Study On The Cnv P20 Function Of Viral Suppressor Of Rna Silencing

Conserved in many eukaryotic cells, RNA silencing is a mechanism that suppresses gene through RNA-mediated sequence-specific interactions. In plants, RNA silencing plays an important role against virus infection. A counter-defensive strategy has evolved by encoding suppressor proteinsviral suppressor of RNA silencing, to overcome RNA silencing in plant viruses. For example, the Tomato Bushy Stunt Virus (TBSV)p19 gene has been identified to encode a viral suppressor of RNA silencing (VSR).Cucumber Necrosis Virus (CNV)is a member of tombusvirus genus in the family Tombusvirida. The genome of CNV is a positive sense single-strand RNA with 4701nt in length that contains five open reading frames (ORF). Among of them, ORF5 encodes a 20KD protein named CNV P20.The CNV p20 shares sequence similarity with the TBSV p19, 75% homology identity in amino acid sequence. The ORFs encoding CNV p20 and TBSV p19 locates similarly in the viral genome. However, the gene function of CNV p20 keeps unknown.In the present study, we study on the gene function of CNV p20 in the plant systematically through the virus-induced gene silencing (VIGS) system and Agrobacterium-mediated gene silencing assay system. Our results identified that CNV p20 worked as a VSR during virus infection, but it fails to show the activity of RNA silencing suppressor effectively in an artificial Agrobacterium-mediated silencing assay system. The main contents were as follows:1: The VIGS system was adopted to identify the role of CNV p20 in the virus infection. Two CNV mutants containing green fluorescence protein (GFP) gene, one CNV p20 knock-out and the other not, were used to inoculate GFP transgenic N. benthamiana, line 16c, and the green fluorescence was monitored under UV illumination. The results showed that the local gene silencing and systemic gene silencing were induced in the 16c plants inoculated with the CNV mutant p20 knock-out, and no gene silencing was observed in the 16c plants of the CNV mutant with p20 gene. Northern blot analysis confirmed that the GFP-transgene mRNA was dramatically reduced in the.16c plants inoculated with CNV mutant containing p20 gene when compared to that CNV p20 knock-out as well as the control mock inoculation. Further study identified that the CNV mutant GFP gene unexpressed still may induce the GFP-transgene silencing in the 16c plants. The CNV p21 sharing the overlap gene sequence with p20 was also examined to be not involved in the RNA silencing suppresses. All of results above identified that CNV p20 is a VSR in plant.2: In an Agrobacterium-mediated silencing assay, CNV p20 failed to play a role of VSR as TBSV p19. Moreover, none of the CNV genes, alone or in combination were found to assist p20 in suppression of GFP-transgene silencing in the Agrobacterium-mediated silencing assay. The mRNA and protein of CNV p20 expressed at a low level, only 1/100~1/50 times of TBSV p19 in infiltrated plants. The Q-PCR result revealed that the GFP transgene RNA kept approximately same level at 4dpi in plants infiltrated by GFP and CNV p20, which was far lower than that of TBSV p19. We changed another expression vector and made the mutants on some amino acid of CNV p20 to increase the protein expression, but the help was limited in the Agrobacterium-mediated silencing assay system. However, CNV p20 could enhance transient exprsssion of a protein co-infiltrated in plant as TBSV p19 did. The results demonstrated that CNV p20 functioned like a VSR in suppression of RNA silencing and its suppression activity was dependent on p20 dosage in plant.3: To investigate the role of p20 in CNV infection, transcripts of wild type CNV and a p20 knockout CNV mutant were inoculated in plants. The viral RNA was examined and the result revealed that the CNV p20 suppressed the RNA silencing induced by virus during CNV infection. Viral proteins were examined from the plants inoculated by equal amounts of TBSV and CNV virions. The result showed that the two proteins are present at an almost equal level in infected plants. This showed that CNV p20 worked as a VSR like TBSV p19 during virus infection.4: Based on the similarity of CNV p20 and TBSVp19 gene sequence and function, we predicted and analyzed the bioinformatics of secondary and tertiary proteins structure. The secondary structure predicted that CNV P20 had similar protein monomer like TBSV P19, including five alpha helices and four beta sheets. The 3-D crystal structure of CNV P20 predicted that CNV P20 and TBSV P19 shared similar monomer and dimmer structure, both of them formed a"caliper"structure binding the siRNA duplex. CNV P20 contained eight of the ten amino acid residues that were demonstrated to interact with the siRNA duplex in TBSV p19. However, one replacement of amino acid residue in CNV p20 might not affect its suppressor activity; the other might be able to compensate the lack of the amino acid residue by its neighbor residue inCNV P20. the data indicated that CNV P20 might bind the siRNA duplex like TBSV P19 to prevent siRNA from incorporation into the RISC complex, thereby suppress RNA silencing induced by virus.