Cloning and characterization of the first serine carboxypeptidase from a plant parasitic nematode, Radopholus similis

Radopholus similis is an important parasitic nematode of plants. Serine carboxypeptidases (SCPs) are peptidases that hydrolyse peptides and proteins and play critical roles in the development, invasion, and pathogenesis of certain parasitic nematodes and other animal pathogens. In this study, we obtained the full-length sequence of the SCP gene from R. similis (Rs-scp-1), which is 1665 bp long and includes a 1461-bp open reading frames encoding 486 amino acids with an 18-aa signal peptide. This gene is a double-copy gene in R. similis. Rs-scp-1 was expressed in the procorpus, esophageal glands and intestines of females and in the esophageal glands and intestines of juveniles. Rs-scp-1 expression levels were highest in females, followed by juveniles and males, and lowest in eggs. Rs-scp-1 expression levels were significantly suppressed after R. similis was soaked in Rs-scp-1 dsRNA for 12 h. Nematodes were then inoculated into Anthurium andraeanum after RNAi treatment. Compared with water treatment, R. similis treated with RNAi were reduced in number and pathogenicity. In summary, we obtained the first SCP gene from a plant parasitic nematode and confirmed its role in the parasitic process.

A phylogenetic tree was constructed based on Rs-SCP-1 and 19 other SCP amino acid sequences (Fig. 3). All nematode SCPs were grouped into three branches. Rs-SCP-1 and SCPs of C. brenneri (GenBank: EGT47621), C. briggsae (XP 002630350), C. elegans (NP 494846), C. remanei (XP 003099200), Haemonchus contortus (CDJ88063), Ancylostoma ceylanicum (EYC11833), Necator americanus (ETN85041), Brugia malayi (EDP30838), and Loa loa (XP 003139679) were grouped on the same branch, suggesting they have a close phylogenetic relationship. However, Rs-SCP-1 was separated from these 9 SCP sequences. This separation may be attributable to the different lifestyle of these nematodes. Southern blot analysis. Southern blot analysis indicated the 489-bp-long, digoxigenin (DIG)-labelled probe hybridized to two fragments from R. similis gDNA digested with EcoR I and Hind III. Only one fragment was detected in pTA2-gscp digested with EcoR I and Hind III (Fig. 4). Based on these results, Rs-scp-1 exists as a double-copy gene in the R. similis genome.

Tissue localization and expression of Rs-scp-1. In situ hybridization results revealed the presence of
Rs-scp-1 mRNA in the procorpus (Fig. 5A), esophageal glands (Fig. 5B,C) and intestines (Fig. 5E) of females and in the esophageal glands and intestines (Fig. 5G,H) of juveniles. No hybridization signals were detected in females (Fig. 5D,F) and juveniles (Fig. 5I) after hybridization with a control sense probe. According to the qPCR results, Rs-scp-1 expression was detectable at all life stages in R. similis. Expression in females was significantly higher (p < 0.05) than at other life stages. Expression in eggs, juveniles and males corresponded to 23%, 80%, and 35% of the expression observed in females, respectively (Fig. 5J). Rs-scp-1 expression in juveniles was significantly higher  (p < 0.05) than that observed in eggs and males. There was no significant difference (p > 0.05) in expression between eggs and males.
RNAi and a pathogenicity test. qPCR was performed to detect Rs-scp-1 expression in R. similis after dsRNA treatment for 4, 12, 24 or 36 h. Soaking in Rs-scp-1 dsRNA solution led to a significant decrease (p < 0.05) in Rs-scp-1 expression compared with controls. No significant difference (p > 0.05) was detected between untreated controls and nematodes treated with enhanced green fluorescent protein (egfp) dsRNA (Fig. 6). Rs-scp-1 had the lowest expression levels among the treatments after 12 h of Rs-scp-1 dsRNA soaking. There were significant differences (p < 0.05) between the 12-h versus 24-and 36-h treatments and no significant difference (p > 0.05) between the 12-h and 4-h treatments. Differences in expression levels also were not significant (p > 0.05) between the 4-h versus the 24-to 36-h treatments. Therefore, Rs-scp-1 dsRNA soaked for 12 h achieved maximal silencing.
Anthurium was inoculated with approximately 1,000 mixed-stage nematodes treated with Rs-scp-1 dsRNA for 12 h. Sixty days after inoculation, nematode numbers in the roots were counted. Nematode numbers in roots inoculated with Rs-scp-1 dsRNA-treated nematodes were significantly lower (284) (p < 0.05) than those observed in anthurium plants inoculated with untreated nematodes (blank control) (2,893) or nematodes treated with egfp dsRNA (2,763) (Fig. 7A). There was no significant difference (p > 0.05) in nematode numbers observed in plants inoculated with blank control nematodes and those inoculated with egfp dsRNA-treated nematodes. Only a few reddish-brown lesions were observed on anthurium plant roots inoculated with nematodes treated with Rs-scp-1 dsRNA, whereas the roots on anthurium plants inoculated with untreated nematodes or nematodes treated with egfp dsRNA were largely rotted ( Fig. 7B,C,D).

Discussion
SCPs have a close relationship with parasitism in animal parasitic nematodes 15,[17][18][19] . The roles that SCPs play in plant parasitic nematodes are not yet clear. In this study, we cloned the SCP gene, Rs-scp-1, from R. similis and identified its structure and features. We confirmed the expression of Rs-scp-1 mRNA in the procorpus, esophageal glands and intestines, and expression differs throughout the life cycle of R. similis. In addition, Rs-scp-1 expression was significantly decreased after RNAi treatment, and R. similis pathogenicity in anthurium was significantly reduced after nematodes were treated with Rs-scp-1 dsRNA for 12 h. This study is the first to investigate an SCP in a plant parasitic nematode.
SCPs are widespread in higher organisms and cleave C-terminal amino acid residues from peptides. Fifty-one proteins homologous to known SCPs have been found in the Arabidopsis thaliana genome. The SCP gene family encodes a diverse group of enzymes whose functions are likely to extend beyond protein degradation and processing to include activities such as the production of secondary metabolites 21 . Several SCPs have been found in nematodes, and some function in parasitism or development 14,17,18,22 . According to a phylogenetic tree analysis, SCPs from different nematodes with different lifestyles separate into three branches by function 17,22 . Therefore, there are likely to be several SCPs with different functions in R. similis. SCP is an excretory/secretory protein in A. cantonensis and H. contortus 14,17 . Rs-SCP-1 is phylogenetically related to the SCP in H. contortus. In this study, we confirmed that Rs-SCP-1 is an esophageal gland-secreted protein with a signal peptide in the N-terminus. Rs-scp-1 was also detected in the procorpus of R. similis. This is consistent with the function of SCPs. Like other proteins that are secreted from the esophageal glands of plant parasitic nematodes, Rs-scp-1 expressed in the esophageal glands of R. similis may help quickly destroy the host defence system and facilitate host invasion and the establishment of a parasitic relationship to obtain nutrients from the host [23][24][25][26] . In addition, R. similis pathogenicity decreased significantly after RNAi treatment. Thus, Rs-scp-1 appears to be involved in R. similis parasitism. This gene may help R. similis degrade plant defence proteins, leading to higher Rs-scp-1 expression levels in infective juveniles than in eggs and males. SCP in B. malayi belongs to the same branch as Rs-SCP-1 and is reportedly associated with developmental processes 22 . Rs-scp-1 is located in the intestines of females and juveniles. Intestinal proteins related to nutritional absorption influence nematode development 6 . Thus, Rs-scp-1 may be associated with nutrient uptake in the intestines of females and juveniles. Rs-scp-1 mRNA hybridization signals were stronger and Rs-scp-1 expression levels were higher in females than in juveniles. Meanwhile signal were not detected and Rs-scp-1 expression was also significantly lower in males. These results were consistent with the biological functions of females, juveniles and males. Females and juveniles require more nutrition to complete development and infection processes. Females also bear the task of breeding and therefore need more nutrition than juveniles. As males are degenerate in the stylet and esophageal glands and have no ability to infect, their nutrient intake and absorption are weakened.
Based on the results of this study, Rs-scp-1 may participate in nematode development and pathogenicity. The feasibility of targeting Rs-scp-1 for the control of R. similis should be further investigated.

Methods
Nematode isolate. R. similis, isolated from Zingiber officinale Roscoe and cultured on carrot disks 27 , was employed in this study. Nematodes were extracted as described elsewhere 20 .
Anthurium andraeanum used in this study was purchased from the Flowers and Plants Research Center, Guangzhou, Guangdong. These plants were grown in a greenhouse at 26 ± 1 °C (16-h light/8-h dark photoperiod) and 60-80% relative humidity. From the SSH library of R. similis 20 , a fragment with a polyA structure was identified as a candidate for Rs-scp-1 by performing a BLAST search of the NCBI database. 5′ RACE primers (GSP and NEST-R) ( Table 1) were designed to amplify the 5′ end of Rs-scp-1 using a SMART RACE cDNA Amplification Kit (Clontech, Takara Biotechnology (Dalian) Co., Ltd., Dalian, China). The two fragments of Rs-scp-1 were then spliced into the complete sequence of Rs-scp-1. The ORF was predicted using ORFfinder (http://www.ncbi.nlm.nih.gov/gorf/orfig.cgi). Specific primers (scp-F/scp-R, Table 1) were designed to amplify the ORF sequence and genomic sequence of Rs-scp-1. These fragments were cloned into pTA2 for sequencing. The positive plasmids pTA2-scp and pTA2-gscp were stored at −20 °C for further use.
Sequence analysis and phylogenetics. The sequence similarity of Rs-SCP-1 was analysed by performing a BLAST search of the NCBI non-redundant protein database (nr). Signal peptide and trans-membrane domains were predicted using SignalP 4.0 (http://www.cbs.dtu.dk/services/SignalP-4.0/) and TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/), respectively. WoLF PSORT and PSORT II were used to predict protein subcellular localization. A phylogenetic tree containing SCPs from 11 species was constructed using the maximum-likelihood method.
Southern blot analysis. The primers 179SB-F/179SB-R (Table 1) were designed to amplify a DIG-labelled probe using a PCR DIG Probe Synthesis Kit (Roche Applied Science, Penzberg, Germany). Approximately 10 μg of R. similis gDNA was digested with EcoR I and Hind III (Thermo Fisher) overnight at 37 °C. The digested DNA was separated via electrophoresis and transferred to a Hybond N + membrane (Amersham Biosciences, GE Healthcare, UK). Hybridization and detection were performed using a Dig High Primer DNA Labeling and Detection Starter Kit I (Roche Applied Science) at 48 °C for 24 h. pTA2-gscp digested with EcoR I and Hind III was used as a control.   (Table 1) were designed to amplify DIG-labelled sense and antisense probes using DIG RNA Labeling Mix (Roche Applied Science). In situ hybridization was performed as previously described 29 . After hybridization at 51.5 °C for 12 h, the nematodes were examined using Nikon Eclipse 90i microscope (Nikon, Kawasaki, Japan).
Expression analysis of Rs-scp-1. RNA samples were extracted from 500 R. similis eggs, juveniles, females and males, respectively. Total RNA was then quantified with a NanoDrop spectrophotometer (Thermo Fisher). The RNA samples were used as templates for cDNA synthesis with HiScript II Q RT SuperMix for qPCR (Vazyme, Nanjing, China). Specific primers (q179-F/q179-R, Table 1) were designed to assay Rs-scp-1 expression. The primers Actin-F/Actin-R (Table 1) 30 were used to amplify β-actin as a reference gene. qPCR was performed using a CFX96 qPCR instrument (Bio-Rad, Hercules, CA, USA) with AceQ qPCR SYBR Green Master Mix (Vazyme). All expression experiments were performed in triplicate with three biological replicates.
RNAi and silencing detection. The primers 179iT7-F/179i-R and 179i-F/179iT7-R (Table 1), which contained a T7 promoter, were designed to amplify Rs-scp-1 sense and antisense single-stranded RNA (ssRNA) using a Script Max TM Thermo T7 Transcription Kit (TOYOBO, Osaka, Japan). Equal amounts of sense and antisense ssRNA were mixed and incubated at 75 °C for 10 min to hybridize and form dsRNA. The dsRNA was purified with a 1/10 volume of NaAc (3 mol/L) and a 2-fold volume of ethanol at −20 °C overnight, washed twice with 70% ethanol, and then dissolved in deionized water. The quantity of dsRNA was measured using a NanoDrop spectrophotometer and analysed by performing gel electrophoresis in a 1.2% agarose gel. Finally, the dsRNA was stored at −80 °C for later use. Non-endogenous control egfp dsRNA was synthesized using the primers GFPiT7-F/GFPi-R and GFPi-F/GFPiT7-R (Table 1).
Approximately 500 mixed-stage nematodes were soaked in Rs-scp-1 dsRNA solution (2.0 μg/μL) for 4, 12, 24, or 36 h. Nematodes soaked in egfp dsRNA solution were used as non-endogenous controls. The soaking times for the controls were the same as those for the Rs-scp-1 dsRNA. In addition, untreated nematodes were used as blank controls. Nematodes treated with dsRNA were washed three times with ddH 2 O, and then RNA was extracted and qPCR was performed using the methods described above to analyse the suppression of Rs-scp-1 mRNA expression in R. similis.
Approximately 1,000 mixed-stage nematodes treated with Rs-scp-1 dsRNA were employed for pathogenicity analysis. Untreated nematodes and those treated with egfp dsRNA solution were used as controls. Sixty days after inoculation, rhizosphere nematodes were isolated and counted. Plant inoculation and nematode counting were performed based on previously described methods 20 . Five biological replicates were performed per trial, and the experiment was performed twice. Data analysis. Statistical analysis was performed using SPSS 23.0. All data in this study were subjected to analysis by one-way ANOVA and tested for differences between treatments at a 5% level using Duncan's Multiple Range Test (DMRT).