Function Of Chemosensory Protein In Cyantraniliprole Resistance Of Aphis Gossypii Glover

Chemosensory proteins(CSPs)are small soluble proteins with four conserved cysteines that are linked to form disulfide bridges,and expressed ubiquitously in chemosensory organs and nonchemosensory tissue of arthropods.Most of the functions of CSPs are related to its binding ability to small molecules,include detecting chemosignals,involving in the regeneration and development,immune regulation,releasing semiochemicals and distinguishing nutrition.Except for the above functions,a lot of research showed that CSPs are related to the insect resistance recent years.Through the transcriptome data,we found there are nine CSPs in Aphis gossypii,CSP1,CSP4,and CSP5 were constitutively overexpressed in the cyantraniliproleresistant(Cy R)strain.The RT-q PCR results of CSP1,CSP4,and CSP5 demonstrated that the expression level of Cy R strain cotton aphid were upregulated significantly,which increased 2.04-,2.69-and 2.94-fold than in susceptible(SS)strain.The analysis of tissue-specific expression showed that CSP1,CSP4,and CSP5 displayed higher expression in broad body tissue than in the gut but without tissue specificity.However,the function of these three upregulated CSPs remains unknown.Here,we investigated the function of CSPs in resistance to the diamide insecticide cyantraniliprole.Firstly,we knockdown the CSPs in Cy R strain cotton aphid using RNA interference(RNAi)assay.Mortality significantly increased from 50%,38% and38% in the corresponding control to 72%,50% and 58% with the suppression of CSP1,CSP4,and CSP5 transcription,respectively,under the cyantraniliprole treatment.Then,we expressed CSP1,CSP4,and CSP5 in Drosophila melanogaster by the UAS/GAL4 system under Act5C(body-specific expression)or Esg(midgut-specific expression)conditions.Bioassay results indicated that the cyantraniliprole tolerance ability of the flies ectopically expressing CSP1 and CSP4 in remaining carcass significantly increased 4.02-and 4.53-fold.And flies ectopically expressing the CSP1,CSP4 and CSP5 genes showed 10.13-,1.88-and 9.01-fold higher tolerance,respectively.CSP4 was more effective in broad body tissue,and CSP1 and CSP5 were more effective in the midgut.To identify the affinity of CSPs to cyantraniliprole,fluorescence competition assays and molecular docking were completed.Heterologous expression of CSPs in Escherichia coli and competitive binding assays with the fluorescent marker1-NPN were carried out for functional validation of the CSPs.The expressed CSP1,CSP4 and CSP5 proteins showed moderate binding affinity for cyantraniliprole which was stronger significantly than other insecticides in this study.Moreover,the results of molecular docking proved that the binding of CSPs to cyantraniliprole mainly depended on hydrophobic interaction.Two hydrogen bonding interactions existed between residues ASP-135 and TYR-144 of CSP1 and cyantraniliprole,with distances of 2.37? and 2.35 ?,respectively.One hydrogen bonding interaction existed between residue GLN-77 of CSP5 and cyantraniliprole,with a distance of 1.87 ?.The three residues which forming hydrogen bonds may be the key sites of CSP1 and CSP4 binding cyantraniliprole.The present results strongly indicate that CSPs participate in xenobiotic detoxification by sequestering and masking toxic insecticide molecules,providing insights into new factors involved in resistance development in A.gossypii.