S-98. Subcellular studies on the functional states of viral factors and on their interactions with plant factors, using bimolecular fluorescence complementation
Abstract
Bimolecular fluorescence complementation (BiFC) is a novel technique that allows the visualisation in real time of protein/protein interactions inside a living cell. With this technique, the biologically active states of some viral suppressors of gene silencing, their interactions with factors from the plant, and the role of subcellular compartimentalisation and trafficking on their functionalities were studied in vivo in epidermal cells of Nicotiana benthamiana.This was made through the transient expression of these factors in the plant from agroinfiltrated T-DNAs, fused to appropriate markers (N- and C-terminal split yellow fluorescence protein), coupled with confocal microscopy.
Using BiFC, the aggregated state of the 2b suppressor from Cucumber mosaic virus (CMV strain Fny) in the cell nucleus was shown for the first time, whereas those previously known of the suppresors P19 of Tomato bushy stunt virus and HCPro from Potato virus Y (PVY) were visualised for the first time in a living cell. Interaction of the suppressor P19 dimer with members of the host family of proteins ALY altered its subcellular distribution (and also that of ALY) and biological activity but did preserve the dimeric state of the former.
Interactions found in heterologous systems between viral suppressors and novel host factors, as well as the role of the cofactor P1 from PVY on the subcellular distribution and activity of the suppressor HCPro were also studied in vivo by BiFC.
