Introduction

Rice (Oryza sativa) is one of the most important food crops in the world. Several plant nematodes infect rice plants. The economic loss of rice caused by plant nematodes is around 10–25% per year, and white tip nematode, Aphelenchoides besseyi, is the most important plant nematode that damage the aboveground parts of rice. A. besseyi is a migratory parasitic nematode, which is found in most rice-growing areas of the world. Its life cycle is 7–10 days at 21–25 °C. Upon infection, rice generally present white tips and spikelets, which can cause up to 50% economic loss due to severe damage1,2,3,4,5.

Plants are frequently attacked by pathogens and have evolved a multilayer self-defense reactions6. Upon pathogen attack, plants respond with production of specific alarm signals salicylic acid (SA), jasmonic acid (JA), bacterial flagellin protein (flg22) and ET, etc., which varies greatly in quantity, composition, and timing, and it is so-called the specificity of the plant’s primary induced defense response. The signaling pathways that are activated upon endogenous accumulation of these signals regulate different defense responses that are effective against partially distinct pathogens7.

Plant kinase genes play a key role in pathogen recognition and plant defense responses. When plants are infected by pathogens, kinase receptors can recognize particular signaling molecules of pathogens, which are further transmitted downstream and activate the expression of transcription factors, initiating self-defense mechanisms to regulate the physiological and biochemical processes in the interaction of plants and pathogens8. Plant defense response to pathogen infection is regulated by a complex network. When attacked by a pathogen, plants recognize signaling molecules and activate their own defense responses. Using signaling molecules to treat target hosts is helpful for understanding the roles of plant target genes in plant defense responses. Mitogen activated protein kinase (MAPK) and leucine rich repeat receptor-like kinases (LRR-RLK) play an important role in the signal pathways of plant defense reactions, which are induced by the defense signal molecules JA and SA. For example, MAPK kinase BnOIPK can be induced to express by JA, but is not sensitive to SA9. MAPK kinase OsSJMK1 and OsBWMK1 can be induced by JA and SA10,11, and LRR-RLK kinase OsGIRL1, OsSalT, and OsPBZ1 can be induced by SA12.

Among plant pathogens, there is a particular group of evolutionarily conserved molecules that can induce the defense response of host plants, known as microbe-associated molecular pattern (MAMP). Currently, reported MAMPs include flg22, EF-Tu protein, chitin, and peptidoglycan (PGN)13. In plants, there is a particular class of kinase proteins containing a lysine motif (LysM domain). These LysM kinases are mainly recognition receptors, which are located at the cell membrane14 and can directly or indirectly recognize MAMPs of pathogens. For example, the plant LysM kinase CERK1, which is found in Arabidopsis and rice, can specifically recognize the MAMP chitin and stimulate a self-defense response through chitin-related pathways15,16. However, during the long periods of co-evolution between plants and plant pathogens, pathogen gradually evolved an infection mechanism that can inhibit or evade the plant defense response, mediated by LysM kinase protein. For example, AvrPtoB, the effector protein of Pseudomonas syringae, can ubiquitinate and degrade LysM kinase CERK1 in Arabidopsis, thus inhibiting LysM protein recognition mechanisms in Arabidopsis17.

More than 1500 kinase genes have been predicted according to the reported rice genome18, but there are no reports on the role of rice kinase in the interaction of rice and A. besseyi. Wang et al.5 reported that the rice kinase gene OsRLK3 (OS01G0741200) was significantly upregulated and downregulated at the early and late stage of rice infected by A. besseyi, respectively. Thus, the authors suggested that OsRLK3 is involved in the recognition process and could stimulate a self-defense response at the early stage, but might be inhibited by effectors secreted by A. besseyi at the late stage during interaction between rice and A. besseyi. In this study, interaction genes were screened through yeast two-hybridization between OsRLK3 and a cDNA A. besseyi library. Related functions were studied to explore the role of the rice kinase in the interaction of rice and A. besseyi, so as to provide a theoretical basis for uncovering the defense mechanisms of rice against A. besseyi. Our findings may provide useful data to study the other interactions of plants and plant migratory parasitic nematodes.

Results

Expression levels of OsRLK3 in rice treated with flg22, JA, SA, and chitin

Expression levels of OsRLK3 in rice treated with Flagellin protein flg22, JA, SA, and chitin were detected by qPCR. After treatment with flg22, the OsRLK3 gene was significantly upregulated at 0.5–3 days (p < 0.05). The highest level was at 2 days and it was downregulated, but not significantly different to the control, at 7 days (p > 0.05). After treatment with JA, the OsRLK3 gene was significantly upregulated at 0.5–2 days (p < 0.05). The highest level was at 1 day and it was downregulated, but not significantly different to the control, at 3 days and 7 days (p > 0.05). After treatment with SA, the OsRLK3 gene was significantly upregulated at 0.5–7 days (p < 0.05), and the highest level was at 2–3 days. After treatment with chitin, the OsRLK3 gene was significantly upregulated at 0.5–2 days (p < 0.05). The highest level was at 0.5–1 days and the expression level decreased over time, but was not significantly different to the control at 3 days and 7 days (p > 0.05) (Fig. 1). These results showed that the expression of OsRLK3 could be induced by flg22, JA, SA, and chitin, and was significantly upregulated at the early stage after treatment. According to the results, OsRLK3 is indicated to be involved in the self-defense reactions of rice, consistent with the report that OsRLK3 is significantly upregulated at the early stage in rice infected by A. besseyi5.

Figure 1
figure 1

Expression of the receptor-like gene OsRLK3 upon treatment of rice with flg22, JA, SA, and chitin at different time points. Bars indicated standard errors of mean data (n = 3) from three biological and two technical replicates each containing a pool of three plants. Data are shown as relative expression levels of treated/untreated tissue in comparison with the control tissue. Gene expression levels were normalized using the internal reference gene OsUBQ5. The asterisk (*) indicates significant differential expression compared with the control group (p < 0.05). flg22: flagellin 22; JA: jasmonic acid; SA: salicylic acid; CHITIN: chitin.

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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). The cDNA sequence was identified as A. besseyi ATP synthase (ATPase) according to the sequence alignment results of blastx, and named Ab-atps, showing highest homology with ATP synthase from Strogyloides ratti (GenBank accession: XP_024505238.1, similarity 88%, identity 47.49%, E value:149), followed by ATP synthase from Necator americanus (Genbank accession: XP_013292314.1, similarity 86%, identity 45.14%, E value 9e−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.

Figure 2
figure 2

Phylogram constructed on the basis of amino acid sequences depicting the evolutionary relationships among ATP synthase (ATPase) from 24 species of nematodes. Aphelenchoides besseyi Ab-ATPS is underlined; Accession numbers of the sequences are shown in brackets; Distances on the X-axis correspond to the grade of sequence homology; Distances on the Y-axis are arbitrary.

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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).

Figure 3
figure 3

Detection of the interactions between rice OsRLK3 and Aphelenchoides besseyi Ab-atps. (A) Interaction of OsRLK3 and Ab-atps. Yeast cells transformed with pGBKT7-OsRLK3 (BD) and pGADT7-Ab-ATPS (AD) were grown on YDPA media plates and then printed onto QDO/XA (SD/-Leu/-Trp/-Ade/-His/X-α-gal/AbA) plates, and the colony turned blue; yeast cells transformed with pGADT7-Lam and pGBKT7-53 were used as positive controls; yeast cells were transformed with pGADT7 and pGBKT7-53 were used as negative controls. (B) Serial dilutions (1, 10–1, 10–2) of the yeast cells with pGBKT7-OsRLK3 (BD) and pGADT7-Ab-ATPS (AD) were cultured on QDO/XA plates for 1 day to detect their interaction abilities.

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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).

Figure 4
figure 4

Expression of Ab-atps in Aphelenchoides besseyi of different developmental stages: egg, juvenile, female, and male. Bars indicate standard errors of mean data (n = 3) and letters indicate significant differences (p < 0.05) between treatments.

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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).

Figure 5
figure 5

Tissue localization of Aphelenchoides besseyi ATPase mRNA Ab-atps using in situ hybridization. (A) Ab-atps mRNA was expressed in the esophagus; (C,E) Ab-atps mRNA was expressed in the reproductive system; (B,D,F) A hybridization signal was observed in the control nematodes using a DIG-labeled sense Ab-atps RNA probe; mb: medium bulb, o: ovary, s: spicules, te: testis, v: vulva.

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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).

Figure. 6
figure 6

Expressions of the Ab-atps mRNA in Aphelenchoides besseyi treated with Ab-atps double-stranded (ds) RNA. G12, G24, G36, and G48: Expressions of Ab-atps mRNA in nematodes that soaked in non-endogenous gfp dsRNA solution for 12 h, 24 h, 36 h, and 48 h, respectively; A12, A24, A36, and A48: Expression of Ab-atps mRNA in nematodes that soaked in Ab-atps dsRNA for 12 h, 24 h, 36 h, and 48 h, respectively. Standard errors of mean data (n = 5) were indicated by bars and significant differences (p < 0.05) between treatments were indicated by letters.

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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 decreased, when treated with Ab-atps dsRNA. And significant differences (p < 0.05) were found between different RNAi treatment time groups except between 12 and 24 h, and 36 h and 48 h (p > 0.05). There was no significant difference between gfp dsRNA treatment groups (p > 0.05). Therefore, Ab-atps RNAi inhibited Ab-atps expression in A. besseyi effectively by soaking the nematodes in Ab-atps dsRNA, and Ab-atps RNAi depressed the reproduction of A. besseyi.

Figure. 7
figure 7

Number of Aphelenchoides besseyi on carrot callus after inoculation of 30 females for 35 days. G12, G24, G36, and G48: Number of A. besseyi after inoculating 30 females soaked in non-endogenous gfp dsRNA solution for 12 h, 24 h, 36 h, and 48 h, respectively; A12, A24, A36, and A48: Number of A. besseyi after inoculating 30 females soaked in Ab-atps dsRNA for 12 h, 24 h, 36 h, and 48 h, respectively. Standard errors of mean data (n = 5) were indicated by bars and significant differences (p < 0.05) between treatments were indicated by letters.

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Expression of OsRLK3 gene in rice tissue at different times after inoculation with A. besseyi treated with dsRNA

Expression levels of OsRLK3 in rice shoots were detected at different times (DAI) 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.

Figure. 8
figure 8

Expression of the OsRLK3 gene in rice tissue at different times after inoculation with Aphelenchoides besseyi treated with dsRNA. Bars indicate standard errors of mean data (n = 3) from three biological and two technical replicates each containing a pool of 3 plants. Data are shown as relative expression levels of treated/untreated tissue in comparison with the control tissue. Gene expression levels were normalized using the internal reference gene OsUBQ5. Uppercase letters indicate significant differences (p < 0.05) among treatments when rice plants were inoculated with A. besseyi treated with Ab-atps dsRNA. Lowercase letters indicate significant differences (p < 0.05) among treatments when rice plants were inoculated with A. besseyi treated with gfp dsRNA. The asterisk (*) indicates significant differential expression compared with healthy rice plants (p < 0.05). s: significant difference (p < 0.05) between rice plants inoculated with A. besseyi treated with Ab-atps dsRNA and gfp dsRNA at the same time points. ns: not significant (p > 0.05) between rice plants inoculated with A. besseyi treated with Ab-atps dsRNA and gfp dsRNA at the same time point. DAI: Days after inoculation; gfp: rice inoculated with A. besseyi treated with gfp dsRNA for 48 h; Ab-atps: rice inoculated with A. besseyi treated with Ab-atps dsRNA for 48 h.

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Transient expression of Ab-ATPS from Aphelenchoide besseyi in tobacco

To confirm the subcellular location of rice OsRLK3 and A. besseyi Ab-ATPS in plant cells, full coding cDNA of Ab-atps and gfp were inserted into the plant transient expression vector pCAMBIA1300 and transiently expressed in tobacco epidermal cells (Fig. 9A). Green fluorescent protein (GFP) and Ab-ATPS-GFP localized at the membrane, plasma, and nucleus in tobacco epidermal cells. The recombinant protein extracts were detected by western blot to confirm expression. A distinct band was observed at 40 kDa, which was GFP (approximately 37 kDa), and at 50–70 kDa, which was Ab-ATPS-GFP (approximately 57 kDa) (Fig. 9B). At 5 days after infiltration, obvious cell death was observed at the infiltrated site with recombinant Agrobacterium of pCAMBIA 1300-Ab-ATPS-GFP, compared with no infiltrated sites with pCAMBIA1300-GFP, pCAMBIA1300 empty vector and 2-(N-morpholino) ethanesulfonic acid hydrate (MES) buffer (Fig. 9C). The results confirmed that Ab-ATPS from A. besseyi triggers plant cell death.

Figure. 9
figure 9

Subcellular localization and transient expression of Ab-ATPS from Aphelenchoides besseyi in tobacco epidermal cells. (A) Ab-ATPS-GFP localized at the membrane, plasma, and nucleus in tobacco epidermal cells. (B) Ab-ATPS-GFP expression was detected by Western-blot. M: Protein Marker (Transgene); 1: Protein mix of GFP extracted from tobacco leaves; 2: Protein mix of Ab-ATPS-GFP extracted from tobacco leaves. (C) Ab-ATPS-GFP triggered cell death. 1: 2-(N-Morpholino) ethanesulfonic acid hydrate (MES) buffer; 2: pCAMBIA1300-GFP; 3: pCAMBIA1300-Ab-ATPS-GFP; 4: pCAMBIA1300 empty vector. n: nuclear, c: cytoplasm, m: membrane. GFP: protein was generated with green fluorescent protein.

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Discussion

OsRLK3 could be induced by chitin, flg22, JA, and SA

The results of this study showed that expression of rice OsRLK3 could be induced by several signal molecules in the plant defense pathway and A. besseyi infection, which means that it plays an important role in the rice defense response to A. besseyi. OsRLK3 is a LysM kinase. Most LysM kinases are induced by chitin10,15. The LysM kinases Lyp1, Lyk7, and LysMe3, play important roles in chitin perception and defense against Verticillium dahliae in cotton. These kinases are 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 generation19. 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, etc20. 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 juveniles20,21,22,23. ATPase expressed in esophagus is essential for survival19. The role of ATPase in nematode development is conserved, and it indicated its role in ovulation and reproduction were also conserved20,24,25. At present, the ATP synthase genes of pathogenic parasitic nematodes are being studied as targets for drug development20. So far, ATP synthase has been reported in more than 10 species of nematodes including animal parasitic nematodes and free-living nematodes20,26,27,28,29.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 plants21,22,23. 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 Ab-atps 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 site31. N. benthamiana was a valuable heterologous system for fast-forward analysis of plant pathogens regardless of their host plant32,33,34,35 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 infection36. Another ATPase gene, MgAPT2 from M. grisea, induces plant resistance and triggers cell death37. Plants also activate self-defense responses by self-secreting ATPase. Lee and Sano38 reported that tobacco ATPase NtAAA1 participates in the self-defense response, and after its silencing, the anaphylactic cell necrosis defense response of tobacco was inhibited by inoculation with the pathogen Pseudomonas syringae. The AAA ATPase AtOM66, localized in the mitochondrial membrane of Arabidopsis thaliana, plays an important role in triggering cell death to resist pathogen infection39. In our study, we also found a similar result in that the ATP synthase gene Ab-atps from A. besseyi could induce plant cell death in tobacco. This indicates that Ab-atps can be recognized by the host plant and is involved in the plant defense response in the interaction of rice and A. besseyi. Based on these results, Ab-atps might be important for developing new methods to control A. besseyi.

Rice OsRLK3 interacts with A. besseyi Ab-atps

The expression of OsRLK3 gene was affected by A. 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 Ab-atps 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 effectors14,15,16,40. Wang et al.5 reported that OsRLK3 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 domains20,29. 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.

Methods

Plants materials, nematode, vectors, and chemically competent cell strains

Rice, Oryza sativa L. cv. “Nipponbare”, was obtained from Prof. Guohui Zhou of the Laboratory of Plant Virus, South China Agricultural University, and cultivated as described by Wang et al.5. Tobacco, Nicotiana benthamiana, was preserved in the laboratory, and cultivated as described by Li et al.41 and Wang et al.4. The A. besseyi used in this study was collected from rice, O. sativa, in Luhe Town, Nanjing, Jiangsu Province, China. Nematodes were isolated, identified, preserved, and cultured by the Laboratory of Plant Nematology, South China Agricultural University. 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.

Expression levels of OsRLK3 in rice treated with flg22, JA, SA, and chitin

At the two-leaf stage, healthy rice plants were treated with flg22, JA, SA, and chitin respectively. Flagellin protein flg22 (amino acid sequence, QRLSTGSRINSAKDDAAGLQIA) and chitin were purchased from Sangon Biotech Co., Ltd (Shanghai, China). SA with purity ≥ 99.5% was purchased from Tianjin Baishi Chemical Co., Ltd (Tianjin, China). JA with purity ≥ 85% was purchased from Shanghai Makclin Biochemical Co., Ltd (Shanghai, China). At the two-leaf stage of rice, 1 ml of 2-μM flg22 protein water solution was infiltrated into the surface of rice plant43; for SA, JA, and chitin treatments, rice plants were sprayed with 100 μM SA, JA, and chitin water suspension separately and evenly10,44. 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/m2/s1 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 template5. 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 software45.

Cloning of OsRLK3 and sequence analysis

RNA from rice plants was used as a template and reverse-transcribed 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/services/SignalP/. Cell location analysis was performed at http://psort.ims.u-tokyo.ac.jp/form2.html.

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 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) (Table S1) were designed for cDNA amplification according to the sequencing results of positive clones selected from the A. besseyi cDNA library. The amplified products were purified and ligated with pMD 18-T vector (Takara) to obtain recombinant plasmid. Recombinant plasmid was transformed into E. coli DH5α competent cells, and then positive clones were selected for sequencing (BGI Company) as described method by Cheng et al.42. According to the sequencing results, primers D2F and D2R (Table S1) were designed for the full-length amplification of the Ab-atps gene from A. besseyi.

Sequence analysis, alignment and phylogenetic analysis of Ab-ATPS

Sequence homology alignments were performed using NCBI blastn and blastx (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Predictions of signal motifs and cleavage sites were performed at http://www.cbs.dtu.dk/services/SignalP/. Predictions of transmembrane regions were performed at http://www.cbs.dtu.dk/services/TMHMM/#opennewwindow. The phylogenetic tree was constructed using the neighbor-joining method46 using the program MEGA 6.0 (Molecular Evolutionary Genetics Analysis, USA) based on Ab-ATPS and other 23 ATP synthase sequences of representative nematodes in the NCBI database.

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 MicroElute 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 situ hybridization was performed as described as De et al.47. Approximately 10,000 nematodes of mixed-stages of A. besseyi were collected and concentrated into 30–50 μL. The nematodes were fixed in 3% paraformaldehyde for 18 h at 5 °C and later at 22 °C for 4 h. DIG-labelled sense and antisense RNA probes (Roche, Mannheim, Germany) were synthesized using sense primers (IS-D2F, IS-D2R) and anti-sense primers (IA-D2F, IAD2R) (Table S1) and the full-length cDNA of Ab-atps as a template. DIG-labeled RNA probes were added to the hybridization solution containing nematode and then rotated for 12 h at 47 °C. Results were examined and photographed by an optical microscopy (Nikon Eclipse 90i, Nikon, Japan).

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 single-stranded 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 method42, 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 N2 transformation49 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 described50. 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.