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

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.

species oxidize DNA, proteins, and lipids, which causes damage to organelles and inhibits cell functions in plant attackers 13 . H 2 O 2 can cross the plasma membrane and activate signaling molecules to induce defensive genes and enzymes. Specifically, H 2 O 2 acts as a marker in pathogen recognition, causal trigger for hypersensitive reaction and apoptosis of host plants. Reactive oxygen species, particularly H 2 O 2 , is an important factor for regulation of host-nematode interactions and partly govern the success or failure of disease 14,15 . Salicylic acid (SA) is an important signaling molecule involved in systemic resistance by inducing the expression of pathogenesis-related (PR) proteins in plants 16 . A feature of SA signaling is its interaction with ROS in plants 17 . The increase of H 2 O 2 titer at the site of nematode penetration could induce the accumulation of SA. Vice versa, SA could regulate H 2 O 2 level through manipulating ROS-scavenging systems 18,19 . Durner et al. propose that H 2 O 2 might play a role in the activation of PR genes by interaction with SA 10 . Therefore, breaking down ROS defense is not only important to the initial invasion, but also facilitate its ongoing and persistent infestations by weakening the resistant in host plants. Neutralizing ROS is essential for parasitic nematodes to overcome plant defenses 20 . Consequently, the survival of nematodes is directly linked to the antioxidant enzymes 21 .
In many parasitic nematodes, 2-Cysteine peroxiredoxins (Prxs) are overexpressed and involved in H 2 O 2 detoxification 22 . In animal parasitic nematodes, the secretion of Prxs is considered an evolutionary adaptation to protect nematodes against host H 2 O 2 23 . Onchocerca volvulus, the parasitic nematode for 'river blindness' , excreted a 2-Cys Prx that localized within the hypodermis and cuticle in the infective larvae 24 . Among plant parasitic nematodes, 2-Cysteine peroxiredoxin (Prx2) has been found on the cuticle surface of infective and post-infective juvenile Globodera rostochiensis 25,26 , and excreted by Meloidogyne incognita when in close contact with plant cells 14 . Furthermore, silencing of Prx2 by RNAi led to the impairment of M. incognita development inside the host 14 . These results suggested that Prxs, especially Prx2, may play a critical role in plant-nematode interactions by counteracting plant defensive H 2 O 2 27 . Previously, a 2-Cys Prx gene from B. xylophilus, BxPrx, was cloned and its expression profile was analyzed 28 . Using an integrative approach, we comprehensively characterized BxPrx to gain a better understanding of its ecological and biological functions in the co-evolution of pine-nematode arm race. The combined results demonstrated that BxPrx plays a key role in the dispersal of B. xylophilus, acting as a scavenger for host-derived ROS attack in the propagative cycle, and may protect against its own metabolic H 2 O 2 in the dispersal cycle.

Results
Nematode, plant defensive compounds and BxPrx. The roles of plant secondary metabolites, including H 2 O 2 and SA, have been investigated in this study. H 2 O 2 titer in pine hosts were significantly increased after inoculation with B. xylophilus for 8 h, peaked at 12 h post inoculation, and high level of H 2 O 2 was sustained for about 16 h before it dropped back to the level of control pines (Fig. 1A). Similarly, SA titer in pine was significantly induced after inoculation with B. xylophilus, and it peaked at 24 h post-inoculation (Fig. 1A).
The stability of four candidate reference genes, including EF 1α (eukaryotic translation elongation factor 1α, accession number: GU130132), α-tubulin (GU130151), β-tubulin (AB500150), and β-actin (EU100952), under an array of hydrogen peroxide treatments were examined by quantitative real-time PCR (qRT-PCR) analysis. GeNorm analysis demonstrated β-actin (0.400) and EF 1α (0.493) had the lowest M-values, suggesting that this pair was the most stably expressed reference genes in B. xylophilus under oxidative stress (Table S1) 29 . BxPrx expression in B. xylophilus was found significantly induced in respond to elevated H 2 O 2 titer (0.1 ~ 1.0 mM) in the environment, although the expression of BxPrx started to decrease when H 2 O 2 concentration reached 1.0 mM (Fig. 1B).
Immunohistochemical localization study showed that BxPrx was abundantly expressed in B. xylophilus and predominantly distributed in excretory canal, and the two lateral glands (Fig. 2B). BxPrx also could be secreted Antioxidant activity of recombinant BxPrx. Recombinant BxPrx exhibited limited thione peroxidase (TPx) activity in a reaction system containing both thioredoxine (produced from E. coli, EcTx) and thione reductase (produced from E. coli, EcTR). In contrast, in a reaction system containing L-glutathione and glutathione reductase (GR), BxPrx showed substantial GPx activity (563.8 ± 41.6 units/mg protein) against H 2 O 2 , however, it did not reduce cumene hydroperoxide (C 9 H 12 O 2 ) (Fig. 3A). In optimal condition assays, BxPrx exhibited GPx activity between pH 5 and 9, with pH 7and 8 recorded the highest activities, whereas pH 3 and 10, respectively, led to a dramatic reduction of the antioxidant activity (Fig. 3C). BxPrx activity was stable across a wide range of temperature (4-60 °C), however, its antioxidant activity was significantly decreased when temperature reached 80 °C (Fig. 3D).
The conformational change of BxPrx from monomeric to dimeric protein was induced by the addition of H 2 O 2 (Fig. 3B). The rate of dimerization is positively correlated with H 2 O 2 concentration. Consequently, BxPrx is a typical 2-Cys Prx with the formation of an inter-molecule dimer to complete the catalytic cycle.
Recombinant BxPrx showed significant tolerance to the toxicity of H 2 O 2 oxidation (30% lower susceptibility than a blank, P < 0.05, Fig. 4A,B). DNA cleavage protection test demonstrated that BxPrx protected plasmid DNA against the free radical attack, and this protection weakened when the amount of BxPrx proteins declined. During a 4 h-observation, supercoiled plasmid DNA was best protected at a BxPrx concentration of 200 μ g/μ l. The damaged DNAs, such as nicked and slowly moving linear DNAs, became evident when less BxPrx was added into the reaction system. When BxPrx concentration reduced to 25 μ g/μ l, only the linear DNAs were detected (Fig. 4C).

Prx2s in nematodes with different ecological niches. Peroxiredoxin was first detected in the yeast
Saccharomyces cerevisiae, where its expression was induced by exposure to oxidative stress 31 . In free living bacterivore, Caenorhabditis elegans, expression of CePrx2 was induced by short-term exposure to t-butyl hydroperoxide (t-BOOH) 32 . In an animal parasitic nematode, Haemonchus contortus, HcPrx2, exhibited high Km toward H 2 O 2 , suggesting that HcPrx2 can protect the nematode against the host immune response 23 . Similarly, in a plant parasitic nematode, G. rostochiensis, a 2-cys peroxiredoxin could breakdown H 2 O 2 and was located on the surface of invasive and post-infective juveniles, suggesting that Prx may play a role in protection against plant defensive responses 25 . MiPrx2, a Prx2 from the other plant parasitic nematode, M. incognita, was located in tissues in close proximity with plant cells during parasitism. The development of nematode was impaired after the silencing of MiPrx2 14 . Bursaphelechus xylophilus has a complex life cycle and unique feeding behavior in comparison to other plant parasitic nematodes which feed primarily on plant tissues for nutrients. It resides two distinctly different habitats: 1) insect vectors for dispersal and foraging, and 2) pine hosts for food, shelter, and proliferation. Moreover, B. xylophilus also exploits a wide range of fungi as food source which occupy the dying pine hosts caused by the infestation of B. xylophilus 7 . Thus, we hypothesize that genetic factors which suppress or counter the innate immune responses from the pine hosts are essential for the infestation and distribution of B. xylophilus. BxPrx, a conserved 2-Cys peroxiredoxin protein with notable antioxidant activity 28 , is likely the genetic factor facilitating B. xylophilus to circumvent the host immune response to ensure a successful infestation.

Host innate immune response to B. xylophilus infestation. H 2 O 2 titers and SA levels were induced in
pine seedlings against the infestation of B. xylophilus. The induction of SA was lagging behind the release of H 2 O 2 , indicating that ROS was likely to be the first line of defense in pine host innate immune response to B. xylophilus infestation. As one of the ROS species, H 2 O 2 is an early stress signal interacting with a network of signal transduction in plants, including SA pathway to promote defensive responses and systematic acquired resistance (SAR) against pathogens 33 Antioxidant activity of BxPrx. BxPrx has significant antioxidant activity toward H 2 O 2 in a reaction system containing L-glutathione and GR, but limit activity in a system composed of EcTx and EcTR or on organic oxidants. Some human and animal parasites can use both reaction systems for regeneration, such as Schistosoma mansoni 37 and Plasmodium falciparum 38 , although HcPrx could not utilize the glutathione system directly 23 . Prxs in the potato cyst nematode G. rostochiensis and plant parasitic root-knot nematode M. incognita exhibited notable antioxidant activity in a system that used both trypanothione and thiocyanate as thione donors 14,25 . MiPrx2, CePrx2 and HcPrx2 all exhibited strong activities for the reduction of H 2 O 2 and organic oxidants. Similar to BxPrx, Prx in G. rostochiensis was not active against organic oxidants 25 . The different substrate specificity exhibited by these Prxs may be correlated with the habitats occupied by these nematodes. In animals, acquired immunity shows its major capacity in suppressing pathogen infection, however, plants mainly rely on innate immune responses, including ROS and accompanied signal molecules to initiate SAR 39,40 . Prxs, with strong antioxidant activity to breakdown H 2 O 2 and protect DNA from oxidative damage, facilitate B. xylophilus infestation by down-regulating the defensive responses in pine hosts.
Bursaphelenchus xylophilus, an invasive nematode with a wide range of distribution 41 , shows strong tolerance toward cold and heat stresses 5,42 (11 to 32 °C). In this study, recombinant BxPrx were active against H 2 O 2 under a broad range of temperature and pH, and protected DNA from oxidative damages. Based on the catalytic mechanisms and the presence of either one or two highly conserved cysteine residues, Prxs are categorized into 1-Cys, typical 2-Cys, and atypical 2-Cys Prxs 43   demonstrated that virulent B. xylophilus isolates survived better than avirulent counterparts under the oxidative stress 9,48 . The bacteria symbiont Serratia spp 9 . and elevated catalase expression in the intestine of B. xylophilus 49 contribute their tolerance towards the oxidative stress and facilitate the infection of pine wood disease. In this study, BxPrx expression was significantly induced by the increase of environmental H 2 O 2 titer. Recombinant BxPrx showed high levels of antioxidant activity to breakdown H 2 O 2 and protect DNAs from oxidative radical attacks. Vice versa, decreased H 2 O 2 level also reduce the accumulation of SA and the subsequent host plant innate immune responses. In addition, BxPrx were broadly expressed under cuticle surface where is in close contact with pine hosts, in excretory canals and two lateral glands which are the main secretory organs in nematodes. BxPrx could also be secreted outside the nematode through stylet. These combined results suggest that BxPrx plays a critical role in protecting B. xylophilus against pine host-derived H 2 O 2 .
Prx2 is also essential for the development and reproduction of nematodes. Silencing of CePrx2 in C. elegans resulted in a stunted development and a significantly reduced brood size 32 . Similarly, knocking down BxPrx significantly reduced the propagation in B. xylophilus 50 . Our previous study found that anti-serum of BxPrx could only detect protein samples extracted from virulent B. xylophilus but not B. mucronatus 28 , suggesting that BxPrx could be used as a molecular marker to identify the virulent B. xylophilus 50 . A shorter life cycle and higher proliferation rate are the fundamental differences between avirulent and virulent B. xylophilus, therefore, BxPrx could play a role in the pathogenicity of B. xylophilus. Different gene clusters were identified between the EST libraries derived from dauer-like and fungi-feeding B. xylophilus 30 . Many homologs of antioxidant genes, including SOD, GPX and CAT, which were involved in longevity in C. elegans 32 , were found with notably reduced number in dauer-like EST library compared to fungi feeding B. xylophilus 30 . In contrast, BxPrx isoforms were similar between dauer-like and fungi-feeding EST libraries. BxPrx expression was consistent in the dauer stage, a life stage that is structurally and physiologically adapted for the long-term survival and dispersal without much food sources, suggesting that BxPrx may play an important role against the nematode's own metabolic ROS for survival during its dispersal cycle.

Summary.
To summarize our finding, we schematically proposed the multiple functions of BxPrx in the nematode-pine arm race (Fig. 5). During the propagative cycle, the infestation of B. xylophilus can induce the production of H 2 O 2 in pine hosts. Besides direct toxicity to B. xylophilus, the accumulation of H 2 O 2 triggers the elevation of SA. It will also induce the expression of resistance genes and initiate the SAR pathway in pine hosts. In the meantime, however, the elevated H 2 O 2 level in pine hosts likely induces BxPrx expression in nematodes. Synchronized secretion of BxPrx, in turn, reduces H 2 O 2 level at the micro-environment surrounding B. xylophilus in pine hosts and thus protecting nematodes from oxidative damages. This can also reduce the SAR response in pine hosts through the suppression of H 2 O 2 to a level that the induction of SA pathway is arrested. Therefore, in the propagative cycle, BxPrx plays a critical role in protecting B. xylophilus against ROS attack from hosts. After infestation and proliferation of B. xylophilus, hosts show symptoms of wilt and nematodes turn to feed on a variety of fungi occupied in the decaying pine materials. When fungal food sources become scarce, the pre-dauer LIII nematodes congregate around the M. alternatus pupae and latch onto the newly emerged adults. The dauer LIV nematodes complete the dispersal cycle through the maturation of the adult pine sawyer beetle on healthy trees. ROS generated from the metabolism can threaten the survivorship of nematodes in the dispersal period. In the dauer stage, antioxidative BxPrx protects nematodes from their own metabolic attacks for a longer life span and a broader distribution during the dispersal cycle.
In conclusion, we suggest that BxPrx is a key genetic factor that facilitates the infestation and distribution of B. xylophilus within pine hosts, and consequently the spread of pine wilt disease. With the advent of functional genomics tools, including RNA interference (RNAi) 51 and transgenic approach 50 , genetic basis governing the transmission and infection of pine wilt disease is a logic choice for future research. A better understanding of how nematodes overcome oxidative stresses generated from their own metabolic processes and from the innate immune responses in pine hosts will provide novel targets for the long-term, sustainable management of this devastating pine disease.

Materials and Methods
Biological materials. The B. xylophilus isolate JSZJ1-6 (Sampled from Jiangsu, Zhejiang province 1 ) was maintained as described previously 52 . Seedlings of 1-2 year-old whitebark pine, Pinus bungean (20-40 cm in height and 5-8 cm in diameter), were maintained in a green house with a long-day photoperiod (light/dark 16:8) at 25 °C for 1.5 months until new shoots start to grow.

Interactions between the pine hosts and nematodes. Change of H 2 O 2 and SA level in pine host
responding to B. xylophilus infestation. The innate resistance of P. bungean was investigated by an artificial infestation (Fig. S1). Pine seedlings were inoculated with 20,000 B. xylophilus per plant 53 using M9 buffer (KH 2 PO 4 30 g/L, K 2 HPO 4 60 g/L, NaCl 50 g/L) as a control. Leaves were sampled at 0, 4,8,12,24, and 36 h after inoculation, and snap frozen at − 80 °C for the subsequent H 2 O 2 and SA analysis. H 2 O 2 concentration was determined as described previously by Brennan and Frenkel 54 . In brief, 0.5 g of frozen pine needles were ground in 1 ml acetone and centrifuged at 3,000 g for 10 min. After mixing the supernatant with 20% TiCl 4 -HCl solution and ammonium hydroxide, the resultant pellet was harvested and resuspended in 2 M H 2 SO 4 . H 2 O 2 concentration was measured by A 410 of the titanium-peroxide complex. Free SA was extracted as described previously 55 . A total of 0.5 g of frozen needles were homogenized in 3 ml of 90% methanol and kept soaking at − 20 °C for overnight. After centrifugation at 4 °C, 8,000 rpm, for 30 min, the supernatants were collected and dried in a speed vacuum with heat (~40 °C). The residue was resuspended in 2.5 ml of 5% trichloroacetic acid and sonicated for 10 min. Free SA was then separated from conjugated SA through organic extraction with 2 volumes of ethyl acetate-cyclohexane-isopropanol (50:50:1). The organic phase containing free SA was then dried using a speed vacuum, resuspended in 400 μ l of acetonitrile, filtered, and measured by reverse-phase HPLC (HP1100 Agilent, USA). An Agilent-C18 column ( BxPrx expression profile under oxidative stresses. To select the suitable reference gene(s) for the subsequent qRT-PCR analysis, the stability of four housekeeping genes, EF 1α (accession number: GU130132), α-tubulin (GU130151), β-tubulin (AB500150), and β-actin (EU100952), under the hydrogen peroxide treatments were evaluated by geNorm 29 . The end point measurement of this conventional statistical software is M-value, the variation of a gene compared to all other candidates. Based on the pair-wise comparison, geNorm automatically eliminates the gene with the highest M-value, and repeats the process until there is only two genes left. The last pair remaining is considered as the optimum pair of reference genes 29 . The PCR primers were listed in Table S2, and the PCR was performed with 40 cycles of 94 °C (30 s), 58 °C (45 s), and 72 °C (30 s).
Bursaphelenchus xylophilus of mixed developmental stages were incubated with various concentrations of H 2 O 2 for 5 min 56 . Total RNA extraction and cDNA synthesis were carried out as described previously 28 . BxPrx (GenBank accession number: EU095848) expression profile under oxidative stresses was investigated using qRT-PCR with primers listed in Table S2. qRT-PCR was performed on a 7300 Real-Time PCR System (Applied Biosystems, Foster City USA). The 2 −ΔΔCt method was used to calculate the expression level of BxPrx. SPSS Statistics 17.0 (SPSS Inc., Chicago, USA) was used for the statistical analyses. Since the data did not fit homoscedasticity, BxPrx expression level under oxidative stress was assessed by one-way ANOVA and followed by a Games-Howell test.
Immunohistochemical localization of BxPrx. Bursaphelenchus xylophilus of mixed developmental stages were collected by centrifugation at 2,000 rpm for 1 min, and washed with M9 buffer for 3 times. The resultant nematode samples were fixed, dehydrated, cut to ultrathin sections, hybridized with antiserum, and observed in transmission electron microscope 28 .

Antioxidant activity of recombinant BxPrx. Biochemical characterization of recombinant
BxPrx. 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 H 2 O 2 and C 9 H 12 O 2 was measured, respectively, at OD 340 57 . The reaction system contained 5 mM potassium phosphate (pH 7.0), 1 mM EDTA, 0.1 mM NADPH, and with 16 μ M EcTx (Sigma) and 0.23 μ M EcTR ( Sigma) for the TPx activity assay; or 1 mM L-glutathione (Sigma) and 0.1 unit/ml GR (Sigma) for the GPx activity assay. To determine the optimum pH, BxPrx activity was measured between pH 3 and 10 at room temperature. For optimal temperatures, 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 (SPSS Inc., Chicago, USA).
Immunoblot analysis. Bursaphelenchus xylophilus was treated with H 2 O 2 and harvested for the assay as previously described by Oláhová 58 . Nematodes were incubated in 0, 1 and 5 mM H 2 O 2 solution separately for 5 min, and then washed with M9 buffer for 3 times. Nematodes were then harvested in 20% trichloroacetic acid (TCA) before snap-frozen in liquid nitrogen. Thawed pellets were washed with acetone to remove TCA, dried and resuspended in SDS-PAGE loading buffer. The resultant samples were then separated through 12% SDS PAGE and analyzed by Western blotting using an anti-BxPrx serum 35 . Band intensity was analyzed using Quantity One 4.6.2 (Bio-Rad, USA).
Antioxidant activity assay. Antioxidant activity of BxPrx transformants was assayed following Isermann 32 with minor modifications. Escherichia coli was spread onto the top of a LB (Lysogeny Broth) agar plate (LB medium supplemented with a 2 mm of 0.8% agarose layer below, 1 mM IPTG, and 100 μ g/ml ampicillin inside), and then incubated at 37 °C for 3 h. Subsequently, oxford disc (1 cm in diameter) was inserted into the middle of the LB medium and positioned upon the agarose layer. A total of 100 μ l of 1 mM H 2 O 2 was then added onto the disc. The resultant plates were placed at the room temperature for 16 h without moving until all the H 2 O 2 infiltrated into the LB medium, and then plates were transferred to 37 °C. After 24 h of incubation, the diameters of the killing zones were measured. The viability of E. coli transformats with or without BxPrx was compared using a paired-samples T test (SPSS17. 0, SPSS Inc., Chicago, USA).
DNA cleavage protection assay. According to Stacy 59 , various concentrations of recombinant BxPrx was first incubated in a reaction mixture containing 250 mM Hepes, 5 mM EDTA, 100 mM DTT, and 600 μ M FeCl 3 , at 37 °C for 30 min, and then 250 ng of pUC19 plasmid was introduced into the reaction system (Tiangen, Beijing, China). The status of pUC19 with different incubation conditions was resolved by agarose gel electrophoresis (1%).
Transcripts abundance of antioxidant enzymes in B. xylophilus. Transcripts abundance of antioxidant enzymes was analyzed by searching and comparing the number of homologs between EST libraries generated from dauer-like and fungi feeding B. xylophilus. The sequence information of the two EST libraries was downloaded from NCBI database, and the retrieval of the homologs was conducted using software BioEdit (Ibis Biosciences, Carlsbad, CA).