Low Temperature Extends the Lifespan of Bursaphelenchus xylophilus through the cGMP Pathway

Abstract

The causal agent of pine wilt disease, pine wood nematode (PWN) (Bursaphelenchus xylophilus), revealed extended lifespan at low temperature. To discover the molecular mechanism of this phenomenon, we attempted to study the molecular characterization, transcript abundance, and functions of three genes of the cyclic guanosine monophosphate (cGMP) pathway from B. xylophilus. Three cGMP pathway genes were identified from B. xylophilus. Bioinformatic software was utilized to analyze the characteristics of the three putative proteins. Function of the three genes in cold tolerance was studied with RNA interference (RNAi). The results showed that the deduced protein of Bx-DAF-11 has an adenylate and guanylate cyclase catalytic domain, indicating an ability to bind to extracellular ligands and synthesizing cGMP. Both Bx-TAX-2 and Bx-TAX-4 have cyclic nucleotide-binding domains and ion transport protein domains, illustrating that they are cGMP-gated ion channels. The transcript level of Bx-daf-11, Bx-tax-2, and Bx-tax-4 increased at low temperature. The survival rates of three gene silenced B. xylophilus revealed a significant decrease at low temperature. This study illustrated that the cGMP pathway plays a key role in low-temperature-induced lifespan extension in B. xylophilus.

Keywords: Bursaphelenchus xylophilus; cGMP pathway; lifespan extension; low temperature; pine wilt disease.

Figure 1

Alignment of B. xylophilus Bx-DAF-11, Bx-TAX-2, and Bx-TAX-4 with homologs identified from NCBI. (A) Comparison for protein homology of Bx-DAF-11 from Brugia malayi (CRZ24751.1), Caenorhabditis briggsae (XP_002636586.1), C. elegans (NP_505960.3), C. remanei (XP_003114238.1), Dictyocaulus viviparus (KJH45027.1), Haemonchus contortus (CDJ86375.1), Loa loa (XP_020304885.1), and Toxocara canis (KHN73536.1); (B) Comparison for protein homology of Bx-TAX-2 from Ascaris suum (ERG83679.1), C. elegans (AAB41492.1), C. remanei (ACQ44009.1), D. viviparus (KJH52997.1), H. contortus (CDJ81705.1), Mesorhabditis sp. (API61682.1), and Acrobeloides sp. (API61683.1); (C) Comparison for protein homology of Bx-TAX-4 from Ancylostoma ceylanicum (EYC38988.1), A. suum (ERG86438.1), L. loa (XP_003138541.1), B. malayi (CDP95269.1), Necator americanus (XP_013302261.1), C. brenneri (EGT33630.1), C. elegans (NP_499033.1), C. remanei (XP_003112989.1), and C. briggsae (XP_002642523.1). Note: Red, yellow, and green indicate variant amino acid residues (identity < 50%), highly conserved amino acid residues (50% ≤ identity < 100%), and invariant amino acid residues (identity = 100%), respectively.

Figure 2

Analysis on phylogenetic trees of three deduced protein sequences with other organisms (same sequences as Figure 1). (A) Phylogenetic trees of the deduced Bx-DAF-11 protein sequences with other organisms’ DAF-11; (B) Phylogenetic trees of the deduced Bx-TAX-2 protein sequences with other organisms’ TAX-2; (C) Phylogenetic trees of the deduced Bx-TAX-4 protein sequences with other organisms’ TAX-4. One thousand bootstrap replicates were performed.

Figure 3

Survival rates of B. xylophilus and transcript abundance of three genes at 5 °C and 25 °C. (A) B. xylophilus showed higher survival rates at 5 °C than at 25 °C; (B) Bx-daf-11 revealed higher transcript levels at 5 °C than 25 °C over 7 days; (C) Bx-tax-2 revealed higher transcript levels at 5 °C than 25 °C over 7 days; (D) Bx-tax-4 revealed higher transcript levels at 5 °C than 25 °C over 7 days. Data represent mean values ± standard deviation (SD) from different repetitions. Asterisks indicate statistically significant differences (** p < 0.001, Student’s t-test) were found between 5 °C and 25 °C.

Figure 4

RNAi efficiency of three genes. (A) B. xylophilus soaked in fluorescein isothiocyanate (FITC) revealed green fluorescent signal; Scale bars = 20 μm; (B) B. xylophilus soaked in ddH₂O (FITC-free) revealed no signal; Scale bars = 20 µm; (C) The Bx-daf-11, Bx-tax-2, and Bx-tax-4 dsRNA had no obvious effect on the transcript level of Actin; (D) Bx-daf-11, Bx-tax-2, and Bx-tax-4 transcript level log₂ (RNAi-treated/RNAi-free) fold of B. xylophilus. Data represent mean values ± SD from different repetitions. Asterisks indicate statistically significant differences (** p < 0.001, Student’s t-test) were found between the dsRNA-treated group and dsRNA-free group.

Figure 5

B. xylophilus revealed significantly different survival rates between the CK (dsRNA-free groups) and dsRNA-treated groups at 5 °C while few differences in survival rates between the CK and dsRNA-treated groups can be detected at 25 °C. (A) Bx-daf-11 dsRNA-treated B. xylophilus showed a decreased survival rate at 5 °C; (B) Bx-tax-2 dsRNA-treated B. xylophilus showed a decreased survival rate at 5 °C; (C) Bx-tax-4 dsRNA-treated B. xylophilus showed a decreased survival rate at 5 °C; (D) CK and Bx-daf-11 dsRNA-treated B. xylophilus showed few differences in survival rate at 25 °C; (E) CK and Bx-tax-2 dsRNA-treated B. xylophilus showed few differences in survival rate at 25 °C; (F) CK and Bx-tax-4 dsRNA-treated B. xylophilus showed few differences in survival rate at 25 °C. Data represent mean values ± SD from different repetitions. Asterisks indicate that statistically significant differences (* p < 0.01, ** p < 0.001, Student’s t-test) were found between the CK and dsRNA-treated groups.