A 2-Cys peroxiredoxin in response to oxidative stress in the pine wood nematode, Bursaphelenchus xylophilus.

Zhen Li,Xuguo Zhou,Qingwen Zhang

doi:10.1038/srep27438

Zhen Li, Xuguo Zhou + Show 1 more

Open Access

https://doi.org/10.1038/srep27438

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Abstract

The pine wood nematode, Bursaphelenchus xylophilus, is the causal agent of pine wilt disease that has devastated pine forests in Asia. Parasitic nematodes are known to have evolved antioxidant stress responses that defend against host plant defenses. In this study, the infestation of whitebark pine, Pinus bungean, with B. xylophilus led to a significant increase in plant hydrogen peroxide (H2O2) and salicylic acid levels. Correspondingly, the expression of an antioxidative enzyme, 2-Cysteine peroxiredoxin (BxPrx), was elevated in B. xylophilus following the H2O2 treatments. Recombinant BxPrx, a thermal stabile and pH tolerant enzyme, exhibited high level of antioxidant activity against H2O2, suggesting that it is capable of protecting cells from free radical attacks. Immunohistochemical localization study showed that BxPrx was broadly expressed across different tissues and could be secreted outside the nematode. Finally, the number of BxPrx homologs in both dauer-like and fungi-feeding B. xylophilus were comparable based on bioinformatics analysis of existing EST libraries, indicating a potential role of BxPrx in both propagative and dispersal nematodes. These combined results suggest that BxPrx is a key genetic factor facilitating the infestation and distribution of B. xylophilus within pine hosts, and consequently the spread of pine wilt disease.

Highlights

Recombinant BxPrx protein was expressed in a Novagen’s pET-28a expression vector (Merck KGaA, Darmstadt, Germany) and purified with Ni-NTA agarose (Qiagen, Hamburg Germany)[28]
We schematically proposed the multiple functions of BxPrx in the nematode-pine arm race (Fig. 5)
The infestation of B. xylophilus can induce the production of H2O2 in pine hosts

Summary

Introduction

Recombinant BxPrx protein was expressed in a Novagen’s pET-28a expression vector (Merck KGaA, Darmstadt, Germany) and purified with Ni-NTA agarose (Qiagen, Hamburg Germany)[28]. Optimal conditions for BxPrx activity assay, including substrate specificity, pH tolerance, and temperature range, were determined enzymatically. The capability of recombinant BxPrx on the reduction of H2O2 and C9H12O2 was measured, respectively, at OD34057. To determine the optimum pH, BxPrx activity was measured between pH 3 and 10 at room temperature. Recombinant BxPrx was incubated for 30 min at a temperature gradient of 4, 20, 40, 60, 80, and 100 °C, respectively, before engaging in a BxPrx activity assay at pH 7. The activities of BxPrx were compared by one-way ANOVA and followed by a Tukey test using SPSS Statistics 17.0

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