RNA silencing suppression activity of the proteins encoded by the brome mosaic virus RNA genome
Weber, Philipp Heinrich
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The purpose of this study was to investigate the RNA silencing suppression activity of the proteins expressed by the Brome mosaic virus (BMV). For this purpose a binary, pCambia-based vector containing a GFP (Green Fluorescence Protein) gene was introduced into leaves of Nicotiana benthamiana (N. benthamiana) by agroinfiltration. The GFP gene gets transiently expressed, visualized by fluorescence activity under UV-light, and partially silenced by the RNA silencing activity (RNAi) of N. benthamiana, as described before. The RNAi-based silencing in turn can be suppressed by a co-infiltrated and also transiently expressed vector, which carries a RNA silencing suppressor-gene. The suppression of RNA silencing can be visualized by a higher intensity of fluorescence under UV-light. Two pROK2-based binary vectors, expressing the open reading frames (ORF) of either only the movement protein (MP) 3a or the coat protein (CP) were constructed. The expression of the genes and therefore the presence of the correspondent proteins in the plant leaves was verified by western blot analysis. The T-DNA constructs expressing the two other BMV proteins 1a (BMV RNA 1) and 2a (BMV RNA 2) were received from Prof. Kao. The vector encoding the GFP-gene, as well as a construct harboring the 2b-gene, used as positive control, was received from Dr. Canto. Every plasmid was introduced separately into Agrobacterium tumefaciens via electroporation. Subsequently, the agrobacteria suspensions were agroinfiltrated into N. benthamiana. The agrobacteria carrying the gene to be tested were always coinfiltrated with agrobacteria carrying the GFP-gene as a reporter gene. After three days, the RNA silencing suppression activity of the transiently expressed gene was determined by the comparison of the intensity of fluorescence under UV-light against a positive respective negative control. All proteins encoded by the BMV RNA genome displayed the same intensity of fluorescence as the negative control. The positive control always showed a much higher intensity of fluorescence than any of the proteins to be tested for RNA silencing suppression activity. The results indicate that none of the BMV proteins has local RNA silencing suppression activity.