Screening And Identification Of Rhopalosiphum Padi Proteins Involved In WYDV-GPV Transmission

Wheat virus diseases often occurred on wheat in the winter wheat production areaworldwide, caused by single or mixed infection of yellow dwarf viruses(YDVs). YDVs arepersistently transmitted by various wheat aphids. Therefore, the virus-aphid interactions arethe key to the disease triangle of virus-aphid-wheat. Aphid proteins, involved in virustransmission, show multiple molecularly interacions with viral proteins, however, were justscreened and found out with the methods in forward genetics. In this study, yeast two hybridtechniques (including GAL4yeast two hybrid systm and split-ubiquitin based mambraneyeast two hybrid system), which belong to the reverse genetic method, were used to screenthe interactors from oat cherry-bird aphid (Rhopalosiphum padi) associated with transmissionof Wheat yellow dwarf virus-GPV (WYDV-GPV). Further, the obtained proteins from yeasttwo hybrid screens were detected and confirmed for the genetic and physical interactions withviral proteins by yeast two hybrid and Chemiluminescent Co-immunoprecipitation(Chemiluminescent Co-IP).For GAL4yeast two hybrid system screen, the bait plasmids pGBKT7-CP andpGBKT7-RTD were constructed by inserting the WYDV-GPV coat protein (CP) gene andreadthrough protein (RTD) gene, which both are aphid transmission-associated viral proteins,into the bait vector pGBKT7-BD. And then, they proved that their protein products werecorrectly expressed in Y2HGold yeast cells, no toxinity to yeast growth, and no autoactication.So these two bait plasmids were suitbale for library screening.The cDNA yeast library wascontructed from whole body total RNA of R. padi, whose transformation efficiency was5×106cfu/ug, indepentant clones4×106/ml，and library titre5.12×107cfu/ml, and wasscreened by two baits. The mating screening procedures were performed for library screeningof two baits: pGBKT7-CP obtained no interactors, and pGBKT7-RTD got three prey ptoteins,hypothetical protein LOC100158933, ribosomal protein large subunit31(Rpl31) andribosomal protein large subunit12(Rpl12). The resulting that the screened prey proteins wasso less in number and class led to an inference: this yeast two hybrid system was unsuitable toresearch the interactions between virus and aphid.Another yeast two hybrid system, split-ubiquitin based mambrane yeast two hybridsystem was tried to find interactors from R. padi with WYDV-GPV CP and RTD. CP and RTD genes were inserted into bait vector pDHB1, yielding two bait plasmids pDHB1-CP andpDHB1-RTD. The functional assays indicated that these two bait plasmids could expresscorrect and functional bait proteins for library screening. The matched NubG-X cDNA librarywas constructed by using the whole body total RNA of R. padi. The resultings of libraryscreening by two baits were that: pDHB1-CP screened9functional proteins and3hypothetical proteins; and pDHB1-RTD screened24functional proteins and5hypotheticalproteins. The prey proteins of interest were selected to carry on the second interactiondetection and X-β-Galactosidase assay with their baits. All5prey proteins of CP, Emi, Pan,LOC, Naca, and CV4, were shown that they had strong interactions with CP. Of22preyproteins of RTD, UL36had few chance to interact with RTD, Nar, TCP, CVa, and pro3ownedweak interaction relationship with RTD, and the remaining17prey proteins, Comx, Gps,Naca, OSD, Cup, Vha, Ft3, LOC, Cu(I), GRIK3, CV4, Pan, p450, Ubi, Jagn1,TrI, and Tsrwere denmonstrated that they strongly interacted with RTD.Chemiluminescent Co-IP was applied to comfirm the physical interactions betweenCP/RTD and their prey proteins screened from split-ubiquitin based mambrane yeast twohybrid system. Among the prey proteins of interest, seven proteins were determined tointeract with RTD, Pan, TrI,Tsr, Comx, Naca, CV4, and p450in the order of strong to weakinteractions; no prey proteins, except Pan, had interaction with CP. We focused on theresearch to Pan, because of its strong gentic and physical interactions with RTD. According tothe deduced amino acids from the full-length cDNA sequence, the N terminus of this proteinpossesses the conserved functional domain of lipase, but C terminus is much longer thancommom lipases, in which part, some amino acid residues strongly interact withWYDV-GPV RTD. So Pan probably involves in the WYDV-GPV virion-coating vesicaformation and transportation in R. padi.To detect the expression profiles and functional assays of the aphid interactors in thefuture, relative RT-qPCR method was established in YDV-viruliferous R. padi. With thismethod, the titre of several YDVs in R.padi and the expression of aphid endogenous genecould be monitored.