Study On Regulation Mechanism Of Soybean Cyst Nematode In Response To Chemical Signals

Soybean cyst nematode(SCN,Heterodera glycines Ichinohe)impacts soybean production by host infection,which causes several billion dollars of annual loss worldwide.Thus,SCN management is urgent for soybean production security.Due to many limitations in current management strategies,the development of safe and effective management strategies based on plant-nematode interaction mechanism has become a hot spot for study.The infective second stage juveniles of plant parasitic nematode can locate host by recognizing root signals.If the recognition and localization to the host are interrupted,the infection fails,resulting in effective SCN management.Root exudates contain sugars,amino acids,salt ions,etc.It has been reported acidic and basic p H and salt ions are key chemotactic signals for SCN.Amino acids as the second most abundant compounds in root exudates are divided into the acidic,the basic,and the neutral amino acids.Whether amino acids are chemotactic factors remains unknown.If they are,whether p H-amino acids synergism drives nematode chemotaxis.Previous study reported that nematode exhibited chemotaxis to p H and ions,but changes in behavior and gene expression at whole genomic level remain unclear.In addition,how genes regulate nematode behavior and parasitism is also poorly understood.In this study,chemotaxis assay,mortality assay,behavior and transcriptome analysis were performed.Subsequently,candidate genes were screened through bioinformatics analysis.Next,gene clone,tissue-specific localization and biological functional verification were conducted.The results are as follows:First,chemotaxis assay was used based on Pluronic gel system with Petri dish confrontation method to analyze chemotaxis response of SCN to 15 types of amino acids.The results showed that 11 amino acids attracted SCN and the average nematode numbers of them were all greater than that of control(11±5).These amino acids includes acidic Aspartic acid(890±50),Glutamic acid(925±74),basic Arginine(tightly accumulated outside the dispenser),Serine(407±69),Lysine(225±17),and Histidine(227±17),neutral Isoleucine,Leucine,Proline,Glycine,and Valine(loosely accumulated outside the dispenser).Compared with results from chemotaxis mode of nematode to acidic and basic p H and p H mortality assay,we considered that the synergism between acidic or basic p H and amino acids resulted in strong nematode attraction.SCN could survive strongly acidic Aspartic acid and Glutamic acid but failed to survive acetic acid with p H 3,revealing that SCN can be attracted to so acidic amino acids and be adapted to live in them.Second,nematode behavioral analysis was performed with Worm Lab software for real-time track on nematode locomotion in attractive acid(p H 5.25),base(p H 8.6),and170 m M Na Cl salt,as well as extreme acid(p H 4.5),and base(p H 10.0).The results showed that nematode locomotion speed in extreme p H environment was greater than that of control p H 7.0,indicating the stressed condition for nematode.In attractive p H solution,nematode locomotion first increased and then decreased,indicating that nematode condition turned into steady adaption from initial stimulation.Third,in order to reveal changes in gene expression at genomic level in various p H and salt solutions,full-length transcriptome sequencing was conducted on 18 samples belonging to 6 treatments.In total,an average of 4.36 Gbp of clean reads were generated for each sample.A total of 3,972 novel genes and 29,529 novel transcripts were identified following alignment with reference genome and functional annotation.The structural analysis of transcripts showed that alternative splicing,alternative polyadenlation,fusion transcripts were changed in structure than control,confirming that posttranscriptional modification was involved in nematode behavioral regulation.The GO and KEGG enrichment analysis on differentially expressed genes and transcripts indicated that transmembrane receptors as GPCR receptors including chemoreceptor SRSX,Wnt receptor MOM-5,dopamine receptor F59D12.1,peptide receptor NPR-18,acetylcholine receptor m ACh R,and hormone thyrotropin receptor TRHR and acetylcholine receptor n ACh R,GABAB receptor,guanylate cyclase receptor GCY-18 can regulate ion channels after stimulation by acid,base and salt.Additionally,ion transporters PMCA and ion channels VGCC and TRP-1(TRPC4)were also activated.However,oxidative phosphorylation pathway and proteins in ribosomal metabolism pathway were inhibited,indicating that energy generation and protein synthesis were inhibited,which decrease energy consumption.The receptor activation and inhibition in growth and development indicated that nematode sustained energy balance by regulating metabolism pathway in favorable condition.A regulatory model responding to p H and salt ion stimulation was established by the combination of differentially expressed genes and protein-protein interaction analysis.Forth,according to results from transcriptome sequencing and q RT-PCR verification,gene cloning,developmental expression analysis,and functional validation were performed for key G protein α subunit gene Hg-goa-1 and TRPV channel genes Hg-osm-9 and Hg-ocr-2.All three genes have the greatest expression level at preparasitic second-stage juveniles(Pre-J2).Silence of any genes can change expression level of the other two genes,indicating interaction among them.The decreased expression level of Hg-osm-9 or Hg-ocr-2 by RNAi,nematode chemotaxis index to the attractive acid p H decreased and that to the attractive base p H increased,indicating that both genes positively regulate chemotaxis to acidic p H and negatively regulate chemotaxis to basic p H.The silence of any one of three genes reduced 17.7-40.5% of infection and 24.7-45% of cyst or female,which indicated the silence of three of each genes casue chemotaxis reduction and infection inhibition,ultimately affects nematode reproduction in soybean.Overall,this study clarified that the type,dosage,and p H of amino acids all affect the chemotaxis of soybean cyst nematodes to amino acids.Soybean cyst nematodes exhibited significant changes in behavior after being stimulated by acid,base and salt,and post-transcriptional modifications,transmembrane receptors,ion channels,ion transport proteins,oxidative phosphorylation metabolic pathways,and ribosome metabolic pathways were involved in this behavioral change process.The interaction between G protein α subunits and TRPV channels jointly regulates the chemotaxis,infection,and reproduction of soybean cyst nematodes.This study enriched the knowledge of nematode chemotaxis and behavior,and the identified transmembrane receptor proteins and TRPV channel key genes can serve as molecular targets for the development of new nematicides.