Jalal Soltani; Jonathan A. Lal; G. Paul H. van Heusden; Paul J.J. Hooykaas
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 ...
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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.
Jalal Soltani; Raymond Brandt; Gerard Paul H. van Heusden; Paul J. J. Hooykaas
Abstract
Agrobacterium tumefaciens is capable to transfer genes across kingdoms. It can genetically transform not only plant cells, but also many other bacterial, algal, fungal, animal and human cells. This depends on the interactions among a variety of both Agrobacterium and host genes. Inside the host cell, ...
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Agrobacterium tumefaciens is capable to transfer genes across kingdoms. It can genetically transform not only plant cells, but also many other bacterial, algal, fungal, animal and human cells. This depends on the interactions among a variety of both Agrobacterium and host genes. Inside the host cell, RAD52 which is involved in DNA repair is a key gene determining integration of T-DNA by homologous recombination. Here, using Saccharomyces cerevisiae haploid strains BY4741 and BY4742, a rad52 diploid deletion strain is constructed in yeast BY4743 background.This model organism was employed to show that RAD52 deletion severely decreases frequencies of Agrobacterium genetic transformation mediated by either an integrative T-DNA or a circular non-integrative T-DNA. Indeed, the frequencies of such Agrobacterium-mediated transformation (AMT) decreased by ca. 25-fold. Hence, host RAD52 deletion might affect AMT by a mechanism which differs from its only involvement in DNA repair in yeast.
