Screening And Functional Validation Of Differentially Expressed Genes Between Host-specialized Biotypes Of Aphis Gossypii Glover

The cotton aphid Aphis gossypii Glover,also known as cotton-melon aphid,is a worldwide horticultural and agricultural pest.It is a typical polyphagous aphid,with different host-specialized types(also called host biotypes)due to their preference for different plants.The most important of biotypes are the cotton biotype,which feeds mainly on Malvaceae plants,and the cucumber biotype,which feeds mainly on cucurbits.Host-specialized biotypes perform poorly or can’t survive when transferred to non-native hosts.The mechanism for host specialization of this aphid is still unknown.Some salivary proteins play an important role in the adaptation of aphids to their hosts.We hypothesize that differences in salivary gland gene expression between different host biotypes may result in different adaptations of A.gossypii to different hosts.In this study,we dissected the salivary glands of different host biotypes with the same genetic background and conducted transcriptome sequencing for screening differentially expressed genes,and validated the functions of 5 differentially expressed genes.This study aimed to reveal the molecular mechanisms of host biotype in this aphid.The results of the study are as follows.1.Screening for differentially expressed genes between different biotypesThe transcriptomes of salivary glands of cotton biotype and cucumber biotype were analyzed.A total of 12,808 expressed genes were detected,of which 523 differentially expressed between the two biotypes.In the differentially expressedgenes,319 were significantly over-expressed in cotton biotype and 204 in cucumber biotype.Most of the differential genes had "catalytic" activity and "binding" functions,and were involved in "cellular processes" and "metabolic processes".Differentially expressed genes were highly enriched in "cuticle structure","carbohydrate process","oxidoreductase activity","glucuronyltransferase" and other enzymatic activity pathways.The expression levels aphid salivary proteins reported in other aphids,such as carbonic anhydrase,peroxidase and glutathione S-transferase,did not differ significantly between cotton biotype and cucumber biotype.The expression levels of the 15 reported homologues of aphid effectors and elicitors did not differ significantly between cotton biotype and cucumber biotype.These results suggested that aphid salivary proteins that had been identified may not be responsible for the biotype formation of A.gossypii.It was found that the cotton biotype could not feed on healthy cucumber leaves but could feed on cucumber leaves which were pre-infested with the cucumber biotype.So,we hypothesized that the cotton biotype is lack of effectors that overcome defenses of cucumber plants.According to the characteristics of secretory proteins,such as small molecular weight,with signal peptide regions and without transmembrane structure,we screened genes with significantly higher expression in the salivary gland of cucumber biotype than in cotton biotype.A total of 14 genes were obtained,including five protein genes of unknown function,three cuticular protein genes,one transcriptional regulatory protein gene,one pro-resilin gene,one adhesive plaque matrix protein gene,one lipase gene,one uclacyanin gene and one yellow protein gene.The qPCR verification showed that C2,M4,M5 and C9 were significantly highly expressed in the cucumber biotype,compared to the cotton biotype.A recent study reported that cathepsin B(Cath B)can act as an elicitor to regulate the adaptation of green peach aphid to tobacco.Therefore,in this study,the expression of three Cath B genes was compared between the two biotypes.The qPCR verification showed that Cath1 and Cath2 were significantly highly expressed in cucumber biotype and Cath3 was significantly highly expressed in cotton biotype.Therefore,C2,M4,M5,C9 and Cath3 were selected as candidate genes.2.Functional validation of candidate genesC2 was annotated as a cuticle protein 7-like,consisting of 171 amino acids,containing a signal peptide sequence and without a transmembrane structure.The expression of C2 in the head of cucumber biotype was 3.3 times higher than that in other tissues.The M4 was annotated as a pro-resilin-like,consisting of 116 amino acids and consistent with secretory protein properties.The expression of M4 in the head of cucumber biotype was 21.64 times higher than that in other tissues.M5 is a protein of unknown function,consisting of 126 amino acids,containing a signal peptide sequence and without a transmembrane structure.The expression of M5 in the head of cucumber biotype was 9.58 times higher than that in other tissues.The expression of Cath1 was significantly higher in the head of cucumber biotype than in the rest tissues,and Cath2 was lower in the head of cucumber biotype than in the rest tissues,and Cath3 was significantly lower in the head of the cotton biotype than in the rest tissues.C2,M4 and M5 RNAi in cucumber biotype and Cath3 RNAi in cotton biotype were carried out,and there were no apparent changes in body appearance,survival rate,reproduction and feeding efficiency.C9 was annotated as an adhesive plaque matrix protein-like,consisting of 202 amino acids,and was predicted to be a secretory protein.The expression of C9 in the head of cucumber biotype was 7.44 times higher than in the rest tissues;it was expressed in all ages of the cucumber biotype,with about four times higher expression in the 1st instar than in other ages.The homologous sequences of C9 were found in Apolygus lucorμm and other 9 aphid species.There was a significant decrease in reproduction,a significant decrease in the efficiency of phloem ingestion when C9 was RNA interfered in cucumber biotype.In summary,salivary proteins,as well as effector and elicitor that have reported other aphid were not significantly different between the two biotypes,suggesting that these the genes of those proteins were not key genes involved in host specialization in A.gossypii.By comparative transcriptome analysis,four genes(C2,M4,M5,C9)that were highly expressed in the cucumber biotype and one gene(Cath3)that was highly expressed in the cotton biotype were obtained.Functional validation showed that the C9 gene had significant effects in the cucumber biotype adapting to cucumber and was promising to determine the adaptation of the cucumber biotype to cucumber.