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; Mahsa Yousefi-Pour H.; Sonbol Nazeri
Abstract
The fungus Colletotrichum gloeosporioides is the causative agent of anthracnose disease of many tropical, subtropical and temperate fruits, and a microbial source of the anticancer drug, Taxol. Here, we introduce an optimized Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for genetic ...
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The fungus Colletotrichum gloeosporioides is the causative agent of anthracnose disease of many tropical, subtropical and temperate fruits, and a microbial source of the anticancer drug, Taxol. Here, we introduce an optimized Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for genetic manipulation of this fungus using hph and gfp-tagged hph genes as selection markers. Results showed that falcate spores can be easily used instead of protoplasts for transformation. Several experimental parameters were shown to affect transformation efficiencies, among which the length of co-cultivation, the ratio of fungal conidia to bacterium during co-cultivation, the kind of membrane during co-cultivation, and the kind of fungal growth medium during transformants selection, showed the highest influences on ATMT frequencies. Results indicated that the optimal ATMT of C. gloeosporioides was achived after 3 days of co-cultivation, at 107 per ml fungal conidia, via the use of Fabriano 808 filter paper and Czapek's culture medium. Successive subculturing of transformants on selective and non-selective media demonstrated the stable expression of transgens, and subsequent PCR based analyses of transformants revealed the presence (100%) of transferred genes. Flourescence microscopy analyses showed a punctuate pattern for localization of an expressed Gfp-tagged Hph protein inside fungal hyphae. The optimized ATMT protocol generated mutants that showed different phenotypes based on their vegetation and pigmentation. This suggests the possible applicability of this technique for functional genetics studies in C. gloeosporioides, through insertional mutagenesis.
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.
