Function And Role Of Salivary OBPs In Feeding Of Nilaparvata Lugens (St(?)l)

Odorant binding proteins(OBPs)are small molecule transporters that bind and transport odorant molecules to odorant receptors.It is an important component of the insect olfactory system.With the deepening of research,it has been found that OBPs not only exist in olfactory organs,but also in the midgut,salivary gland,legs,ovary,testis,and other non-olfactory organs of insects,and new physiological functions of OBPs have been found in non-olfactory organs.According to brown planthopper Nilaparvata lugens(St(?)l),BPH salivary glands transcriptome sequencing,the NlugOBPs and Nlug CSPs were discovered in the salivary glands.The location of these proteins indicated that OBPs might be involved in the interaction between BPH and rice Oryza sativa L.Therefore,this study used the BPH and rice as the research system.Based on the analysis of the BPH salivary gland transcriptome,the RNA interference technology was used to analyze the feeding behavior and growth and development status of the BPH after the interference.Through western blotting,rice protoplasts,tobacco instantaneous expression of leaves and exogenous expression in rice verified its role in feeding and regulating plant defense responses.At the same time,transgenic technology was used to cultivate rice plants containing dsRNA,and the development and survival rate of BPH on transgenic plants were determined.The results will provide new research for the OBP research and provide a theory for pest control of BPH.The main findings are as follows:1.The temporal and spatial expression of NlugOBPs in the BPH and their distribution in the salivary glandsFour secretory NlugOBPs were obtained from the salivary glands of BPH by transcriptome sequencing,namely NlugOBP2(FJ215306.2),NlugOBP4(KC516720.1),NlugOBP9(XM?022348448),and NlugOBP11(MW813955).The expression levels of these genes in the development stages and tissues of BPH were detected by q PCR.Our findings showed that these genes were expressed at all stages of BPH and more highly expressed in salivary glands than in other tissues.The distribution of NlugOBPs in the salivary glands of BPH were determined.The results showed that NlugOBP2 and NlugOBP9 were widely distributed in the principal glands,accessory glands,and salivary ducts;NlugOBP4 was mainly distributed in salivary ducts and accessory glands but less distributed in the main glands;NlugOBP11 is mainly distributed in the principal glands and salivary ducts,but not in the accessory glands.2.The effect of NlugOBPs on the survival and feeding behavior of BPHThe NlugOBPs of BPH were silenced by injection of dsRNA.The results showed that the expression of NlugOBPs were decreased after injected dsRNA and lasted for more than7 days.Silencing NlugOBP2,NlugOBP4,NlugOBP9 did not impact the survival of BPH,while silencing NlugOBP11 the survival rate of BPH decreased at 4 d,and all died at 7 d.Electrical penetration graph(EPG)was used to measure the feeding behavior of the BPH after interference.The experimental results showed that the frequency of non-probing(np)waveform increased after silenced NlugOBP2.There was not any change in the feeding behaviors when silenced NlugOBP4.Silenced NlugOBP9,the frequency of np and total duration of N4-b(phloem sap ingestion)waveform increased,while the total duration of N4-b(sieve element salivation)decreased.When compared with the control group silenced NlugOBP11 will increase the frequency and total duration of the non-probing waveforms,but the total duration of the N4-a and N4-b were reduced.Transcriptome sequencing was used to analyze the gene expression levels of the whole BPH 1d,2d,and 3d after interference with NlugOBP11.The results showed that at 1d,a total of 10 BPH genes were changed;at 2d,a total of 254 genes were changed.At 3d,a total of 731 genes were changed.Enrichment analysis of the KEGG pathway found that these altered genes were highly concentrated in the signal pathways of biodegradation and metabolism,carbohydrate metabolism,lipid metabolism,amino acid metabolism,and signal transduction system.3.The effect of NlugOBPs on rice hormonesTo investigate whether NlugOBPs are secreted into rice plants during feeding,the western blot was used to detect rice after BPH feeding.The results showed that except NlugOBP4 the other NlugOBPs will be secreted into plants during BPH feeding.The content of jasmonic acid(JA)and salicylic acid(SA)in rice induced by BPH after interference was detected by LC-MS.The results showed that silenced NlugOBP2-and NlugOBP9-BPH would reduce the JA and SA content in rice plants.the NlugOBP4 silenced BPH would increase the content of SA in rice plants but had little effect on JA.The NlugOBP11 silenced BPH would decrease the content of JA,but increase of SA content in rice.However,as time increases,the JA content eventually becomes consistent with the control group.By constructing the eukaryotic expression vector of NlugOBPs and transforming the vector into rice protoplasts,the effects of NlugOBPs on rice JA and SA synthesis-related genes were explored by q PCR.The results showed that NlugOBP2 up-regulated SA synthesis-related genes EDS1,PAD4,PAL,ICS1 and JA synthesis-related genes LOX,AOS2.NlugOBP4 up-regulated of SA synthesis-related genes EDS1,PAD4,PAL,ICS1,and JA synthesis-related genes AOS2 but down-regulated JA synthesis-related genes LOX.NlugOBP9 up-regulated SA synthesis-related genes EDS1,PAD4,PAL,ICS1 and JA synthesis-related genes LOX,AOS2.NlugOBP11 down-regulated SA synthesis-related genes EDS1,PAD4,PAL,ICS1 and JA synthesis-related genes LOX,AOS2.The results about the effect of NlugOBPs on plant hormones were confirmed again in the transient expression system of Nicotiana benthamiana leaves by Agrobacterium tumefaciens.The results showed that NlugOBP2 upregulated SA synthesis-related genes ICS,NbPR1,and JA synthesis-related genes LOX,NbPR3,NbPR4 when overexpressing this protein in N.benthamiana leaves at 24 h and 48 h.NlugOBP4 up-regulated SA synthesis-related genes ICS and NbPR1 and the down-regulated of the JA synthesis-related gene NbPR3 at 24 h when overexpressing this protein in N.benthamiana leaves.At 48 h NlugOBP4 up-regulated the SA synthesis-related gene ICS and the JA synthesis-related gene NbPR4.NlugOBP9 upregulation the SA synthesis-related genes ICS and JA synthesisrelated genes LOX and NbPR3 at 24 h and up-regulation of SA synthesis-related genes NbPR1 and JA synthesis-related genes LOX,NbPR3,NbPR4 at 48 h.Overexpressing NlugOBP11 in N.benthamiana leaves downregulated the SA synthesis-related genes Nb ICS and NbPR1 at 24 and 48 h and the JA-related genes Nb LOX,NbPR3 and NbPR4 at24 h but increased at 48 h when compared with the control group.To explore the effects of NlugOBP11 on rice JA and SA synthesis-related genes and the feeding behavior of BPH the NlugOBP11 was expressed in rice plants.The results showed that the NlugOBP11 downregulated the SA synthesis-related genes EDS1,PAD4,PAL,ICS1,but the JA synthesis-related genes LOX and AOS2 increase.EPG results showed that the NlugOBP11 in rice plants significantly reduced the total duration of N4-a waveform during the BPH feeding.4.Effects of dsNlugOBP11 rice on the growth and development of BPHAccording to the characteristics that the injection of dsNlugOBP11 caused the death of BPH,the partial sequence of NlugOBP11 was selected to design the corresponding dsRNA expression vector,and the RNA interference vector was introduced into rice plants.The insect resistance of the obtained transgenic plants was tested and showed that the developmental of BPH on the transgenic plants was prolonged and the survival rate was significantly reduced.The results also showed that the second BPH generation survival rate was significantly lower than the first.In this study,four NlugOBPs highly expressed in the salivary glands of BPH were obtained,and their distribution characteristics were clarified by analyzing the transcriptome of the salivary glands of BPH.It was confirmed that three of the salivary gland NlugOBPs would enter the rice plant during BPH feeding.It was proved that the salivary gland NlugOBPs had a significant effect on the main plant hormones SA and JA metabolic pathway genes.The dsRNA rice material containing the lethal gene of BPH was cultivated,and it was confirmed that it inhibited the growth and development of BPH.The results of the study laid an important foundation for further probing the physiological functions of OBPs in BPH,broadened the functions of insect OBPs,and provided new ideas for the use of OBPs to prevent and control BPH.