Screening And Identification Of Aphis Citricida Proteins Interacting With Coat Proteins Of Citrus Tristeza Virus

Tristeza disease caused by Citrus tristeza virus(CTV)is an important economic disease destructing citrus industry worldwide.CTV can be transmitted to most citrus species,cultivars and hybrids by grafting and CTV-infected propagating material play a role in long-distance transmission.CTV can also be transmitted by several aphids in a semi-persistent manner,among which Aphis citricida is most efficient vector to CTV.After feeding on CTV-infected plant,the particles of CTV were attached on the foregut of Aphis citricida and then inoculated to a new healthy citrus by saliva.However,the molecular mechanism of transmission process has not been clarified.Therefore,to assess the molecular mechanisms of the interaction between CTV and Aphis citricida,the interaction proteins between CTV and Aphis citricida were screened and identified.The results will provide a theoretical foundation for further research on control tristeza disease by blocking the spread of CTV.Studies showed that virus-encoded structure protein,especially coat protein has played an important role on the interaction between virus and vector insects.In previous studies,several Aphis citricida proteins which may potentially interact with CTV coat proteins(CP and CPm)were screened by using far-Western blot method,yeast two hybrid technology and differential proteome between CTV-free and viruliferous A.citricida.But the interaction between those proteins of A.citricida and coat proteins of CTV should be further confirmed by other methods,such as GST-pull down.In this study,20 transmembrane proteins or secreted proteins which expressed in head and chest of A.citricida were selected and identified by yeast two hybrid technology and GST-pull down.The main research results are as follows:1.Proteins potentially interacted with CTV coat protein(CP and CPm)were screened.Fifteen transmembrane proteins or secretory proteins were selected from 78 proteins which obtained previous studies,by subcellular localization with subcellular prediction website.Furthermore,5 homologous sequences of cuticle proteins that may be involved in vector transmission of non-persistent viruses were screened from the transcriptomic database of A.citricida.RT-q PCR analysis showed that the genes of these 20 proteins were expressed in the head and chest of A.citricida,and the expression levels of MPCP1,MPCP2,MPCP4,Sylin02,FLCPR2,PDR,AC9786,Cup20 and NP9398 were higher than that of the housekeeping geneα-Tubulin.Nucleotide sequence alignment analysis showed that the similarity of the 20 genes in A.citricida and the homologous genes in other aphid species was higher than 80%.Phylogenetic tree results showed that the homologous genes of these 20 genes from A.citricida and A.gossypii which another effective CTV vector,may be evolutionarily farther than the homologous genes from non-CTV-vector aphids.2.The interaction between NP9398 and CP,NP9147 and CPm was confirmed by yeast two hybrid assay.Prey plasmid was constructed by fusing the 20 transmembrane proteins or secreted proteins genes into p PR3 N.Prey plasmid was respectively co-transformed into the yeast strain NMY51 with bait vector(p DHB1-CP or p DHB1-CPm).The yeasts transformed with p PR3N-NP9398 and p DHB1-CP were grown well on SD/-Trp-Leu-HisAde medium.The yeasts transformed with p PR3N-NP9147 and p DHB1-CPm were grown poorly on SD/-Trp-Leu-His-Ade medium.The interaction between above bait proteins and prey proteins was further confirmed by β-Galactosidase assay.The yeasts transformed with other prey plasmid and bait plasmid could not grow on SD/-Trp-LeuHis-Ade medium.Yeast two hybrid assay showed that the interaction between NP9398 and CP is strong,while the interaction between NP9147 and CPm is weak.The other 18 proteins did not interact with CP or CPm.3.The interaction between NP9398 and CP was proved by GST-pull down.Four prokaryotic expression vectors p GEX-4T-NP9398,p GEX-4T-NP9147,p ET-28a-CP and p ET-28a-CPm were constructed and then respectively transformed into Escherichia coli strain Rosetta(DE3).The fusion proteins were induced by IPTG and purified by GST or His tag purification resin.GST-pull down assay proved that NP9398 could interact with CP.The result of GST-pull down was accord with the result of yeast two-hybrid assay.Even using different prokaryotic expression vectors,optimizing conditions include inducer concentration and temperatures and codon optimization,the recombinant protein NP9147 could not be expressed in Escherichia coli.Therefore,the interaction between NP9147 and CPm cannot be verified by GST-pull down assay.