The Functional Analysis Of Odor-binding Proteins And Chemosensory Proteins In Diaphorina Citri (Hemiptera:Psyllidae)

In the face of the devastating disease of the citrus industry,citrus Huanglong disease,the oligophagous insect Diaphorina citri is the main vector of Candidatus Liberibacter spp..We believe that in new control strategies,attractants may be promising broad-spectrum alternatives for pesticides.Studying its olfactory molecular mechanism related to host recognition can provide a theoretical basis for the design and screening of attractants for Diaphorina citri.Based on the published results of sequencing of the abdomen and antennae transcriptome,this study re-identified and annotated the Diaphorina citri genome(V2.0)in order to comprehensively study the OBP and CSP of Diaphorina citri.The results showed that there are 9 Diaphorina citri.OBP(8 Classic and 1 Plus-C OBP)and 12 CSPs have complete open reading frames.The results were consistent with previous transcriptome sequencing results.Diaphorina citri lacks the olfactory glomeruli in the primary olfactory central antennae,and has only a small number of OBPs and CSPs.Therefore,we speculate that Diaphorina citri may have a relatively simple and specific olfactory system.The expression of Diaphorina citri OBP and CSP in different tissues(the antennaes,salivary glands,midgut,hindgut,wings,feet)and different developmental stages(the egg,nymph five ages,newly emerged female,male and sexual mature female,male stage)was analyzed by real-time quantitative PCR(qRT-PCR).Four OBP(DictOBP2,DictOBP7,DictOBP8,DictOBP9)and four CSP(DcitCSP1,DcitCSP4,DcitCSP8,DcitCSP12)genes were enriched in the antennae.It is worth noting that DcitOBP9 and DcitCSP(1,8)are highly expressed in the hindgut compared to other tissues.This suggests that odor-binding proteins and chemosensory proteins may be involved in other non-olfactory-related physiological functions in addition to olfactory sensory functions.Compared with adults,the expression level of DictOBP2 was significantly up-regulated in the nymphal stage and increased with the growth of nymphs.In contrast,the adult stages of DictOBP8,DictOBP9,DictCSP1,DictCSP4 and DictCSP12 were significantly up-regulated compared to the nymphal stage.DcitCSP8 is mainly highly expressed in the newly emerged adult stage,and its expression level is very low in other developmental stages.Two DcitOBPs(2,7)and two DcitCSPs(4,12)were significantly up-regulated during the sexual maturation phase compared to the unsexual maturity phase.Based on the results of Real-time Quantitative PCR Detecting System,4 OBPs and 4 CSPs with high specific expression of antennae were screened.Through the prokaryotic expression system,7 recombinant proteins without fusion tags(DictOBP2,DictOBP 7,DictOBP8,DictOBP9,DictCSP1,DictCSP8 and DictCSP12)were successfully obtained in this experiment.In order to study the characteristics of OBP and CSP and host plant volatiles,sex pheromone components and repelling plant volatiles and the function of OBP and CSP in the process of olfactory recognition,we completed the fluorescence competition binding assay and molecular docking experiments.the results showed that the host plant's shoot bud volatile ?-caryophyllene had strong binding ability to DcitOBP8(Ki ? 10).The bud volatiles E-?-ocimene of the host plant and the repellent plant volatile dimethyl trisulfide have high binding affinity to DcitOBP9(Ki ? 10).DcitCSP1 and DcitCSP12 can be combined with all test compounds.DcitCSP12 has high binding affinity to the host plant volatiles cymene,?-caryophyllene,?-terpinene and myrcene(Ki ? 10).In particular,?-caryophyllene showed the highest affinity(Ki = 2.54).The dissociation constant and free binding energy of phytochemical volatiles of dimethyl trisulfide are generally lower than that of dimethyl disulfide;methyl salicylate specifically released from the diseased strain of Candidatus liberibacter asiaticus DcitCSP1 and DcitCSP12 have higher affinity(Ki = 10.9 and Ki = 11.2).We hypothesize that DcitCSP may be widely perceived to recognize common volatiles in host plants.In contrast,all DcitOBP showed specific binding properties,suggesting that DcitOBP may narrowly perceive the recognition of specific odor volatiles from host plants.In summary,our current study presents for the first time the ligand binding profile of OBP and CSP specifically expressed in the antennae of Diaphorina citri,and also provides a set of potential odorants for the new control strategy of Diaphorina citri.