The cathepsin S cysteine proteinase of the burrowing nematode Radopholus similis is essential for the reproduction and invasion
© The Author(s) 2016
Received: 25 February 2016
Accepted: 2 June 2016
Published: 10 June 2016
The nematode Radopholus similis is an important migratory endoparasite of plants. Cysteine proteinases such as cathepsin S (CPS) play key roles during embryonic development, invasion, and pathogenesis in nematodes and many other animal parasites. This study was designed to investigate the molecular characterization and functions of a cathepsin S protease in R. similis and to find new targets for its control.
Rs-CPS of R. similis, Hg-CPS of Heterodera glycines and Ha-CPS of H. avenae are closely genetically related and share the same branch of the phylogenetic tree. Rs-cps is a multi-copy gene that is expressed in the esophageal glands, ovaries, testes, vas deferens, and eggs of R. similis. Rs-cps mRNA transcripts are expressed at varying levels during all developmental stages of R. similis. Rs-cps expression was highest in females. The neurostimulant octopamine did not significantly enhance the ingestion of the dsRNA soaking solution by R. similis but instead had a detrimental effect on nematode activity. The dsRNA soaking solution diffused into the body of R. similis not only through the esophageal lumen but also through the amphids, excretory duct, vagina, anus and cloacal orifice. We confirmed that RNAi significantly suppressed the expression level of Rs-cps and reproductive capability and pathogenicity of R. similis.
Our results demonstrate that Rs-cps plays important roles in the reproduction, parasitism and pathogenesis of R. similis and could be used as a new potential target for controlling plant parasitic nematodes.
The burrowing nematode Radopholus similis is a migratory endoparasite of plants. R. similis is one of the most destructive plant pathogenic nematodes in the world and is listed as a quarantine pest in many countries and regions [1, 2]. R. similis has a wide host range and attacks more than 250 plant species . R. similis severely harms banana, citrus, pepper, coffee and other agronomic and horticultural crops [4, 5] and is the most serious plant pathogenic nematode in most banana-growing areas of the world . Despite extensive attention and research, controlling R. similis remains problematic worldwide, and effective approaches must be explored and established.
Proteolytic enzymes can be classified into four main groups: cysteine, serine, aspartyl and metallo proteinases. Cysteine proteinases are the most extensively studied . Cysteine proteinases (EC 3.4.22) have been identified in a variety of organisms . Most cysteine proteases are expressed and proteolytically active in the intestines, and these enzymes are the main digestive enzymes in nematodes and animal parasites . Cysteine proteinases play important roles in embryogenesis and development, infection, parasitism, pathogenesis and immune evasion in nematodes and many other animal parasites [9–12]. Nematode cysteine proteinases mainly include cathepsin B-, L-, S-, K- and Z-like cysteine proteinases, and cathepsin L (CL) and cathepsin B (CB) have been extensively studied in recent years. CL is essential for embryogenesis and development in Caenorhabditis elegans [10, 13]. Guiliano et al.  demonstrated that CL proteinases in filarial nematodes are associated with larval molting and cuticle and eggshell remodeling. CB plays important roles in molting, the successful development of Onchocerca volvulus fourth-stage larvae  and in the invasion and pathogenesis of Fasciola hepatica and Angiostrongylus cantonensis [16, 17]. At present, CB genes have rarely been cloned in plant parasitic nematodes, and only CB of Bursaphelenchus xylophilus (GenBank No: GU130153) and R. similis (GU360972) are cloned. However, many CL genes have been cloned in plant parasitic nematodes, such as Heterodera avenae (ACJ13100), H. glycines (Y09498), H. schachtii (ACJ13098), Globodera virginiae (ACJ13094), G. Mexicana (ACJ13096), Meloidogyne incognita (CAD89795), Rotylenchulus reniformis (AAY45870) and B. xylophilus (ACH56225) [7, 18, 19]. Li et al. [19, 20] reported that Rs-cb-1 plays key roles in reproduction, development, hatching and pathogenesis in R. similis. However, the cathepsin S gene (cps) has rarely been reported, and only the cps genes of H.glycines , R. similis (EU659125) and H. avenae  have been cloned. The functions of cps in plant parasitic nematodes have not been explored. In this study, the expression and tissue localization of Rs-cps in R. similis were investigated using qPCR and in situ hybridization, and the roles of Rs-cps during reproduction and pathogenesis were studied using RNAi combinated with inoculation of carrot callus and tomato plants in pots. This study is the first to examine the functions of cps in plant parasitic nematodes and suggests a promising new target for controlling R. similis.
RNAi is a means by which dsRNA (double stranded RNA) induces sequence-specific posttranscriptional gene silencing . This method was first developed in C. elegans and has subsequently been used in organisms ranging from lower fungi to higher mammals [22–26]. RNAi is also a very powerful tool for examining the functions of genes in plant nematodes and other organisms. For plant parasitic nematodes, in vitro RNAi (performed by soaking the nematodes in a solution of dsRNA in vitro) is the most widely used method, but the soaking time required to obtain optimal RNAi efficiency differs greatly among nematode species [19, 27–33]. The feeding mechanisms of plant parasitic nematodes vary. The infective second-stage juveniles of sedentary endoparasitic nematodes (such as cyst and root-knot nematodes) feed only following the establishment of a feeding site inside the root. Therefore, the primary barrier to successful in vitro RNAi is ensuring the ingestion of the dsRNA by the non-feeding second-stage juveniles of plant parasitic nematodes. In second-stage juveniles of G. pallida, H. glycines and M. incognita, dsRNA uptake can be induced by adding the neurotransmitter octopamine or resorcinol to successfully silence the targeted genes [27, 28]. In vitro RNAi-induced gene silencing has been achieved in B. xylophilus without exogenous neurotransmitter [31, 32]. The in vitro dsRNA soaking method has been used to induce RNAi to R. similis [19, 20, 34], but whether neurotransmitters facilitate the uptake of dsRNA by R. similis and affect nematode activity have not been examined. This study examined these questions using FITC as a visual marker.
Animals were treated in strict accordance with the Animal Ethics Procedures and Guidelines of the People’s Republic of China. All animal procedures were approved by the Animal Ethics Committee of the South China Agricultural University.
Nematode inoculum and plant growth conditions
Radopholus similis was collected from the roots of the ornamental plant Anthurium andraeanum and cultured in vitro on carrot disks at 25 °C . At 50 d after inoculation, the cultured nematodes were extracted from the carrot disks according to the method described by Zhang et al. . The tomato seeds used in this study were purchased from Guangzhou Changhe Seed Limited Company, Guangdong, and surface sterilized as described by Arshad et al. . The sterilized seeds were sown in 1.5 L of sterilized soil and cultured in a 25 °C growth cabinet (16 h light/8 h dark photoperiods) for 30 d .
RNA extraction, PCR amplification of Rs-cps and phylogenetic analysis
Primers used in this study
5′- CCGTTCTCCTCGATGTAGTCA -3′
The amino acid sequences of the Rs-CPS protein and other CPS proteins were aligned using ClustalW. Based on the amino acid sequences of 25 CPS proteins from 17 species, a phylogenetic tree was constructed using the neighbor-joining method in MEGA 5.1 . Bootstrap values were calculated from 1000 replicates.
Southern blot hybridization
Approximately 10 μg of gDNA was obtained from R. similis and digested with NdeI and EcoRI. The digested DNA products were separated by 0.8 % (w/v) agarose gel electrophoresis and transferred to a Hybond N + membrane (Amersham) . A 438-bp DIG-labeled probe was prepared using a PCR DIG Probe Synthesis Kit (Roche) with the specific primers SF and SR (Table 1). The membrane was hybridized for 18 h at 54.5 °C with the probe. Hybridization was performed using a Dig High Primer DNA Labeling and Detection Starter Kit I (Roche) according to the manufacturer’s instructions. After hybridization, the membrane was washed with 2 × SSC/0.1 % SDS for 15 min at 25 °C followed by 0.5 × SSC/0.1 % SDS for 30 min at 65 °C and examined. An equal amount of carrot callus gDNA was used as a control.
In situ hybridization
In situ hybridization was performed as described by De Boer et al.  and Cheng et al. . Specific sense (Ish-T7S1, Ish-A1) and antisense (Ish-S2, Ish-T7A2) primers (Table 1) were designed to amplify a 478-bp fragment based on the full-length sequence of Rs-cps. The purified PCR product served as the template to synthesize DIG-labeled sense and antisense RNA probes using DIG RNA labeling mix (Roche) according to the manufacturer’s instructions. Following fixation, the intact nematodes were cut into 2–5 fragments and hybridized with the DIG-labeled RNA probes (300 ng/mL). After hybridization, the stained nematode sections were examined and photographed using a 90i differential interference microscope (Nikon).
Expression analysis of Rs-cps and qPCR
qPCR was used to detect the expression levels of Rs-cps in R. similis at different developmental stages. Total RNA samples were extracted from 100 R. similis females, males, eggs and juveniles using an RNeasy Micro kit (Qiagen), respectively. The extracted RNA was treated and quantified as previously described . cDNA was synthesized using an iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer’s instructions. Specific primers, qPCR-A and qPCR-S (Table 1), were designed according to the full-length sequence of Rs-cps to detect Rs-cps expression levels in R. similis. β-actin was amplified as a reference gene using the primers Actin-F/Actin-R (Table 1) . qPCR was performed on a CFX-96 qPCR machine using iTaq Universal SYBR Green Supermix (Bio-Rad). The initial data analysis was performed using CFX-96 manager software, which created Ct values and extrapolated the relative levels of PCR products from standard curves. Melt curves were obtained routinely, which allowed the possibility of both contamination and primer dimers to be discounted [34, 38, 40]. All experiments were performed in triplicate with three biological replicates .
Synthesis of Rs-cps dsRNA of R. similis
The specific primers CPS-T7S/CPS-A and CPS-S/CPS-T7A (Table 1) were designed to amplify a 438-bp fragment containing the T7 promoter. The purified PCR product was used to transcribe Rs-cps sense and antisense single-stranded RNA (ssRNA) using a ScriptMAXTM Thermo T7 Transcription Kit (TOYOBO). The corresponding dsRNA was synthesized and purified as described by Hannon . The non-endogenous control dsRNA (the enhanced green fluorescent protein gene, egfp) was synthesized using the specific primers eGFP-T7S/eGFP-A and eGFP-S/eGFP-T7 (Table 1).
Effects of soaking R. similis with FITC and octopamine
The effects of FITC and octopamine on nematode activity and the effect of octopamine on dsRNA solution uptake by R. similis were assessed using FITC as a visual marker. The RNAi soaking method was performed as previously described . Approximately 20,000 mixed-stage nematodes were collected from carrot disks and soaked in M9 buffer. The following compounds respectively were added to the above soaking solution at the indicated concentrations: (I) 0 (CK), 0.1, 0.2, 0.4, 0.8, 1.0 or 2.0 mg/mL fluorescein isothiocyanate (FITC) (Sigma-Aldrich) (stock made up at 20 mg/mL in DMF) ; (II) 0 (CK), 10, 25, 50, 75, 100 or 200 mM neurostimulant octopamine (Sigma-Aldrich); (III) FITC (0.8 mg/mL) or FITC(0.8 mg/mL) plus octopamine (50 mM). Rs-cb-1 dsRNA was added to the soaking solution at a final concentration of 2.0 mg/mL . Nematodes not treated with FITC or octopamine were used as controls. The nematodes were maintained in 1 mL of soaking solution with gentle agitation (100 rpm) in a dark rotary incubator at 25 °C for 4, 8, 12 and 24 h. After incubation, the nematodes were transferred to a 10-mL centrifuge tube and washed six times with sterile water to remove the soaking solution. FITC uptake was measured based on fluorescence intensity, and the effects of FITC and octopamine on nematode activity were detected using a fluorescence microscope (Nikon 90i) with appropriate filters. The nematodes were considered dead if they did not move after being pricked with a platinum wire . For each treatment, 100 nematodes were effectively counted to quantify the effects of soaking R. similis with FITC and octopamine. Five biological replicates were performed.
Effects of Rs-cps silencing on R. similis
Approximately 1000 mixed-stage nematodes were washed with DEPC water and then soaked in Rs-cps dsRNA solution (2.0 mg/ml) with gentle agitation in a dark rotary incubator (100 rpm, 25 °C) for 12, 24, 36 and 48 h, respectively. The treated nematodes were used in the following experiments. (I) Total RNA was extracted from 100 nematodes in each treatment group after they were washed with DEPC water, and qPCR was used to detect the silencing efficiency of Rs-cps in R. similis, as described above. These experiments were performed in triplicate with three biological replicates. (II) The phenotypic changes of nematodes were observed after soaking in Rs-cps dsRNA solution for 36 h. (III) A total of 30 female nematodes were inoculated onto carrot disks and cultured for 50 d at 25 °C, and then the reproductive rates (reproductive rate = final nematodes ⁄ initial nematodes) of the nematodes were calculated. Non-endogenous egfp dsRNA treated nematodes (2.0 mg/mL) were used as a control. The soaking times for the controls were the same as those used for the Rs-cps dsRNAs. Untreated nematodes were used as a blank control (CK).
To detect the effect of Rs-cps silencing on the pathogenicity of R. similis, 1000 mixed-stage nematodes treated with Rs-cps dsRNA (2.0 mg/mL) for 36 h were inoculated onto each of the selected tomato plantlets. The selected plantlets were identical in height (approximately 20 cm) and growth conditions and were cultivated in a greenhouse as described above . Nematodes treated with egfp dsRNA for 36 h were used as the control. Untreated nematodes were used as a blank control. The plantlets were managed as usual except that they were not watered for the first 5 days . After 60 days, the plant heights, fresh shoot weights and fresh root weights of the plants were measured and recorded. The symptoms of infected roots were photographed. The nematodes in the rhizosphere were isolated and quantified as described elsewhere [34, 43]. Five biological replicates were performed.
All data in this study were analyzed using SAS 9.2 (SAS Institute, Cary, NC, USA) and subjected to one-way analysis of variance (ANOVA), and differences between treatments were compared using Duncan’s Multiple Range Test at p = 0.05.
PCR amplification of Rs-cps and phylogenetic analysis
Southern blot hybridization
Tissue localization and expression of Rs-cps mRNA in R. similis
Effects of FITC and octopamine on R. similis
After 8 h of incubation, there was no significant difference (p > 0.05) in the activity of R. similis in the groups soaked with octopamine at concentrations of 50 mM or less and the control group (without octopamine). Significant differences were observed in the activity of R. similis soaked with octopamine at concentrations greater than 50 mM and the control group (p < 0.05). However, activity of nematodes was reduced by only 10.2 % in the group soaked with 200 mM octopamine compared to the control group. When the incubation time was extended to 12 h, the activity of R. similis soaked in different concentrations of octopamine did not differ from that observed after incubation for 8 h (Fig. 4h). These results indicate that the detrimental effects of octopamine on the activity of R. similis are very small. Therefore, the ability of 50 mM octopamine to enhance the ingestion of the soaking solution by R. similis was assessed.
Detection of RNAi efficiency
Phenotype and reproduction of R. similis after RNAi
The pathogenicity of R. similis decreases significantly after RNAi
Cysteine proteinases play important biological roles in nematodes and many other animal parasites [9–12, 17]. In vitro RNAi-induced gene silencing has been successfully applied to free-living nematode C.elegans and the migratory endoparasitic plant-parasitic nematode B. xylophilus, without the addition of a neurostimulant [31, 32]. Delivery of dsRNA via ingestion is difficult in sedentary endoparasites such as cyst nematodes and root-knot nematodes because these second-stage juveniles (J2s) feed only following the establishment of a feeding site inside the root, and do not ingest substances prior to this stage . However, uptake of dsRNA soaking solution has been induced in these nematode J2s by adding neurostimulant [27, 28, 44]. RNAi has been applied to R. similis by soaking the nematodes in dsRNA solution that does not contain neurostimulant [19, 20, 34]. The ability of the neurostimulant octopamine to enhance the ingestion of soaking solution by R. similis and the potential detrimental effects of octopamine on nematode activity have not been evaluated. Tan et al.  reported that the percentage of activity Pratylenchus thornei was reduced by only 12 % after the worms were incubated with 100 mM octopamine for 16 h compared to the control group. Tan et al. also demonstrated that more than 90 % of both P. thornei and P. zeae were dead after only 4 h of incubation in 1 % resorcinol (another neurostimulant) . In this study, using FITC as a marker, we demonstrated that R. similis can ingest dsRNA soaking solution without stimulation by a neurostimulant, similar to C. elegans and B. xylophilus [27, 31, 32]. We also confirmed that the neurostimulant octopamine did not significantly enhance the ingestion of dsRNA soaking solution by R. similis but instead had a detrimental effect on nematode activity. In addition, the dsRNA soaking solution may diffuse into the body of R. similis not only via the esophageal lumen but also via the amphids, excretory duct, vagina, anus, cloacal orifice and egg shells.
The cb gene is mainly expressed in the intestines in C. elegans , A. cantonensis  and Haemonchus contortus  and in the cecal epithelial cells, digestive tract and reproductive system in F. gigantic . Li et al.  reported that Rs-cb-1 is expressed in the esophageal glands, intestines and gonads of females, the testes of males, and juveniles and eggs in R. similis. Hashmi et al.  reported that Ce-cl-1 is widely expressed in the intestines, hypodermal cells and eggshells of C. elegans. Guiliano et al.  confirmed that cl is highly expressed in the esophageal glands of B. malayi and B. pahangi infective third-stage larvae. In the plant parasitic nematode M. incognita, Mi-cl-1 is expressed in the intestines of young and mature female nematodes . In this study, the expression and localization of Rs-cps in R. similis were associated with the biological functions of cathepsin. The expression of Rs-cps in the esophageal glands of R. similis may facilitate the disruption of plant defensive responses, the establishment of a parasitic relationship, and the rapid digestion of host cells to obtain the nutrients necessary for metabolism and other physiological functions. These findings are also consistent with the functions of esophageal secretions that have been documented in other plant parasitic nematodes. The secretions of esophageal glands produced by plant parasitic nematodes are thought to play key roles throughout the process of parasitism [48, 49]. Rs-cps was located in the eggs and reproductive system of R. similis, possibly because CPS plays important roles in development, reproduction and cell differentiation in R. similis. The females of R. similis are responsible for both infection and reproduction; Rs-cps expression is therefore highest in females. Thakur et al.  reported that the expression level of Ha-cps was also highest in females of H. avenae. Rs-cps expression was significantly higher in infective juveniles than in eggs, likely because the successful destruction of host defense responses and the establishment of a parasitic relationship are a precondition for other functions to be implemented by R. similis. In this study, Rs-cps expression was lowest in eggs, 42.5 % of the expression level in females. This result reveals that CPS may also play important roles in embryo formation and cell differentiation in R. similis. Previous studies have shown that Rs-cb-1 plays vital roles in reproduction, development and pathogenesis in R. similis [19, 20]. B-, L- and S-like cathepsin belong to the cysteine protease family, and they share a close genetic relationship and similar structures. Therefore, these proteases may have similar biological functions during processes such as infection and pathogenesis in nematodes.
To further define the functions of Rs-cps and to explore the possibility of using this promising target for controlling R. similis, an RNAi experiment was performed in this study. Treatment with Rs-cps dsRNA significantly decreased the expression levels of Rs-cps and the reproductive rates of R. similis compared to the control groups. The silencing efficiency of Rs-cps was highest and the reproductive rate of R. similis was lowest on carrot disks after treatment with Rs-cps dsRNA for 36 h. In the subsequent pot experiments, the pathogenicity of R. similis to tomato plants was also significantly reduced after treatment with Rs-cps dsRNA for 36 h. These results are consistent with the tissue localization of Rs-cps in R. similis. Therefore, we confirmed that Rs-cps plays key roles in reproduction and pathogenesis in R. similis and that CPS might be a promising target for controlling this nematode. Here we first report the use of RNAi in studying the functions of cps in plant parasitic nematodes and suggests a promising new target for controlling R. similis. An RNAi effect could be generated in nematodes by feeding on transgenic plants expressing a specific target gene dsRNA [20, 27, 38]. Therefore, these findings support applications aimed at controlling plant parasitic nematodes by constructing Rs-cps plant RNAi vectors and obtaining transgenic plants that express specific hairpin dsRNAs of reproduction-, parasitism- and pathogenesis-related genes and merit further investigation.
This is the first work to examine the functions of cps from R. similis. Rs-cps mRNA was expressed in the esophageal glands and ovaries of females, the esophageal glands, testes and vas deferens of males and the eggs of R. similis. Rs-cps was expressed at varying levels in all developmental stages of R. similis. The expression level of Rs-cps was significantly suppressed in nematodes and that the reproductive capability and pathogenicity of R. similis were significantly reduced after RNAi. These results indicated that Rs-cps plays important roles in the reproduction, parasitism and pathogenesis of R. similis and could be used as a promising target for controlling plant parasitic nematodes.
KW, YL and HX conceived and designed the experiments; KW, YL and DWW performed the experiments; KW, YL, XH, CLX and HX analyzed the data; KW, YL and HX wrote the manuscript. All authors read and approved the final manuscript.
This work was funded by National Natural Science Foundation of China (No. 31071665 and No. 31371920).
The authors declare that they have no competing interests.
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