 0.001, SCBN). Similarly, Bxy011316 showed significantly higher normalized expression than Bmu010594 in females (Fig 3H; P < 0.001, SCBN). Structural modeling indicated high similarity between BXY007358/BMU009603 and BXY011316/BMU010594, with TM score of 0.94 and 0.73, respectively) (Fig 3I and 3L).
To verify the role of DGAT in lipid droplet synthesis in B. xylophilus and B. mucronatus, RNAi was performed on several DGAT genes. Sequence alignment result revealed that the extremely low DNA sequence similarity among four DGAT genes (Bxy007358, Bxy0011316, Bmu009603, and Bmu010594) in their homologous regions of the homologous genes (S13A, S13C, S13E, and S13G Fig). High similarity signals confined to the diagonal, indicating a negligible risk of off-target effects. The qRT-PCR confirmed significant downregulation of all four genes in respective RNAi groups, with average inhibition efficiencies of 55% to 70% (S13B, S13D, S13F, and S13H Fig).
At the J2 stage, lipid droplet in Bxy011316 and Bmu010594 dsRNA-treated groups did not differ significantly from controls. However, lipid droplet areas in the Bxy007358 and Bmu009603 dsRNA-treated groups were significantly reduced (Fig 4A and 4C). In both male and female stages, lipid droplet areas were significantly smaller in all four DGAT dsRNA-treated groups compared with controls (Fig 4A and 4C). Knockdown of Bxy011316 and Bxy007358, the lipid droplet diameters in both female and male B. xylophilus were significantly smaller than those in the control group (Fig 4B and 4D). Notably, lipid droplets in males treated with Bxy011316 dsRNA-treated group nearly disappeared under starvation and cold conditions. Under normal conditions, lipid droplet diameter decreased significantly in both sexes after Bmu010594 knockdown, whereas no significant changes were observed in other treatment groups. The diameter in the Bmu009603 dsRNA group significantly decreased only under starvation and cold stress, with no effect under normal conditions (Fig 4B and 4D).
(A) Whole-body lipid droplet staining of two nematodes from different treatment groups. (B) Localized lipid droplet staining of two nematodes from different treatment groups. (C) Percentage of lipid droplet area of two nematodes from different treatment groups. (D) Statistics on diameter of lipid droplets of two nematodes from different treatment groups. Boxplot displays the median and interquartile range. Upper and lower whiskers extend to data no more than 1.5 × the interquartile range from the upper and lower edges of the box, respectively. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 as determined by two-tailed Student’s t-test. 1S&C, starvation and cold treatment.
In the control group, oocytes of females were large numerous, and neatly arranged, filling the proximal gonadal arm (S14A Fig). In contrast, Bxy011316 dsRNA-treated groups, the oocytes were fewer in number, smaller in volume, irregular in shape, and retarded in development (S14B Fig). Quantitative analysis revealed a significant reduction in mature oocyte (control: 7.5 ± 1.2; Bxy011316 dsRNA: 4.5 ± 1.1) (S14 Fig C). Under starvation, survival was significantly reduced in Bxy011316-silenced females compared to controls (median survival: 20 d vs. 30 d) (S14D Fig).
Compared with controls, mating rate (number of mated groups/ total number of experimental groups) and effective mating rate (number of egg-laying groups/number of mating groups) in Group 2 (CK♀ + RNAi♂) and Group 3 (RNAi♀ + CK♂) showed no significant differences (mating rate: 96.67 ± 4.71% vs. 86.67 ± 4.71% and 80.00 ± 8.17%; effective mating rate: 90.00 ± 8.16% vs. 80.00 ± 8.16% and 76.67 ± 4.71%). Male silencing did not significantly reduce mating frequency or duration (0.90 ± 0.08 times; 13.34 ± 1.01 min) relative to controls (1.07 ± 0.05 times; 14.60 ± 1.13 min). However, female silencing significantly decreased both metrics (0.80 ± 0.08 times; 11.58 ± 0.29 min). Additionally, the average egg production in Group 2 (CK♀ + RNAi♂) and Group 3 (CK♂ + RNAi♀) was 11.63 ± 1.21 and 11.97 ± 0.73, respectively, both significantly lower than the controls (16.83 ± 0.45) (S24 Table).
PLCP-driven virulence of Bursaphelenchus sp. nematode
PLCPs are a large and highly diverse superfamily of proteins involved in numerous essential biological functions, including protein turnover, deubiquitination, tissue remodeling, blood clotting, virulence, defense, and cell wall remodeling [19]. With 37 members, the PLCP gene family was significantly expanded in B. xylophilus compared with nine reference nematode species (3–15 members) (Fig 5A). PLCPs in the eight nematode species clustered into three clusters (PLCP L1, PLCP L2, and PLCP L3) (Fig 5B). PLCP L1 underwent specific expansion in B. xylophilus and B. mucronatusby multiple gene duplication. PLCP L2 was restricted to three Aphelenchoidea species (B xylophilus, B. mucronatus, and B. okinawaensis). PLCP L3 was present in nine nematode species (Figs 5B and S15). Notably, PLCP L1 was exclusive to B. xylophilus and B. mucronatus, and both nematodes had the highest expression in PLCP L3 (S15 Fig). Therefore, the focus was on analyzing PLCP L1 and PLCP L3 in both two nematodes.
(A) Number of PLCP genes in Bursaphelenchus species and other species. Bars show means ± SD. n denotes the number of species in each group (n = 3 for Bursaphelenchus species; n = 7 for the other species shown in S15 Fig). Species names are indicated in S15 Fig, and summary statistics are provided in the Sheet X in S1 Data. (B) Simplified phylogenetic ML tree of PLCP proteins from eight nematodes (B. xylophilus, B. mucronatus, B. okinawaensis, D. destructor, G. pallida, M. hapla, C. briggsae, and C. elegans). Genes from B. xylophilus are marked in dark red, while clades from other species are collapsed. (C) PLCP expression of B. xylophilus and B. mucronatus at different developmental stages. FPKM values of genes at different stages are scaled by rows to emphasize the stage with the highest expression. Source data are provided in the Sheet Y in S1 Data. (D) Total expression levels of B. xylophilus and B. mucronatus PLCP at different stages. It represents the cumulative transcriptional abundance of the entire gene family. (E) Comparison of the expression levels of PLCP genes between PWN infestation and fungus-eating stages. Significantl DEGs (log2 fold change >0 at 6, 12, and 24 h) are colored red.(F) Expression levels of genes significantly upregulated during the invasion of PWNs into pine trees. The top three highest expression levels are highlighted in red.(G) SCBN-normalized expression of the orthologous pair Bxy004826 and Bmu006654 at the J2 (P = 1.01e-201) and J3 (P = 2.38e-302) stages. P-values were calculated using the SCBN method. (H–K) Structural diversity and electrostatic properties of BXY007574 (H), BMU008640 (I), BXY014241 (J), and BXY013180 (K). The core secondary structural elements are color-coded, while other regions are shown in grayscale. The electrostatic potential surfaces of the active sites and corresponding pockets are depicted for each PLCP protein. (L–O) Cell death triggered by PLCP proteins. Green fluorescent protein-tagged PLCPs (BXY007574, BMU008640, BXY014241, and BXY013180) were transiently expressed in Nicotiana benthamiana. Infiltrated leaves were photographed 3 days post-agroinfiltration (dai) (n = 6).
Transcriptional profiling revealed that total PLCP expression in B. xylophilus and B. mucronatus was highest at mated females and J3 stage, respectively (Fig 5C and 5D). Specifically, PLCP L1 was highly expressed at the J2 and male stages of B. xylophilus, while B. mucronatus showed high expression in the male stage (S16A Fig). The expression pattern of PLCP L2 in both species mirrored that of total PLCP expression (S16B Fig). The expression of PLCP L3 in both nematodes was significantly higher in the J3 stage than at the other stages (S16C Fig). Transcriptome analysis across feeding stages showed that 31% of PLCP genes (50% of PLCP L1, 20% of PLCP L2, 30% of PLCP L3) were significantly upregulated during pine infection by B. xylophilus (Fig 5E). In particular, the expression of Bxy013180 in PLCP L1, as well as Bxy007574 and Bxy014241 in PLCP L3, was remarkably high when pine nematodes were infesting pine trees (Fig 5F). Bxy007574 and Bmu008640 are orthologs and represent the most highly expressed PLCP L3 members in B. xylophilus and B. mucronatus, respectively (Fig 5F). In cross-species SCBN analysis, Bmu008640 showed higher normalized abundance at J2, whereas Bxy007574 was more abundant at J3 (P < 0.001, SCBN; Fig 5G). Transcriptome profiling confirmed significant upregulation of PLCP genes during pine infection compared with fungal feeding stage (Fig 5E). Among these, Bxy007574, Bxy014241, and Bxy013180 showed the highest expression levels during pine infection and were selected for further functional characterization (Fig 5F). Both Bxy014241 and Bxy013180 are rapidly evolving genes in B. xylophilus with detectable no homologs in B. mucronatus (S15 and S17 Figs).
Four PLCP proteins, BXY014241, BXY007574, BXY013180, and BMU008640, shared a conserved structural core typical of papain-like cysteine proteases, consisting of an α-helix followed by an antiparallel β-sheet of five β-strands (Fig 5H–5K). The catalytic site lies between the helix (contributing provides the catalytic cysteine at its N-terminus) and the β-strands (providing histidine and polar residues). These strands are slightly bent inward, forming a convenient pocket for aligning the incoming catalytic substrates (Fig 5H–5K). The typical catalytic sites of BXY007574 and BMU008640 were formed by a cysteine-histidine-threonine triad, and feature a negatively charged active site pocket (Figs 5H, 5I, and S18). The catalytic sites of BXY014241 and BXY013180 consist of a cysteine-histidine-serine triad and a cysteine-histidine-asparagine triad, respectively, with positively charged active site pockets (Figs 5J, 5K, and S18).
We injected leaves of Nicotiana benthamiana with Agrobacterium strains carrying Bxy013180, Bxy007574, Bxy014241, and Bmu008640 without the signal peptide for PLCP genes. After 3 days of infiltration indicated that Bxy007574 and Bxy014241 induced cell death in N. benthamiana, Bxy013180 and Bmu008640 did not (Fig 5L-5O). The result of in situ hybridization suggested that Bxy007574 is expressed in the dorsal esophageal gland in females (S19A Fig) and the subventral glands in males (S19B Fig). Bxy014241 is only expressed in the intestine of nematodes (S19C and S19D Fig). These findings suggested that Bxy0075741 and Bxy014241 are secreted into host plant tissues and play essential roles during the parasitic phase of B. xylophilus.
To further evaluate the contribution of PLCP genes to the virulence of B. xylophilus and B. mucronatus, Bxy007574, Bxy014241, Bxy013180, and Bmu008640 were silenced by RNAi. qRT‐PCR analysis confirmed efficient and specific knockdown of each target genes (S20 Fig). At 44 dpi, 60% of Pinus thunbergii seedlings inoculated with wild-type B. xylophilus exhibited severe needle wilting. In contrast, disease incidence in the Bxy0075741, Bxy014241, and Bxy013180 dsRNA-treated groups was reduced to 40%, 30%, and 0%, respectively. For B. mucronatus, the disease incidence in the control group was 20%, but it dropped to 0% in the Bmu008640 dsRNA-treated group (Figs 6 and S21). By 67 dpi, all needles in the wild-type B. xylophilus had wilted, and 80% of seedlings had died. In comparison, mortality rates in the Bxy007574, Bxy014241, and Bxy013180 dsRNA-treated groups were 40%, 40%, and 50%, respectively. In the control group of B. mucronatus-inoculated, the mortality rate was 30%, and no reduction was observed in the Bmu008640 dsRNA-treated group. These results demonstrated that PLCP genes are critical for the pathogenicity of B. xylophilus (Figs 6 and S21).
Treatments were monitored throughout the pre-disease (0 d), mid-disease (44 d), and late-disease (67 d) phases, and data were systematically recorded at each stage.
Discussion
High-quality and high-coverage (> 120 ×) genomes of B. xylophilus and B. mucronatus were assembled using a PacBio Sequel sequencer in this study. The first reference genome of B. xylophilus, generated using the 454 FLX platform, was released in Japan in 2011 [20]. However, the previous reported assembly (74.5 Mb) was smaller than the 76.32 Mb genome obtained in this study. The first Chinese B. xylophilus genome was sequenced using second-generation methods and published in 2020 [13]. The genome of B. xylophilus in this study was completed using a three-generation sequencing method. Compared to second-generation sequencing, three -generation sequencing technology has obvious advantages in terms of read length, data splicing, and accuracy [19]. Our B. xylophilus genome assembly exhibits numerous structural differences (e.g., breakpoints, translocations, inversions, SNPs) relative to the BXYJv5 reference, which may be attributed to genomic structural variation among distinct geographic lineages. The genome size of B. mucronatus spans 80.39 Mb in this study, which was larger than that reported by Sun et al. (73 Mb) [21,22]. A larger genome size may reflect better handling of repetitive sequences by the assembly algorithm. Comparative genomic analyses will help elucidate genetic basis of the competitive displacement between B. xylophilus and B. mucronatus, providing a foundation for uncovering molecular mechanisms underlying environmental adaptation in PWN.
Gene family expansion and contraction often reflect molecular mechanisms associated with environmental adaptation and evolutionary processes [23]. Phylogenetic analysis of single-copy orthologous genesacross nematode genera revealed a more rapid expansion of gene families in B. mucronatus compared with B. xylophilus (Fig 1A). Both species are interspecific competitors. The rapid reproduction and migration of the PWN, along with its strong pathogenicity, are key factors driving PWD spread [24,25, 26] B. mucronatus is a weakly pathogenic nematode that affects pine species in South Korea and Japan [9,27,28]. In China, B. xylophilus exhibits far greater pathogenicity and population size than B. mucronatus. To better understand functional implications of gene family expansions, we analyzed the differences between the two species. Both nematodes showed rapid expansion in detoxification-related gene families, such as cytochrome P450 (S16 Table). Rapid expansion of the genes for cytochrome P450, flavin monooxygenase FMO and glutathione-S-transferase GST in B. mucronatus could boost the size of the detoxification enzyme system to efficiently metabolise terpene toxins produced by pine trees [29]. This expansion strategy enables the nematode to tolerate persistent host chemical defenses, establishing chronic parasitic homeostasis within resin ducts that manifested, as low virulence and long-term coexistence. This evolutionary strategy may be the reason why B. mucronatus has adapted to its environment and could parasitic to pine trees. In contrast, the expanded gene families of B. xylophilus are functionally specialized toward pathogenicity and stress resistance. Rapid expansion of DGAT and BolA-like gene families enhances population stability under adverse conditions. PLCP genes facilitate cell wall degradation in resin tract, disrupting host barriers [30].The functional integration of these three gene classes enables rapid disease epidemics. Differences in gene expansion patterns across species may represent a crucial molecular basis driving the diversification of their survival strategies and environmental adaptability. However, the specific molecular mechanisms underlying this hypothesis require further validation and elucidation through subsequent research.
B. mucronatus and B. xylophilus both show strong adaptability, which is manifested as a short life cycle, high fecundity, and accelerated developmental and reproductive rates across various temperatures [31]. However, the underlying molecular mechanisms remain unclear. In the present study, the BolA1 gene family was screened as an important gene family at the genomic level to determine the advantages of B. mucronatus and B. xylophilus in terms of environmental adaptation. The BolA-like gene family is primarily involved in cell formation, growth, and division [32–34]. The J2 and J3 stages of B. mucronatus and B. xylophilus are periods of intense growth and development, coinciding with peak cell division and growth peaking. It was found that the expression of Bxy004826, Bxy015400, Bxy02668 and Bmu006654 exhibited significantly high expression levels. The RNAi experiments demonstrated that silencing each of these four genes impaired nematode development. Simultaneous disruption of all four genes markedly slowed individual development in B. xylophilus (Fig 2P and 2Q), that Bxy004826, Bxy015400, Bxy02668, and Bmu006654 play critical roles in the growth and development of B. xylophilus and B. mucronatus.
The distribution of B. mucronatus and B. xylophilus spans five major climatic zones across China, covering tropical, subtropical, warm temperate, temperate, and cold temperate regions, suggesting an enhanced adaptation to severe climates. For successful invasion into colder regions, B. mucronatus and B. xylophilus must overcome low-temperatures stress, but the molecular mechanisms underlying its survival in these conditions are poorly understood. In our study, we knocked out the Bxy004826, Bxy015400, Bxy02668, and Bmu006654 and exposed the nematodes to 40°C, -10°C, and 15 mM H2O2. Mortality in the disrupted groups was significantly lower than in control. Stress-response genes play a major role in the adaptation to adverse environments. The BolA-like superfamily, a stress-responsive gene family in Escherichia coli, is under various stresses (e.g., starvation, heat shock, and oxidative stress) [35]. Phylogenetic analysis indicated that the nematode BolA-like superfamily is most closely related to bacterial homologs. Combined with previous findings, our results suggest that the BolA-like super family is crucial for stress resistance in B. mucronatus and B. xylophilus. In addition, nematodes with disrupted BolA-like genes developed a wrinkled skin when placed in 3% NaCl solution. Under stress, BolA1-like gene family expression levels increased, leading to thickening of the biofilm [1], which functions as a protective membrane in bacteria. Similarly, in B. mucronatus and B. xylophilus, the cuticle serves as the outermost protective layer. Thus, B. mucronatus and B. xylophilus may regulate epidermal thickness by BolA-like expression to withstand harsh external environments.
DGAT enzymes are widely conserved across animals, plants, and microorganisms [15]. DGATs are essential for intestinal absorption (DGAT1) and adipose tissue formation (DGAT2) [9]. The phylogeny of the DGAT gene families in mammals and nematodes suggests that nematode DGAT genes cluster with DGAT2, indicating their role in adipose tissue formation. Lipids serve as energy reserves [36], help organisms withstand external stress, maintain body temperature, and minimize heat loss [37] In B. mucronatus and B. xylophilus, the dauer stage enables survival under unfavorable conditions, such as low temperature and starvation, by lipid accumulation [38]. Phylogenetic analysis of DGAT genes across five nematode genera showed that Bursaphelenchus sp. have the highest number of DGAT genes, likely due to their dauer stage and adaptation to hostile environments. In contrast, root-knot nematodes enter hosts at the J2 stage and remain relatively immobile, while cyst nematodes develop energy-storing sporocarps that store energy and retain heat [39].
Under starvation conditions, the lipid content in B. xylophilus was significantly higher compared with its normal state and to B. mucronatus under similar conditions (Fig 4D). Transcriptome analysis further confirmed that DGAT gene expression was significantly elevated in dispersing nematodes compared with propagative nematodes, suggesting that B. xylophilus adapts to environmental challenges and maintains population size by enhanced lipid accumulation. DGAT2 expression was notably higher in the J2 stage and in females of both species. The J2 stage is characterized by active feeding and growth, supporting substantial lipid consumption. Elevated DGAT2 expression in females likely reflects increased energy demands associated with mating and egg production. RNAi experiments targeting three highly expressed gene pairs (Bxy007358/ Bmu009603, Bxy011316/ Bmu010594, and Bxy013071/ Bmu007080) confirmed their involvement in lipid synthesis in both species. DGAT2 is crucial for lipid biosynthesis in animals, as evidenced by the absence of triacylglycerol in DGAT2-knockout mice [40]. Notably, RNAi-mediated suppression of Bxy011316 led to reduced size and number of lipid droplets in female gonads. This lipid depletion resulted in fewer, smaller oocytes and a pronounced decrease in starvation survival (median survival: 20 d vs. 30 d in controls; S14 Fig), underscoring a direct link between lipid reserves and reproductive capacity as well as stress resistance. Similar phenotypes have been observed in C. elegans, where DGAT deficiency causes diminished lipid stores, defective oogenesis, and shortened lifespan [41]. Comparable effects occur in female silkworms, where loss of DGAT function inhibits mature egg formation [42]. These findings provide a theoretical foundation for the developing targeted inhibitors of lipid synthesis in B. xylophilus.
PLCPs are a large class of protein hydrolases associated with digestion, immune escape, and development in plant nematodes [43,44] The PLCP phylogeny of ten species (nine nematodes and Drosophila) revealed that the genes in B. xylophilus and B. mucronatus predominantly are belonged to Cluster 1 and Cluster 2, while Cluster 3 genes are present in all species. It is hypothesized that B. xylophilus and B. mucronatus expanded key genes in Cluster 1 and Cluster 2 to aid in immune evasion during host invasion. Analogous to active serine proteases in Blomia tropicalis, which induce human skin allergies by cleaving tight junctions, expanded PLCPs may contribute to immune escape [45].Comparative transcriptomic analyses revealed that 31% of the PLCPs were significantly upregulated during nematode invasion of pine compared with the fungus-feeding stage. Moreover, the total PLCPs gene expression was higher in B. xylophilus than in B. mucronatus. Although previous comparative proteomic studies suggested a crucial role for cysteine peptidases in the pathogenicity of B. xylophilus, this finding requires functional validation [10].
In situ hybridization revealed that Bxy013180 transcripts are localized specifically to intestinal cells (S19C and S19D Fig), whereas Bxy007574 is restricted to esophageal gland cells (S19A and S19B Fig). A similar intestinal localization pattern was observed in Meloidogyne incognita, where silencing the MiCpl1 gene (a cathepsin L-like protease) decreased protein hydrolysis activity to levels comparable to oryzacystatin, implicating its role in digestion [10]. The function of Bxy007574 and Bxy014241 in promoting nematode infestation and inducing necrotic responses in plant cells was verified using results of BAX assay and inoculation assays (Fig 5L). Notably, the third residue of the catalytic triad—typically a threonine in PLCPs—is conserved as Thr in BXY007574 but is substituted by Ser in BXY014241. Given the key role of this hydroxyl‐bearing side chain in stabilizing the oxyanion transition state and orienting the catalytic histidine, the Thr to Ser substitution in BXY014241 is likely to alter its catalytic efficiency and substrate specificity. This subtle change may account to differential abilities of these two PLCPs to trigger plant cell death upon delivery into host tissues, highlighting the critical importance of the third catalytic residue in PLCP pathogenicity. Similarly, in viral polyproteins and effector proteins, only the cysteine and histidine dyad is retained, and this simplification alters the enzymatic reaction rates or substrate preferences [19]. PLCPs have also been implicated in host tissue invasion and immune evasion by parasitic nematodes [11]. In the present study, PLCPs are reported for the first time as pathogenic genes in pine wood nematodes. The results of this study might provide a theoretical basis for understanding the pathogenic mechanism of PWD and reveal the complexity of nematode–host interactions.
This study presents the first comparative genomic analysis of B. xylophilus and B. mucronatus. The results of this study demonstrate that the BolA-like, DGAT, and PLCP gene families play critical roles in the environmental adaptation of both nematode species. The correlation between the expression levels of these gene families under pathogenic and stress conditions revealed a high degree of co-expression (S22 Fig). Specifically, BolA-like regulates developmental progression and cuticle thickness in nematode to resist harsh conditions and ensure population survival. DGAT mediates lipid synthesis, enabling energy storage for survival under adverse conditions. The expansion of the BolA-like and DGAT gene families contributes to the ongoing spread of nematode into cooler climates. Pathogenicity is a specialized form of environmental adaptation, representing an evolutionary strategy pathogens employ to survive, transmit, and overcome pressures within the host environment [46]. PLCP genes contribute to the pathogenesis of B. xylophilus, which possesses a larger PLCP gene repertoire and greater pathogenic potential than B. mucronatus. The results of this study demonstrated that PLCP genes in B. mucronatus do not contribute to its pathogenicity. A specific amino acid substitution in the catalytic site of PLCPs, unique to B. xylophilus, may enhance its pathogenic capability relative to B. mucronatus (Fig 7).
Materials and methods
Sampling and genome sequencing
B. xylophilus NXY61 and B. mucronatus ANL7 were isolated from diseased Pinus massoniana in Ningbo, Zhejiang Province and Wuhu, Anhui Province, China. Synchronized nematode of B. xylophilus and B. mucronatus at different developmental stages were obtained using previously described methods [22,47]. DNA extracted from B. xylophilus and B. mucronatus were used for PacBio sequencing by BGI Genomics Co. (Shenzhen, China). DNA samples were randomly fragmented using E220 Focused-ultra-sonicator (Covaris). Fragmented DNA underwent damage repair and end repair, followed by ligation of stem-loop sequencing adapters. Fragments with unsuccessful ligation were removed using an exonuclease. The final library was sequenced using the PacBio Sequel platform.
Genome survey and assembly
The Jellyfish software (v2.0) [48] was used to calculate k-mer frequency (k-mer length 17). Genomescope (v1.0.0) [49] was used to estimate genome heterozygosity, repeat content, and size from the sequence reads. Genome assembly of B. xylophilus was performed using MECAT [50] default parameters based on PacBio long reads. Because of the large number of heterozygous sites in B. mucronatus, assembly was done using Falcon [51], which better handles heterozygosity. After obtaining the corrected genome assembly, the final version was produced by removing contaminants using the method reported by Wheeler [52].
Assessment of the genome assembly
To assess structural accuracy and continuity, BxV1.0 was aligned with the previous BXYJv5 (NCBI, currently the most widely used B. xylophilus reference genome) assembly using MUMmer [53]. BUSCO (v5.0.0) [54] was used to evaluate genomic integrity using the parameter set for the nematoda_odb9 -m genome. The transcriptome sequencing data were aligned to the genome using Bowtie2 (v2.4.2) [55]. Raw Illumina data were filtered using fastp (v0.20.0) [56] to remove splice sequences, low-quality sequences, and sequences that were too short. Finally, SAMtools (v1.10) [57] were used to determine the alignment rate.
Repeat analysis and gene annotation
Repeat sequences were annotated using structural prediction and homology comparisons. Repetitive sequences of both nematode genomes were predicted ab initio using RepeatModeler2 (v2.0.1) [58]. Species-specific repetitive sequence libraries were identified by RepeatMasker (v4.1.0) [59] with default parameters and the Repbase 60 and Dfam [61] databases.
The structural annotations included homology-, ab initio-, and transcript-based predictions. Homology-based predictions were performed for six closely related species (Ascaris suum, Brugia malayi, Caenorhabditis briggsae, Caenorhabditis elegans, Clonorchis sinensis, and M. incognita). Augustus, Genescan, and SNAP were used for ab initio prediction. Full-length transcript sequences from the PacBio reads were used for gene structure prediction. These gene predictions were integrated using Glean and Maker software, combining homology-based, ab initio, and cDNA-based predictions. Final gene structure predictions were performed using the full-length cDNA sequences and GFF annotation files.
Functional annotation involved comparing predicted gene sequences against public databases, including InterProScan (v54.0), GO, KEGG, Swiss-Prot, TrEMBL, and non-redundant (nr). BLASTP (v2.9.0) [62] with an E-value cutoff of 1e-5 was used for sequence comparison. Conserved protein domains were identified with InterProScan, while GO terms and KEGG pathways were linked with an E-value filter of 1e-10.
Family evolutionary analysis and GO enrichment analysis
For an evolutionary analysis of gene families, we generated two proteomes from our annotated genomes, along with eight publicly available proteomes from Aphelenchoidea, Sphaerularioidea, Tylenchoidea, and Rhabditoidea nematodes were assigned into orthogroups (or gene families) according to amino-acid sequence similarities (S13 Table). The proteome of Drosophila melanogaster was also included for orthogroup (gene family) assignment based on amino acid sequence similarities.
Based on the 674 single-copy orthologs identified by OrthoFinder (v2.4.0) [63], a phylogenetic tree for the nine nematode species (S13 Table) was constructed using the maximum likelihood method with RAxML-NG (v1.1.0) [64]. Multiple sequence alignments for each single-copy ortholog were performed using MAFFT (v7.505) [65] with parameters set to maxiterate 1,000 gene pairs. Next, multiple sequence alignment results for each single-copy ortholog were filtered using trimAl (v1.4) [66] with the parameters set to -automated1. ModelTest-NG (v0.1.7) [67] was used to estimate the best amino acid substitution model, which was determined to be the GTR + I + G4 model.
Bayesian phylogenomic dating analysis was conducted on the selected genes using the MCMC tree, part of the PAML package (v4.10.0) [68]. Branch lengths were approximated using the likelihood method. The molecular clock model employed an independent rates model, and the site substitution model used the HKY85 substitution model with an alpha value of 0.5 for site-specific evolutionary rates. The analysis was run for one million iterations, including a burn-in of 400,000 iterations, a sampling frequency of 10, and 100,000 samples. The divergence time between C. elegans and D. melanogaster (mean time: 727 MYA) was obtained from the TIMETREE website (http://www.timetree.org/) [69]. CAFÉ software (v4.2.1) [70] was used to compute gene family evolution based on the phylogenetic tree and orthologous groups.
Estimating gene family dynamics
The clustered gene families (mcl output) were processed with CAFE, which, in addition to estimating counts of gene family members for each node, identifies gene families that are evolving rapidly [71]. In all CAFÉ analyses, gene families were classified as rapidly evolving if they attained a family-wide P-value of 0.05 or less. For these families, branch-specific “Viterbi” P-values were then automatically calculated to identify the branches driving the significant evolutionary rate changes. These “Viterbi” P-values were then used to list all rapidly changing gene families along a given branch (Viterbi P < 0.05).
GO enrichment analysis was performed using DAVID (david.ncifcrf.gov). Rapid-evolving gene families and species-specific orthogroups were collected, and the genes were mapped to the proteome of C. elegans to acquire UniProt accession identifiers for GO enrichment using BLASTP with an E-value cutoff of 1e-5.
Transcriptome analyses
We collected 48 samples from eight life stages (J2, J3, and J4 females, J4 males, virgin females, virgin males, mated females, and mated males) of B. xylophilus and B. mucronatus, with six replicates for each life stage. Sequencing was performed by BGI Co. (Shenzhen, China). RNA-seq libraries with an insert size of approximately 150 bp were constructed. The qualified library was pooled based on a pre-designed target data volume and sequenced on an Illumina (HiSeq 2500) platform. Protein-coding genes for all other species were determined using BLASTp, and RBH pairs were selected as orthologous genes. Clean reads were mapped to the respective genomes using Bowtie2 (v2.4.2) [55], and gene expression levels in each of the 48 samples were quantified with RSEM, which generated gene-level expected counts and FPKM values. The raw expected counts were then used as input to DESeq2 [71] for normalization and differential expression analysis; FPKM values are used only for within-species visualization to highlight the stage with maximal expression for each gene. Transcriptomic data for the dauer stage of B. xylophilus and data for various infection stages (6 h, 12 h, and 24 h) in pine trees were obtained from the sources PRJDB3458 and PRJNA397001, respectively.
Differential expression analysis of all cross samples
The scale-based normalization (SCBN) method by Zhou et al. was applied for the comparison of orthologous gene expression between B. xylophilus and B. mucronatus [72]. For each life stage of nematodes, we first constructed a matrix of one-to-one orthologs containing gene lengths and raw read counts in B. xylophilus (species 1) and B. mucronatus (species 2). A subset of conserved orthologs was used as the reference set (H) to estimate the SCBN scaling factor c by minimizing the deviation between the empirical and nominal type I error, as implemented in the Bioconductor package SCBN.
Gene cloning
The total RNA from all developmental stages of B. xylophilus was extracted, using the TRIZOL method. Reverse transcription was performed by PrimeScript RT reagent Kit and gDNA Eraser (TaKaRa) kit to obtain cDNA. The target genes included Bxy004826, Bxy002668, Bxy015400, Bmu006654, Bxy007358, Bxy011316, Bmu009603, Bmu010594, Bxy007574, Bxy014241, Bxy013180, and Bmu008640. All cloning primers are included in S25 Table. These genes were cloned into the pGEM-T Easy vector using the Easy-TOPO TA Cloning Kit (Invitrogen). Individual bacterial colonies were screened via PCR to confirm the presence of the insert, and selected clones were verified by sequencing. The primers are listed in S25 Table.
Construction of plant expression vectors
Homologous recombination (Novoprotein, #NR005-01B) was used to ligate the Bxy007574, Bxy014241, Bxy013180, and Bmu008640 genes into the pBINGFP expression vector to generate GFP-tagged constructs. Positive clones were confirmed via PCR, followed by sequence verification of recombinant plasmids. The primers are listed in S25 Table.
In situ hybridization
PCR products were amplified from cDNA of B. xylophilus and used as templates for preparation of antisense and sense DIG-labeled probes via unidirectional PCR. In situ hybridization was performed as previously described [73] using a DIG RNA labeling kit (Roche, Mannheim, Germany). Nematodes were observed under an inverted optical microscope (Carl Zeiss AG, Germany).
RNAi experiment
The dsRNA for the target genes Bxy004826, Bxy002668, Bxy015400, Bmu006654, Bxy007358, Bxy011316, Bmu009603, Bmu010594, Bxy007574, Bxy014241, Bxy013180, Bmu008640, and the negative control gfp was synthesized using the MEGA script T7 High Yield Transcription Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. To evaluate potential off-target effects, each dsRNA fragment was compared with all other members of its gene family. Percent identity values were extracted to build a similarity matrix, which was visualized as a heatmap using the pheatmap package in R. The quality of synthesized dsRNA was determined by a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific, USA) and gel electrophoresis. Nematodes were soaked in a solution containing target gene dsRNA (final concentration 1 μg/μL), gfp dsRNA (final concentration 1 μg/μL), and soaking buffer (0.05% gelatin, 5.5 mM KH2PO4, 2.1 mM NaCl, 4.7 mM NH4Cl, 3 mM spermidine). Incubation was performed at 25°C in a shaking incubator at 180 rpm for 24 h.
Quantitative Real-Time PCR
The specific primers were designed using Primer Premier 5.0 software (S25 Table). β-Actin (GenBank accession: EU100952) was used as an internal reference gene. The qPCR was performed on a qTOWER 2.2 system (Analytik Jena AG, Germany). The experiment was conducted with three biological and technical replicates for each sample. The 2-ΔΔCT method was used to calculate the relative expression of genes in different treatment.
Stress tolerance test
To investigate the role of BolA-like in nematode stress adaptation, J3-stage juveniles (approximately 200 nematodes per sample, with four replicates) of B. xylophilus and B. mucronatus were soaked for 24 h in a buffer containing dsRNA targeting Bxy004826, Bxy002668, Bxy015400, Bmu006654, and gfp as controls.
For the temperature stress experiments, nematodes were exposed to extreme temperatures reflective of natural conditions: 40°C for heat stress and –10°C for cold stress, both for 24 h. Afterward, samples were transferred to 25°C for a 12 h recovery period. Survival rates were determined based on the proportion of nematodes that exhibited movement in response to mechanical stimulation under an optical microscope. For the oxidative stress experiment, nematodes were incubated in a 15 mM H₂O₂ solution for 24 h, and survival was assessed similarly by observing movement after mechanical stimulation. To evaluate the role of BolA-like genes in cuticle composition, which is critical for maintaining osmotic balance, J2-stage B. xylophilus and B. mucronatus juveniles (approximately 200 per sample, four replicates) were soaked in dsRNA-containing buffer for 24 h. Nematodes were then transferred to a 3% NaCl solution for 3 h. The percentage of abnormal nematodes was recorded using an optical microscope.
Observation of the developmental progress of nematodes
To explore the role of BolA-like genes in nematode development, J2-stage of B. xylophilus and B. mucronatus (approximately 200 nematodes per sample, with four replicates) were soaked in a buffer containing dsRNA targeting Bxy004826, Bxy002668, Bxy015400, Bmu006654, and gfp for 24 h. Following dsRNA treatment, the J2-stage nematodes were transferred to plates containing Botrytis cinerea as a food source.
Developmental stages were assessed using an optical microscope after 72 h for B. xylophilus and 82 h for B. mucronatus, as these times correspond to the transition from J2-stage juveniles to adults in each species. The number of nematodes in each life stage was recorded to determine the effect of BolA -like silencing on development of nematodes.
Lipid droplet staining
To investigate the role of DGAT genes in lipid droplet synthesis, J2-stage juveniles, adult males, and adult females of B. xylophilus and B. mucronatus were incubated in a buffer containing dsRNA targeting Bxy007358, Bxy011316, Bmu009603, and Bmu010594 for 24 h. After soaking, the nematodes were stained with Oil Red O to visualize lipid droplets. Images were captured using an optical microscope. The proportion and diameter of the lipid droplets were quantified using ImageJ software, with images taken from 10 nematodes per group for analysis.
For the starvation and cold stress experiments, adult male and female B. xylophilus and B. mucronatus were soaked in dsRNA against Bxy007358, Bxy011316, Bmu009603, and Bmu010594 for 24 h. Nematodes were then transferred to ddH₂O and incubated at 25°C for 15 d. Subsequently, nematodes were stained with Oil Red O and imaged under an optical microscope. Lipid droplet diameters were quantified using ImageJ software, with images taken from 10 nematodes per group.
Observation of oocyte in B. xylophilus
Adult females of B. xylophilus were immersed in 1% paraformaldehyde for 1 h at 25°C to fix nematodes. The nematodes were transferred to a glass slide with a drop of ddH₂O and gently covered with a coverslip to avoid compression artifacts. The oocytes in the proximal gonadal arm were examined under a compound light microscope, and images were recorded with a digital camera. Three independent biological replicates were performed.
Starvation survival assay
For each treatment, 50 individuals (females or males) were placed per well in a 24-well plate containing ddH₂O and incubated at 25°C in the dark. The number of live nematodes was counted every 5 days under a stereomicroscope; nematodes were considered alive if they responded to gentle mechanical stimulation. Survival curves were generated from three independent biological replicates.
Assay of mating and reproductive behavior
Virgin adults were obtained by separating females and males at the J4 stage and culturing them individually to adulthood. Three pairing groups were set up: control (CK♀ + CK♂), female-RNAi cross (RNAi♀ + CK♂), and male-RNAi cross (CK♀ + RNAi♂). One female and one male were placed together in a small Petri dish containing a drop of ddH₂O. For each group, 10 pairs were tested per replicate, with three independent replicates. Mating behavior was monitored intermittently over 8 h under a stereomicroscope, and behaviors were recorded for analysis. Eggs laid per female were counted 24 h after pairing.
Agroinfiltration assays in N. benthamiana
The Agrobacterium-mediated transient expression was determined following established protocols [74]. The recombinant plasmids were transformed into Agrobacterium tumefaciens strain GV3101 via liquid nitrogen freeze-thaw transformation and plated on Luria–Bertani agar containing kanamycin. The plates were incubated at 30°C for 48–72 h. Single colonies were picked and verified for successful transformation. Verified Agrobacterium GV3101 cultures were inoculated into LB liquid medium containing kanamycin and incubated overnight at 30°C in a shaking incubator at 200 rpm. After overnight incubation, bacterial cultures were centrifuged at 5,000 rpm for 3 min to collect the cells. Bacterial cultures were resuspended in an agroinfiltration buffer (1 M MgCl₂, 0.5 M MES, 1 M acetosyringone, pH 5.7) and adjusted to an OD of 0.6, followed by three washes with the same buffer. Bacterial suspension carrying the PLCP genes (Bxy007574, Bxy014241, Bxy013180, and Bmu008640) was infiltrated into the abaxial side of N. benthamiana leaves using a needleless 1 mL syringe. Each experiment was conducted on six leaves from three independent plants and was repeated at least three times. Agrobacterium carrying XEG1 was used as a positive control, whereas Agrobacterium carrying GFP was used as a negative control. Photographs of the infiltrated leaves were taken 3 d post-infection.
Inoculation assays
For the pine tree infection assay, J2-stage juveniles B. xylophilus and B. mucronatus (approximately 2,000 nematodes per sample) were soaked in dsRNA targeting Bxy007574, Bxy014241, Bxy013180, and Bmu008640 for 24 h. Wild-type B. xylophilus and B. mucronatus served as controls. Nematodes were inoculated into 10 black pine (P. thunbergii) trees per treatment group.
Data analysis
The mean ± standard error (SE) of triplicate measurements for each group was calculated using Microsoft Excel software (Office Excel 2016, Microsoft Corp, Redmond, WA, USA). Statistical analysis was conducted using Prism 5.0 (IBM, Armonk, NY, USA). Data were analyzed using one-way ANOVA with Tukey’s multiple comparisons (three or more groups) or t-test (two groups), with P < 0.05 considered significant.
Supporting information
S1 Fig. Genome size estimation based on k-mer statistics for B. xylophilus.
https://doi.org/10.1371/journal.ppat.1014033.s001
(TIF)
S2 Fig. Divergence distribution diagram of four tandem repeat sequences in B. xylophilus sample.
https://doi.org/10.1371/journal.ppat.1014033.s002
(TIF)
S3 Fig. Whole-genome alignment of BxV1.0 and BXYJv5 assemblies using MUMmer.
https://doi.org/10.1371/journal.ppat.1014033.s003
(TIF)
S4 Fig. Genome size estimation based on k-mer statistics for B. mucronatus.
https://doi.org/10.1371/journal.ppat.1014033.s004
(TIF)
S5 Fig. Divergence distribution diagram of four tandem repeat sequences in B. mucronatus sample.
https://doi.org/10.1371/journal.ppat.1014033.s005
(TIF)
S6 Fig. Phylogenetic analysis of 317 BolA-like proteins across various organisms.
https://doi.org/10.1371/journal.ppat.1014033.s006
(TIF)
S7 Fig. Phylogenetic analysis of BolA-like superfamily across eight nematodes.
https://doi.org/10.1371/journal.ppat.1014033.s007
(TIF)
S8 Fig. Multiple sequence alignment of the BolA-like family (related to Fig 2).
(A–D) Structural core of BXY004826 (A), BMU006654 (B), BXY002668 (C), and BXY015400 (D). Secondary structural elements defining the core are rendered in colors, whereas remaining parts of the structure are bleached. (E) Multiple sequence alignment of the BolA1 L1family. (F) Multiple sequence alignment of the BolA1 L2 subfamily. α, α-helix. β, β-sheet.
https://doi.org/10.1371/journal.ppat.1014033.s008
(TIF)
S9 Fig. Heatmap analysis of stage-specific expression of BolA-like superfamily genes in B. xylophilus and B. mucronatus (related to Fig 2).
https://doi.org/10.1371/journal.ppat.1014033.s009
(TIF)
S10 Fig. Knockdown efficiency of four BolA-like superfamily genes.
Potential off-target effects of RNAi fragments targeting Bxy004826 (A), Bxy002688 (C), Bxy015400 (E), and Bmu006654 (G) were assessed via DNA sequence similarity heatmaps across the BolA-like gene families of B. xylophilus (A, C, E) and B. mucronatus (G), respectively. High similarity (purple) indicates potential off-target risk, while low similarity (yellow) suggests specificity of the RNAi fragment. (B, D, F, H) Quantification of target gene expression levels by qRT-PCR after RNAi treatment. Relative expression of Bxy004826 (B), Bxy002688 (D), Bxy015400 (F), and Bmu006654 (H). Error bars represent standard deviation (n = 3). Statistical significance was determined by two-tailed Student’s t test: *P < 0.05; **P < 0.01.
https://doi.org/10.1371/journal.ppat.1014033.s010
(TIF)
S11 Fig. Phylogenetic analysis of DGAT proteins from various organisms (related to Fig 4).
https://doi.org/10.1371/journal.ppat.1014033.s011
(TIF)
S12 Fig. Heatmap representation of gene expression levels of DGAT members in different life stages from B. xylophilus and B. mucronatus (related to Fig 4).
https://doi.org/10.1371/journal.ppat.1014033.s012
(TIF)
S13 Fig. Knockdown efficiency of four DGAT family genes.
DNA sequence similarity heatmaps showing the potential off-target effects of RNAi fragments designed for Bxy007358 (A), Bxy0011316 (C), Bmu009603 (E), and Bmu010594 (G) across the DGAT gene family in B. xylophilus (A, C) and B. mucronatus (E, G). High similarity (purple) indicates potential off-target risk, while low similarity (yellow) suggests specificity of the RNAi fragment. (B, D, F, H) Quantification of target gene expression levels by qRT-PCR after RNAi treatment. Relative expression of Bxy007358 (B), Bxy0011316 (D), Bmu009603 (F), and Bmu010594 (H) was significantly reduced in both J2 and J4 stages after dsRNA-treated, compared with gfp dsRNA and untreated controls. Error bars represent standard deviation (n = 3). Statistical significance was determined by two-tailed Student’s t test: *P < 0.05; **P < 0.01.
https://doi.org/10.1371/journal.ppat.1014033.s013
(TIF)
S14 Fig. Knockdown of Bxy011316 reduces oocyte number and accelerates female mortality under starvation at 25°C.
(A) Representative bright-field images of oocytes (white outlines) in the gonad of control females incubated at 25°C. (B) Representative bright-field images of oocytes in the gonad of Bxy011316 dsRNA-treated females at 25°C. (C) Quantification of oocyte number per female in control versus Bxy011316 dsRNA groups. Data are mean ± SEM (**** P < 0.0001, unpaired t-test). (D) Kaplan–Meier survival curves of control (blue circles) and Bxy011316 dsRNA (tan squares) females under starvation at 25°C; curves show probability of survival ± SEM.
https://doi.org/10.1371/journal.ppat.1014033.s014
(TIF)
S15 Fig. Phylogenetic analysis of PLCP proteins from eight nematodes (B. xylophilus, B. mucronatus, B. okinawaensis, D. destructor, G. pallida, M. hapla, C. briggsae, C.elegans), related to Fig 5.
https://doi.org/10.1371/journal.ppat.1014033.s015
(TIF)
S16 Fig. The overall expression patterns of PLCP subfamily genes in B. xylophilus and B. mucronatus (related to Fig 5).
(A) Expression pattern of PLCP L1. (B) Expression pattern of PLCP L2. (C) Expression pattern of PLCP L3.
https://doi.org/10.1371/journal.ppat.1014033.s016
(TIF)
S17 Fig. Heatmap of gene expression levels of PLCP members in different life stages from B. xylophilus and B. mucronatus (related to Fig 5).
https://doi.org/10.1371/journal.ppat.1014033.s017
(TIF)
S18 Fig. Multiple sequence alignment of the PLCP L1 and L3 family (related to Fig 6).
(A) Multiple sequence alignment of the PLCP L1family. (B) Multiple sequence alignment of the PLCP L3 subfamily. α, α-helix. β, β-sheet.
https://doi.org/10.1371/journal.ppat.1014033.s018
(TIF)
S19 Fig. In situ Hybridization of Bxy007574 and Bxy014241 in B. xylophilus.
(A) In situ hybridization of Bxy007574 in females. (B) In situ hybridization of Bxy007574 in males. (C) In situ hybridization of Bxy014241 in females. (D) In situ hybridization of Bxy014241 in males.
https://doi.org/10.1371/journal.ppat.1014033.s019
(TIF)
S20 Fig. RNAi specificity and knockdown efficiency of four PLCP family genes.
(A, C, E, G) DNA sequence similarity heatmaps showing the potential off-target effects of RNAi fragments designed for Bxy007574 (A), Bxy014241 (C), Bxy013180 (E), and Bmu008640 (G) across the PLCP gene family in B. xylophilus (A, C, E) and B. mucronatus (G). High similarity (purple) indicates potential off-target risk, while low similarity (yellow) suggests specificity of the RNAi fragment. (B, D, F, H) Quantification of target gene expression levels by qRT-PCR after RNAi treatment. Relative expression of Bxy007574 (B), Bxy014241 (D), Bxy013180 (F), and Bmu008640 (H) was significantly reduced in J2 stage after dsRNA-treated, compared with gfp dsRNA and untreated controls. Error bars represent standard deviation (n = 3). Statistical significance was determined by two-tailed Student’s t test: *P < 0.05; **P < 0.01.
https://doi.org/10.1371/journal.ppat.1014033.s020
(TIF)
S21 Fig. Infection rates of pine seedlings after inoculation with wild-type and PLCP-RNAi nematodes.
https://doi.org/10.1371/journal.ppat.1014033.s021
(TIF)
S22 Fig. Correlation network diagram of gene expression related to the rapid expansion of B. xylophilus.
https://doi.org/10.1371/journal.ppat.1014033.s022
(TIF)
S2 Table. Statistics of the assembly process of B. xylophilus genome.
https://doi.org/10.1371/journal.ppat.1014033.s024
(DOC)
S3 Table. BUSCO statistics of B. xylophilus genome.
https://doi.org/10.1371/journal.ppat.1014033.s025
(DOC)
S4 Table. Organization of repetitive sequences in B. xylophilus genome.
https://doi.org/10.1371/journal.ppat.1014033.s026
(DOC)
S5 Table. Statistics of protein-coding genes in B. xylophilus genome.
https://doi.org/10.1371/journal.ppat.1014033.s027
(DOC)
S6 Table. Statistics of identified non-coding RNAs in B. xylophilus genome.
https://doi.org/10.1371/journal.ppat.1014033.s028
(DOC)
S8 Table. Statistics of the assembly process of B. mucronatus genome.
https://doi.org/10.1371/journal.ppat.1014033.s030
(DOC)
S9 Table. BUSCO statistics of B. mucronatus genome.
https://doi.org/10.1371/journal.ppat.1014033.s031
(DOC)
S10 Table. Organization of repetitive sequences in B. mucronatus genome.
https://doi.org/10.1371/journal.ppat.1014033.s032
(DOC)
S11 Table. Statistics of protein-coding genes in B. mucronatus genome.
https://doi.org/10.1371/journal.ppat.1014033.s033
(DOC)
S12 Table. Statistics of identified non-coding RNAs in B. mucronatus genome.
https://doi.org/10.1371/journal.ppat.1014033.s034
(DOC)
S13 Table. Proteomes of 9 nematode species (related to Fig 1).
https://doi.org/10.1371/journal.ppat.1014033.s035
(DOC)
S14 Table. GO enrichment results of 12 rapidly expanding gene family of Sphaerularioidea and Tylenchoidea (related to Fig 1).
https://doi.org/10.1371/journal.ppat.1014033.s036
(DOC)
S15 Table. GO enrichment results of 19 rapidly expanding gene family of Aphelenchoidea (related to Fig 1).
https://doi.org/10.1371/journal.ppat.1014033.s037
(DOC)
S16 Table. GO enrichment results of 54 rapidly expanding gene family of B. xylophilus and B. mucronatus (related to Fig 1).
https://doi.org/10.1371/journal.ppat.1014033.s038
(DOC)
S17 Table. Rapidly contracting gene families of B. xylophilus and B. mucronatus (related to Fig 1).
https://doi.org/10.1371/journal.ppat.1014033.s039
(DOC)
S18 Table. GO enrichment results of 61 rapidly expanding gene family of B. xylophilus (related to Fig 1).
https://doi.org/10.1371/journal.ppat.1014033.s040
(DOC)
S19 Table. GO enrichment results of 54 rapidly contracting gene family of B. xylophilus (related to Fig 1).
https://doi.org/10.1371/journal.ppat.1014033.s041
(DOC)
S20 Table. GO enrichment results of 209 rapidly expanding gene family of B. mucronatus (related to Fig 1).
https://doi.org/10.1371/journal.ppat.1014033.s042
(DOC)
S21 Table. GO enrichment results of 48 rapidly contracting gene family of B. mucronatus (related to Fig 1).
https://doi.org/10.1371/journal.ppat.1014033.s043
(DOC)
S22 Table. GO enrichment results of 26 rapidly expanding gene family of B.okinawaensis (related to Fig 1).
https://doi.org/10.1371/journal.ppat.1014033.s044
(DOC)
S23 Table. Rapidly contracting gene families of B.okinawaensis (related to Fig 1).
https://doi.org/10.1371/journal.ppat.1014033.s045
(DOC)
S24 Table. Mating behavior of B. xylophilus after Bxy011316 RNAi.
https://doi.org/10.1371/journal.ppat.1014033.s046
(DOC)
S1 Data. Raw data for all main and supplementary figures.
The Excel file titled S1 Data serves as the supporting information for this manuscript, containing 34 independent worksheets corresponding to the raw data of all main figures and supplementary figures. The details of each worksheet are as follows: Main Figure Data: Sheets A-B1 include the complete raw data for main Figs 1–5. This data covers experimental measurements, quantitative results, and replicate records that support the conclusions presented in the main text. Supplementary Figure Data: Sheets C1-D1 provide the raw data for supplementary figures. This includes additional experimental validations, extended analyses, and control group data not fully displayed in the main manuscript. Data Format: Each worksheet contains clearly labeled columns (e.g., “FamilyID”, “2–ΔΔCT”, “q-MEAN”, “actin-MEAN”) to indicate data types, with no missing critical information. All data points represent direct experimental observations or calculated values from three independent biological replicates, ensuring reproducibility.
https://doi.org/10.1371/journal.ppat.1014033.s048
(XLSX)
References
- 1. Pereira F, Moreira C, Fonseca L, van Asch B, Mota M, Abrantes I, et al. New insights into the phylogeny and worldwide dispersion of two closely related nematode species, Bursaphelenchus xylophilus and Bursaphelenchus mucronatus. PLoS One. 2013;8(2):e56288. pmid:23409167
- 2. Mota MM, Braasch H, Bravo MA, Penas AC, Burgermeister W, Metge K, et al. First report of Bursaphelenchus xylophilus in Portugal and in Europe. Nematology. 1999;1(1):727–34.
- 3. Mamiya Y. History of pine wilt disease in Japan. J Nematol. 1988;20(2):219–26. pmid:19290205
- 4. Eizo K. Observations on the intratracheal existence and cuticle surface of the pine wood nematode, Bursaphelenchus xylophilus, associated with the Cerambycid beetle, Monochamus carolinensis. Appl Entomol Zool. 1986;21(1):340–6.
- 5. Nobuo O, Tadakazu N. In vitro occurrence of dispersal fourth stage juveniles in Bursaphelenchus xylophilus co-incubated with Monochamus alternatus. J Nematol. 2002;32(2):53–9.
- 6. Ichihara Y, Fukuda K, Suzuki K. Early Symptom Development and Histological Changes Associated with Migration of Bursaphelenchus xylophilus in Seedling Tissues of Pinus thunbergii. Plant Dis. 2000;84(6):675–80. pmid:30841110
- 7. Liu Z, Li Y, Pan L, Meng F, Zhang X. Cold adaptive potential of pine wood nematodes overwintering in plant hosts. Biol Open. 2019;8(5):bio041616. pmid:31023716
- 8. Zhao LL, Wei W, Kulhavy DL, Zhang XY, Sun JH. Low temperature induces two growth-arrested stages and change of secondary metabolites in Bursaphelenchus xylophilus. Nematol. 2007;9(5):663–70.
- 9. Son JA, Jung CS, Han HR. Migration and Attacking Ability of Bursaphelenchus mucronatus in Pinus thunbergii Stem Cuttings. The Plant Pathology Journal. 2016;32(4):340–6.
- 10. Cardoso JMS, Anjo SI, Fonseca L, Egas C, Manadas B, Abrantes I. Bursaphelenchus xylophilus and B. mucronatus secretomes: a comparative proteomic analysis. Sci Rep. 2016;6:39007. pmid:27941947
- 11. Wang S, Wang S, Luo Y, Xiao L, Luo X, Gao S, et al. Comparative genomics reveals adaptive evolution of Asian tapeworm in switching to a new intermediate host. Nat Commun. 2016;7:12845. pmid:27653464
- 12. Liu X, Feng Y, Bai X, Wang X, Qin R, Tang B, et al. Comparative multi-omics analyses reveal differential expression of key genes relevant for parasitism between non-encapsulated and encapsulated Trichinella. Commun Biol. 2021;4(1):134. pmid:33514854
- 13. Yen CLE, Stone SJ, Koliwad S, Harris C, Farese RV1. Thematic review series: glycerolipids. DGAT enzymes and triacylglycerol biosynthesis. J Lipid Res. 2008;49(11):2283–230.
- 14. Galego L, Barahona S, Romão CV, Arraiano CM. Phosphorylation status of BolA affects its role in transcription and biofilm development. FEBS J. 2021;288(3):961–79. pmid:32535996
- 15. Shingles J, Lilley CJ, Atkinson HJ, Urwin PE. Meloidogyne incognita: molecular and biochemical characterisation of a cathepsin L cysteine proteinase and the effect on parasitism following RNAi. Exp Parasitol. 2007;115(2):114–20. pmid:16996059
- 16. Yang N, Matthew MA, Yao C. Roles of Cysteine Proteases in Biology and Pathogenesis of Parasites. Microorganisms. 2023;11(6):1397. pmid:37374899
- 17. da Silva AA, Galego L, Arraiano CM. New Perspectives on BolA: A Still Mysterious Protein Connecting Morphogenesis, Biofilm Production, Virulence, Iron Metabolism, and Stress Survival. Microorganisms. 2023;11(3):632. pmid:36985206
- 18. Chen G, Harwood JL, Lemieux MJ, Stone SJ, Weselake RJ. Acyl-CoA:diacylglycerol acyltransferase: Properties, physiological roles, metabolic engineering and intentional control. Prog Lipid Res. 2022;88:101181. pmid:35820474
- 19. Ozhelvaci F, Steczkiewicz K. Identification and classification of papain-like cysteine proteinases. J Biol Chem. 2023;299(6):104801. pmid:37164157
- 20. Kikuchi T, Cotton JA, Dalzell JJ, Hasegawa K, Kanzaki N, McVeigh P, et al. Genomic insights into the origin of parasitism in the emerging plant pathogen Bursaphelenchus xylophilus. PLoS Pathog. 2011;7(9):e1002219. pmid:21909270
- 21. Midha MK, Wu M, Chiu K-P. Long-read sequencing in deciphering human genetics to a greater depth. Human Genetics. 2019;138(11-12):1201–15.
- 22. Wu S, Gao S, Wang S, Meng J, Wickham J, Luo S, et al. A Reference Genome of Bursaphelenchus mucronatus Provides New Resources for Revealing Its Displacement by Pinewood Nematode. Genes. 2020;11(5):570.
- 23. Baroncelli R, Amby DB, Zapparata A, Sarrocco S, Vannacci G, Le Floch G, et al. Gene family expansions and contractions are associated with host range in plant pathogens of the genus Colletotrichum. BMC Genomics. 2016;17:555. pmid:27496087
- 24. Ichihara Y, Fukuda K, Suzuki K. Early Symptom Development and Histological Changes Associated with Migration of Bursaphelenchus xylophilus in Seedling Tissues of Pinus thunbergii. Plant Dis. 2000;84(6):675–80. pmid:30841110
- 25. Ishida K, Hogetsu T, Fukuda K, Suzuki K. Cortical responses in Japanese black pine to attack by the pine wood nematode. Canadian Journal of Botany. 1993;71(11):1399–405.
- 26. Kiyohara T, Suzuki K. Nematode population growth and disease development in the pine wilting disease. European Journal of Forest Pathology. 1978;8(5–6):285–92.
- 27. Son JA, Moon Y-S. Migrations and Multiplications of Bursaphelenchus xylophilus and B. mucronatus in Pinus thumbergii in Relation to Their Pathogenicity. Plant Pathol J. 2013;29(1):116–22. pmid:25288937
- 28. Woo KS, Yoon JH, Woo SY, Lee SH, Han SU, et al. Comparison in disease development and gas exchange rate of Pinus densiflora seedlings artificially inoculated with Bursaphelenchus xylophilus and B. mucronatus. For Sci Technol. 2010;6(2):110–7.
- 29. Zhang W, Yu H, Lv Y, Bushley KE, Wickham JD, Gao S, et al. Gene family expansion of pinewood nematode to detoxify its host defence chemicals. Molecular Ecology. 2020;29(5):940–55.
- 30. Hotson A, Mudgett MB. Cysteine proteases in phytopathogenic bacteria: identification of plant targets and activation of innate immunity. Curr Opin Plant Biol. 2006;7(4):384–90.
- 31. Futai K. Population dynamics of Bursaphelenchus lignicolus (Nematoda: Aphelenchoididae) and B. mucronatus in pine seedlings. Appl Entomol Zoolog. 1980;15:458–64.
- 32. Santos JM, Lobo M, Matos APA, De Pedro MA, Arraiano CM. The gene bolA regulates dacA (PBP5), dacC (PBP6) and ampC (AmpC), promoting normal morphology in Escherichia coli. Mol Microbiol. 2002;45(6):1729–40. pmid:12354237
- 33. Lee JK, Park EJ, Chung HK, Hong SH, Joe CO, Park SD. Isolation of UV-inducible transcripts from Schizosaccharomyces pombe. Biochem Biophys Res Commun. 1994;202(2):1113–9. pmid:8048925
- 34. Kim SH, Kim M, Lee JK, Kim MJ, Jin YH, Seong RH, et al. Identification and expression of uvi31+, a UV-inducible gene from Schizosaccharomyces pombe. Environ Mol Mutagen. 1997;30(1):72–81. pmid:9258332
- 35. Santos JM, Freire P, Vicente M, Arraiano CM. The stationary-phase morphogene bolA from Escherichia coli is induced by stress during early stages of growth. Mol Microbiol. 1999;32(4):789–98. pmid:10361282
- 36. Zhang Y, Zou X, Ding Y, Wang H, Wu X, Liang B. Comparative genomics and functional study of lipid metabolic genes in Caenorhabditis elegans. BMC Genomics. 2013;14(1):164–72.
- 37. Cohen P, Kajimura S. The cellular and functional complexity of thermogenic fat. Nat Rev Mol Cell Biol. 2021;22(6):393–409. pmid:33758402
- 38. Liu Z, Li Y, Pan L, Meng F, Zhang X. Cold adaptive potential of pine wood nematodes overwintering in plant hosts. Biol Open. 2019;8(5):bio041616. pmid:31023716
- 39. Jones JT, Haegeman A, Danchin EGJ, Gaur HS, Helder J, Jones MGK, et al. Top 10 plant-parasitic nematodes in molecular plant pathology. Mol Plant Pathol. 2013;14(9):946–61. pmid:23809086
- 40. Li T, Jin Y, Wu J, Ren Z. Beyond energy provider: multifunction of lipid droplets in embryonic development. Biol Res. 2023;56(1):38. pmid:37438836
- 41. Chitraju C, Walther TC, Farese RV Jr. The triglyceride synthesis enzymes DGAT1 and DGAT2 have distinct and overlapping functions in adipocytes. J Lipid Res. 2019;60(6):1112–20. pmid:30936184
- 42. Xing Z, Lu T, Deng Y, Zhang Y, Dong H, Li Z, et al. DGAT1-mediated lipid metabolism is essential for female reproduction in the silkworm, Bombyx mori. Pest Manag Sci. 2025;81(12):8096–105. pmid:40781918
- 43. Dutta TK, Papolu PK, Banakar P, Choudhary D, Sirohi A, Rao U. Tomato transgenic plants expressing hairpin construct of a nematode protease gene conferred enhanced resistance to root-knot nematodes. Front Microbiol. 2015;6:260. pmid:25883594
- 44. Xue Q, Wu X-Q, Zhang W-J, Deng L-N, Wu M-M. Cathepsin L-like Cysteine Proteinase Genes Are Associated with the Development and Pathogenicity of Pine Wood Nematode, Bursaphelenchus xylophilus. Int J Mol Sci. 2019;20(1):215. pmid:30626082
- 45. Xiong Q, Wan AT-Y, Liu X, Fung CS-H, Xiao X, Malainual N, et al. Comparative Genomics Reveals Insights into the Divergent Evolution of Astigmatic Mites and Household Pest Adaptations. Mol Biol Evol. 2022;39(5):msac097. pmid:35535514
- 46. Turner WC, Kamath PL, van Heerden H, Huang Y-H, Barandongo ZR, Bruce SA, et al. The roles of environmental variation and parasite survival in virulence-transmission relationships. R Soc Open Sci. 2021;8(6):210088. pmid:34109041
- 47. Wang J, Liu H, Guo K, Yu H, Ye J, Hu J. Molecular characterization and functional analysis of Bxy-octr-1 in Bursaphelenchus xylophilus. Gene. 2022;823:146350. pmid:35189249
- 48. Marçais G, Kingsford C. A fast, lock-free approach for efficient parallel counting of occurrences of k-mers. Bioinformatics. 2011;27(6):764–70. pmid:21217122
- 49. Vurture GW, Sedlazeck FJ, Nattestad M, Underwood CJ, Fang H, Gurtowski J, et al. GenomeScope: fast reference-free genome profiling from short reads. Bioinformatics. 2017;33(14):2202–4. pmid:28369201
- 50. Xiao C-L, Chen Y, Xie S-Q, Chen K-N, Wang Y, Han Y, et al. MECAT: fast mapping, error correction, and de novo assembly for single-molecule sequencing reads. Nat Methods. 2017;14(11):1072–4. pmid:28945707
- 51. Chin C-S, Peluso P, Sedlazeck FJ, Nattestad M, Concepcion GT, Clum A, et al. Phased diploid genome assembly with single-molecule real-time sequencing. Nat Methods. 2016;13(12):1050–4. pmid:27749838
- 52. Wheeler D, Redding AJ, Werren JH. Characterization of an ancient lepidopteran lateral gene transfer. PLoS One. 2013;8(3):e59262. pmid:23533610
- 53. Marçais G, Delcher AL, Phillippy AM, Coston R, Salzberg SL, Zimin A. MUMmer4: A fast and versatile genome alignment system. PLoS Comput Biol. 2018;14(1):e1005944. pmid:29373581
- 54. Manni M, Berkeley MR, Seppey M, Simão FA, Zdobnov EM. BUSCO update: novel and streamlined workflows along with broader and deeper phylogenetic coverage for scoring of eukaryotic, prokaryotic, and viral genomes. Mol Biol Evol. 2021;38:4647–54.
- 55. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9(4):357–9. pmid:22388286
- 56. Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018;34(17):i884–90. pmid:30423086
- 57. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009;25(16):2078–9. pmid:19505943
- 58. Flynn JM, Hubley R, Goubert C, Rosen J, Clark AG, Feschotte C, et al. RepeatModeler2 for automated genomic discovery of transposable element families. Proc Natl Acad Sci U S A. 2020;117(17):9451–7. pmid:32300014
- 59. Tarailo-Graovac M, Chen N. Using RepeatMasker to identify repetitive elements in genomic sequences. Curr Protoc Bioinformatics. 2009;Chapter 4:4.10.1-4.10.14. pmid:19274634
- 60. Jurka J, Kapitonov VV, Pavlicek A, Klonowski P, Kohany O, Walichiewicz J. Repbase Update, a database of eukaryotic repetitive elements. Cytogenet Genome Res. 2005;110(4):462–7.
- 61. Hubley R, Finn RD, Clements J, Eddy SR, Jones TA, Bao W, et al. The Dfam database of repetitive DNA families. Nucleic Acids Res. 2016;44(D1):D81-9. pmid:26612867
- 62. McGinnis S, Madden TL. BLAST: at the core of a powerful and diverse set of sequence analysis tools. Nucleic Acids Research. 2004;32(2):W20–5.
- 63. Emms DM, Kelly S. OrthoFinder: solving fundamental biases in whole genome comparisons dramatically improves orthogroup inference accuracy. Genome Biol. 2015;16(1):157. pmid:26243257
- 64. Kozlov AM, Darriba D, Flouri T, Morel B, Stamatakis A. RAxML-NG: a fast, scalable and user-friendly tool for maximum likelihood phylogenetic inference. Bioinformatics. 2019;35(21):4453–5. pmid:31070718
- 65. Katoh K, Misawa K, Kuma K, Miyata T. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 2002;30(14):3059–66. pmid:12136088
- 66. Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics. 2009;25(15):1972–3. pmid:19505945
- 67. Darriba D, Posada D, Kozlov AM, Stamatakis A, Morel B, Flouri T. ModelTest-NG: A New and Scalable Tool for the Selection of DNA and Protein Evolutionary Models. Mol Biol Evol. 2020;37(1):291–4. pmid:31432070
- 68. Yang Z. PAML 4: phylogenetic analysis by maximum likelihood. Mol Biol Evol. 2007;24(8):1586–91. pmid:17483113
- 69. Kumar S, Suleski M, Craig JM, Kasprowicz AE, Sanderford M, Li M, et al. TimeTree 5: An Expanded Resource for Species Divergence Times. Mol Biol Evol. 2022;39(8):msac174. pmid:35932227
- 70. De Bie T, Cristianini N, Demuth JP, Hahn MW. CAFE: a computational tool for the study of gene family evolution. Bioinformatics. 2006;22(10):1269–71. pmid:16543274
- 71. Love M, Anders S, Huber W. Differential analysis of count data–the DESeq2 package. Genome Biol. 2014;15:10–1186.
- 72. Zhou Y, Zhu J, Tong T, Wang J, Lin B, Zhang J. A statistical normalization method and differential expression analysis for RNA-seq data between different species. BMC Bioinformatics. 2019;20(1):163. pmid:30925894
- 73. Tang J, Ma R, Zhu N, Guo K, Guo Y, Bai L, et al. Bxy-fuca encoding α-L-fucosidase plays crucial roles in development and reproduction of the pathogenic pinewood nematode, Bursaphelenchus xylophilus. Pest Manag Sci. 2020;76(1):205–14. pmid:31140718
- 74. Wang Q, Han C, Ferreira AO, Yu X, Ye W, Tripathy S, et al. Transcriptional programming and functional interactions within the Phytophthora sojae RXLR effector repertoire. Plant Cell. 2011;23(6):2064–86. pmid:21653195