Screening And Identification Of Aphis Citricida Proteins Interacted With The Coatproteins Of Citrus Tristeza Virus

Tristeza,caused by Citrus tristeza virus(CTV),is one of the most important citrus diseases in the world.CTV is mainly spread by grafting and aphids in a semi-permanent way.Brown citrus aphid(Aphis citricida)is the most important vector to CTV in China,and can effectively transmit most CTV isolates.Until now,the mechanism of aphid transmission of CTV is still unknown.Therefore,to explore the interaction mechanism between CTV and A.citricida is of great significance for further understanding the aphid transmission mechanism of CTV,and prevention and control of tristeza.Previous studies suggested that coat protein(CP)and minor coat protein(CPm)of CTV are correlated with its aphid transmissibility.In this study,the cephalothorax of A.citricida was used to construct nuclear system yeast library and membrane system yeast library.The potential interaction proteins to CP and CPm of CTV were screened and identified by yeast two-hybrid.Furthermore,the differential expressed proteins of nonviruliferous A.citricida and viruliferous A.citricida were also analysised by proteomic sequencing.The main research results are as follows:1.Nucleus system yeast library of A.citricida was constructed by using total RNA from the cephalothorax of A.citricida.The titer of this c DNA library was4.5×10~8cfu/m L,and the recombinant rate was 95.6%,which meets the requirements of high-quality c DNA library and can be used in subsequent screen library tests.CP and CPm genes of CTV were inserted into the bait vector plasmid p GBKT7-BD,respectively,to construct the bait plasmids p GBKT7-CP and p GBKT7-CPm.These bait vector plasmids were non-toxic to yeast cells and don’t have self-activating activity.The bait plasmid screening library experiment was conducted by mating method.Circadian clocked-controlled protein-like(Cir)and pre-m RNA-processing factor 40 homolog B isoform X1(pre)were obtained by using p GBKT7-CP from the c DNA library.Twenty-three proteins were obtained by using p GBKT7-CPm screen library,and 3proteins(Argonaute 1 region(Ago),Actin(Act)and ubiquitin(Ubi))were co-transformed with corresponding bait proteins to conduct secondary interaction verification.The results showed that both Cir and Pre could interact with p GBKT7-CP,and Ago could interact with p GBKT7-CPm,while Act and Ubi could not interact with p GBKT7-CPm.2.Membrane system yeast library of A.citricida was constructed by using total RNA from the cephalothorax of A.citricida.The titer of this library was 3.6×10~6cfu/m L and the recombinant rate was 100%,which could be used for the subsequent screen library tests.By comparing the bait vector plasmids p BT3-N、p BT3-STE and p DHB1 in the split-ubiqitin membrane yeast two-hybrid system,it was found that p DHB1 could be used to construct the bait plasmids p DHB1-CP and p DHB1-CPm with CP and CPm genes and no self-activation,respectively.Fifty-five and 17 of proteins were screened from the yeast library of membrane system.Twenty-three proteins screened by p DHB1-CP and 7 proteins screened by p DHB1-CPm were co-transformed with corresponding bait proteins to conduct secondary interaction verification.The results showed that CP interacted with other 21 prey proteins except for protein bric-a-brac 1(Pro1)and RNA-binding protein squid-like isoform X2(Rbp X2).CPm interacts with 14other prey proteins,except for LOC114129262(LOC262),uncharacterized protein LOC114122708(LOC708)and PREDICTED:voltation-gated potassium channel subunit beta-2 isoform X1(Vol).3.Using Tandem mass tags(TMT)quantitative technology to analyze the difference among the healthy A.citricida,the viruliferous A.citricida and the A.citricida that lost ability to transmit virus after being infected.The results showed that the viruliferous group compared with the healthy group(DD/WD),517 proteins were differentially expressed,and the viruliferous group compared with the lost the ability to transmit virus after being infected group(DD/QSHWD),344 proteins were differentially expressed.Furthermore,the lost the ability to transmit virus after being infected group compared with the healthy group(QSHWD/WD),1259 proteins were differentially expressed.By comparing the differentially expressed proteins of DD/WD and DD/QSHWD,after removal of the uncharacterized proteins,there were 47 common differentially expressed proteins(CDEPs),and mainly enriched in 5 pathways,such as starch and sucrose metabolism,mitophagy-animal,phototransduction-fly,arginine and proline metabolism,citrate cycle(TCA cycle).4.Calcium ion transport ATPase was found by using yeast two-hybrid and the TMT.