Meloidogyne graminicola protein disulfide isomerase may be a nematode effector and is involved in protection against oxidative damage

The rice root-knot nematode, Meloidogyne graminicola, is a serious pest in most rice-growing countries. Usually, nematodes employ antioxidants to counteract the harm of reactive oxygen species (ROS) and facilitate their infection. Here the gene encoding M. graminicola protein disulphide isomerase (MgPDI) was identified. The deduced protein is highly conserved in the putative active-site Cys-Gly-His-Cys. In situ hybridization showed that MgPDI was specifically localized within esophageal glands of pre-parasitic second stage juveniles (J2s). MgPDI was significantly up-regulated in the late parasitic J2s. Characterization of the recombinant protein showed that the purified MgPDI exhibited similar activities to other oxidases/isomerases such as the refolding of the scrambled RNase and insulin disulfide reductase and the protection of plasmid DNA and living cells from ROS damage. In addition, silencing of MgPDI by RNA interference in the pre-parasitic J2s lowered their multiplication factor. MgPDI expression was up-regulated in the presence of exogenous H2O2, whereas MgPDI silencing resulted in an increase in mortality under H2O2 stress. MgPDI is localized in the apoplast when transient expression in Nicotiana benthamiana leaves. The results indicated that MgPDI plays important roles in the reproduction and pathogenicity of M. graminicola and it also contributes to protecting nematodes from exogenous H2O2 stress.


Results
Cloning and sequence analysis of MgPDI. The full-length MgPDI was identified through RACE-PCR and was deposited in GenBank under accession number MH392200. The ORF of the MgPDI was from nucleotide 170-1666, encoding a putative protein of 498 amino acids with a deduced signal peptide cleavage site between residues 20 and 21. The predicted isoelectric point and molecular weights were 7.02 and 56.96 kDa, respectively. Further domain analysis revealed that the putative protein contained four conserved thioredoxin domains (a, b, b' , a') with two catalytic domains (CGHC) (Fig. 1a,b). Protein disulphide isomerase has been found in humans and other animals and plant-parasitic and free-living nematodes. Results of a BLASTP search showed that MgPDI shared 79%, 71%, 69%, 32% and 33% identity to PDI from Heterodera schachtii, Caenorhabditis elegans [NP_491995], Ascaris suum [ERG84937], Leishmania major [AAN75008], and Brugia malayi [XP_001899304], respectively.
In their tertiary structure, the four domains are found to be spatially organized in the shape of a twisted "U", in which the a and a' domains are located at the ends while the b and b' domains formed the base of the "U"; Four conserved thioredoxin domains (a, b, b′, a′) are highlighted in purple, blue, yellow and red, respectively; The C-terminal extension is in green; Two catalytic domains (CGHC) are highlighted in the MgPDI tertiary structure as colored spheres. One sphere represents one of the atoms in the corresponding amino acids (Fig. 1b).
In situ hybridization and expression analysis. In situ hybridization was performed to localize the expression of MgPDI in the pre-parasitic juveniles of M. graminicola. The DIG-labelled antisense probe of MgPDI specifically hybridized with transcripts in the subventral gland cells of the pre-parasitic J2s (pre-J2s), while no hybridization was observed in the negative control, the labeled sense probe (Fig. 2a). To further investigate the expression pattern of MgPDI during different developmental stages of M. graminicola, we used cDNA generated from nematode RNA isolated at different pre-parasitic (eggs and freshly hatched J2s) and parasitic developmental stages (par-J2s, par-J3s/par-J4s and young females) in qRT-PCR analyses. The expression level of MgPDI increased during the sedentary stages of nematode development, reaching its maximum in the late parasitic J2s (5 days post-infection, 5dpi) with a 6-fold increase compared with pre-parasitic J2s. In J3/J4s and young females, expression decreased but was still higher when compared with unhatched J2s in eggs and hatched pre-parasitic J2s (Fig. 2b).

Recombinant MgPDI: optimization and validation.
We expressed MgPDI in E. coli BL21 (DE3) cells ( Supplementary Fig. S1). Isopropyl-B-D-thiogalactoside induced the E. coli expression of the recombinant protein (Fig. 3, lane 2). To exclude the possible effects of the tags on the activity of MgPDI, thrombin was used to cleave N-terminal fusion tags and the recombinant protein yielded an enriched nontagged protein band after purification (Fig. 3, lane 3). The final concentration of MgPDI was 2 mg/ml after removing the thrombin. Mass spectrometry analysis was used to validate the identity of the expressed protein. The purified recombinant protein exhibited high sequence homology with the peptide fragments deduced from MgPDI where the score was 11512.77% and coverage was 72.09% (Supplementary Table S1).
Oxidoreductase activity of MgPDI. Oxidase/isomerase activity was analyzed using an RNase A renaturation assay, and the results indicated that MgPDI exhibited the properties of oxidative folding on RNase A, similar to the properties of human PDI. The activity of MgPDI induced a time-dependent increase in denatured RNase A refolding; the velocity of cCMP hydrolysis catalyzed by renatured RNase A increased with the time allowed for the refolding reaction (Fig. 4a). The human PDI used as a positive control showed marginally greater oxidase/ isomerase activity than the MgPDI in the present study. For the MgPDI reductive assay, the decline in bovine insulin disulfide bonds with the increase in MgPDI concentrations was detected by the increasing turbidity in the MgPDI function as antioxidants. The influence of MgPDI on viability of HEK293 cells under H 2 O 2 stress was determined using a cell proliferation assay kit. Overall, viability of cells in the MgPDI-treated groups was higher than that of the BSA-treated control when exposed to the H 2 O 2 stress. Moreover, viability increased with the increase of MgPDI concentration (Fig. 4c). The degree of DNA damage was indicated by distinct mobility-shift patterns produced by the two DNA bands after electrophoretic resolution on a gel; MgPDI appears     (Fig. 5b). Next, qRT-PCR was used to analyse the expression of MgPDI in response to the H 2 O 2 stress. A significant increase in transcript abundance of MgPDI was observed when comparing juveniles that were exposed to H 2 O 2 (20 mM-80 mM) for 30 min with J2s that were treated with water ( Fig. 5c).
Influence of RNAi silencing of MgPDI on nematode parasitism. In vitro RNAi targeting of MgPDI was performed to analyze whether MgPDI plays an important role in parasitism. The results of qRT-PCR experiments showed a decrease in the transcript abundance of MgPDI when the nematodes were soaked with dsRNA against MgPDI, indicating the effective silencing of MgPDI (Fig. 6a). We examined the mortality rates of MgPDI or GFP dsRNA-treated J2s after soaking them in various concentrations of H 2 O 2 like those used in the transcript expression experiment. A significantly lower percentage in survival of MgPDI dsRNA-treated J2s was observed than of GFP dsRNA-treated J2s where both groups were exposed to H 2 O 2 (Fig. 6b).
The infection studies showed that the treatment of J2 with MgPDI dsRNA significantly reduced the reproductive ability of nematodes (MF = 6.22) compared to that of the treatment of J2 with GFP dsRNA (MF = 11.46) (Fig. 6c).

MgPDI is localised in the plant apoplastic space.
To investigate the subcellular localization of MgPDI in plant cells, the reporter protein GFP was fused to the C-terminus of the MgPDI protein. The GFP signal from the MgPDI:GFP fusion expressed in N. benthamiana was observed at the cell periphery. However, the GFP signal was observed in the apoplastic space when the plant cells were plasmolysed by treating with 1 M NaCl 2 which indicated that the localization of MgPDI was in the apoplast (Fig. 7).

Discussion
The present study identifies MgPDI encoding a typical PDI in the endoparasitic nematode M. graminicola, and MgPDI contains a 20-amino acid signal peptide at its N-terminal and two catalytic thioredoxin-like domains (active sites), each containing the canonical CGHC motif, and two non-catalytic domains (Fig. 1a,b). PDI is specifically responsible for folding proteins into the endoplasmic reticulum 18 . In addition, due to the active motifs in PpPDI1, its enzymatic function purportedly causes cell death in P. parasitica 17 . Our results also showed that the www.nature.com/scientificreports www.nature.com/scientificreports/ active catalytic motifs are highly conserved in the sequences under investigation (Fig. 1a). The described sequence was annotated as MgPDI for its arrangement of PDI classical domains.
In situ hybridization showed that MgPDI was expressed in oesophageal gland cells of M. graminicola pre-parasitic J2s, which is said to be nematode secretory effector proteins origin and presumed to be involved in the early parasitic stages of Mg. In addition, the expression analysis in different life stages of MgPDI found that transcription of MgPDI was up-regulated during early parasitic stages and peaked at 5 dpi, which is similar with the expression pattern of HsPdi in H. schachtii 14 .
To explore other functional characteristics of MgPDI, a large quantity of active recombinant MgPDI was obtained by induction at optimal conditions (20 °C overnight with 0.5 mM IPTG). Previous studies demonstrated that the enzymatic activity of PDI lies in its roles in oxidization, isomerization, and reduction 9 . In addition, the oxidase/isomerase activity of MgPDI utilizes the refolding of scrambled RNase 19 . A classical assay of insulin disulfide bridge-reduction in the presence of DTT was performed to analyze the enzymatic activity of PDI reduction, and the results indicated that MgPDI catalysed the reduction of insulin disulfide bonds, as seen in previous studies 9 . Protein disulphide isomerase is a member of the thioredoxin superfamily, which is composed of several redox proteins that play key roles in many essential antioxidant and redox-regulatory processes 20,21 . As for the enzymatic activity of antioxidants, first, our results indicated that MgPDI functioned in HEK293 cells to reduce oxidative stress by H 2 O 2 , and in particular, the recombinant protein exerted its protective influence in a dose-dependent manner (Fig. 4c). To further confirm the ability of MgPDI to function as an antioxidant, the plasmid DNA nicking assay was performed to demonstrate the ability of MgPDI to protect super-coiled DNA from nicking. Results indicated that MgPDI (at 0.1, 1, 10 and 100 μg/ml) effectively protects the super-coiled DNA from damage (Fig. 4d). DNA protection against oxidative damage by thioredoxin has also been demonstrated in Haemonchus contortus 22 . This is the first report that a functional recombinant PDI protein was obtained from the plant parasitic nematode M. graminicola and confirmed that MgPDI encodes a functional protein disulfide isomerase.
To determine the importance of MgPDI in the parasitism of M. graminicola, MgPDI expression was knocked down using RNAi, which has been effectively used to research both sedentary and migratory endoparasites, such as plant-parasitic nematodes and the function of their pathogenicity factors 23 . Knocking down the expression of MgPDI in preparastitic nematodes reduced the MF by more than 45% compared with the MF when in the non-specific presence of dsRNA (GFP). Our results are in accordance with Habash et al., who reported that RNAi  Studies have shown that ROS causes damage to cellular organelles and inhibits cell functions by oxidizing DNA, proteins, and lipids 24 . However, plant-parasitic nematodes can encode various antioxidant enzymes including superoxide dismutase (SOD), catalase, peroxiredoxins, glutathione peroxidases and protein disulfide isomerase to protect against ROS 14,[25][26][27][28] . Results of the present study support that the primary role of MgPDI is to protect Mg from damage by ROS. Previous research reported that the production of ROS during the oxidative burst reflected the defense response in plants 29 . In order to test whether the reactive oxygen species of rice infected with Mg were affected. The H 2 O 2 level was determined in rice roots, and we found that H 2 O 2 levels in knots were lower at 7 dpi and 14 dpi compared with the root tips without Mg infection. What's more, we observed a significant increase in MgPDI expression in response to the exogenous stress of H 2  Previous research of immunolocalization indicated that effector proteins were translocated to the plant apoplast 30 . The HaEXPB2 from Heterodera avenae containing the signal peptide could also target the protein to the apoplast when overexpressed in N. benthamiana 31 . Our studies of subcellular localization showed that MgPDI is localized in apoplast, but how it functioned in the apoplast need to be researched in the further studies.
In conclusion, the combined results from the gene discovery, expression, localization, silencing, and recombinant protein characterization suggest that MgPDI potentially plays an important role in combating the burst of ROS in host plants during the infection process. In brief, MgPDI is a putative virulence factor facilitating M. graminicola infestation of rice hosts. MgPDI gene discovery and sequence analysis. Total RNA was extracted from about 10000 freshly hatched second-stage juveniles using the TRIzol reagent (Invitrogen, California, USA). Based on M. graminicola transcriptome data 34 , the full-length cDNA sequence of MgPDI was obtained by rapid amplification of cDNA ends using the SMART RACE cDNA Amplification kit (Clontech, USA) according to the manufacturer's instructions. PCR-amplified DNA fragments were cloned into pGEM-T Easy Vector (Promega, USA) and then sequenced by Sangon Biotech (Shanghai, China). All primers used in this study were synthesized by Invitrogen Biotechnology Co. Ltd. and are listed in Table 1.
The deduced protein sequence, pI (isoelectric point) and MW (molecular weight) of MgPDI were predicted by DNAMAN V.6 (Lynnon Biosoft, Que-bec, Canada). An online protein structure homology-modeling server SWISS-MODEL was used to predict the protein tertiary structure. The signal peptide of MgPDI was determined using SignalP 4.1 Server at http://www.cbs.dtu.dk/services/SignalP/ 35 . Homologous PDI protein sequences from various species were retrieved from the NCBI database and aligned using the program of DNAMAN 6.0.
In situ hybridization. The specific primers PDI-T7-P/PDI-AP and PDI-P/PDI-T7-AP (Table 1) were designed to synthesize DIG-labelled sense and antisense RNA probes (307 bp) using DIG RNA labelling mix (Roche, Basel, Switzerland). In situ hybridization was performed according to a modified method of De Boer et al. on about 8000 freshly hatched J2s 36 . After hybridization, the probe was detected with an anti-digoxin-FITC monoclonal antibody (diluted 1:1000), and nematodes were imaged using a Laser Scanning Confocal Microscope (Zeiss LSM800, Oberkochen, Germany).

Developmental expression analysis.
Total RNA was extracted from M. graminicola nematodes at different life stages as described previously 37 using the TRIzol method (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The cDNA was synthesized using the ReverTra Ace qRT-PCR RT kit (Toyobo, Osaka, Japan). Quantitative RT-PCR was performed with the primer pair MgqRT-PCR-F/MgqRT-PCR-R (Table 1) and actin genes of M. graminicola were amplified as a reference with the primers Mg-ACT-Q-F/ Mg-ACT-Q-R 34 . The qRT-PCR reactions were performed on a CFX Connect Real-Time System (BIO-RAD, USA) using THUNDERBIRD qRT-PCR Mix (Toyobo, Osaka, Japan). Three technical replicates for each reaction were performed and three independent experiments were performed under the following thermal cycler conditions: 95 °C for 60 sec, 40 cycles at 95 °C for 15 sec, and 60 °C for 30 sec. The relative changes in gene expression were determined using the 2 −ΔΔCT method 38 .

Expression, purification, and validation of recombinant MgPDI. The cDNA fragments encoding
MgPDI without signal peptide were amplified by PCR primers with restriction sites ( Table 1). Products of the PCR assembly were ligated into the pET-32a vector (Novagen, USA) using T4 DNA ligase (Promega, USA). The resulting recombinant vector was validated by PCR product sequencing and was transformed into an Escherichia  www.nature.com/scientificreports www.nature.com/scientificreports/ coli BL21 (DE3) competent cell (TaKaRa, China) for protein expression. Transformed E. coli was cultured at 37 °C and agitated at 180 rpm in lysogeny broth (LB) containing ampicillin (50 μg/ml) until optical density 600 (OD600) reached 0.5. Isopropyl-B-D-thiogalactoside (IPTG) was added to the final concentration of 0.5 mM and then incubated at 20 °C, while shaking at 180 rpm. After incubating for 1 day, the bacterial cells were precipitated by centrifugation and expression analyzed on 12% SDS-PAGE. Thrombin (Sigma, MO, USA) was used to cleave the tags from MgPDI in HisTALON ™ Gravity Columns (Clontech, USA) to purify the recombinant proteins, and p-aminobenzamidine-agarose (Sigma, MO, USA) was applied to bind thrombin after cleavage. Lastly, the supernatant was collected and the concentration was measured by absorbance at 280 nm and stored at −80 °C 39 . The purified MgPDI protein band was cut out from the gel after SDS-PAGE and staining by coomassie blue. In-gel proteins were digested overnight in 12.5 ng/mL trypsin in 25 mmol/L NH 4 HCO 3 . The peptide mixtures were injected into the trap column, with a flow rate of 10 μl/min, of a Thermo Scientific Easy nanoLC 1000 (LTQ-Orbitrap Elite, Thermo Fisher Scientific, Waltham, MA, USA) for 2 min to analyze the purified proteins according to the method described by Xu et al. 40 .
PDI activity assays. Isomerization activity of PDI was determined by a standard assay using the modified method previously reported by Hong & Soong 9 . A preparation of 8 μM scrambled RNase A (Sigma) was incubated with 1.4 μM purified recombinant MgPDI protein in a buffer containing 4.5 mM cytidine 2′,3-cyclic monophosphate (cCMP) (Sigma), 1 mM reduced glutathione (Sigma), 0.2 mM oxidized glutathione (Sigma), 2 mM EDTA, and 100 mM Tris-Cl (pH 8.0). The reduction of cCMP by active RNase into CMP was monitored by the absorbance at 296 nm for 60 min in a Microplate Reader (Thermo, Varioskan Flash). For the PDI reductive assay, the MgPDI enzyme activity to reduce insulin was determined by a modified method 41,42 . Reaction mixtures (200 μl) included 100 mM Tris-Cl (pH 6.8), 2 mM ethylenediaminetetraacetic acid (EDTA), 0.13 mM insulin from bovine pancreas (Sigma), 0.33 mM dithiothreitol (DTT) and increasing concentrations (ranging from 2.5 to 10 μM) of purified MgPDI protein. The turbidity of the reaction mixture (Dithiothreitol-mediated insulin reduction by MgPDI) was monitored by measuring the increase of absorbance at 650 nm using a Microplate Reader (Thermo, Varioskan Flash). The reduction of insulin by DTT was recorded in a solution without MgPDI as a negative control.

Human cell viability assay. The method which assessed cell viability activity of MgPDI on Human
Embryonic Kidney 293 (HEK293) cells was modified from the previously described study 43 . The HEK293 cells were adjusted to 3 × 10 4 cells/well and cultured overnight at 37 °C. Afterwards, the final concentration of MgPDI added to the medium was 0.1, 10 or 1000 ng/ml. After a further 6 h of incubation, H 2 O 2 was added to a final concentration of 100 μM and cells were incubated for another 30 min in order to induce oxidative stress. Cell viability was measured using a CCK-8 Cell Counting Kit (Vazyme, China) at a wavelength of 450 nm. Cells incubated with 5 μg/ml bovine serum albumin (BSA) were used as a control. Each group was tested in triplicate.
Protective effect of MgPDI against oxidative damage by an MFO system. A mixed function oxidase (MFO) system was used to generate thiol radicals to damage the plasmid DNA 44 . The MFO system consisting of 1.65 mM DTT, 16.5 mM FeCl 3 and different concentrations of MgPDI (from 0.1 to 100 μg/mL) were pre-incubated at 37 °C for 2.5 h. Subsequently, pGEM-T Easy plasmid DNA (500 ng, Promega) was added and incubated for 1 h at 37 °C. Nicking of DNA was evaluated by ethidium bromide staining after electrophoresis in 0.8% agarose gels 45 . Hydrogen peroxide content determination. The H 2 O 2 level was determined according to Ji et al. 46 .
Each sample consisted of approximately 0.1 g fresh roots, collected from knots or tips. Root samples were collected at 3, 7 and 14 dpi, The experiment was performed twice, each time with three replicate samples.
Double stranded RNA (dsRNA) and infection assay. The forward and reverse primers contained T7 promoter sequences at their 5′ ends for in vitro RNA synthesis (Table 1). Double stranded RNA (dsRNA) was synthesized and purified using the T7 RiboMAXTM Express kit (Promega, USA) according to the manufacturer's instructions. The GFP template was used for the synthesis of a dsRNA construct as a negative control.
RNAi soaking was performed using the modified method of Rosso et al. and Huang et al. 47,48 . Twenty-five thousand freshly hatched J2s of M. graminicola were soaked in the dsRNA solution (2 mg mL −1 dsRNA, 3 mM spermidine, 50 mM octopamine, and 0.05% gelatin, adjusted with 0.25 × M9 buffer) for 36 h at room temperature in the dark on a rotator. For each reaction, approximately 8000 J2s were used for the plant infection assay; approximately 3000 J2s were used to determine nematode survival after the H 2 O 2 stress, Freshly hatched J2s of M. graminicola were soaked in MgPDI dsRNA or GFP dsRNA (control), dsRNA-treated nematodes were soaked in 20, 40 and 80 mM H 2 O 2 or in sterile water (0 mM) and live nematodes were counted after 30 min; and all the remaining J2s were used for the qRT-PCR to evaluate the level of MgPDI silencing.
For the infection assay, Pluronic F-127 (PF-127) (Sigma-Aldrich) gel was used as previously reported 49,50 . Eighty second-stage juveniles were soaked in dsRNA targeted against MgPDI or GFP and then inoculated each rice seedling according to Tian 51 . Roots of rice plants were stained with acid fuchsin 52