Screening Of RSV-CP Interaction Protein Factors In Small Brown Planthopper Using Yeast Two-hybrid System

Rice stripe disease, which is caused by rice stripe virus, has brought huge loss ofrice production in China. As a plant virus, it is transmitted by the small brownplanthopper (Laodelphax striatellus Fallén, SBPH), and RSV can propagate and movein insect vector. The mechanisms how RSV specially recognized SBPH, how RSVmoved and propagated in vector, and how virus is transmitted through eggs, are stillunclear. Researching these questions is the key to elucidate transmission mechanismsof RSV and control transmission of RSV. Studying molecular mechanism ofinteractions between RSV and SBPH can provide new strategy and theory to controlrice stripe disease. A yeast two-hybrid cDNA library of high viruliferous small brownplanthopper populations was constructed. To find the special receptors in interactionwith RSV, RSV CP was used as a bait to screen the library in this paper.Firstly, pGBKT7-CP vector was constructed. The vector was transformed intoyeast AH109cells, and the CP did not exhibited toxicity and self-activation in AH109cells. AH109competent cells which contained pGBKT7-CP plasmids were preparedin large scale, and then the cDNA library vectors were transformed into AH109competent cells. The transformants were incubated on SD/-Trp-Leu-His medium. Thetransform efficiency was6.25×105cfu/μg. Yeast colonies which grew well onSD/-Trp-Leu-His medium were picked and transfered into SD/-Trp-Leu-His-Ademedium (plus X-a-gal). After several days, yeast colonies which grew well andchanged blue on medium were cultured in liquid medium, and plasmids in thesecolonies were extracted. The cDNA inserts in pGAD vectors were detected usingPCR. The colonies which contained inserts above500bp were assigned as positivecolonies, and their vectors were transformed into E.coli DH5a and sequenced.Sequences of inserts in positive vectors were blasted in Genbank database. The results,these cDNA inserts belonged to11different gene sequences, with four completeORF.Two genes of11genes were choosed for farther study, including ribosomalprotein L18(RPL18) gene and cuticular protein (CPR) gene. Yeast two-hybrid assaywas used to confirm interactions of CP and L18, CP and CPR. Vectors pGADT7-L18,pGBKT7-L18, pGADT7-CPR and pGBKT7-CPR were constructed and respectivelycotransformed into yeast AH109cells with different pairs AD-CP/BD-L18, AD-CP/BD-CPR, BD-CP/AD-L18and BD-CP/AD-CPR. The results showed RSVCP could interact with L18and CPR in yeast two-hybrid system.pET32a-L18vector were constructed, and induced to generate L18protein inE.coli BL21via IPTG inducing. Because CPR protein can not be expressed in E.coliBL21by using pET32a-CPR vector, we cut off150bp sequences at CPR's N-end,and constructed pET32a-CPR47(residued sequences of CPR as CPR47) to expresseCPR protein. Expressed L18and CPR proteins were separated via SDS-PAGE, andtransfered on to NC membrane. After renaturation of blots, virus-binding experimentswere perform to detect virus-binding capacities of two proteins. The results showedthat L18was able to bind to RSV particles, but CPR protein did not bind to RSV.To confirm whether RSV's infection influences expression of L18gene, theexpression levels of L18gene in RSV-free and viruliferous SBPH were detected byusing real-time quantitative PCR. The results showed that no different expression ofL18gene exhibited in RSV-free and viruliferous SBPH, which was considered thatexpression of L18gene was not excited by RSV's infection.