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

Abstract

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.

Whole-organism Transcriptomic Insight Into the Effects of Cytco, A Novel Nematotoxic Protein on the Pine Wood Nematode Bursaphelenchus Xylophilus 

“…The present study showed that the nematocidal protein toxin, CytCo, displayed diverse effects on PWN development, reproduction, infectivity, motility, and immune defenses based on whole-organism transcriptome pro ling, in consistent with the reported bioassay results [16].…”

“…Many DEGs annotated as "kinase and phosphatase" were downregulated (Tables S9 and S10), which may cause multiple effects on development, reproduction, metabolism, and immune responses. For example, serine/threonine-protein phosphatase 1 (PP1) is essential for sperm meiosis and motility in C. elegans [30], in agreement with the previous report that the reduced fecundity of the PWN under CytCo treatment [16]. The abovementioned genes may favor developing new molecular targets to control PWN.…”

“…Dafachronic acids play multiple functions in suppressing dauer formation, lipid metabolism, and inducing reproductive growth in host trees, by binding directly to the nuclear hormone receptor daf-12 [24,25]. The DEG encoding DHS16 (3 βhydroxysteroid dehydrogenase), which is involve in the production of Δ 7 -dafachronic acid from cholesterol, was found to be downregulated, in agreement with the lowering lipid droplet formation in CytCo-treated PWN [16]. Nipi-3, a tribbles-like kinase is required in the response of C. elegans to fungal infection [26].…”

“…In our study, the upregulated DEGs related to sodium/sulphate symporter and potassium channel proteins (Table S5) might also cause ionic imbalance besides that produce consequences similar to the pore-forming effects of CytCo on cell membrane. Moreover, the effects of CytCo in the bioassays were analogous to the adverse effects of the chemical nematicide, emamectin benzoate (EB), including lowered fecundity, hatching rate, and thrashing frequency [8,16]. However, some of the observed DEGs were unique, and even the shared DEGs had different expression patterns (Fig.…”

“…However, the large molecular masses and dimensions of Cry proteins decrease their control e cacy on plant-parasitic nematodes. A Cyt-like protein from an entomopathogenic fungus Conidiobolus obscurus (named as CytCo) has been found to hold high nematicide activity against PWN and an inhibitory effect on egg hatching, and also signi cantly suppresses the reproductive and thrashing behaviors of PWN in bioassays [16]. CytCo (less than 22 kDa) characterized with a single domain of the center of β-strands being wrapped with a layer of α-helices can be easily taken up by PWN, presenting a potential of nematode control [16,17].…”