Transcriptomic insights into the effects of CytCo, a novel nematotoxic protein, on the pine wood nematode Bursaphelenchus xylophilus.

BACKGROUND: The pine wood nematode Bursaphelenchus xylophilus is a destructive pest of Pinus trees worldwide and lacks effective control measures. Screening for nematotoxic proteins has been undertaken to develop new strategies for nematode control. RESULTS: The results of the present study provided initial insights into the responses of B. xylophilus exposed to a nematotoxic cytolytic-like protein (CytCo) based on transcriptome profiling. A large set of differentially expressed genes (DEGs = 1265) was found to be related to nematode development, reproduction, metabolism, motion, and immune system. In response to the toxic protein, B. xylophilus upregulated DEGs encoding cuticle collagens, transporters, and cytochrome P450. In addition, many DEGs related to cell death, lipid metabolism, major sperm proteins, proteinases/peptidases, phosphatases, kinases, virulence factors, and transthyretin-like proteins were downregulated. Gene Ontology enrichment analysis showed that the CytCo treatment substantially affected DEGs involved in muscle contraction, lipid localization, and the mitogen-activated protein kinase cascade. The pathway richness of the Kyoto Encyclopedia of Genes and Genomes showed that the DEGs were concentrated in lysosomes and involved in fatty acid degradation. Weighted co-expression network analysis indicated that the hub genes affected by CytCo were associated with the nematode cuticular collagen. CONCLUSIONS: These results showed that CytCo toxin interferes with gene expression to exert multiple nematotoxic effects, thereby providing insights into its potential use in pine wood nematode control.

Ruthenium Complex-Incorporated Two-Dimensional Metal-Organic Frameworks for Cocatalyst-Free Photocatalytic Proton Reduction from Water.

Ultrathin two-dimensional (2D) nanosheets with efficient light-driven proton reduction activity were obtained through the exfoliation of novel metal-organic frameworks (MOF), which were synthesized by using a bis(4'-carboxy-2,2':6',2″-terpyridine) ruthenium complex as a linker and 3d transition-metal (Mn, Co, Ni, and Zn) anions as nodes. The nanosheet of the Ni2+ node exhibits a photocatalytic hydrogen evolution rate of 923 ± 40 µmol g-1 h-1 at pH = 4.0, without the presence of any cocatalyst or cosensitizer. A combined experimental and theoretical study suggests a reductive quenched pathway for the photocatalytic hydrogen evolution by the nanosheet. The transition-metal nodes at the edge of the nanosheets are proposed as the active sites. Density functional theory (DFT) calculations attributed the different catalytic activities of the nanosheets to the discrepancy of H adsorption free energy at various transition-metal nodes.

Characterization of a cytolytic-like gene from the aphid-obligate fungal pathogen Conidiobolus obscurus.

Cytolytic (Cyt)-like genes are known by omics analyses to exist widely in bacterial and fungal pathogens, but their insecticidal activities in fungi remains unknown. A full-length coding sequence of a Cyt-like gene was first amplified from Conidiobolus obscurus (an obligate aphid-pathogenic fungus) through RACE (rapid-amplification of cDNA ends). The deduced protein structure was structurally characterized by a single Cyt-typical α/ß domain. The expression level of the Cyt-like gene in conidia correlated well with the fungal virulence against aphids (r2 = 0.97). The results demonstrate the Cyt-like gene acts as an important virulence factor of C. obscurus against aphids, and has potential for exploitation in aphid control.

Calcium leakage involved in nematotoxic effects of the Conidiobolus obscurus CytCo protein on the pine wood nematode, Bursaphelenchus xylophilus.

BACKGROUND: The pine wood nematode Bursaphelenchus xylophilus, a severe invasive species, is responsible for causing widespread pine wilt disease. The CytCo protein, a pore-forming toxin derived from Conidiobolus obscurus, exhibits nematotoxicity towards B. xylophilus. RESULTS: Our present study reveals the expression variation of a range of gene products in B. xylophilus that respond to the effects of CytCo using the isobaric tags for relative and absolute quantification proteomics technology. Functional enrichment analysis indicates that many differentially expressed proteins are linked to calcium signaling system, proteasome, energy production and conversion, and the determination of adult lifespan. It suggests that the dysregulation of calcium homeostasis, energy metabolism, and apoptosis contribute to the CytCo nematotoxicity. Using the calcium ion (Ca2+)-indicator calcein, we detected changes in Ca2+ levels in B. xylophilus, with a significantly increase in fluorescence in the nematode's intestine and pseudocoelom following CytCo treatments. Meanwhile, the apoptosis and reactive oxygen species (ROS) assays showed an enhancement of fluorescence in B. xylophilus cells, with increased CytCo concentrations. CONCLUSION: The protein toxin CytCo triggers Ca2+ leakage, disrupts Ca2+ balance in B. xylophilus, and induces apoptosis and ROS outburst, thereby intensifying its nematotoxic effects. This finding facilitates our understanding of the modes of action of nematotoxic proteins, and contributes to the development of innovative nematode control strategies. © 2024 Society of Chemical Industry.

Nematotoxicity of a Cyt-like protein toxin from Conidiobolus obscurus (Entomophthoromycotina) on the pine wood nematode Bursaphelenchus xylophilus.

BACKGROUND: The pine wood nematode Bursaphelenchus xylophilus is a destructive pest on Pinus trees and lacks effective control measures. The present study identified a novel nematotoxic cytolytic (Cyt)-like protein originating from the entomopathogenic fungus Conidiobolus obscurus. RESULTS: The protein was successfully purified using heterologous expression in Escherichia coli and affinity chromatography. N-hydroxysuccinimide-rhodamine-labeled Cyt-like protein was used to establish the route of toxin uptake, and revealed that the toxin can enter the nematode via the stylet. In bioassays, the purified protein had high nematicide activity against B. xylophilus, with a median lethal concentration at 24 h of 15.8 and 29.4 µg mL-1 for juveniles and adults, respectively. Compared with the deionized water control, fecundity, thrashing, and egg hatching were significantly reduced by 97%, 98%, and 83%, respectively, with 40 µg mL-1 Cyt-like protein at 24-36 h. Staining with Oil-Red-O showed a decrease in large lipid droplet formation in the protein-treated adult nematodes. CONCLUSION: The Cyt-like protein toxin possesses high nematicide activity against B. xylophilus with effects on nematode vitality and fecundity. The potential exists to use the Cyt-like protein for the control of B. xylophilus.

Transcriptomic profiling of effects of emamectin benzoate on the pine wood nematode Bursaphelenchus xylophilus.

BACKGROUND: Emamectin benzoate (EB) has recently been successfully applied as a trunk injection for preventative control of the pine wilt disease (PWD) caused by Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle. Here, a whole-organism transcriptomic analysis provides comprehensive insights into the adverse effects of EB on B. xylophilus. RESULTS: A large set of differentially expressed genes (DEGs) were found, demonstrating the antagonistic effects of EB on B. xylophilus embryonic and larval development, reproduction, nervous and motor systems, and pathogenesis. In toxicity assays with EB, the number of eggs laid, hatching rate, thrashing frequency, and developmental rate of B. xylophilus were significantly suppressed at low concentrations (0.1 µg mL-1 ). Moreover, the transcriptional changes validated by real-time quantitative PCR showed downregulated transcript levels of the genes encoding pectate lyases, ß-1,4-endoglucanases, and upregulated the genes encoding glutamate-gated chloride channel, γ-aminobutyric acid type ß receptor, uridine 5'-diphospho-glucuronosyl transferase, ATP-binding cassette transporter. The potential responses of B. xylophilus to EB included the upregulation of several genes putatively contributing to oocyte protection, stem cell renewal, and xenobiotic degradation, implying the potential for drug resistance to develop. CONCLUSION: Our findings further our understanding of the effects of EB for managing the PWD and may help to improve the pesticide-use strategies for controlling B. xylophilus. © 2019 Society of Chemical Industry.