Identification And Functional Analysis Of Regulatory Factors Of Wing Dimorphism Of Brown Planthopper,Nilaparvata Lugens

Wing polymorphism of insects is a form of biological polymorphism,which is a result of their evolutionary adaptation to constantly changing environments.It is also an important manifestation of plasticity development and epigenetic inheritance.The brown planthopper（BPH）,Nilaparvata lugens（St?l,1854）（Hemiptera:Delphacidae）,the most important insect pest of rice,is a typical insect with wing polymorphism.Although the adult BPHs have the same genotype,they exhibit two forms of wings:long wings and short wings.The former is good at migration while the latter has a stronger reproductive ability,which exacerbates the damage caused by BPH.Studying the wing polymorphism of BPH may provides new ideas for pest control using its migration.In recent years,with the application of various genetic research methods,the genes that regulate the wing polymorphism of BPH have been gradually discovered.Among them,the most representative is the insulin signaling pathway,which plays a key role in regulating the wing polymorphism.However,the specific mechanism of the regulation of wing dimorphism remains unclear.In this study,we used transcriptome and proteome analysis,RNAi,CRISPR/Cas9 and other methods to investigate the key regulatory factors that regulate the wing plasticity of brown planthopper,and obtained the following research results:（1）Screening and identification of a new transcriptional regulator of wing dimorphism of BPH,Rotund（NlRn）.By transcriptome analysis in the long-winged and short-winged populations of BPH,123 differentially expressed transcription factors were screened,of which 59 were up-regulated in long-winged BPH and 64were down-regulated.Among these transcription factors,77 genes belonged to zinc finger protein family.17 transcription factors belong to the Homeobox family and 4belong to the Forkhead box family.RNAi analysis showed that only silencing NlRn led to the transformation of long-winged BPH into intermediate-winged type,suggesting that NlRn was involved in wing dimorphism regulation.Silencing of 4genes resulted in wing deformities in adult insects,while silencing 19 genes caused mortality.This indicates that although these 23 genes do not affect wing dimorphism,they may be involved in the regulation of wing or individual development.（2）NlRn was discovered co-regulating wing dimorphism in coordination with the insulin signaling pathway.Through RNAi experiments,we found that silencing NlRn in the Nl In R2^E4 mutant reversed the long-wing phenotype observed in Nl In R2^E4.BPHs after NlRn RNAi developed intermediate wings instead of long wings.These results suggest that NlRn may play a role downstream the insulin pathway in regulating wing dimorphism.To further investigate the position of NlRn in the insulin signaling pathway,CRISPR/Cas9 was used to modify the second exon of Forkhead boxO（NlFoxO）which caused a deletion of 4 base pairs and an insertion of 14 base pairs,leading to a frameshift mutation and loss function of NlFoxO.A homozygous mutant line,NlFoxO^E2,was obtained through crossing with wildtype and selfing.The NlFoxO^E2 displayed a consistent long-wing phenotype,aligning with the previously reported phenotype of NlFoxO RNAi.Notably,RNAi of NlRn in the NlFoxO^E2 also resulted in a reversal of the long-wing phenotype,indicating that the action of NlRn may occur downstream of NlFoxO or in an equivalent position.（3）NlRn and NlFoxO form a protein interaction complex.Using the 293T cell expression system,co-immunoprecipitate assays were conducted,revealing that the Flag antibody was able to co-immunoprecipitate the NlFoxO-Flag and NlRn-His fusion proteins.This result indicates the interaction between NlRn and NlFoxO.Additionally,bimolecular fluorescence complementation assays were performed.Co-transfection of the e Yfp^N-NlFoxO and e Yfp^C-NlRn plasmids into 293T cells resulted in the observation of yellow fluorescence from the Yfp protein,as confirmed by laser scanning microscopy.Conversely,the control group transfected with e Yfp^N-NlFoxO and e Yfp^C did not exhibit fluorescence.This further confirms the interaction between NlRn and NlFoxO,indicating that they co-regulate wing dimorphism through their interaction.（4）Exploration of transcriptional complex components involved in wing dimorphism.Flag and HA tags were inserted into the 5’end of the stop codons of NlRn and NlFoxO,respectively,using CRISPR/Cas9-mediated homology-directed repair.Two homozygous insertion mutants,NlRn-Flag and NlFoxO-HA,were obtained through hybridization and selfing.However,the NlRn-Flag homozygous mutant showed poor survival rates.Immunoprecipitation-mass spectrometry analysis of the nuclear proteins from the NlFoxO-HA mutant identified 118 interacting proteins of NlFoxO.GO functional annotation of these interacting proteins shows that their functions mainly focus on protein binding,ATP binding and nucleic acid binding.KEGG functional annotation shows that their functions are mainly located in transport and metabolism,signal transduction,translation.After screening these 118 interacting proteins,10 transcription factors and 21 genes with unknown functions were selected for RNAi analysis.Among these 31 genes,two were found regulating wing development in the BPH,but no other proteins involved in wing dimorphism regulation were identified.Results in this study is not only beneficial for deepening our understanding of biological polymorphism,but also provides new ideas for pest control in rice production.