The Interactions Between P3 Proteins Of Rice Gall Dwarf Virus And Pns 9 Proteins Functional

Rice gall dwarf disease is one of the most widely occurred viral diseases of rice in Southern China. It is iduced by Rice gall dwarf virus (RGDV). In recent years, the genome of RGDV has been sequenced and the functions of the proteins encoded by some RNA segments have been known. However, the mechanisms underlying the interactions between RGDV proteins and their interactions with host factors remain unclear. The genome of RGDV consists of 12 dsRNA segments. Except S9, which contains two ORFs, each of the genome segment of RGDV encodes only one protein.The yeast two hybrid and BiFC expremements were used in this study to investigate the interaction between P3 and Pns9 and to map the interaction rigion of the two proteins. In addition, P3 was usd as a bait protein to screen the cDNA library of rice. It was found that P3 interacted with a rice protein which containd a conserved eukaryotic aspartyl protease domain.To confirm the interaction between P3 and Pns9, RNA was extracted from RGDV infected rice. RT-PCR was carried out to obtain the full-length ORF of s3 and s9, which were then ligated into the Y2H vectors pGADT7 and pGBKT7, resulting the recombinants pGAD-S9 , pGAD-S3, pGBK-S3 and pGBK-S9. The four recombinant plasmids were transformed into the yeast strain AH109 and different dropout mediums were used to test the interaction between P3 and Pns9.The results indicated that P3 encoded by S3 could inteacted with Pns9 speciefied by S9.Secondly, The plasmids pGBK-S3 and pGBK-S9 were used as templates to amplify YGFP contained S3 and S9. The PCR products were ligated into BiFC vectors pSPYNE-35S and pSPYCE-35S, resulting the recombinant vectors pSPYNE-S3, pSPYCE-S3, pSPYNE-S9 and pSPYCE-S9,which were transfomed into Agrobacterium strain GV1301, the Agrobacterium-treated with different combinations of injections of tobacco. The Agrobacterium strain GV1301 were infiltrated into leaves of Nicotiana benthamiana to observe protein interactions. The results of the BiFC experiments were consistent with those of Y2H, i.e. P3 could interact with Pns9.A panel of N-terminal deletion mutants of P3 ( lacking 20, 40 and 60 animo acids respectively) and Pns9 ( lacking 50, 100 and 170 animo acids respectively) were created by RT-PCR to map the rigions required for the interaction between P3 and pns9. The results indicated that the N-terminal 20th to 40th animo acids of P3 and the N-terminal 1 to 50th animo acids of Pns9 were important for the interaction between P3 and Pns9.Finnaly, rice cDNA library was screened by Y2H experiments using p3 as a bait, which resulted in the finding of ASP. However, the interaction between P3 and ASP was not supported by subsequent BiFC expreiments. Taken together, Y2H and BiFC were used in this study to investigate the interaction between P3 and pns9 of RGDV. It was found that the N-terminal 20th to 40th animo acids of p3 and the N-terminal 1 to 50th animo acids of pns9 were important for the interaction between P3 and pns9. The results of this study are important for further studies of RGDV.