Analysis Of Salivary Proteome Of Aphis Gossypii And Identification Of Suspected Effectors

Cotton aphid,Aphis gossypii Glover(Hemiptera: Aphididae),is a worldwide pest that can damage many hortic?ltural and agric?ltural crops.The host plant of A.gossypii is up to 285 species in 76 families.There are several host-associated biotypes in this aphid,mainly cotton-biotype and cucumber-biotype.The fitness reduced greatly when A.gossypii were transfer to novel host plants.However,the mechanism of host biotypes of A.gossypii remains elusive.The salivary effector proteins play an important role in the interaction between piercing-sucking insects and host plants.We hypothesized that the host biotypes of A.gossypii may be due to the differences of salivary effector proteins between host-associated biotypes.In this paper,we studied the fitness of cucumber-biotype and cotton-biotype in reciprocal host transfer experiences.Then we collected salivary proteins and salivary glands of cucumber and cotton-biotype aphid and analyzed the salivary proteome and salivary gland transcriptome.The suspected effectors which were potentially related to host adaptation were screened according to the differences of salivary protein composition and gene expression.Two suspected effectors were selected for functional verification.The res?lts are as follows:1.Fitness of cucumber-and cotton-biotype in reciprocal host transfer experiencesCucumber-biotype aphid survived and established pop?lation in cotton,with pop?lation increasing 10.7 folds in 14 days.While the pop?lation of cucumber-biotype aphid increased 19.5 folds in the original host in 14 days.The body size of cucumberbiotype aphids on cotton was as 80% big as the normal cucumber-biotype aphids.The body color of cucumber-biotype aphid on cotton turned yellow green,compared to dark green of the normal cucumber-biotype aphid.The pop?lation of cotton-biotype aphid on cucumber increased 3.3 folds in 14 days,compared to 18.4 folds on the original in 14 days.The body size of cotton-biotype aphid on cucumber decreased to 20% of the normal cotton-biotype aphid.The body color turned yellow.So,cotton-biotype aphid co?ld not adapt to cucumber and establish normal pop?lation in cucumber.This shows that the cucumber-biotype aphid can adapt to cucumber and cotton,while the cottonbiotype cotton aphid only can adapt to cotton and cannot adapt to cucumber.After a month's survival on cotton,the cucumber-biotype aphid was transferred back to the cucumber,and the reproduction was not significantly different from that of the normal cucumber-biotype aphid on cucumber.Previous studies showed that cotton-biotype and cucumber-biotype aphid co?ld not survive after transferred reciprocal.Our res?lts showed that the cucumber-biotype aphid can establish pop?lation in cotton and the cotton-biotype aphid cannot establish pop?lation in cucumber.2.Transcriptome and proteome analysis of salivary gland and saliva proteinsA total of 59303 unigenes gene were identified in transcriptome analysis of salivary gland.There are 1153 genes with significant difference in TPM value between cucumber-biotype and cotton-biotype salivary gland.Compared to cucumber biotype,532 genes were up-reg?lated in cotton biotype and 621 genes were down-reg?lated in cotton biotype.A total of 90 genes have binding function and 110 genes have catalytic function.Watery saliva of cotton-biotype,cucumber-biotype and cucumber-biotype transferred to cotton was collected using double-layer Parafilm.Label-free quantification was used to analyze salivary proteins,including process of raw protein ?ltrafiltration,SDS-PAGE,FASP,LC-MS and Max Quant.A total of 129 proteins were identified,and they were enzymes,transporters,cytoskeleton-related proteins,DNA\RNA\chromatin-binding proteins,transcription factors,agonists,heat shock proteins,non-enzyme proteins and proteins with unknown functions.A total of 80 peptides have catalytic activity and 70 peptides have binding function.The proteome analysis of saliva proteins showed that there were 85 salivary proteins shared but significantly differed in abundance between cucumber-biotype and cotton-biotype,and 36 proteins were only expressed in the cucumber-biotype and 19 proteins were only expressed in the cotton-biotype.Signal P 5.1 was used to predict signal peptide and TMHMM was used to predict transmembrane structure,and Target P 1.1 was used to predict protein subcell?lar localization.We got 7 suspected effector proteins with secretory properties from 144 salivary proteins.3.The functional of the suspected effector Ag22814 and Ag18415 gene.In this experiment,Ag22814 and Ag18415 were selected from the 7 candidates for function verification.Transcriptome analysis of salivary glands showed that the expression of Ag22814 and Ag18415 genes in cucumber-biotype was 2.8 and 3.5 folds higher than that in cotton-biotype,respectively.The res?lts of q PCR showed that the expression of Ag22814 and Ag18415 genes in the salivary glands of cucumber-biotype was 4.5 and 238 folds higher than that in the salivary gland of cotton biotype.Transcriptome and q PCR analysis showed that the expression of Ag22814 and Ag18415 genes in salivary gland of cucumber-biotype was significantly higher than that in cotton-biotype.Compared with cucumber-biotype aphids feeding cucumber,the expression of Ag22814 and Ag18415 genes increased in the cucumber-biotype aphid feeding cotton.These res?lts suggest that the two genes are potentially related to host adaptation of cucumber-biotype aphids.In the 1st and 2nd instars,the expression of Ag22814 and Ag18415 genes was higher than that of 3th,4th and 5th instars.Polyclonal antibodies were synthesized by using prokaryotic expression Ag22814 and peptide synthesis Ag18415.Ag22814 protein was detected by Western blot in the cucumbers damaged by cucumber-biotype aphids.This indicated that Ag22814 protein was secreted into host plants by aphids.But the protein Ag18415 was not detected in cucumber plants,which may be due to the failure of antibody synthesis.The function of Ag22814 and Ag18415 in host adaption was studied by silencing the gene of 4-daysold cucumber-biotype aphids with synthetic ds RNA.The expression of Ag22814 and Ag18415 genes decreased to 46% and 43% of the control on the third day after RNAi.The mortality of aphids was less than 10% after 72 hours of ds RNA feeding.The mortality of aphids was 34% on the 13 th day after Ag18415 RNAi,and there was no significant difference between ds Ag18415 and control.Ag22814 and Ag18415 had no significant effects on aphid body size and reproduction after ds RNA feeding within one week.EPG(Electrical Penetration Graph)was used to monitor the feeding process of aphids.The res?lts showed that the duration of E1 wave in ds Ag22814 was significantly longer than that of control,and the duration of E2 wave(phloem feeding wave)was significantly shorter than that of the control.This indicated that ds Ag22814 caused obstacles in aphid feeding.The silencing of Ag18415 gene had no effect on the feeding process of aphids.Although Ag22814 protein can be secreted into the host plant and interferes with aphid feeding,but the Ag22814 and Ag18415 genes are not the key effectors for the host adaptation of cucumber and cotton-biotype aphids.