The significance of C-terminal NLS sequences of VirD2 in its nuclear localization in Saccharomyces cerevisiae

Abstract

Agrobacterium tumefaciens is capable of gene transfer to both plant and non-plant organisms. Indeed, upon infection of eukaryotic cells, Agrobacterium tumefaciens transfers a piece of its tumor inducing (Ti)-plasmid, called T-DNA, to the host cell nucleus, which subsequently integrates into the host genome. The VirD2 virulence protein which has relaxase endonuclease activities covalently binds to the 5'end of T-DNA and facilitates its transfer, nuclear localization and integration into the host genome in collaboration with the interacting proteins of the host cell. The VirD2 is essential for Agrobacterium–mediated transformation of both plants and non-plant cells. Here, using yeast Green Flourescent Protein (yGFP) technology, we studied the subcellular localization of VirD2, expressed in the model eukaryote Saccharomyces cerevisiae. Fluorescence microscopy showed that an N-terminal yGFP fusion of VirD2 (i.e. 5' GFP-VirD2 3'), was located in the nucleus of yeast. With C-terminal fusions of VirD2 to yGFP (i.e. 5' VirD2-GFP 3'), no particular subcellular concentration of fluorescence was seen. This further confirms nuclear localization of VirD2 in eukaryotic cells and more importantly highlights the role of Nuclear Localization Signal sequences (NLS) of the C-terminal of VirD2 in this phenomenon.

Keywords

• vird2
• nuclear delivery
• gfp
• agrobacterium
• saccharomyces cerevisiae