The Self-interaction Of Rice Gall Dwarf Virus P3 Protein And The Interaction Between RGDV P2 And PC1/PC3 Of Oryza Sativa

The epidemics of Rice gall dwarf virus in some regions have caused great harm to agricultural production. On the traditional and molecular biology, much work about RGDV has been done. In this study, the interaction between Rice Gall Dwarf Virus proteins and proteins of Oryza sativa were studied by yeast two-hybrid system and bimolecular fluorescence complementation technology.RGDV P2,P3,P7,P8,Pn9,Pn10,Pn11 and Pn12 genes were amplified by RT-PCR and then inserted into yeast two-hybrid system bait vector pGBKT7 and prey vector pGADT7 respectively. In order to detect the transcriptional activation of P2,P3,P7,P8,Pn9,Pn10,Pn11 and Pn12 in the yeast cell, the recombinants pGAD-S2,pGAD-S3,pGAD-S7,pGAD-S8,pGAD-S9,pGAD-S11,pGAD-S12,pGBK-S2,pGBK-S3,pGBK-S7,pGBK-S8,pGBK-S9,pGBK-S10,pGBK-S11 and pGBK-S12 were transformed into yeast strain AH109 individually, and the transformants were plated on the different synthetic dropout nutrient medium. The results showed: RGDV Pn10 showed transcriptional activation in yeast.In order to detect the self-activation of RGDV P3, the recombinants pGAD-S3 and pGBK-S3 were co-transformed into yeast strain AH109, and the transformants were plated on the different synthetic dropout nutrient medium. The results showed: RGDV P3 can interact with itself. In order to further verify the experiment results of the self-activation of RGDV P3 detected by yeast two-hybrid system, the bimolecular fluorescence complementation technology was used. P3 fused with HA tag were amplified by PCR with the recombinants pGAD-S3 as the template; P3 genes fused with c-Myc tag were amplified by PCR with the recombinants pGAD-S3. The fusion genes above were inserted into fluorescence expression vector pSPYCE-HA and pSPYNE-Myc. The recombinants pSPYCE-HA-S3 and pSPYNE-Myc-S3 were transformed into Agrobacterium tumefaciens GV1301, respectively. The Agrobacterium tumefaciens injection were prepared and injected into tobacco leaves which the foreign proteins expressed in. Cutting the tobacco leaf of the injection site, we can observe the tobacco epidermal cells undering the aser scanning confocal microscope (Leica TCS STED, Germany) by the yellow fluorescent and take the picture. The detection results were consistent with the results of yeast two-hybrid experiments: RGDV P3 can interact with itself.In order to find the host proteins which can interact with RGDV P2, the rice cDNA library plasmids and the recombinants containing bait plasmids pGBK-S2 were co-transformed into yeast strain AH109. The transformants were plated on different synthetic dropout nutrient medium and positive clones which can interact with bait proteins were screened. The cDNA insert fragments of positive clones were identified by PCR. The yeast plasmids containing different cDNA inserts were transformed into E.coli DH5αand sequenced. The sequencing results were blasted. Accordng to the annotation information of homologous sequences, the size, sequence integrity and open reading frames correctness of the cDNA inserts were analysised. The results show: 2 kinds of positive clones were acquired by screening rice cDNA library using RGDV P2 as bait protein. Accordng to the function annotation of homologous sequences and reports about the protein function, the role of the interaction between viral proteins and host proteins in the pathogenic process of RGDV and the process of host response to virus infection was predicted.Furthermore, 2 positive clones PC1 and PC3 were selected from the yeast two-hybrid screening results. In order to detect the interaction of PC1 and PC3 with RGDV P2, the full-length PC1 and PC3 genes were amplified by RT-PCR and sequenced. PC1 and PC3 genes were inserted into yeast two-hybrid prey vector pGADT7. The recombinants pGAD-PC1 and pGAD-PC3 were constructed. The recombinants pGAD-PC1 and pGAD-PC3 were co-transformed into yeast cell AH109 with bait plasmids pGBK-S2, respectively. And then the co-transformants were plated on different synthetic dropout nutrient medium. The results showed that PC1 and PC3 both can interact with RGDV P2.In order to further verify the experiment results of the interactivation of PC1 or PC3 with RGDV P2 detected by yeast two-hybrid system, the bimolecular fluorescence complementation technology was used. P2 fused with HA tag were amplified by PCR with the recombinants pGAD-S2 as the template; PC1 and PC3 genes fused with c-Myc tag were amplified by PCR with the recombinants pGAD-PC1 and pGAD-PC3. The fusion genes HA-P2 were inserted into fluorescence expression vector pSPYCE-HA. The fusion genes Myc- PC1 and Myc- PC3 were inserted into fluorescence expression vector pSPYNE-Myc. The recombinants pSPYCE-HA- P2,pSPYNE-Myc- PC1 and pSPYNE-Myc- PC3 were transformed into Agrobacterium tumefaciens GV1301, respectively. The Agrobacterium tumefaciens injection were prepared and injected into tobacco leaves which the foreign proteins expressed in by pSPYCE-HA-P2+ pSPYNE-Myc-PC1 and pSPYCE-HA-P2 +pSPYNE-Myc-PC3. Cutting the tobacco leaf of the injection site, we can observe the tobacco epidermal cells undering the aser scanning confocal microscope (Leica TCS STED, Germany) by the yellow fluorescent and take the picture. The results show: PC3 can interact with RGDV P2; The detection result was consistent with the results of yeast two-hybrid experiments. PC1 can not interact with RGDV P2. The detection result was not consistent with the results of yeast two-hybrid experiments.In summary, the transcriptional activation of RGDV P2,P3,P7,P8,Pn9,Pn10,Pn11 and Pn12 in the yeast cell were studied ; the self-interaction of RGDV P3 were studied by yeast two-hybrid system and the bimolecular fluorescence complementation technology; The host proteins which can interact with RGDV P2 were screened from the rice cDNA librarie by yeast two-hybrid system, and the interaction of RGDV P2 with host proteins PC1 and PC3 were further verified by yeast two-hybrid system and the bimolecular fluorescence complementation technology. Results of this study can provide reliable evidences for further clarifying the function of RGDV proteins, and the mechenisms of RGDV pathogenesis and host responses to RGDV infection.