Transcriptome-Based Analysis Reveals a Crucial Role of BxGPCR17454 in Low Temperature Response of Pine Wood Nematode (Bursaphelenchus xylophilus)

Abstract

Background: The causal agent of pine wilt disease is the pine wood nematode (PWN) (Bursaphelenchus xylophilus), whose ability to adapt different ecological niches is a crucial determinant of their invasion to colder regions. To discover the molecular mechanism of low temperature response mechanism, we attempted to study the molecular response patterns under low temperature from B. xylophilus with a comprehensive RNA sequencing analysis and validated the differentially expressed genes (DEGs) with quantitative real-time polymerase chain reaction (qRT-PCR). Bioinformatic software was utilized to isolate and identify the low-temperature-related BxGPCR genes. Transcript abundance of six low-temperature-related BxGPCR genes and function of one of the BxGPCR genes are studied by qRT-PCR and RNA interference.

Results: The results showed that we detected 432 DEGs through RNA sequencing between low-temperature-treated and ambient-temperature-treated groups nematodes. The transcript level of 6 low-temperature-related BxGPCR genes increased at low temperature. And, the survival rates of BxGPCR17454 silenced B. xylophilus revealed a significant decrease at low temperature.

Conclusion: in conclusion, this transcriptome-based study revealed a crucial role of BxGPCR17454 in low temperature response process of pine wood nematode. These discoveries would assist the development of management and methods for efficient control of this devastating pine tree pest.

Keywords: Bursaphelenchus xylophilus; GPCR; RNAi; low temperature; pine wilt disease; transcriptome.

Figure 1

General transcription patterns of all samples (A). Clustering of the biological duplicates for each RNA sequencing condition reveals conserved alignment between replicates. (B). Pearson correlation between low-temperature-treated and CK nematodes.

Figure 2

Differentially expressed genes (DEGs) analysis. (A). DEGs between low-temperature-treated and CK group nematodes. Red dots represent up-regulated genes and green dots represent down-regulated genes. (B). Heat map of all the DEGs between low-temperature-treated and CK group nematodes. (C). Gene Ontology (GO) categories of DEGs between low-temperature-treated and CK group nematodes.

Figure 3

Quantitative real-time polymerase chain reaction (qRT-PCR) identification of differentially DEGs between low-temperature-treated and CK group nematodes. The relative gene expressions were normalized with the internal control gene Bx28S. The putative function of each gene is as follows: BXY_0069900, NH127; BXY_0262500, SERA; BXY_0413400, ST2B1; BXY_0508700, LIP1; BXY_0596900, ASK12; BXY_0688500, NHR54; BXY_0794300, NPY2R; BXY_0799000, DUS9; BXY_0959300, ADH3; BXY_1220600, SOCS4; BXY_1423800, AATC; BXY_1722500, CYSK2.

Figure 4

Identification and transcript pattern analysis of six low-temperature-related BxGPCRs. (A). Analysis on phylogenetic trees of six deduced protein sequences with other organisms. Labels at the end of each branch are gene id/NCBI accessions of each gene. Different shapes stand for the Latin name of each organism. (B). Transcript abundance analysis of six BxGPCRs under low temperature.

Figure 5

RNAi validaton of BxGPCR17454. (A). B. xylophilus soaked in fluorescein isothiocyanate (FITC) revealed green fluorescent signal under ultraviolet light. (B). BxGPCR17454 RNAi efficiency was validated by expression changes between BxGPCR17454 dsRNA-treated and dsRNA-free nematodes. BxGPCR17454 gene was significant silenced after dsRNA treatment, while dsRNA had no obvious effect on the transcript level of internal control gene β-actin. (C). BxGPCR17454 dsRNA-treated nematodes showed significate decreased survival rate under low temperature. Data represent mean values ± SD from different repetitions. Asterisks indicates statistically significant differences (* p < 0.05, ** p < 0.01, Student’s t-test) was found between the dsRNA-treated and dsRNA-free groups.