Functional analysis of 3 genes in xenobiotic detoxification pathway of Bursaphelenchus xylophilus against matrine.

Bursaphelenchus xylophilus is the causative agent of pine wilt disease. It has caused devastating damage to ecosystems worldwide, owing to the characteristic of being widely spread and uncontrollable. However, the current methods of control are mainly based on pesticides, which can cause irreversible damage to the ecosystem. Therefore, the search for new drug targets and the development of environmentally friendly nematicides is especially valuable. In this study, three key genes of the xenobiotic detoxification pathways were cloned from B. xylophilus, which were subsequently subjected to bioinformatic analysis. The bioassay experiment was carried out to determine the concentration of matrine required for further tests. Subsequently, enzyme activity detection and three gene expression pattern analysis were performed on matrine treated nematodes. Finally, RNA interference was conducted to verify the functions carried out by the three genes in combating matrine. The results indicated that cytochrome P450 and glutathione S-transferase of B. xylophilus were activated by matrine, which induced high expression of BxCYP33C4, BxGST1, and BxGST3. After RNA interference of three genes of B. xylophilus, the sensitivity of B. xylophilus to matrine was increased and the survival rate of nematodes was reduced to various degrees in comparison to the control group. Overall, the results fully demonstrated that BxCYP33C4, BxGST1, and BxGST3 are valuable drug targets for B. xylophilus. Furthermore, the results suggested that matrine has value for development and exploitation in the prevention and treatment of B. xylophilus.

Exploring the role of detoxification genes in the resistance of Bursaphelenchus xylophilus to different exogenous nematicidal substances using transcriptomic analyses.

Bursaphelenchus xylophilus (Pine wood nematode, PWN) has become a worldwide forest disease due to its rapid infection ability, high lethality and difficulty in control. The main means of countering B. xylophilus is currently chemical control, but nematicides can present problems such as environmental pollution and drug resistance. The development of novel environmentally-friendly nematicides has thus become a focus of recent research. In this study, BxUGT3 and BxUGT34, which might be related to detoxification, were investigated by comparing transcriptomic and WGCNA approaches. Three other genes with a similar expression pattern, BxUGT13, BxUGT14, and BxUGT16, were found by gene family analysis. Further bioassays and qPCR assays confirmed that these five genes showed significant changes in transcript levels upon exposure to α-pinene and carvone, demonstrating that they respond to exogenous nematicidal substances. Finally, RNAi and bioassays showed that B. xylophilus with silenced BxUGT16 had increased mortality in the face of α-pinene and carvone stress, suggesting that BxUGT16 plays an important role in detoxification. Taken together, this study used novel molecular research methods, explored the detoxification mechanism of B. xylophilus at a transcriptomic level, and revealed a molecular target for the development of novel biopesticides.

Molecular characterization and functional analysis of glutathione S-transferase genes of pine wood nematode (Bursaphelenchus xylophilus) for avermectin.

Pine wilt disease (PWD), caused by Bursaphelenchus xylophilus (pine wood nematodes, PWNs), is a forest disease that seriously threatens the health of Pinus forestry. Glutathione S-transferases (GSTs) play important roles in xenobiotic metabolism, lipophilic compound transport, antioxidative stress reactions, anti-mutagenesis, and antitumor activity. The analysis and investigation of the specific functions of GSTs in the metabolism of toxic substances in nematodes are important for identifying potential target genes to control the spread and transmission of B. xylophilus. In this study, 51 Bx-GSTs were found in the genome of B. xylophilus. Two key Bx-gsts (Bx-gst12 and Bx-gst40) were analyzed when B. xylophilus was exposed to avermectin. The expression of Bx-gst12 and Bx-gst40 was significantly increased when B. xylophilus was exposed to 1.6 and 3.0 mg/mL avermectin solutions. Notably, combined silencing of both Bx-gst12 and Bx-gst40 did not further increase the mortality rates under avermectin exposure. Mortality rates were significantly increased in nematodes treated with dsRNA compared to control nematodes (p < 0.05) after RNAi. The feeding ability of nematodes was also significantly reduced after treatment with dsRNA. These results suggested that Bx-gsts are associated with the detoxification process and feeding behavior of B. xylophilus. Silencing Bx-gsts leads to increased susceptibility to nematicides and reduces the feeding ability of B. xylophilus. Therefore, Bx-gsts will be a new control target of PWNs in the future.

Functional Study on Cytochrome P450 in Response to L(-)-Carvone Stress in Bursaphelenchus xylophilus.

Bursaphelenchus xylophilus (PWN) causes pine wilt disease (PWD), which is one of the most devastating pine diseases worldwide. Cytochrome P450 (CYP) catalyzes the biosynthetic metabolism of terpenoids and plays an important role in the modification of secondary metabolites in all living organisms. We investigated the molecular characteristics and biological functions of Bx-cyp29A3 in B. xylophilus. The bioinformatics analysis results indicated that Bx-cyp29A3 has a transmembrane domain and could dock with L(-)-carvone. The gene expression pattern indicated that Bx-cyp29A3 was expressed in 0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL L(-)-carvone solutions. The Bx-cyp29A3 expression increased in a dose-dependent manner and peaked at 24 h of exposure when the L(-)-carvone solution concentration was 0.8 mg/mL. However, the gene expression peaked at 0.6 mg/mL after 36 h. Furthermore, RNA interference (RNAi) indicated that Bx-cyp29A3 played an essential role in the response to L(-)-carvone. The mortality rates of the Bx-cyp29A3 knockdown groups were higher than those of the control groups in the 0.4, 0.6, 0.8, and 1.0 mg/mL carvone solutions after 24 h of exposure or 36 h of exposure. In summary, bioinformatics provided the structural characteristics and conserved sequence properties of Bx-cyp29A3 and its encoded protein, which provided a target gene for the study of the P450 family of B. xylophilus. Gene silencing experiments clarified the function of Bx-cyp29A3 in the immune defense of B. xylophilus. This study provides a basis for the screening of new molecular targets for the prevention and management of B. xylophilus.