The new effector AbSCP1 of foliar nematode (Aphelenchoides besseyi) is required for parasitism rice

HUANG Xin,CHl Yuan-kai,Addisie Abate BlRHAN,3,ZHAO Wei,Ql Ren-de,PENG De-liang

1 Institute of Plant Protection and Agro-products Safety,Anhui Academy of Agricultural Sciences,Hefei 230001,P.R.China

2 State Key Laboratory for Biology of Plant Diseases and Insect Pests,Institute of Plant Protection,Chinese Academy of Agricultural Sciences,Beijing 100193,P.R.China

3 Agricultural Biotechnology Directorate,Ethiopian Biotechnology Institute,Addis Ababa 5954,Ethiopia

Abstract Plant parasitic nematodes secrete effector proteins to parasitize hosts successfully.Of these proteins,serine carboxypeptidases have critical roles in pathogenicity.This study investigated the role of new effector AbSCP1 in Aphelenchoides besseyi pathogenicity.In situ hybridization and qRT-PCR analyses indicated that AbSCP1 was exclusively expressed in the esophageal glands and upregulated in juveniles.Subcellular localization assays indicated that the protein was expressed in the nucleus.The ability to hydrolyze C-terminal amino acid residues was proven for AbSCP1.Moreover,RNAi significantly reduced the expression of AbSCP1 and RNAi-treated nematodes’ reproductive potential.Pathogenicity assays on rice showed that RNAi-treated nematodes were less pathogenic than the untreated control groups.These results suggest the important role of AbSCP1 in the A.besseyi infection process.

Keywords:Aphelenchoides besseyi,serine carboxypeptidases,effector,nematode,pathogenicity

1.lntroduction

The rice white tip nematode (RWTN)Aphelenchoides besseyiis considered to be one of the ten most economically important plant parasitic nematodes (PPNs)(Joneset al.2013).These nematodes can infect more than 200 plant species,including rice,wheat,soybean and cotton (Bridgeet al.2005;Meyeret al.2017;Favoretoet al.2018).The RWTN is widely distributed in major rice-growing areas worldwide (EPPO 2012).The RWTN contributes to causing an estimated 16 billion USD worth of annual damage to rice crops globally (Lilleyet al.2011).This nematode is also among the most economically important pathogens in rice production in China (Peng 1998).Several control strategies have been attempted to reduce the damage caused by this important nematode.Nonetheless,to date,soaking seeds with hot water or chemical nematicides is the main method used to control RWTN (Chenget al.2013).The use of chemical nematicides is restricted mainly because of their negative impact on human health,nontarget organisms and the environment.Therefore,an environmentally friendly and sustainable control strategy is required.In this regard,one of the recent advances in PPN control is the use of molecular techniques to target the proteins secreted by these nematodes.

To successfully infect and continuously obtain nutrients from the host plant,PPNs need to secrete numerous effectors into the host plant.First,these nematodes secrete many cell wall-degrading enzymes.Subsequently,the stylet punctures the host cell wall and injects many effectors,including peptidases,into the host.One recent study demonstrated that animal parasitic nematodes (APNs) secrete peptidases,which are important virulence factors,in host tissue to successfully parasitize their host (Alvarezet al.2012).Studies have shown that peptidases of many parasites play a significant role in nutrient uptake,embryonic development of the egg and molting (Malagónet al.2013).The major types of peptidases in parasitic nematodes are aspartate peptidase,cysteine peptidase,metalloproteinase and serine peptidase (Sakanari and McKerrow 1990).Serine peptidases are believed to play the largest role in the invasion of human and animal tissues (Sakanari and McKerrow 1990).Serine proteases can also weaken the host defense response by inhibiting the activity of macrophages (Auriaultet al.1983).Serine carboxypeptidase (SCP) is a member of the serine protease family,belonging to the S10 family of carboxypeptidases (Breddam 1986).These SCPs secreted by parasites are involved in the penetration progress of nematodes and have been defined as potential drug targets (Marikovskyet al.1988;Sakanariet al.1989).Several SCPs have been identified in the secretions of APNs,such asHeligmosomoides polygyrus,Strongyloides rattiandHaemonchus contortus,with proteomic methods (Yatsudaet al.2003;Hewitsonet al.2011;Sobliket al.2011).However,very few studies have been performed on the role of SCP in PPNs.For example,one putative SCP gene was more highly expressed in the juveniles ofHeterodera glycinesRace 3 than in Race 4 using RNA-seq (Wanget al.2014).Another study reported that theSCPgene secreted byRadopholus similisplayed a significant role in the reproduction and pathogenesis of this nematode(Huanget al.2017).However,the role of SCP inA.besseyiand the interaction between nematodes and host plants have not yet been studied.

Therefore,the objective of this study was to investigate the potential role of the SCP gene (AbSCP1) in the development and infection process ofA.besseyi.In this study,the full-lengthAbSCP1was amplified based on the transcriptome data ofA.besseyi.The expression and localization ofAbSCP1were investigated by performingin situhybridization,quantitative real-time PCR (qPCR),eukaryotic expression and transient expression analyses.In addition,the function of theAbSCP1gene was investigated by RNA interference (RNAi).

2.Materials and methods

2.1.Experimental materials

Aphelenchoides besseyiwere isolated from rice leaves and cultured on carrot disks according to previously described methods (Sunet al.2009;Tuleket al.2009).Rice (Oryza sativaL.japonicacv.Nipponbare) was used for the nematode infection experiment.The rice was grown in a 0.5-L plastic cup filled withca.400 g of sterilized soil in a greenhouse at 28°C (16 h light/8 h dark photoperiod) and 60-80% relative humidity.

Nematode DNA/RNA extraction was performed according to Huanget al.(2017).First-strand cDNA was synthesized using a cDNA Synthesis Kit (Thermo Fisher,Waltham,MA,USA).

2.2.Gene amplification and phylogenetic analysis

From the transcriptome data ofA.besseyi,one putative gene coding for serine carboxypeptidase was screened out.Amplification of the gene was performed using a SMART RACE cDNA Amplification Kit (Clontech,TaKaRa Biotechnology (Dalian) Co.,Ltd.,Dalian,China)with 865GSP1/UPM for the 5′ end and 865GSP2/UPM(Appendix A) for the 3′ end.The two sequences were spliced into the complete sequence ofAbSCP1(GenBank ID:MN200102).The online NCBI server ORFfinder(http://www.ncbi.nlm.nih.gov/gorf/orfig.cgi/) was used for ORF prediction.The specific primers Ab-scp-f/Ab-scp-r(Appendix A) were designed to amplify the full-length sequence ofAbSCP1.PCR was performed according to the instructions of KOD Plus Neo (TOYOBO,Osaka,Japan) at an annealing temperature of 60°C usingA.besseyicDNA and genomic DNA as templates.Then,the cDNA and genomic DNA sequences ofAbSCP1were confirmed by sequencing.SignalP 4.0 (http://www.cbs.dtu.dk/services/SignalP-4.0/) and TMHMM Server v.2.0(http://www.cbs.dtu.dk/services/TMHMM/) were used to predict the signal peptide and transmembrane domains in AbSCP1.To predict the subcellular localization of the protein sequence,the PSORT server (https://psort.hgc.jp/) was used.

To construct the phylogenetic tree,the amino acid sequence ofAbSCP1 was blasted against the NCBI nonredundant protein database (nr),and 19 amino acid sequences from 11 species were used for comparative purposes.Phylogenetic analyses of the amino acid sequence data were conducted using MEGA 7 Software(Kumaret al.2016).

2.3.In situ hybridization

Gene-specific primers SCP-T7F/SCP-R and SCP-F/SCPT7R (Appendix A) were designed to amplify two fragments with the T7 promoter sequence at the 5′ and 3′ ends,respectively.DIG-labeled sense and antisense probes were synthesized with these two fragments as templates using DIG RNA Labeling Mix (Roche,Basel,Switzerland)according to the manufacturer’s procedures.In situhybridization was performed as previously described (de Boeret al.1998).After hybridization at 47°C for 14 h,the nematodes were examined using a Leica DM1000 LED(Leica,Weztlar,Germany).

2.4.Expression analysis of AbSCP1

RNA from different life stages ofA.besseyi(eggs,juveniles,females and males) was extracted using the miRNeasy Micro Kit (Qiagen,Dusseldorf,Germany).cDNA templates for qPCR were synthesized with HiScript II Q RT SuperMix (Vazyme,Nanjing,China).qPCR was performed using a CFX96 qPCR instrument with primers scp-qf/scp-qr (Appendix A) following the instructions of the AceQ qPCR SYBR Green Master Mix Kit (Vazyme).Furthermore,act-f/act-r and ubc-f/ubc-r were designed to amplify two internal reference gene (Actinandubc)fragments,respectively.The 2-ΔΔCtmethod was used to calculate the relative expression level ofAbSCP1in different life stages ofA.besseyi.Three biological replicates were set.

2.5.Subcellular localization of AbSCP1

To observe the subcellular localization of AbSCP1 in the host plant,theAbSCP1gene sequence without a signal peptide was amplified with the specific primers SCPSPBamHi-F/SCP-EcoRi-R (Appendix A) and fused to the N-terminus of an enhanced green fluorescent protein (eGFP)in pYBA1132.The recombinant vector pYBA1132:SCP:GFP and the nuclear marker vector (pYBA1132:NLS:mKate)were transformed intoAgrobacterium tumefaciensEHA105.Then,tobacco leaves were injected with equal amounts ofA.tumefacienscontaining these two vectors following the procedure described by Zhuet al.(2017).Two days after injection,the leaves were examined under a Nikon C2-ER microscope (Nikon,Tokyo,Japan) in the dark at room temperature.The empty vectors that expressed eGFP were set as controls.

2.6.RNAi

Before the RNAi experiment,short interfering RNAs(siRNAs) inAbSCP1were predicted by BLOCK-iT RNAi Designer (http://rnaidesigner.thermofisher.com/rnaiexpress/),and a 240-bp fragment from theAbSCP1sequence that has four predicted siRNAs was chosen.Then,AbSCP1dsRNA was synthesized as described by Huanget al.(2017).Approximately 500 mixed-stageA.besseyiwere soaked in dsRNA (2.0 μg μL-1),and the expression ofAbSCP1was measured at different time intervals (6,12,24,and 36 h).Nematodes treated withegfpdsRNA solution (2.0 μg μL-1) and ddH2O for the same time periods were used as positive and blank controls,respectively.The HiScript II Q RT SuperMix Kit (Vazyme) mentioned above was used for cDNA synthesis from different RNAi samples.qPCR was used to analyze the expression change ofAbSCP1after RNAi as described above.

To confirm the impact ofAbSCP1silencing onA.besseyireproduction,nematodes were cultured onBotrytis cinerea.The fungi were grown on PDA plates at 25°C for 10 days,and approximately 200 mixed-stageA.besseyitreated withAbSCP1dsRNA,egfpdsRNA and ddH2O were inoculated at the center of the plate.TheegfpdsRNA-and ddH2O-treated nematodes were used as controls.Three replications were set for each treatment.The feeding ofA.besseyiwas observed at three,seven and ten days post inoculation.After 10 days,the nematodes were collected with a Baermann funnel and counted with a Leica DM1000 LED (Leica).

In the pathogenicity test,approximately 1 000 mixedstageAbSCP1RNAi-treatedA.besseyiwere inoculated onto two-leaf stage rice seedlings per plastic cup(Wanget al.2019).Untreated andegfpdsRNA-treated nematodes were used as controls.Nematodes were counted after 60 days of inoculation based on previously described methods (Liuet al.2008).Symptom severity on rice was rated based on methods described by Wanget al.(2016).Five biological replicates were set for each treatment,and the experiment was repeated two times.

2.7.Enzyme activity of AbSCP1

To express AbSCP1 in the baculovirus/Sf9cell system,the full-length sequence ofAbSCP1was inserted into pFastBac1 between theBamHI andEcoRI sites.The presence of the construct was confirmed by sequencing.Plasmid purification and transposition were performed according to the Bac-to-Bac?Baculovirus Expression System protocol (Invitrogen,Carlsbad,USA).

Sf9cells were transfected with bacmid DNA using Cellfectin?II Reagent (Thermo Fisher).The recombinant virus was harvested 72 h post transfection and further amplified to obtain more recombinant protein.This recombinant protein was first filtered through a 0.22-μm filter and then bound to the column overnight with a flow rate of 1 mL min-1,and the proteins were eluted with imidazole.The purification of recombinant protein was determined by Western blot using an anti-6×His tag mouse monoclonal antibody (Sangon Biotech,Shanghai,China).The protein concentration was measured with a BCA Protein Assay Reagent Kit (Thermo Fisher).The purified protein was dissolved in protein storage buffer(20 mmol L-1Tris,300 mmol L-1NaCl,10% Glycerol,pH 7.5).

Furyl acryloyl (FA)-Phe-Phe-OH was used as the substrate in the enzyme activity analysis.Carboxypeptidase activity was measured with the same method described by Parussiniet al.(2003) with Cytation5(Bio-Tek,Vermont,USA) at 25°C.The enzyme solution(1 μL) was added to 99 μL of substrate solution (50 mmol L-1sodium acetate,1 mmol L-1EDTA buffer containing 0.2 mmol L-1substrate).Enzyme activity was measured under different pH values.

2.8.Data analysis

GraphPad Prism 7 was used to analyze the data and layout graphs.All data were analyzed by one-way ANOVA and Tukey’s multiple comparison tests (at a 5% level).

3.Results

3.1.Gene amplification and phylogenetic analysis

The full-length sequence ofAbSCP1was amplified with Abscp-f/Ab-scp-r and confirmed by sequencing.TheAbSCP1gDNA was 2 001 bp in length with eight introns (Fig.1-A).The ORF ofAbSCP1was 1 425 bp in length (Fig.1-B),encoding a 474-aa sequence containing a signal peptide(Fig.1-C),but did not have a transmembrane domain.Sequence alignment showed thatAbSCP1has 65.84%similarity withRs-SCP-1fromR.similis.Maximum likelihood(ML) analysis ofAbSCP1showed a close phylogenetic relationship withRs-SCP-1(Fig.2).

Fig.1 PCR amplification and sequence analysis of Aphelenchoides besseyi AbSCP1.A,PCR amplification of the AbSCP1 gDNA sequence.M,DS2000 marker;1,PCR product of the AbSCP1 gDNA sequence.B,PCR amplification of AbSCP1 ORF sequence.M,DS2000 marker;2,PCR product of AbSCP1 ORF sequence.C,the ORF sequence of AbSCP1;the signal peptide is marked in red.

Fig.2 Maximum-likelihood phylogenetic tree inferred from sequences of serine carboxypeptidases (SCPs).

3.2.AbSCP1 was expressed in the esophageal gland at the parasitic stages

qPCR analysis of eggs,juveniles,females and males showed the expression ofAbSCP1in all life stages (Fig.3).AbSCP1had the highest relative expression level in juveniles,followed by females and males.No significant difference (P＞0.05)was found in expression between females and males.However,low expression was detected in the eggs.In situhybridization showed thatA.besseyihybridized with the DIG-labeled anti-sense probe showed a strong signal in the esophageal glands (Fig.4-A and B) ofA.besseyi.No hybridization signals were detected in the nematodes and eggs hybridized with the control sense probe (Fig.4-C).

Fig.3 Relative expression of AbSCP1 in Aphelenchoides besseyi at different life stages.Bars indicate the standard error of the mean (n=3),and different letters indicate significant differences (P＜0.05) between treatments.

Fig.4 Tissue localization of AbSCP1 in Aphelenchoides besseyi.A and B,AbSCP1 was expressed in the esophageal glands.C,no hybridization signals were observed in the control nematodes.eg,esophageal glands.Scale bars=20 μm.

3.3.AbSCP1 was localized to the nuclear in the plant cell

PSORT did not predict any putative nuclear localization signals (NLSs) in AbSCP1.To clarify the subcellular localization ofAbSCP1in host cells,SCP:eGFP,eGFP and the colocalization marker NLS:mKate were transformed into tobacco leaf cells.In the GFP control,the green fluorescence was limited to the plasma membrane system and nucleus (Fig.5-A).When transformed with the target protein containing NLS:mKate,

AbSCP1 was localized in the nucleus (Fig.5-B).

Fig.5 Subcellular localization of AbSCP1 in tobacco leaf cells.A,fluorescence signal of eGFP.B,fluorescence signal of AbSCP1 merged with NLS:mKate.Scale bar is equal to 20 μm.

3.4.AbSCP1 degrade host proteins from C-terminus

AbSCP1 expressed and purified from the baculovirus/Sf9cell system was used in catalytic activity analysis.Western blot analysis clearly showed an extra protein band (～55 kDa) in the infectedSf9cells and supernatant compared with the negative controls (Fig.6-A).The molecular mass of this protein (55 kDa) is consistent with the molecular mass of AbSCP1 (53.6 kDa) with an additional nine histidine residues (HHHHHHHHH) at its C-terminal end.The final concentration of purified protein was 0.185 mg mL-1.The enzyme had higher activity under acidic conditions,and the optimum pH of the enzyme activity was 4.5 (Fig.6-B).

Fig.6 Eukaryotic expression of AbSCP1 and enzyme activity analysis.A,Western blot of AbSCP1 in different samples.M,protein marker;1-6,six cell samples transfected with positive AbSCP1 bacmid clones;7-12,samples prepared from the supernatant of culture medium corresponding to six cell samples (1-6);NC,blank control.B,enzyme activity of AbSCP1 under different pH values.

3.5.AbSCP1 RNAi decreased the infectivity of nematodes

qPCR was used to clarify the expression change ofAbSCP1after RNAi.Compared with the relative expression level of the controls,it decreased significantly afterAbSCP1dsRNA treatment (P＜0.05).There were no significant differences (P＞0.05) in the expression abundance between the water-treated and positive controls (Fig.7-A).The expression ofAbSCP1decreased to the lowest level after 24 h ofAbSCP1dsRNA soaking.There were no significant differences between treatmentsat 6,12 and 36 h (P＜0.05).However,the expression level ofAbSCP1was significantly lower after 24 h of RNAi treatment than at 6 and 12 h.Therefore,24 h ofAbSCP1dsRNA soaking achieved the best silencing effect.

Ten days after inoculation of the nematodes ontoB.cinerea,there was a significant difference in the feeding area between the treatment and control groups.The feeding areas of nematodes treated withAbSCP1dsRNA were smaller than those of nematodes treated with ddH2O andegfpdsRNA (Fig.7-B).There was also a significant difference (P＜0.05) in the number of nematodes betweenAbSCP1dsRNA-treated (n=317)and control groups (ddH2O-treated,n=949;egfpdsRNAtreated,n=909) (Fig.7-C).There was no significant difference (P＞0.05) between the two control treatments.In addition,the severity of symptoms on rice leaves inoculated withAbSCP1-silenced nematodes (average symptom severity=1.909) was significantly lower than that of rice leaves treated with control nematodes (4.455 for ddH2O-treated nematodes,4.282 foregfpdsRNA-treated nematodes) (Fig.7-D and E).

Fig.7 Changes in the expression of AbSCP1,and the reproduction and pathogenicity of Aphelenchoides besseyi after RNAi.A,expression of AbSCP1 in A.besseyi under different treatments.B,RNAi-treated (dsGFP and dsSCP) and untreated (CK) A.besseyi fed on Botrytis cinerea.The inoculation point is indicated by the red arrow.C,number of A.besseyi from B.cinerea plates counted 10 days after RNAi treatments.D,effects of AbSCP1 RNAi on the pathogenicity of A.besseyi.E,symptom grade of rice leaves infected by A.besseyi infection.CK,water-treated control;G-6-36 and dsGFP,dsGFP-treated;I-6-36 and dsSCP,dsSCP-treated.Bars indicate the standard error of the mean,and different letters indicate significant differences (P＜0.05) between treatments.

4.Discussion

Proteases are among the most abundant effectors present in migratory parasitic nematodes (Huanget al.2019;Mathew and Opperman 2019).Of these,serine carboxypeptidase is one of the most important effectors that play a significant role in the pathogenic process of PPNs.Based on the results ofin situhybridization,qPCR,RNAi and transient expression analyses,the present study confirmed that SCP is a secreted protein that plays an important role in the pathogenesis ofA.besseyi.In a study by Huanget al.(2017),SCP was involved in the pathogenicity ofR.similis.However,information on the infection mechanism of SCP is still unknown in foliar nematode.

In situhybridization and qPCR analysis revealed thatAbSCP1was expressed in the esophageal glands and upregulated in juveniles.Previous studies indicated that the SCP protein was present in the secretion ofAngiostrongylus cantonensis(Fanget al.2010).TheSCPgene was located in the esophageal gland and intestine ofR.similis,and the expression ofRs-scp-1in females and juveniles was significantly higher than that in males and eggs (Huanget al.2017).Secreted proteins produced by the esophageal glands play key roles in the parasitism process of PPNs (Daviset al.2000).Most of the previously reported effectors are highly expressed in juveniles (Vieira and Gleason 2019).After the juveniles hatch from eggs,nematodes begin to search for hosts to establish and maintain parasitism,so a high expression level of a large number of effectors is needed.

This study confirmed that AbSCP1 was localized in the nucleus even though no NLS was predicted.Effectors secreted by PPNs target diverse subcellular structures,such as the cell wall (Liuet al.2016),nucleus(Cabralet al.2020),cytoplasm (Chenet al.2017),and peroxisomes (Thorpeet al.2014),enabling the successful infection of nematodes.In some cases,hostmediated posttranslational modification is very important for effectors to function in the host.It has been reported that MgGPP undergoes glycosylation before finally being transported to the nucleus (Cabralet al.2020).Effectors such as MgMO237 and Hg16B09 use a noncanonical transport mechanism to enter the nucleus (Chenet al.2018;Huaet al.2019).This suggests that a similar mechanism may be used for AbSCP1 delivery into the nucleus.This study confirmed the characteristics of SCP by the expression and localization ofAbSCP1.The AbSCP1 protein produced by the esophageal glands and secreted into the nucleus of the plant cells may accelerate the degradation of host protein,which aidsA.besseyifeeding and parasitism.

SCPs are highly specific for hydrophobic C-terminal amino acid residues (Mortensenet al.2013).The carboxypeptidase activity of serine carboxypeptidase fromT.cruziyhas been confirmed with Fa-Phe-Phe-OH as the substrate,and the optimum pH for this enzyme activity is acidic at 4.5 (Parussiniet al.2003).In this study,AbSCP1 could also catalyze the degradation of FA-Phe-Phe-OH under the same conditions,and the optimal pH was the same as that of serine carboxypeptidase fromT.cruziy.AbSCP1 secrete byA.besseyi.The nematode-secreted SCP protein enters the host cell and is transferred to the nucleus.In the nucleus,AbSCP1 RNAi is an effective tool in gene characterization research.AbSCP1was effectively silenced byin vitroRNAi,and the expression of the gene was reduced to the lowest level after 24 h of RNAi treatmentviasoaking.SCP is very important for nematodes to parasitize host plants (Sobliket al.2011;Huanget al.2017),and downregulation ofAbSCP1may lead to a decrease in nematode viability in host plants and cause reduced reproduction inA.besseyi.This result further supports the role ofAbSCP1in the pathogenicity ofA.besseyi.

5.Conclusion

This study confirmed the expression and function ofAbSCP1.These results showed thatAbSCP1played an essential role in parasitism.Characterization ofAbSCP1adds to the knowledge of the infection mechanism ofA.besseyi.It opens an opportunity to establish a safe and efficient control method forA.besseyiin the future.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31701774) and the Natural Science Foundation of Anhui Province,China (2008085QC123).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendixassociated with this paper is available on http://www.ChinaAgriSci.com/V2/En/appendix.htm