Catalases Induction in High Virulence Pinewood Nematode Bursaphelenchus xylophilus under Hydrogen Peroxide-Induced Stress

Considered an EPPO A2 quarantine pest, Bursaphelenchus xylophilus is the causal agent of the pine wilt disease and the most devastating plant parasitic nematode attacking coniferous trees in the world. In the early stages of invasion, this nematode has to manage host defence mechanisms, such as strong oxidative stress. Only successful, virulent nematodes are able to tolerate the basal plant defences, and furthermore migrate and proliferate inside of the host tree. In this work, our main objective was to understand to what extent B. xylophilus catalases are involved in their tolerance to oxidative stress and virulence, using as oxidant agent the reactive oxygen species hydrogen peroxide (H2O2). After 24 hours of exposure, high virulence isolates of B. xylophilus could withstand higher H2O2 concentrations in comparison with low virulence B. xylophilus and B. mucronatus, corroborating our observation of Bxy-ctl-1 and Bxy-ctl-2 catalase up-regulation under the same experimental conditions. Both catalases are expressed throughout the nematode intestine. In addition, transgenic strains of Caenorhabditis elegans overexpressing B. xylophilus catalases were constructed and evaluated for survival under similar conditions as previously. Our results suggest that catalases of high virulence B. xylophilus were crucial for nematode survival under prolonged exposure to in vitro oxidative stress, highlighting their adaptive response, which could contribute to their success in host conditions.


Introduction
From 1905 until the present time, pine wilt disease (PWD) has been one of the most threatening diseases for worldwide forests [1]. PWD is considered a complex disease resulting from the interaction of three main elements: the pathogenic agent Bursaphelenchus xylophilus (PWN, pine wood nematode), the insect-vector Monochamus spp. (responsible for PWN dissemination), and the host tree, mostly from Pinus spp. [2][3]. In a B. xylophilus population, there is intra-specific variability in the ability to cause PWD (virulence) [4]. The existence of different levels of virulence among PWN isolates, and their relation to PWN reproductive ability in vitro and in vivo were already reported [5][6]. In this context, the low virulence nematodes, often designated as avirulent, show lower reproduction rates and increased generation times that eventually affect their host invasion and colonization [7][8][9].
In the early stage of PWD, PWN has to cope with different levels of plant immune responses. The first, non-specific, host tree reaction to nematode invasion (or other biotic/ abiotic stress) is oxidative burst, an excessive production of reactive oxygen species (ROS). The predominant ROS in plant oxidative burst is H 2 O 2 [10][11]. H 2 O 2 is relatively stable, cell wall diffusible, and found to be transversal in different plant-pathogens systems, being a fundamental signal for inducing plant resistance (i.e. involved in cell-wall reinforcement or induction of defence-related genes in healthy adjacent tissues) [12]. H 2 O 2 is particularly important since it is extremely reactive and can lead to the formation of radical hydroxyl OH -, for which no specific scavenger exists [12]. Generally, parasitic nematodes respond to an oxidant threat by increasing their protective antioxidant enzyme levels [13]. The major antioxidant enzyme families considered are: superoxide dismutases (SOD, EC 1.15.1.1) for conversion of superoxide anion to H 2 O 2 , glutathione peroxidases (GPX, EC 1.11.1.9), catalases (CTL, E.C. 1.11.1.6) and peroxiredoxins (PRDX, EC 1.11.1.15) for the conversion of H 2 O 2 into water [14]. Furthermore, many parasites employ many enzyme groups under oxidative stress conditions [14]. In plant parasitic nematodes, Molinari and Miacola [15] showed the presence of many antioxidant enzymes and the relation to its lifestyle stages of Meloidogyne incognita and M. hapla, Globodera rostochiensis and G. pallida, Heterodera schachtii and H. carotae, and Xiphinema index. Robertson et al. [16] described an important role of PRDX and GPX in G. rostochiensis. Bellafiore et al. [17] reported the presence of several detoxifying enzymes, in particular several glutathione S-transferases (GST, EC 2.5.1.18) in the secretome of M. incognita as means for controlling the global oxidative status and potential nematode virulence. PWN also has an efficient antioxidant system to reduce the deleterious effects of tree oxidative burst [18], mostly present in the nematode secretome [19] and expressed in the cuticle [20].
Pine oxidative burst was found intrinsically related with the production of ROS in the first stages of B. xylophilus invasion [21][22]. He et al. [23] evaluated the content of H 2 O 2 of P. thunbergii, P. massoniana and P. taeda at the early stage of B. xylophilus invasion, and found that H 2 O 2 content was higher in the susceptible pines than the resistant P. taeda. In this study, we specifically analyzed the tolerance of B. xylophilus isolates with different levels of virulence (high and low virulence) under in vitro oxidative stress conditions by assessing the effects of H 2 O 2 exposure to nematode survival, catalase activity, catalase gene expression and their spatial localization. Additionally, we expressed B. xylophilus in the nematode model C. elegans to explore the efficiency of B. xylophilus catalases in promoting survival.

B. xylophilus isolates and culturing
Two B. xylophilus isolates with high virulence (Bx Ka4 and Bx T4) and one isolate with low virulence (Bx C14-5) [6] were used in this study. In addition, one isolate of B. mucronatus (Un1), considered a low virulence nematode, was also used. All isolates were supplied by the FFPRI (Forestry and Forest Products Research Institute). The origin of nematode isolates Bx Ka4 and Bx C14-5 is described by Aikawa and Kikuchi [6]. The isolate Bx T4 was collected from dead Pinus densiflora in 1992, in Iwate prefecture, Japan and B. mucronatus Un1 was obtained from Monochamus alternatus, in the Kyoto prefecture.
Nematodes were cultured in Botrytis cinerea on autoclaved barley seed at 25°C. Prior to experiments, nematodes were extracted overnight using Baermann funnel technique at 25°C. Nematodes were washed three times with sterilized distilled water with 10 min. centrifugations at 1,000 rpm between washes, surface cleaned with 3% L-lactic acid for 30s, and washed once with sterilized distilled water [9]. Mix-stages nematodes were used in all experiments.
Oxidative stress tolerance test H 2 O 2 was used as oxidative agent at concentrations ranging between 15 and 40 mM for Bursaphelenchus isolates. After nematode surface sterilization, the concentration of Bursaphelenchus was adjusted to 150 nematodes per 50 μl of sterile water. Mortality of nematodes was scored after 24h-stress exposure. Nematodes were considered dead if no movements were observed after mechanical stimulation. This experiment was repeated three independent times, with two technical replications for each.

H 2 O 2 neutralization
H 2 O 2 neutralization was inferred using an H 2 O 2 Assay Kit (abcam, Massachusetts, USA). For this purpose, 150 nematodes were exposed for 24h in 15 mM H 2 O 2 . Afterwards, nematodes were pelleted at 1,000 g for 15 min and the supernatant was used for analysis following the manufacture's protocol. This assay was repeated three independent times with two technical replications.

Relative gene expression of catalases under oxidative stress
Relative gene expressions of CTL (catalase) enzymes (Bxy-ctl-1 and Bxy-ctl-2), previously predicted by Vicente et al. [24], were analyzed by qRT-PCR using SYBR green assay. Total RNA was extracted from 24h-stressed nematodes (approximately 2,500) in 15 mM H 2 O 2 using Cel-lAmp Direct RNA Prep Kit for RT-PCR (Takara Bio Inc., Japan) and following manufacture's instructions. The concentration was quantified using NanoVue plus spectrophotometer (GE Healthcare Life Sciences, USA). Total RNA (adjusted to a concentration of 50 ng/μl) was reverse transcribed using oligo dT primer and PrimeScript RT enzyme from PrimeScript RT reagent Kit (Perfect Real Time, Takara Bio Inc., Japan). qRT-PCR was performed using CFX96 Real-Time (Bio-Rad Laboratories, Inc., California), and SYBR Premix Ex Taq II (Tli RnaseH Plus) kit (Takara Bio Inc., Japan). Actin (Bxy-act-1) was used as a reference gene for calculating relative expression levels of CTLs genes [25]. Primers were designed using Prime 3 software [26] and tested for specificity prior to qRT-PCR (Table 1). For each treatment, two experimental replications were conducted with two technical replications each. Controls with no template added were prepared for each qPCR run. Thermal cycling conditions were: initial denaturation at 95°C for 30 sec; 39 cycles of denaturation at 95°C for 5 sec, annealing and extension at 60°C for 30 sec; followed by the melting curve. A single peak at the melting temperature of the PCRproduct confirmed primer specificity.

In-situ hybridization
Single-stranded DNA probes (sense and antisense) labelled with digoxigenin (Roche, United States of America) were used to detect mRNA in mix-stage B. xylophilus as described by De Boer et al. [28]. Table 1 presents the primers used to generate Bxy-CTL-1 and Bxy-CTL-2 probes ( Table 1). As positive control for successful hybridization, ENG (cellulase) probe was also prepared as described by Kikuchi et al. [29]. Images were acquired on an Olympus Bx50 light microscope at 10x-40x magnifications.
Transgenic C. elegans construction C. elegans culturing and handling were carried out at 20°C as described by Brenner [30]. Strains used in this experiments were N2 (Bristol strain) and DP38 unc-119(ed3)III. To make reporter constructs, all PCRs were performed with Takara PrimeSTAR GXL DNA polymerase (Takara Bio Inc., Japan). B. xylophilus ctl-1 and -2 cDNAs were fused with C. elegans ctl-1 or -3 promoters and ligated into the gfp vector pPD95.77 (kindly provided by A. Fire, Stanford University) with In-Fusion HD Cloning system (Takara Bio Inc., Japan). We cloned a 1,940 bp sequence upstream from the Ce-ctl-3 start codon for Ce-Pctl-3, and a 1,600 bp sequence between Ce-ctl-3 and Ce-ctl-1 CDS (coding sequences) for Ce-Pctl-1. Fusion genes created in this experiments were Pctl-1::Bxy-ctl-1::gfp and Pctl-3::Bxy-ctl-2::gfp, and PCR primer sequences are listed in Table 2. Each reporter construct (100 μg/ml) was co-injected with an equal concentration of pDP#MM016B into the gonadal arms of unc-119(ed3) adult hermaphrodites to obtain  fluorescence expression patterns with a Nikon SMZ800 dissection microscope equipped with a fluorescence filter and a ZEISS LSM710 confocal laser-scanning microscope.

Oxidative stress tolerance in C. elegans
Synchronized L1 stage C. elegans were obtained by treating egg-containing/or gravid adults with sodium hypochlorite [30] and allowed to grow on NGM plates seeded with E. coli OP50 at 20°C for 48 hours until the late L4 stage. L4 stage C. elegans were washed with M9 buffer and adjusted to 20 nematodes per 50 μl of M9 buffer. Nematodes were transferred to a 96-well plate containing each concentration of H 2 O 2 (0 to 250 μM). The plate was incubated at 20°C for 24 hours, and then examined for animal viability under a stereomicroscope. Since our transgenic nematodes used in this experiment retained transgenes as an extrachromosomal DNA array, non-transgenic uncoordinated nematodes appeared at a certain proportion (around 30%). We excluded non-transgenic uncoordinated animals when performing this experiment. All tests were carried out at 20°C. This experiment was repeated three independent times, and in each experimental repetition, two technical replications were used.

Data analysis
Statistical analysis was performed using SPSS version 11.5. Relative gene expressions of CTLs were analysed using the 2 -ΔΔCT method [25]. The data were analysed with C T (cycle threshold) values in normal and stress conditions and using Eq. (1), where ΔΔC T = (C T,Target -C T,Actin ) normal -(C T,Target -C T,Actin ) stress . The fold change of CTL genes, normalized to β-act and relative to the expression at normal conditions, were calculated for each sample using Eq. (1). Data represent the mean ± standard deviation (SD). Statistical differences at 95% and 99% confidence levels were inferred by Student t-test by comparison between relative expression of both CTLs for each B. xylophilus isolate and the control treatment without H 2 O 2 .

Oxidative stress tolerance and H 2 O 2 neutralisation
B. xylophilus (high virulence: Bx Ka4 and Bx T4; low virulence: Bx C14-5) and B. mucronatus (low virulence: Bm Un1) isolates were tested in increasing concentrations of H 2 O 2 (0, 15, 20, 30 and 40 mM). After a 24h-exposure to this oxidant, nematode survival was examined (Fig 1) In terms of H 2 O 2 neutralization (Fig 2), high virulence Bx Ka4 and Bx T4 were more efficient at H 2 O 2 reduction (p < 0.05) in comparison with the low virulence Bm Un1 and Bx C14-5 (no statistical differences between each pair).  Gene expression analysis B. xylophilus catalases (Bxy-ctl-1 and Bxy-ctl-2; Table 1) were previously analyzed and found to be conserved among Nematoda [24]. The CDS of both CTLs were similar between high and low virulence B. xylophilus isolates (data not shown). Because the lack of B. mucronatus genome in databases prevented the analyses of gene expression for the Bm Un1 isolate, our studies about levels of virulence will only focus on the Bx Ka4, Bx T4 and Bx C14-5 isolates. Relative gene expression of the CTLs was analyzed after a 24h-exposure to 15 mM H 2 O 2 (Fig 3). High virulence isolates Bx Ka4 and Bx T4 presented a higher induction of CTL expression than low virulence Bx C14-5. In fact, the relative expression of both Bxy-ctl-1 and Bxy-ctl-2 of Bx C14-5 in stress conditions was not significantly different (p > 0.05) from expression in non-stress conditions. In oxidative conditions, Bxy-ctl-1 and Bxy-ctl-2 from both high virulence isolates Bx Ka4 and Bx T4 were significantly induced (0.01< p < 0.05) in comparison with non-stress conditions ( Fig  3A and 3B). Both Bxy-ctl-1 and Bxy-ctl-2 of Bx Ka4 were 2.5-fold up-regulated. Bxy-ctl-1 of Bx T4 was significantly induced, nearly 2-fold ( Fig 3A). Bxy-ctl-2 was also significantly up regulated (p < 0.05) in stress, nearly 4-fold more than in normal conditions (Fig 3B).
Under the control of C. elegans promoters, B. xylophilus Bxy-ctl-1 and Bxt-ctl-2 showed different spatial expression patterns; ctl-1 was mainly detected in the cytosol of the intestine, and ctl-2 was detected as high levels along the nervous system and pharynx (Fig 5A and 5B). These expression patterns were the same with those previously reported [31]. Although these transgenic C. elegans were extrachromosomal lines with unstable transgene transmission, the expression patterns were similar between the transgenic lines KHA149 and 150, and between KHA151 and 152 (data not shown). We used KHA149 and 151 for further experiments.
To gauge C. elegans (wild-type and transgenic) tolerance to oxidative stress conditions, they were exposed to H 2 O 2 concentrations between 50 and 500 μM, substantially lower than the concentrations used for B. xylophilus. The survival of wild-type C. elegans N2 decreased significantly (p < 0.05) with increasing concentrations of H 2 O 2 (Fig 6). Interestingly, both of the transgenic C. elegans overexpressing B. xylophilus catalases were resistant to H 2 O 2 stress conditions. KHA149 and KHA151, expressing Bxy-ctl-1 and Bxy-ctl-2 respectively, were not statistically different (p > 0.05) in the H 2 O 2 conditions tested.

Discussion
Depending on their lifestyle, sedentary or migratory, plant parasitic nematodes possess different repertoires of enzymatic and non-enzymatic molecules involved in oxidative stress metabolism that are a reflection of their pathogenicity [15] In some organisms, like bacteria, these enzymes are defined as virulence factors [32]. Recent work [18] indicates that B. xylophilus has an enzymatic inventory to counterattack the pine oxidative burst. A total of 12 anti-oxidant enzymes were found in the B. xylophilus secretome, including PRX, CTL, GST, SOD, nucleoredoxin-like protein and thioredoxin [19]. In the present study, we investigated the tolerance of B. xylophilus isolates with different levels of virulence (high and low virulence) under in vitro oxidative stress conditions using H 2 O 2 as an oxidizing agent. We were able to observe a contrasting performance between high and low virulence B. xylophilus and its relative B. mucronatus, corroborating previous results in other plant parasitic nematodes [33][34]. Emphasizing the results of the oxidative stress tolerance and H 2 O 2 neutralization tests, the CTL (Bxy-ctl-1 and Bxy-ctl-2) gene expression indicated that high and low virulence B. xylophilus respond differently towards H 2 O 2 stress. High virulence nematodes showed an up-regulation of CTL genes under induced oxidative stress conditions, corroborating our preliminary results [24]. In addition, since all B. xylophilus isolates share protein sequence similarity between CTLs, the contrasting responses to H 2 O 2 stress may indicate different regulation in the detoxification of ROS. This correlation between catalase levels and in vitro H 2 O 2 tolerance has also been described in other parasitic nematodes [35][36]. Previously, Shinya et al. [20] showed the accumulation of anti-oxidant and detoxifying enzymes on the body surface of the nematode. Here, we were able to determine that both B. xylophilus catalases (Bxy-ctl-1 and Bxy-ctl-2) are expressed in the intestine. We also accounted for other antioxidant enzymes (SODs and GXPs) under H 2 O 2 stress (S1 Fig), and found that, in our experimental conditions, only catalase expressions were significant. Although we could ascertain the importance of CTLs (Bxy-ctl-1 and Bxy-ctl-2) in the protection against H 2 O 2 induced-stress, we cannot rule out the effect of other antioxidant proteins in their response. Another important observation in this study is the maintenance of CTL induction in high virulence B. xylophilus even after 24h of H 2 O 2 exposure. Kotze [37] observed similar CTL induction in parasitic nematode Haemonchus contortus against in vitro H 2 O 2 stress. This feature is crucial and highlights the adaptive response and antioxidant protection of high virulence B. xylophilus in host conditions. Further studies are still needed to completely understand the role of all protectant molecules (enzymatic or non-enzymatic) of B. xylophilus against the massive ROS production of susceptible pines.