Study On The Interaction Between Rice Stripe Virus And Host Rice By Yeast Two-hybrid System

In the recent years, rice stripe disease caused by Rice stirpe virus (RSV) breaks out in china. It has caused great harm to agricultural production and brought huge loss to people. The biology and molecular biology about RSV were studied widely and systematically by scholars at home and abroad. The geographical distribution, symptom, property of pathogen, serology, pathogenicity differentiation, molecular variation, cytopathology and variety resistance were made clear, while the function of some viral proteins, pathogenic mechanism of the virus, and molecular mechanisms of plant resistance and susceptibility to RSV have not been known. Therefore, the studies, which on the interaction relationships of viral proteins, and host proteins with viral proteins at molecular level, has important significance for elucidating these questiones.RSV CP, SP and NSvc4 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 self-activation of CP, SP and NSvc4 and toxicity to yeast cell, the recombinants were transformed into yeast strain AH109 individually, and the transformants were plated on the different synthetic dropout nutrient medium. The results showed: CP, SP and NSvc4 don't self-activate and have no toxictiy to yeast cell; CP can interact with itself, but no interaction of CP with SP , CP with NSvc4, SP with SP, SP with NSvc4, and NSvc4 with NSvc4 was detected.The total RNA of seeding leaves of two rice cultivars (strains) "Wuyujing 3" and "KT95-418" , which have basically consistent genetic background while the difference of resistance to RSV is significant, were extracted. The double-strand cDNA were synthesized by SMART technology and inserted into modified vector NpGADT7 through suitable enzyme sites, and yeast two-hybrid cDNA libraries of two rice cultivars (strains) were constructed. The results of detection showed that the two libraries both contained 1.0×10⁶ independent clones; the titer of primary library were 1.0×10¹⁰cfu/mL and 5.0×10¹⁰ cfu/mL respectively; and the titer of amplified library were l.0×10¹¹ cfu/mL; The recombination rates were both above 95%; The size of most inserts were 700-800bp in the cDNA library of "Wuyujing 3", and 750-1,000 bp in the cDNA library of "KT95-418"; Small-scale sequencing results and bioinformatics analysis showed that the percentage of full-length cDNA were both above 60%. The two cDNA libraries plasmids were extracted and transformed into yeast strain AH109 containing bait plasmids pGBK-CP, pGBK-SP and pGBK-NSvc4, respectively. 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 DH5?and 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 showed the positive clones with different varieties and number were acquired by screening different cDNA libraries using different bait proteins: 4 positive clones were acquired by screening "Wuyujing 3" cDNA library using RSV CP as bait protein; 2 positive clones were acquired by screening "KT95-418" cDNA library using RSV CP as bait protein; 5 positive clones were acquired by screening "Wuyujing 3" cDNA library using RSV SP as bait protein; 6 positive clones were acquired by screening "KT95-418" cDNA library using RSV SP as bait protein; 9 positive clones were acquired by screening "Wuyujing 3" cDNA library using RSV NSvc4 as bait protein; 9 positive clones were acquired by screening "KT95-418" cDNA library using RSV NSvc4 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 RSV and the process of host response to virus infection was predicted. Furthermore, 3 interesting clones CW8, NB2 and NB5 were selected from the yeast two-hybrid screening results. The full-length gene was amplified by RT-PCR and sequenced. The results showed NB2 and NB5 full-length gene were acquired, while PCR products of CW8 were shorter than homologous sequences. NB2 and NB5 genes were inserted into yeast two-hybrid prey vector pGADT7. The recombinants pGAD-NB2 and pGAD-NB5 were constructed. In order to detect the interaction of NB2 or NB5 with RSV CP, SP and NSvc4, the recombinants pGAD-NB2 or pGAD-NB5 were co-transformed into yeast cell AH109 with bait plasmids pGBK-CP, pGBK-SP and pGBK-NSvc4, respectively, and then the co-transformants were plated on different synthetic dropout nutrient medium. The results showed that NB2 and NB5 both can interact with NSvc4 except CP and SP.In order to further verify the experiment results of protein interaction detected by yeast two-hybrid system, CP, SP and NSvc4 genes fused with c-Myc tag were amplified by PCR with the recombinants pGBK-CP, pGBK-SP and pGBK-NSvc4 as the template; CP, SP, NSvc4 and NB5 genes fused with HA tag were amplified by PCR with the recombinants pGAD-CP, pGAD-SP, pGAD-NSvc4 and pGAD-NB5, respectively. The fusion genes above were inserted into plant expression vector pEGAD and pKYLX71:35S2. The recombinants were transformed into Agrobacterium tumefaciens EHA105. The Agrobacterium tumefaciens injection were prepared and injected into tobacco leaves which the foreign proteins expressed in. Total proteins of tobacco leaves were extracted and used for the co-immunoprecipitation with HA or c-Myc antibody. The immunocomplexes were collected and then examined by SDS-PAGE. The proteins were detected by Western-blot with c-Myc or HA antibody. The detection results were consistent with the results of yeast two-hybrid experiments: CP can interact with itself, but no interaction of CP with SP , CP with NSvc4, SP with SP, SP with NSvc4, and NSvc4 with NSvc4 was detected; NB5 can interact with NSvc4 , except CP and SP.The expression of CW8, NB5 and SB12 mRNA in the health and RSV-infected rice leaves were examined by Real-Time PCR. The difference of mRNA expression were compared, and the effect on mRNA expression, which caused by RSV infection and interaction of viral proteins with host proteins, were analysised. The results showed: the expression of CW8 mRNA in the RSV-infected rice leaves was down-regulated compared with which in the health rice leaves; The expression of NB5 mRNA had no difference; The expression of SB12 mRNA in the RSV-infected rice leaves was up-regulated compared with which in the health rice leaves.Besides above research, construction of fusion gene RSV CP and Rubisco SSU leader peptide of rice chloroplast and its prokaryotic expression were studied: The Rubisco SSU leader peptide and RSV CP genes were amplified by PCR. The fusion genes PR-CP and PR-S-CP were constructed by means of the expression vector pGEX-4T-1 and pET-29a. The recombinants pGEX-PR-CP and pET-PR-S-CP were transformed into E.coli BL21(DE3) and induced to express. The expression products were detected by SDS-PAGE which is in same size as expected. The identification results of Western-blot showed the expression products of two fusion genes both can bind with CP antibody specifically. This suggested that RSV CP maintained antigen activity of itself, and its expression was not effected by Rubisco SSU leader peptide. We could conclude that Rubisco SSU leader peptide and RSV CP in the fusion proteins maybe could maintain its own structure and function when the fusion genes PR-CP and PR-S-CP express in the organism.In summary, the self-interaction and interaction each other of RSV CP, SP and NSvc4 were studied by yeast two-hybrid system and co-immunoprecipitation technology; The host proteins which can interact with CP, SP or NSvc4 were screened from two cDNA libraries of two rice cultivars (strains) by yeast two-hybrid system, and the interaction of viral proteins with host proteins were further verified by co-immunoprecipitation; The mRNA expression of several proteins which can interact with viral proteins in the health and RSV-infected rice leaves were examined by Real-Time PCR; Furthermore, fusion genes of RSV CP with Rubisco SSU leader peptide were constructed , and the expression products in E.coli were detected by SDS-PAGE and Western-blot. This study can provide reliable evidences for further clarifying the function of RSV proteins, understanding the pathogenic mechanism and molecular mechanisms of host resistance and susceptibility to RSV, and exploiting resistant germplasm resources.