Transcriptional Regulation Mechanism Of NlObp8 And NlCsp10,Recognizing Linalool In Nilaparvata Lugens(ST(?)L)

A sensitive and specific olfaction is essential for insects to achieve multiple behavioral responses,such as locating host,foraging,mating,oviposition,and evading predators.In insect peripheral olfactory system,odorant-binding proteins（OBPs）or chemosensory proteins（CSPs）efficiently and selectively bind and transport hydrophobic odorants across water-soluble sensillar lymph,which are thought to play an important role in insect olfaction.In response to odorant stimulation,functional OBPs or CSPs genes in peripheral system can be induced to undergo expression changes,and such changes can mediate insects’behavioral plasticity and enhance their adaptation to the environment.However,it is unclear how such induced changes in the expression of OBPs or CSPs genes are regulated.Transcriptional level regulation,a key step in the regulation of gene expression,regulate the transcriptional rate of genes,alter the level of deduced protein,and ultimately impact physiological functions mainly through the interplay of transcription factors and cis-acting elements.Therefore,the identification of key transcription factors involved in regulating the induced expression of OBPs or CSPs genes by odorant would provide a deeper understanding of the study and utilization of insect olfaction.The brown planthopper（BPH）Nilaparvata lugens（St?l）is the typeⅠmajor migratory pest in rice and threats the security of rice production seriously.Bioassays in greenhouse have found that the feeding of BPHs can induce rice plants to release large quantities of linalool volatile and this volatile can elicit significant repellent response of BPHs.However,in the field,monophagous BPHs didn’t leave the rice plant which is releasing linalool due to the feeding of BPHs and continue to feed and damage.We speculate that the behavioral adaptation to linalool exhibited by BPHs while feeding on rice plant may be achieved by regulating changes in the expression of peripheral olfactory function genes.In a previous study,our group identified 10 OBPs and 11 CSPs coding genes in the antenna of BPHs by RNA sequencing,among which the genes encoding several proteins,such as NlOBP2,NlOBP8 and NlCSP10,were specifically highly expressed in the antenna of BPHs and may perform olfactory-related functions.Therefore,this research selected three proteins,NlOBP2,NlOBP8 and NlCSP10,whose coding genes are specifically and highly expressed in the antenna of BPHs,analyzed their functions during linalool perception,and explored the transcriptional regulatory mechanisms underlying expression change of the functional Obps and Csps induced by linalool.The results of this research will lay theoretical foundation for the olfactory adaptation mechanism of BPHs to linalool and provide theoretical support for the use of linalool as semiochemical for controlling BPHs.The main findings are as follows:1.NlOBP8 and NlCSP10 play coordinative roles in the chemoreception of BPHs to linaloolThe cDNA sequences of NlObp2,NlObp8 and NlCsp10（Gene Bank accession numbers:ACI30680.1,KC663687 and AGZ04900.1,respectively）were cloned,and the tissue expression pattern of the three genes were analyzed by q RT-PCR.It was found that NlObp2,NlObp8 and NlCsp10 were specifically and highly expressed in antennal tissues;NlObp2 was weakly expressed in the head without antenna,abdomen,and wing tissues,NlObp8 was weakly expressed in the head without antenna and wing tissues,and NlCsp10 was weakly expressed in the wing tissues.There were no significant differences in the expression of the three genes between the sexes and different wing forms.The function of predicted signal peptides of the three proteins were validated using the yeast Saccharomyces cerevisiae signal peptide screening system and Drosophila melanogaster S2 cell line,respectively.It was found that the signal peptide regions of both NlOBP2,NlOBP8 and NlCSP10 exhibited secretory activity and all three proteins belong to secretory proteins.The recombinant vectors p ET-32b-NlObp2,p ET-32a-NlObp8 and p ET-32b-NlCsp10 were constructed,and recombinant NlOBP2,NlOBP8 and NlCSP10proteins were successfully obtained using the Escherichia coli expression system.The binding affinity of the three recombinant proteins to linalool were determined using fluorescent competitive binding assay.It was found that at p H 7.4 condition,NlOBP2exhibited no binding affinity to linalool with K_i value greater than 50μmol/L,NlOBP8exhibited strong binding affinity to linalool with K_i value of 14.88±5.56μmol/L,and NlCSP10 exhibited weak affinity to linalool with K_i value of 24.34±0.74μmol/L.The interactions of NlOBP8 and NlCSP10 with linalool were predicted using homology modeling and molecular docking techniques,respectively.It was found that the binding interaction of both NlOBP8 and NlCSP10 with linalool was mainly hydrogen bonding and hydrophobic interaction,and the Phe114 and the Asp99 residues in NlOBP8 and NlCSP10 might form hydrogen bonds with linalool,respectively.The physiological functions of NlOBP8 and NlCSP10 in the chemoreception of BPHs to linalool were investigated using H-tube olfactometer bioassays combined with RNAi.The results revealed that BPHs still exhibited repellent behavior to 1%linalool when NlObp8 or NlCsp10 expression was knocked down separately;the repellent behavior of BPHs to 1%linalool was significantly impaired when NlObp8 and NlCsp10expression were knocked down simultaneously.2.The Homeotic protein distal-less（Dll）regulates the expression of NlObp8 and NlCsp10in the response of BPHs to persistent linaloolThe expression level of NlObp8 and NlCsp10 after induction by linalool were detected using q RT-PCR.The relative m RNA level of NlObp8 was significantly decreased after induction with 1%linalool for 12 h compared with liquid paraffin control;the relative m RNA level of NlCsp10 was significantly decreased after induction with 1%linalool for 5 h,and after induction with 0.1%or 1%linalool for 12 h compared with control liquid paraffin,respectively.The promoter region（2527 and 2502 bp,respective）upstream of NlObp8 and NlCsp10 were obtained,and 614 bp active fragment of NlObp8promoter was obtained by Dual-luciferase assays in Drosophila S2 cells.Through the JASPAR targeting insect transcription factor database,there were a total of 44transcription factors with scores above 10 predicted to potentially interact with 10 CREs.Based on the reported expression patterns and functions of these transcription factors,Dll,a transcription factor probably acting in the-456 to-450 region,was selected to further explore the transcriptional regulatory role on NlObp8 and NlCsp10.Subsequently,a total of 13 CREs that potentially bind to Dll were predicted on the NlObp8 and NlCsp10promoters with score greater than 7.The NlDll＿X1 was cloned from BPHs,and the interaction of NlDll＿X1 with the predicted 13 CREs was verified in vitro using yeast-one hybrid（Y1H）,Electrophoretic mobility shift assay（EMSA）and Dual-luciferase reporter gene systems.The results of Y1H assay suggested that among the 13 predicted CREs,transformants harboring p GADT7-NlDll＿X1 and triplet of p CRE2-Ab Ai,p CRE3-Ab Ai,p CRE4-Ab Ai,p CRE5-Ab Ai,p CRE12-Ab Ai or p CRE13-Ab Ai grew normally on SD/-Ura/-Leu plates containing 800 ng/m L and 1000 ng/m L Ab A,respectively.The recombinant NlDll＿X1protein harboring a SUMO tag was obtained by E.coli expression system,and the EMSA suggested that there were distinct shifted bands appeared when recombinant NlDll＿X1-SUMO protein was incubated with the biotin-labeled probes exhibiting positive reaction in Y1H assay and the shifted band was competitively eliminated by adding into the reaction 1000-fold molar excess of unlabeled probes,but not the mutated unlabeled probes.A eukaryotic expression vector p AC5.1/Myc-NlDll＿X1 was constructed and NlDll＿X1 was successfully expressed in Drosophila S2 cells.Dual-luciferase reporter assays showed that over-expression of NlDll＿X1 in Drosophila S2 cells significantly increased the luciferase activity driven by P_Obp8-698（～2.14-fold）and P_Csp10（～28.70-fold）,respectively.Moreover,when the CREs on P_Obp8-698 and P_Csp10 were mutated to“GTGACCA”,the relative luciferase activity significantly reduced compared with mutation before.The relative m RNA levels of both NlObp8 and NlCsp10 in the antenna of BPHs were significantly reduced after knocking down NlDll expression by RNAi;Western blot and WM-FISH suggested that the protein level of NlObp8 in the antenna of BPHs was apparently decreased compared with ds GFP-injected control.The q RT-PCR results showed that the relative m RNA level of NlDll in the antenna of BPHs was significantly reduced after induction with 0.1%and 1%linalool for 5 and 12 h compared with the control liquid paraffin,respectively.3.NlDll mediated the chemoreception of BPHs to linalool through modulating the expression of several olfactory-related genesUsing RNAi technology,NlDll expression was successfully knocked down in BPHs nymph.And injection of ds NlDll at the 3rd instar nymphs caused high mortality of BPHs,while injection of ds NlDll at the 5th instar nymphs did not.ds NlDll was injected at different instar of nymph,and the antennal sensilla morphology of newly emerged adults was observed using SEM.After injection of ds NlDll at the 3^rd instar nymph,it was found that the number of sensilla trichodea on the pedicel were significantly decreased in newly emerged adults,and the diameter of newly plaque organ were significantly reduced,and the sensilla placodea constituting plaque organ was absent.Meanwhile,injection of ds NlDll at the 5^th instar nymph had no significant effect on the number of sensilla trichodea and the diameter of newly plaque organ in the newly emerged adults.Adults emerged within two to three days after injection of ds NlDll at the 5^th instar nymph were used to test the effects of ds NlDll on the responses of BPHs to linalool.After knocking down NlDll expression,BPHs exhibited no preference for 1%linalool,while a significant repellent behavior was still observed upon ds GFP injection.RNA-seq was performed to identify the differentially expressed genes（DEGs）in the antenna of BPHs adults injected ds NlDll.Compared with ds GFP-injected control,270 and 75 genes were down-and up-regulated in the antenna of ds NlDll-injected BPHs adults,respectively.The down-regulated DEGs were significantly enriched to the GO terms related to olfactory process,the most two enriched were odorant binding and olfactory receptor activity including 96 DEGs.To investigate whether NlDll regulate these 96 DEGs expression directly,we obtained 2000 bp candidate promoter regions upstream all the 96 DEGs CDS from the BPH genome and identified the CREs in each gene promoter region,respectively.The results suggested that the promoter region of 89 out of the 96 DEGs contained several CREs that could bind to NlDll＿X1.In summary,this study found that when encountering persistent linalool stimulation,BPHs may reduce the sustained response to linalool by down-regulating the expression of NlDll in the peripheral system to reduce the abundance of NlOBP8 and NlCSP10 and other olfactory functional proteins.The results of this study could provide a theoretical foundation for deeply understanding of the regulatory mechanisms of insect behavioral plasticity and provide theoretical support for pest control strategies utilizing the insect olfactory system.