Studies On SiRNA Interfering TMV Infection And Its Molecular Mechanism

RNA interference (RNAi) is a phenomenon in which double-strand RNA (dsRNA) specifically suppresses the expression of target protein by degrading the target mRNA. It is thought to be an ancient cellular defense system acting against different molecular parasites, including transgenes, viruses and transponsons. Small interference RNAs (siRNAs) of 21 nt in size, an intermediated of the RNA-interfrence pathway, are effective in suppression of gene expression and virus infection in animal systems and human cells. However, siRNA-directed interference with virus infection was seldom been reported in plants. The Agrobacterium tumefaciens-mediated transient expression system is a versatile tool to rapidly introduce gene into plant tissue. This system enables gene expression within a short period of time and without the requirement for regenerating transgenic plants. A useful feature of this system is the ability to introduce multiple genes simultaneously into a patch of leaf tissue. This system will be potent for high-throughput functional genomic and proteomic analysis. The tobacco mosaic virus (TMV) has a single-stranded RNA genome and as an ancient model virus in plants. The usage of siRNA, an intermediate in the silencing pathway, to target the TMV genomic would be a valuable tool to study antiviral theory and therapy in other virus. Therefore, we envisaged the use of siRNA to target the open reading frame (ORF) of 126kDa protein, coat protein and movement protein, which could be an effective way to counter TMV by Agrobacterium-mediated transient expression system. Oligonucleotides expressed siRNA were designed and synthesized according to the target protein gene of Tobacco Mosaic Virus (TMV) and subcloned to the binary vector pBI121. Then, these constructs were introduced into A.tumefaciens strain EHA105 by direct transformation. Our results showed: 1. Constructs targeting the open reading frame (ORF) of TMV containing 126kDa protein, coat protein and movement protein, which transiently expressed siRNA in plant, were performed respectively. 2. The Agrobacterium tumfaciens-mediated transiently expression of siRNA targeting 126 kDa protein of TMV could interfere with TMV infection in intact plant tissue. All plants infiltrated with pBI/siRNA1519-constructs and pBI121 displayed disease symptoms in upper leaves at 14 days postinoculation (dpi), whereas 28 of 34 (83%) plants that were agro-infiltrated with the pBI/siRNA1519 constructs was immune. A similar frequency (85%) of the plants agro-infiltrated with pBI/siRNA2129 was free of symptoms. Consistent with the above results, Northern blot analysis showed TMV RNA level in tobacco leaves was significantly reduced when infiltrated with pBI/siRNA1519 and pBI/siRNA2129 constructs. In contrast, viral RNA was abundant in plants infiltrated with A.tumfaciens containing empty vector pBI121 and pBI/siRNA1519-constructs. Moreover, the interference observed is sequence specific, time and site dependent. SiRNA-directed interference with TMV infection can't take place, when transiently expressed siRNA( five different nucleotides from siRNA1519). Also, transiently expressed siRNA corresponding to TMV 126 kDa protein can't inhibit cucumber mosaic virus (CMV), an unrelated the genus tobamovirus. 3. Transiently expressed siRNA targeting coat protein could specifically interfere with TMV infection. The upper leaves of Nicotiana tobacum infiltrated with Agrobacterium tumefaciens cultures containing pBI121/siRNA were free of mosaic symptom after 14 dpi. Northern blot assays also showed there were no or much less TMV RNA accumulation in these leaves. As for local lesion host, Nicotiana glutinosa, ttransiently expressed siRNA could reduce the lesion number resulting from TMV infection. 4. Transiently expressed siRNA targeting movement protein gene could specifically interfere with TMV infection. When inoculated with TMV, the upper leaves of Nicotiana tobacum infiltrated with Agrobacterium tumefaciens cultures containing pBI121/siRNA were free of mosaic symptom. ELISA assays also showed there were a few TMV in these leaves. This indicated that siRNA could interfere with TMV spread and movement in tobacco plants. As for local host, Nicotiana glutinosa, ttransiently expressed siRNA could significantly reduce the lesion number and even no lesion generated resulting from TMV infection. 5. Co-infiltration assay showed siRNA-directed interference effect was completely blocked by co-infiltration with HC-Pro plus siRNA constructs in both systemic and hypersensitive hosts. In the system host, all plants agro-infiltrated with HC-Pro plus siRNA constructs displayed the same mosaic symptoms as the negative control. On the contrary, plants agro-infiltrated with the siRNA construct alone were free of symptoms. Meanwhile, TMV RNA accumulation was found to be abundant in the upper leaves using Reverse transcriptase-PCR (RT-PCR) and Northern blot assays. Therefore, our study could be in favor of open out some issues about mechanism of PTGS. In order to detect the antiviral effects of siRNA in plant, the combination of siRNA technique and agrobacterium-mediated transient expression system was used to interfere with TMV infection. This approach could shed light on the potent application as an antiviral treatment in plants.