Characterization Of The Biological Functions Of Desaturase Nldesat10 And Salivary Protein NlSEF1 Of Brown Planthopper,Nilaparvata Lugens

The rice brown planthopper(Nilaparvata lugens(Stal),BPH)is one of the most economically devastating pests in the world.BPH infestation reduces the photosynthetic rate,leaf and stem nitrogen concentrations,chlorophyll content,and organic dry weight in rice,thereby dramatically decreasing rice yield.It has been well documented that desaturases(desat)play an important role in the biosynthesis of semiochemicals in insects.However,whether or how these enzymes affect the growth,development and reproduction of insects and the relationship of insects with their host plants remain largely unknown.In this study,we identified and annotated the desaturase family genes in BPH genome.By combining sequences alignment,phylogenetic construction,expression pattern analysis and RNA interference(RNAi),the functions of these desaturase genes,especially the gene Nldesat10 which has an obvious influence on the survival and fecundity of BPH,in the growth,development and fecundity of BPH,as well as in the interaction between BPH and rice were explored.In addition,the role of a BPH salivary protein N1SEF1(salivary EF-hand protein)in mediating defense responses of rice was also investigated.The results are as follows:1)Nldesat10 plays an important role in the growth,development,fecundity of BPH as well as in BPH-rice interactionBased on bioinformatics,we identified 10 genes encoding putative desaturases,including eight △9-desaturases,one cytochrome b5 fused desaturase and one A4-sphingolipid-desaturase in the genome of BPH.Developmental and tissue-specific expression pattern analysis revealed that Nldesatl was mainly expressed in the head and integument;Nldesat2,Nldesat3 and Nldesat8 were mainly expressed in the integument;Nldesat4,Nldesat5 and Nldesat6 had constant expression levels in different tissues;Nldesat7 and Nldesat9 were mainly expressed in the fatbody,integument and head;and NldesatlO was mainly expressed in the ovary and integument.The transcipt level of each desaturase gene varied with developmental satge and each gene showed a specific developmental expression pattern.In general,Nldesat1-Nldesat6 had relatively constant expression levels at diffierent developmental stages;Nldesat7 and Nldesat9 had low expression levels at egg stage;Nldesat8 showed low mRNA levels at egg and female adult stage;and Nldesatl 0 had a low expression level during the nymph stage.Compared with dsGFP-BPH(BPH that had been injected with the dsRNA of GFP)and C-BPH(non-injected BPH),BPH that one of desaturases was silenced besides Nldesat3-,Nldesat5-and Nldesat8-silenced BPH,had a significantly higher mortality rate 7 days post-injection.Of the ten desaturase genes,silencing of Nldesat10 resulted in the highest mortality rate of BPH.Moreover,silencing of Nldesat10 reduced the protein and triglyceride content,food intake,body mass and fecundity of female adults and caused female adults abdomen atrophy and ovarian agenesis.Knockdown of Nldesat10 decreased the transcript levels of Vg(Vitellogenin),VgR(Vitellogenin Receptor),TOR(Target of Rapamycin)and Lpp(Lipophorin)of female adults,whereas the transcript levels of JHAMT(juvenile hormone acid methyltransferase),JHE(juvenile hormone esterase),Met(Methoprene-tolerant),Rheb(Ras homologue enriched in brain),S6K(S6 kinase),AKH(adipokinetic hormone)and AKHR(adipokinetic hormone receptor)were not affected.The results indicate that decreases in Vg transcript levels and fecundity of BPH female adults caused by Nldesat 10-knockdown were mainly because of attenuation of TOR pathway and Lpp.Compared with that of Nldsatl0-knockdown BPH nymphs raised on artificial diet,the corrected survival rate of BPH nymphs with silenced Nldesat10 raised on rice was significantly lower.The levels of JA and JA-Ile were similar among rice plants infested with Nldesat10-silenced 1-d-old female adults(dsdesat10),1-d-old female adults that had been injected with the dsRNA of GFP(dsGFP)and non-injected 1-d-old female adults,whereas SA levels were significantly higher in the dsdesat10-BPH-infested plants than in the dsGFP-BPH and C-BPH-infested plants at 8 h.The data suggest that Nldesat10,in addition to its important role in the growth,development and fecundity of BPH,might also be involved in regulating the interaction between BPH and rice.2)The BPH salivay protein N1SEF1 mediates defense responds in riceWe cloned a gene N1SEF1,which is highly expressed in the salivary glands of BPH.The N1SEF1 protein has EF-hand Ca2+-binding activity and can be secreted into rice plants when BPH feed.Infestation by BPH nymphs whose N1SEF1 was knocked down elicited higher levels of Ca2+ and H2O2 but not JA,JA-Ile and SA in rice than did infestation by control nymphs;Consistently,wounding plus the recombination protein N1SEF1 had no influence on JA,JA-Ile and SA biosynthesis,but suppressed the production of H2O2 in rice.Together,our results indicate that the BPH salivary protein N1SEF1 functions as an effector and plays important roles in interactions between BPH and rice by mediating the plant’s defense responses.