A novel ATPase gene, Ab-atps, plays an important role in the interaction of rice and white tip nematode, Aphelenchoides besseyi

Plant kinases containing the LysM domain play important roles in pathogen recognition and self-defense reactions. And it could recognize microbe-associated molecules including chitin and other polypeptides. The white tip nematode Aphelenchoides besseyi is a migratory parasitic nematode that infects plant shoots. It is distributed over almost all rice-producing areas and causes up to 50% economic losses. The rice OsRLK3 gene was a defense-related LysM kinase gene of rice. This study showed that the rice LysM kinase OsRLK3 could be induced by flg22, jasmonic acid, salicylic acid, and chitin. An interaction gene, Ab-atps from A. besseyi, was identified by screening the interaction between the rice gene OsRLK3 and an A. besseyi cDNA library using yeast two-hybrid screening. Ab-atps is a novel ATP synthase gene with a full length of 1341 bp, coding for 183 amino acids. The mRNA of Ab-atps was located in the esophagus and reproductive system of A. besseyi. The expression of Ab-atps was assessed at different developmental stages of the nematode and found to be the highest in the juvenile, followed by the egg, female, and male. Reproduction was significantly decreased in nematodes treated with Ab-atps double-stranded RNA (dsRNA) (p < 0.05). Transient expression experiments showed that Ab-ATPS-GFP was distributed in the nucleus, cytoplasm, and cell membrane, and Ab-ATPS-GFP triggered plant cell death. OsRLK3 was expressed significantly higher at 0.5 day and 1 day (p < 0.05) in rice plants inoculated with nematodes treated with Ab-atps dsRNA and gfp dsRNA for 0.5–7 days, respectively. Further, OsRLK3 expression under Ab-atps dsRNA treatment was significantly lower than with gfp dsRNA treatment at 0.5 day (p < 0.05) and significantly higher than with gfp dsRNA treatment at 1 day (p < 0.05). These results suggest that rice OsRLK3 could interact with A. besseyi Ab-atps, which plays an important role in growth, reproduction, and infection of the nematode. Our findings provide a theoretical basis to further understand the parasitic strategy of A. besseyi and its interaction mechanism with host plants, suggesting new ideas and targets for controlling A. besseyi.


Results of OsRLK3 cloning and sequence analysis.
A full coding cDNA sequence of 2064 bp in length was amplified from rice using the specific primers RP3F and RP3R (Table S1) and confirmed by sequencing. This sequence is 100% similar to the reported OsRLK3 gene sequence (GenBank accession: OS01G0741200) available in the database of the Rice Genome Annotation Project. This cDNA sequence encodes 687 amino acids, including a signal peptide (between residues 1 and 30) and a transmembrane helix (between residues 261 and 283). The theoretical molecular mass of OsRLK3 was 73.30 kDa, and the molecular mass without the signal peptide was 70.30 kDa. The results of bioinformatic analysis showed that OsRLK3 had the typical characteristics of a LysM kinase family member, including a LysM domain (between residues 175 and 219) and a kinase domain (between residues 376 and 664) (Fig. S1). The predicted location of OsRLK3 was the cell membrane.
OsRLK3 of rice interacts with Ab-atps of A. besseyi. To understand the role of the rice OsRLK3 gene in the interaction of rice and A. besseyi, full coding cDNA of the OsRLK3 gene was constructed as a bait construct, and used to screen potential interaction genes in a cDNA library of A. besseyi using the yeast two-hybrid system. Only one positive clone was identified, and its EST sequence was 301 bp including a poly A in the downstream 3′ untranslated region. Subsequently, a 1341 bp full length cDNA sequence from A. besseyi was amplified by RACE (Fig. S2) and confirmed by sequencing. The cDNA sequence included a 552 bp ORF, encoding for 183 amino acids (Fig. S2) 48), and ATP synthase from Caenorhabditis elegans (Genbank accession: NP_001021420.1, similarity 88%, identity 44.69%, E value 4e−47). The phylogenetic tree was constructed based on protein sequences of Ab-ATPS and other synthase sequences from 23 species of nematodes in the NCBI database (Fig. 2). The result showed that Ab-ATPS has the closest generic relationship with ATP synthase from Strogyloides ratti, which was consistent with the results of alignment analysis obtained by blastx. Some sequence had a lower homology with a higher similarity but a lower identity. And it led to some difference in the homology analysis between NCBI Blast and Mega 6.0. The interaction of OsRLK3 and Ab-ATPS was tested using the yeast two-hybrid assay. Yeast cells transformed with pGBKT7-OsRLK3 and pGADT7-Ab-ATPS exhibited the same blue coloration compared to the positive controls of yeast cells transformed with pGBKT7-53 and pGADT7-Lam. Yeast cells harboring pGBKT7-OsRLK3 co-transformed with the pGADT7 empty vector and those harboring the pGBKT7 empty vector co-transformed with pGADT7-Ab-ATPS could not grow on the QDO/XA (SD/-Leu/-Trp/-Ade/-His/X-α-gal/AbA) plate, and did not show interactions. The results showed that OsRLK3 interacted with Ab-atps (Fig. 3).

Expressions of Ab-atps at different developmental stages of A. besseyi. Ab-atps expression levels
in eggs, juveniles, females, and males were detected by qPCR. The results showed that Ab-atps relative expression level was highest in juveniles, and the expression in juveniles, eggs, and females accounted for 26.79, 13.10, and 4.86 times the expression level in males, respectively (Fig. 4). Significant differences existed among the different developmental stages (p < 0.05), but not between females and males (p > 0.05).
In situ hybridization of Ab-atps. The results of in situ hybridization suggested that Ab-atps was present in the esophagus and reproductive system (Fig. 5A,C,E). No hybridization signal was detected in nematodes when the control sense Ab-cb-1 DIG-Labeled RNA probe was used (Fig. 5B,D,F).

RNAi of Ab-atps.
After nematodes were treated with Ab-atps double-stranded RNA (dsRNA), qPCR was used to detect the RNA interference (RNAi) efficiency of Ab-atps. The expressions of Ab-atps in these nematodes decreased significantly, when compared to those in treated with the gfp dsRNA. And the expression levels were 86.7%, 80.7%, 86.3%, and 75.4%, in nematodes treated with Ab-atps dsRNA for 12 h, 24 h, 36 h, and 48 h (p < 0.05), respectively. Expression of Ab-atps was not significantly different among any of the Ab-atps dsRNA treatments (p > 0.05), neither among any of the gfp dsRNA treatments used as controls in this experiment (p > 0.05), respectively (Fig. 6).
The effect of Ab-atps RNAi on the reproduction of A. besseyi was examined by culturing the nematodes on carrot disks, which had been soaked in Ab-atps dsRNA. After culturing for 35 days, reproductions of nematodes treated with Ab-atps dsRNA for 12, 24, 36 and 48 h were significantly lower, than those of nematodes treated with gfp dsRNA, respectively (p < 0.05) (Fig. 7). With the treatment time of RNAi, reproduction of nematodes   after rice plants were inoculated with nematodes soaked in Ab-atps and gfp dsRNA for 48 h. According to the results, the expression of OsRLK3 gene in rice inoculated with nematodes soaked in Ab-atps dsRNA was significantly lower than in those soaked in gfp dsRNA at DAI 0.5 (p < 0.05), significantly higher than in those soaked in gfp dsRNA at DAI 1 (p < 0.05), and not significantly different to any soaked in gfp dsRNA at DAI 2-7 (p > 0.05) (Fig. 8). The highest OsRLK3 expression was at DAI 1 among those soaked in Ab-atps dsRNA (p < 0.05), and the highest was at DAI 0.5 among those soaked in gfp dsRNA (p < 0.05). Therefore, rice inoculated with A. besseyi treated with Ab-atps RNAi affected the expression levels of the rice OsRLK3 gene, which indicated its interaction with Ab-atps. In addition, expression of OsRLK3 gene in all treatments was significantly different from that in healthy rice plants (p < 0.05), except in rice inoculated with nematodes soaked in Ab-atps dsRNA at DAI 1.  Standard errors of mean data (n = 5) were indicated by bars and significant differences (p < 0.05) between treatments were indicated by letters. www.nature.com/scientificreports/ induced by chitin, SA, JA, and reactive oxygen species generation, and silencing these genes drastically impairs the defense reactions induced by SA, JA, and reactive oxygen species generation 19 . In the present study, the LysM kinase OsRLK3 from rice was also induced by chitin, flg22, JA, and SA. These findings hint at a potential involvement of OsRLK3 in the chitin, flg22, JA signaling pathways, especially the SA signaling pathway because the up-regulation of OsRLK3 persisted longer when induced by SA than that induced by others. More studies should be carried out in the future to clarify the regulation of OsRLK3 expression.

The characteristics and function of Ab-atps in A. besseyi and its interaction with rice
OsRLK3. The Ab-atps gene interacting with rice OsRLK3 was identified from A. besseyi by yeast two-hybrid screening. We identified this gene as coding for a novel ATP synthase gene, and it was subsequently cloned. The characteristics of this gene were confirmed. Most ATPase genes are expressed in the esophagus, the intestine, the hypodermis, reproduction system, etc 20 . The nematode ATP synthase gene is closely related to the growth, development and reproduction of nematodes, and RNAi of ATP synthase genes led to the death of embryos and diapause of juveniles [20][21][22][23] . ATPase expressed in esophagus is essential for survival 19 . The role of ATPase in nematode development is conserved, and it indicated its role in ovulation and reproduction were also conserved 20,24,25 . At present, the ATP synthase genes of pathogenic parasitic nematodes are being studied as targets for drug development 20 . So far, ATP synthase has been reported in more than 10 species of nematodes including animal parasitic nematodes and free-living nematodes 20,[26][27][28][29] .In plant parasitic nematodes, only the ATP synthase gene of Meloidogyne incognita has been reported to be closely related to the pathogenicity of M. incognita to host plants [21][22][23] . In the present study, we found that A. besseyi Ab-atps mRNA was located in the esophagus and reproductive system. Ab-atps RNAi depressed the reproduction of A. besseyi, and rice inoculated with A. besseyi treated with Ab-atps RNAi affected the expression levels of the rice OsRLK3 gene. The Ab-atps relative expression level was highest in juveniles, followed by eggs, and lowest in adult nematodes. The transient expression of Abatps in plants triggered cell death. Therefore, the results indicated that Ab-atps from A. besseyi may be related to the growth, development, reproduction, and infection of the plant nematode A. besseyi. Plants have developed multiple pathways for defense response against pathogens. One example is rapid death of challenged cells upon pathogen attack, leading to the formation of local lesion. This is termed as the hypersensitive response (HR) 30 . If there is an incompatible pathogen-plant interaction, local programmed cell each (PCD) reaction results in mobility arrest, growth retardation and confinement of invading pathogen at the infection site 31 . N. benthamiana was a valuable heterologous system for fast-forward analysis of plant pathogens regardless of their host plant [32][33][34][35] with an in planta transient expression assay. The fact that Ab-ATPS-GFP localized at the membrane, plasma, and nucleus in tobacco epidermal cells indicated that Ab-atps is recognized by the host plant, possibly inducing plant self-defense responses. The ATPase gene is related to plant cell death. The ATPase gene PDE1 of Magnaporthe grisea was closely related to appressorium formation under infection 36 . Another ATPase gene, MgAPT2 from M. grisea, induces plant resistance and triggers cell death 37 . Plants also activate self-defense responses by self-secreting ATPase. Lee and Sano 38 reported that tobacco ATPase NtAAA1 participates in the self-defense response, and after its silencing, the anaphylactic cell necrosis defense response  besseyi Ab-atps. At DAI 0.5, OsRLK3 expression was highest in rice inoculated with nematodes soaked in gfp dsRNA (p < 0.05), and was significantly higher than that of rice inoculated with nematodes soaked in Ab-atps dsRNA (p < 0.05). Meanwhile, OsRLK3 expression was highest in rice inoculated with nematodes soaked in Abatps dsRNA (p < 0.05) at DAI 1. The expression of OsRLK3 was induced by flg22, SA, JA, and chitin. The highest upregulation of OsRLK3 was observed at 0.5-1 days in rice treated with JA and chitin, and at 2 day and 2-3 days in rice treated with flg22 and SA, respectively. These results indicate that Ab-atps might be related to the rice defense induced by chitin and JA, which are initiated by A. besseyi attack. Rice OsRLK3 was able to recognize A. besseyi Ab-atps and activated a self-defense response. However, the regulation of OsRLK3 is complex, as it was induced by several signaling molecules and further study is needed. Many studies have shown that LysM kinases recognize MAMP molecules of pathogen, and stimulate self-defense responses through chitin-related pathways, including CERK1 of rice and Arabidopsis; LYP4 and LYP6 of rice; and AtLYK1, AtLYK4, AtYLK5, and AtLYP1-3 of Arabidophsis. LysM kinases are also the target of pathogen effectors [14][15][16]40 . Wang et al. 5 reported that OsRLK3 www.nature.com/scientificreports/ was significantly upregulated at the early stage and downregulated at the late stage of rice infected by A. besseyi through transcriptome sequencing, and our study confirmed these results. Therefore, we speculate that through the interactions of rice and A. besseyi, OsRLK3 recognizes Ab-atps and stimulates self-defense responses at the early stage of infection, although A. besseyi may secrete effectors to suppress OsRLK3 at the late stage, given that OsRLK3 was inhibited at the late stage after infection in our study. However, the mechanism by which A. besseyi inhibited or evaded the defense response mediated by OsRLK3 requires further study. In addition, it has been reported that nematode ATPase showed much similarity and share conserved domains 20,29 . The interaction between OsRLK3 and Ab-atps provide some insight into understanding the interaction mechanism between rice and nematode A. besseyi.

Conclusions
Our results suggest that rice OsRLK3 could interact with A. besseyi Ab-atps, which plays an important role in growth, reproduction, and infection of the nematode. Our findings provide a theoretical basis to further understand the parasitic strategy of A. besseyi and its interaction mechanism with host plants, suggesting new ideas and targets for controlling A. besseyi. Our finding also suggested new ideas and targets for developing new methods to control A. besseyi. The preservation and cultivation methods for nematodes were as described by Cheng et al. 42 . As for the vectors, pMD-18T was purchased from Takara (Shiga, Japan); pGBKT7 and pGADT7 were purchased from Clontech (CA, USA); and pCAMBIA1300 was preserved in the laboratory. The Escherichia coli chemically competent cell strain DH5α was purchased from Transgene Biotech (Beijing, China). The yeast (Saccharomyces) chemically competent cell strains Y2H and Y187, and the Agrobacterium tumefaciens chemically competent cell strain GV1301 purchased from Shanghai Weidi Biotechnology (Shanghai, China). All test materials were used under approved protocols and guidelines at South China Agricultural University.  10,44 . The water used for solution preparation had been sterilized before use. The treated rice plants were grown in the growth chamber at 30 °C. They were watered once a week, and were under a 16 h: 8 h light: dark regime, 150 μmol/m 2 /s 1 light density, and 70-75% relative humidity. Rice shoots treated by flg22, JA, SA, and chitin for 0.5, 1, 2, 3, and 7 days were used for RNA extraction. The expression pattern of OsRLK3 was detected. RNA extraction was conducted using the RNAprep Pure Plant Kit (Tiangen, Beijing, China). The extracted RNA was diluted to 100 ng/μl using RNase-free water as a template for cDNA reverse transcription, after examination by electrophoresis for integrity and Nanodrop spectrophotometer for purity. Reverse transcription was performed following the instructions of the HiScript Q RT SuperMix for qPCR (+ gDNA wiper) kit (Vazyme, Nanjing, China). The relative expression levels of target defense-related genes in rice at different times were detected by qPCR using the reverse transcribed cDNA as a template 5 . The primers of target genes OsRLK3 and OsUBQ5 that were used are shown in Table S1. Each treatment included three biological replicates composed of three rice plants. All PCRs were performed in two technical replicates. qPCRs were performed in a CFX96 (Bio-Rad, CA, USA), and data were analyzed using the Bio-Rad CFX 96 Manager (Version 1.5 534.0511) and REST 384 software 45 . Cloning of OsRLK3 and sequence analysis. RNA from rice plants was used as a template and reversetranscribed into cDNA using the PrimeScript II 1st Strand cDNA Synthesis Kit (Takara). Primers RP3F and RP3R (Table S1) were designed for the full coding cDNA amplication of the OsRLK3 gene (GenBank accession: OS01G0741200) according to its sequence in the database of the Rice Genome Annotation Project. Sequence analysis was performed using DNAman 6.0 (Lynnon Biosoft, CA, USA). Protein bioinformatic analysis was performed using Protein Machine software (http:// www. expasy. ch/ tools/), including predictions of protein transmembrane region, amino acid sequence, isoelectric point analysis, molecular weight and hydrophobicity analysis. Predictions for signal peptide and cleavage site analysis were performed at http:// www. cbs. dtu. dk/ servi ces/ Signa lP/. Cell location analysis was performed at http:// psort. ims.u-tokyo. ac. jp/ form2. html.

Expression levels of
Yeast two-hybrid screening. Construction of pGBKT7 recombinant. The yeast two hybrid system (Clonech) was used for screening interaction genes between rice kinase OsRLK3 and a cDNA library of A. besseyi. According to the protocol described for the ClonExpress II One Step Cloning Kit (Vazyme), full coding cDNA of the OsRLK3 gene was connected to the DNA binding domain (BD) of pGBKT7. The full coding www.nature.com/scientificreports/ cDNA of OsRLK3 gene obtained in previous OsRLK3 gene cloning was used as the template, primers BDRP3F and BDRP3R (Table S1) were used for amplification, and the restriction enzymes used were NcoI and BamHI. According to the protocol of the Matchmaker two-hybrid system (Clontech), the full coding region of OsRLK3 was cloned into the GAL4 binding domain vector pGBKT7 as a bait construct after sequence confirmation, and was transformed into the Y2H yeast strain.
Construction of the A. besseyi cDNA library. Total RNA of approximately 20,000 mixed-stages nematodes were extracted using the Invitrogen TRIzol Reagent kit (Invitrogen, Carlsbad, CA, USA). The cDNA library of A. besseyi was constructed in the GAL4 activation domain vector pGADT7 according to the manufacturer's protocol, and transformed into the Y187 yeast strain.
Yeast two-hybrid screening. The Y2H and Y187 yeast strains were co-transformed. The transformants were screened according to the manufacturer's protocol. Interaction screening was carried out on QDO (SD/-Leu/-Trp/-Ade/-His) and QDO/XA plates. The blue colored clone, which was cultured on the QDO/XA plate for 3-5 days at 30 °C, was considered to have an interaction with rice kinase OsRLK3. Plasmids of positive clones were extracted using the HiPure Yeast Plasmid Mini Kit (Magen, Guangzhou, China), transferred into E. coli DH5α competent cells, and selected for sequencing (BGI Company, Shenzhen, China) using primers T7 and 3′AD (Table S1).
Cloning of the full-length Ab-atps gene from A. besseyi. Total RNA of nematodes was reverse transcribed into cDNA using the BD SMARTTM PCR cDNA Synthesis Kit (Takara). 5′ RACE primers (IA-D2R, D2RACER1) (

Confirmation of the interaction between OsRLK3 and Ab-ATPs. The full coding region of Ab-atps
was cloned into the GAL4 activation domain vector pGADT7, according to the protocol of the ClonExpress II One Step Cloning Kit (Vazyme). The primers used were ADD2F and ADD2R (Table S1), and the restriction enzymes used were EcoRI and BamHI. The recombinant plasmid was extracted as a prey construct after sequencing (BGI Company). According to the instructions of Matchmaker two-hybrid system (Clotech), the Y2H yeast strain was co-transformed with the pGADT7-Ab-ATPS and pGBKT7-OsRLK3 vectors. Yeast cells were co-transformed with vectors pGBKT7 and pGADT7-Ab-ATPS, and vectors pGBKT7-OsRLK3 and pGADT7 were used as negative controls. Yeast cells co-transformed with pGBKT-Lam and pGADT7-53 served as positive controls. Interaction screening was carried out on QDO plates and QDO/XA plates. The blue colored clone, which was cultured on QDO/XA plates for 3-5 days at 30 °C, was considered the bait construct that interacted with the prey constructs.

Expression of Ab-atps at different development stages of A. besseyi.
RNA of different developmental stages was extracted from 500 each of females, males, juveniles, and eggs of the nematode using Micro-Elute total RNA kit (OMEGA, GA, USA), respectively. The extracted RNA was reverse transcribed into cDNA using the RQ1 Rnase-Free Dnase (Promega, WI, USA) reverse transcription kit as described above. The expression levels of Ab-atps in four development stages were detected on a CFX-96 (Bio-Rad) qPCR machine with cDNA as a template, using the SYBR Green Real-time PCR Master Mix-plus kit (TOYOBO, Osaka, Japan). Specific primers QD2F and QD2R (Table S1) were designed to detect Ab-cb-1 expression. The 140 bp of 18S rRNA (AY508035) was amplified as a reference gene using the primers Ab18sF and Ab18sR (Table S1). qPCR data was analyzed using CFX manger software provided by Bio-Rad. All experiments were performed in three replicates.
In situ hybridization of Ab-atps. In  www.nature.com/scientificreports/ dsRNA synthesis and RNAi efficiency of Ab-atps. RNA interference (RNAi) of the Ab-atps gene was carried out by soaking the nematodes with dsRNA of Ab-atps synthesized by in vitro transcription. Two primer pairs of IS-D2F/ISD2R and IA-D2F/IA-D2R (Table S1) were designed to amplify the sense and antisense singlestranded RNA (ssRNA) products. Ab-atps dsRNA was synthesized according to the instructions of the Script MaxTM Thermo T7 Transcription kit (TOYOBO). The obtained dsRNA synthesis product was purified using the previously described method 42 , then examined for integrity by electrophoresis, detected for concentration and quality using a Nano-drop spectrophotometer, and stored at − 80 °C until further use. The non-endogenous control dsRNA (125 bp) (green fluorescent protein gene, gfp) was generated with using the specific primers G-T7S, G-A, and G-S (Table S1) 48 . Five hundred mixed-stages nematodes were separated from carrot callus and collected in a DEPC-treated centrifuge tube. 50 μl of Ab-atps dsRNA (2 μg/μL) was added into to the tube for soaking the nematodes at 25 °C. The nematodes were soaked for 12, 24, 36, and 48 h. Non-endogenous gfp dsRNA solution (50 mL; 2 μg/μL) was used as a control. There were total of eight treatments, all performed in triplicate. RNA of nematodes treated with dsRNA solution soaking was extracted after washing the nematodes three times with DEPC water. RNAi efficiency was examined through determination of Ab-atps expression levels by qPCR.
Effect of Ab-atps RNAi on nematode reproduction. Female nematodes were treated with Ab-atps dsRNA for 12, 24, 36, and 48 h, and also treated with gfp dsRNA as a control. Thirty female nematodes were selected from each treatment and inoculated on carrot callus, and each treatment was repeated five times. Carrot callus dishes inoculated with nematodes were incubated at 25 °C in the dark for 35 days, then nematodes on carrot callus were separated and counted.
Expression of OsRLK3 gene in rice tissue at different times after inoculation with A. besseyi treated by dsRNA. Rice plants were inoculated with nematodes of mixed stages treated with Ab-atps dsRNA and gfp dsRNA for 48 h. Control treatments were healthy plants that were inoculated with 50 μL sterilized water. The inoculation method was as previously described by Wang et al. 5 . Rice shoots were collected at 0.5, 1, 2, 3, and 7 days after inoculation (DAI). RNA extraction, cDNA reverse transcription and OsRLK3 expression determination were carried out as described in the previous section. Each DAI treatment included three biological replicates and each replicate was composed of three rice plants. All PCRs were performed in two technical replicates.

Transient expression of rice OsRLK3 and A. besseyi Ab-ATPS in tobacco.
According to the protocol described in the ClonExpress II One Step Cloning Kit (Vazyme), full coding cDNA of gfp was amplified using primers GF/GR (Table S1) and inserted into vector pCAMBIA1300 after SacI/BamHI digest. Full coding cDNA of Ab-atps was amplified using primers PD2F/PD2R (Table S1), and inserted into pCAMBIA1300-GFP after SalI/PstI digest. Recombinant plasmids were transformed into A. tumefaciens GV3101 by N 2 transformation 49 after sequence confirmation (BGI company). Positive clones were screened by LB plates containing both 50 μg/ mL kanamycin and rifampicin, and cultured for 2 days at 28 °C. Growth of recombinant Agrobacterium and vacuum infiltration of tobacco leaves was performed as previously described 50 . Cell death was observed in tobacco leaves infiltrated with recombinant Agrobacterium of pCAMBIA 1300-Ab-ATPS-GFP, pCAMBIA1300-GFP, pCAMBIA1300 empty vector and MES buffer. Total protein was extracted using a plant protein extraction kit (KeyGen Biotech, Nanjing, China), separated by 10% SDS-polyacrylamide gel electrophoresis and detected by western blot with green GFP antibody (Transgene).
Statistical analysis. Data in this study were subjected to analysis of variance (ANOVA) and multiple comparisons of means were conducted using Duncan's Multiple Range Test at p = 0.05 using SAS (Release 8.01), including expression levels of qPCR and nematodes separation counting.
Ethics approval and consent to participate. 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. Plant materials including the collection were used under approved protocols and guidelines at South China Agricultural University.

Data availability
All data generated or analyzed during this study are included in this published article and its supplementary information files.