The Mechanism Of Rice Stripe Virus RNA-dependent RNA Polymerase Regulating The Odorant Binding Protein LstrOBP7 Of Laodelphax Striatellus And Its Behavioral Effects

Insect vector-borne plant viruses have caused serious crop losses worldwide and posed a great threat to agricultural production.The spread range and speed of plant viruses are closely related to behaviors of insect vectors,such as host selection.The latest studies suggested that plant viruses can manipulate the behavior of insect vectors in favor of their own transmission.Rice stripe disease caused by Rice stripe virus(RSV)can lead to severe yield reduction in rice regions of Southeast Asian,which is transmitted by small brown planthopper,Laodelphax striatellus,in a persistent propagative pattern.Our previous studies found that nonviruliferous L.striatellus have no preference for choice between healthy and RSV-infested rice,while viruliferous L.striatellus have a stronger tendency to healthy rice than to RSV-infected rice.However,the underlying mechanism of RSV infection altering host selection behavior of L.striatellus remains unclear,so we carry out the following research work:1.Transcriptome analysis was used to screen differentially expressed genes between viruliferous and nonviruliferous chemoreceptor organs of L.striatellus,such as antennae,heads and feet.Compared to nonviruliferous organs,it was found that LstrOBP4,LstrCSP2/5,LstrGR28a,LstrIR1/NMDA1,LstrOR67/85e,LstrSNMP2/2-2 were up-regulated and LstrOBP9,LstrGR43a/43a-1,LstrIR2,LstrOR56a/94 were down-regulated in the antennae from viruliferous L.striatellus;LstrCSP3/5/10,LstrIR1/2/Delta2,LstrOBP3/4/12/13,LstrOR12 were up-regulated and LstrCSP1,LstrIR1/kainate2/NMDA2/Delta2-1,LstrOR21/31/68,LstrORco and LstrSNMP14/2/2-2-2 were down-regulated in the foots from viruliferous L.striatellus;LstrOBP2,LstrSNMP2-2 were up-regulate dand LstrCSP1,LstrOR67 were down-regulated in the heads from viruliferous L.striatellus.Transcriptome data showed that RSV infection mainly altered three types of olfactory genes in olfactory organs:odor-binding protein(LstrOBP),chemoreceptor protein(LstrCSP)and sensory membrane protein(LstrSNMP).Furthermore,the expression levels of LstrOBPs was detected using RT-qPCR.The results showed that LstrOBP2/3 were up-regulated in the heads and foots,LstrOBP4/7 were up-regulated in the antennae,and LstrOBP9 was downregulated in the antennae of viruliferous L.striatellus,which was consistent with the transcriptome analysis results.Based on the above results,LstrOBP4 and LstrOBP7 with high expression in antennae were selected for subsequent mechanism study.2.RNA interference was used to inhibit the expression of LstrOBP4/7 in viruliferous L.striatellus.Then,the selection behavior between RSV-infested and healthy rice of dsLstrOBP4/7 treated L.striatellus was observed using "H" olfactory instrument.The results showed that inhibition of LstrOBP7 led to a change in the host selection behavior of viruliferous L.striatellus,which have a tendency to RSV-infested rice.But inhibition of LstrOBP4 had no significant change in the host selection behavior of viruliferous L.striatellus,which still have a tendency to healthy rice.Furthermore,The approach behavior of dsLstrOBP7 treated viruliferous L.striatellus to five volatile terpenoids from rice(R-(+)-limonene,β-ionone,1-octen-3 alcohol,cis-3-hexene and citronellol)was obtained using "H" olfactory instrument.We found that the approach behavior of dsLstrOBP7 treated viruliferous L.striatellus have changed only at low concentrations of R-(+)limonene.The above results suggest that RSV may regulate the recognition of LstrOBP7 to R-(+)limonene for affecting the host selection behavior of viruliferous L.striatellus.3.Yeast two-hiybird and Pull-down technology were used to detect whether RSV virus proteins directly interacted with LstrOBP4/7 proteins.The results showed that the CP,SP,NS2,NS3 and LstrOBP4/7 of RSV did not interact directly with LstrOBP4/7.Then,dual-luciferase reporter experiment was used to check the regulation of RSV viral proteins on LstrOBP4/7 promoters.The results in vitro showed that the 5410～7163 region peptide chain of RSV RNAdependent RNA polymerase(RdRp)could bind to the promoter region of LstrOBP7 for initiating its expression.But,the RSV protein does not regulate transcriptional initiation of the LstrOBP4.In summary,our thesis has systematically investigated the differential expression of olfactory-related genes such as LstrOBPs,LstrCSPs and LstrSNMPs in the chemoreceptors of L.striatellus mediated by RSV infection,and screened LstrOBP4 and LstrOBP7 for further study.Behavioral experiments showed that the recognition of LstrOBP7 to rice volatiles(R-(+)-limonene)may be involved in determining the preference of L.striatellus for healthy rice,while LstrOBP4 did not show related functions.Finally,experiments in vitro revealed the mechanism by which RSV RdRp transcription regulates the up-regulated expression of LstrOBP7 in L.striatellus.The results of this research anlyzed the mechanism by which plant viruses change insect behavior by directly control the mediator insect olfactory system,This makes up for the theory that plant viruses directly regulate the behavior of mediator insects,and provides a new idea for the use of mediator insect behavior regulation technology to control plant virus diseases.