Prokaryotic Expression Of Rice Stripe Virus Genes And Functional Analysis Of NS3 And NSvc4

Rice stripe disease caused by Rice stripe virus(RSV)spread very rapidly in China in recent years,and has been caused severe damage to rice production.It is difficult to control this disease because the virus is transmitted by Laodelphax striatellus in a circulative,propagative manner and transovarially by viruliferous female planthoppers to their offspring.To understand the mechanisms of pathogenicity of RSV,RSV genes were expressed in Escherichia coli and the functions of NS3 and NSvc4 genes were analyzed in this dissertation.The NS2,NS3,NSvc4 and SP genes of RSV were expressed in E.coli strain BL21(DE3)pLys S using pET32a vector,and recombiant proteins were purified with Ni²⁺-NTA agarose affinity chromatography.The polyclonal antibodies against NS2, NS3,NSvc4 and SP were produced in rabbits,respectively.These polyclonal antibodies can be used to detect the relevant proteins in the RSV infected L.striatellus and rice leaves.RNA silencing in plants is a natural immune system against virus infection.To determine if RSV encoded genes were involved in suppression of RNA silencing, co-infiltration assay in GFP transgenic Nicotiana benthamiana line 16c was carried out.The results revealed that NS3 protein could suppress GFP local silencing induced by GFP construct,inhibit GFP systemic silencing and prevent spread of silencing signal,suggesting that NS3 protein of RSV is a suppressor of RNA silencing.The local GFP mRNA and GFP protein accumulations were increased significantly in N. benthamiana line 16c plants at six days post co-infiltration with NS3 and GFP when tested by Northern blot and Western blot analysis,while the decrease of siRNA accumulation was observed in the same tissues.Co-infiltration of a series of deletion mutants of NS3 with GFP show the 5' terminal and 3' terminal of NS3 are crucial for suppressing RNA silencing.Invert repeat construct(dsGFP)of GFP was produced.The co-infiltration assay with NS3 and GFP and dsGFP in N.benthamiana revealed that NS3 protein can suppress the initiation of RNA silencing induced by dsGFP. The RNA binding ability of recombinant NS3 protein was determined by Electrophoresis mobility shift assay(EMSA).The results showed that NS3 protein could bind single-stranded RNA(ssRNA)but not double-stranded RNA(dsRNA), and its ability of ssRNA binding was related with protein concentration.NS3 protein could bind both siRNA duplex and single-stranded siRNA.These results show that NS3 inhibits the siRNA-initiated RISC assembly pathway via binding to siRNAs and preventing RNA silencing initiator complex formation.To determine the subcellular localization of NS3 protein,NS3 gene was fused with GFP gene and inserted into a plant expression vector.The construct containing NS3 fused with GFP was introduced into the onion epidermis monolayer cells by particle bombardment.The results revealed that NS3 fusion protein accumulated in the cell cytoplasm and nucleus,but had the high accumulation in the nucleus. Accumulation of GFP fluorescence primarily to the nucleus was also observed by confocal microscopy in N.benthamiana leaves infiltrated with Agrobacterium tumefaciens containing the fusion protein of NS3 with GFP.In contrast,the NS3 mutant,NS3/3A:GFP,in which the three basic amino acids in the putative nuclear localization sequence(NLS)¹⁷³KKR¹⁷⁵were replaced with AAA substitutions,failed to target in the nucleus of tobacco cells,suggesting that NS3 protein may have a functional NLS.Immuno-gold labeling also showed that the NS3 protein accumulated mainly in the nuclei of rice plants infected with RSV while no gold labeling was observed in healthy rice leaf cells.The N.benthamiana,N.tabaccum and Oryza sativa plants were transformed with Agrobacterium tumefaciens containing the RSV NS3 gene under the control of cauliflower mosaic virus 35S promoter.PCR and Northern blot analysis confirmed the NS3 gene had integrated into the genomes of these plants.Transgenic rice and tobacco plants expressed NS3 protein did not induce any apparent phenotype alterations. Therefore,we conclude that RSV NS3 protein is not a symptom determinant.To determine the movement protein of RSV,a plasmid containing an infectious clone of potato virus X(PVX)defective in cell-to-cell movement and expressingβ-glucuronidase(GUS)was used for co-bombardment with plasmids containing ORFs from RSV onto N.benthamiana leaves.GUS staining showed cell-to-cell movement of the movement-defective PVX was restored only by co-bombardment with a plasmid containing NSvc4,not by plasmids containing other genes.When the construct containing NSvc4-GFP fuse gene was introduced into the tobacco epidermis cells by particle bombardment,the movement of fluorescence to the neighboring cells and the accumulation of GFP fluorescence to the nucleus and periplasm were observed by confocal microscopy.These results showed that NSvc4 of RSV is a movement protein.When the construct containing NSvc4-GFP fuse gene was introduced into the onion epidermis monolayer cells by particle bombardment or into the tobacco epidermis cells by infiltrating with A.tumefaciens,GFP fluorescence was observed near or within plant cell walls and nucleus.Immunocytochemistry observation showed NSvc4 was localized to plasmodesmata in RSV infected rice leaves or transgenic rice leaves expressing NSvc4.Thus,NSvc4 is a cell-to-cell movement of RSV with characteristics identical to those of other viral cell-to-cell movement proteins.The interaction between NSvc4 and coat protein(CP)was studied using yeast two-hybrid system and NSvc4 was not found to interact with CP in yeast cells,indicating that CP may not be required for cell-to-cell movement.The RT-PCR was use for detection of RSV in wheat leaf samples with yellow stripe symptoms,and specific products were amplified with specific primers for NS2, NS3 and nucleoprotein genes of RSV.Sequence analysis of PCR products(GenBank accession numbers AM397832-34)showed that they shared 97.3%,97.5%and 97% sequence identity at the nucleotide level and 99%,97.6%and 98.5%sequence identity at amino acid level with the NS2,NS3 and nucleoprotein genes of RSV isolate T(Acc.No.NC003754 and NC003776),respectively.The above results show that RSV is the pathogen of wheat yellow stripe disease.