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Tzvi Tzfira

Publications and source records attributed to Tzvi Tzfira.

At least 19 recordsLinked to original sources
PubMed2007

Biological systems of the host cell involved in Agrobacterium infection.

Genetic transformation of plants by Agrobacterium, which in nature causes neoplastic growths, represents the only known case of trans-kingdom DNA transfer. Furthermore, under laboratory conditions, Agrobacterium can also transform a wide range of other eukaryotic species, from fungi to sea urchins to human cells. How can the Agrobacterium virulence machinery function in such a variety of evolutionarily distant and diverse species? The answer to this question lies in the ability of Agrobacterium to hijack fundamental cellular processes which are shared by most eukaryotic organisms. Our knowledge of these host cellular functions is critical for understanding the molecular mechanisms that underlie genetic transformation of eukaryotic cells. This review outlines the bacterial virulence machinery and provides a detailed discussion of seven major biological systems of the host cell-cell surface receptor arrays, cellular motors, nuclear import, chromatin targeting, targeted proteolysis, DNA repair, and plant immunity--thought to participate in the Agrobacterium-mediated genetic transformation.

Active Transport, Cell Nucleus↗
PubMed2006

C2H2 zinc finger-SET histone methyltransferase is a plant-specific chromatin modifier.

Histone modification represents a universal mechanism for regulation of eukaryotic gene expression underlying diverse biological processes from neuronal gene expression in mammals to control of flowering in plants. In animal cells, these chromatin modifications are effected by well-defined multiprotein complexes containing specific histone-modifying activities. In plants, information about the composition of such co-repressor complexes is just beginning to emerge. Here, we report that two Arabidopsis thaliana factors, a SWIRM domain polyamine oxidase protein, AtSWP1, and a plant-specific C2H2 zinc finger-SET domain protein, AtCZS, interact with each other in plant cells and repress expression of a negative regulator of flowering, FLOWERING LOCUS C (FLC) via an autonomous, vernalization-independent pathway. Loss-of-function of either AtSWP1 or AtCZS results in reduced dimethylation of lysine 9 and lysine 27 of histone H3 and hyperacetylation of histone H4 within the FLC locus, in elevated FLC mRNA levels, and in moderately delayed flowering. Thus, AtSWP1 and AtCZS represent two main components of a co-repressor complex that fine tunes flowering and is unique to plants.

Amino Acid Sequence↗
PubMed2006

Plant transformation by Agrobacterium tumefaciens: modulation of single-stranded DNA-VirE2 complex assembly by VirE1.

Agrobacterium tumefaciens infects plant cells by the transfer of DNA. A key factor in this process is the bacterial virulence protein VirE2, which associates stoichiometrically with the transported single-stranded (ss) DNA molecule (T-strand). As observed in vitro by transmission electron microscopy, VirE2-ssDNA readily forms an extended helical complex with a structure well suited to the tasks of DNA protection and nuclear import. Here we have elucidated the role of the specific molecular chaperone VirE1 in regulating VireE2-VirE2 and VirE2-ssDNA interactions. VirE2 alone formed functional filamentous aggregates capable of ssDNA binding. In contrast, co-expression with VirE1 yielded monodisperse VirE1-VirE2 complexes. Cooperative binding of VirE2 to ssDNA released VirE1, resulting in a controlled formation mechanism for the helical complex that is further promoted by macromolecular crowding. Based on this in vitro evidence, we suggest that the constrained volume of the VirB channel provides a natural site for the exchange of VirE2 binding from VirE1 to the T-strand.

Agrobacterium tumefaciens↗
PubMed2006

Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta.

Bimolecular fluorescence complementation (BiFC) represents one of the most advanced and powerful tools for studying and visualizing protein-protein interactions in living cells. In this method, putative interacting protein partners are fused to complementary non-fluorescent fragments of an autofluorescent protein, such as the yellow spectral variant of the green fluorescent protein. Interaction of the test proteins may result in reconstruction of fluorescence if the two portions of yellow spectral variant of the green fluorescent protein are brought together in such a way that they can fold properly. BiFC provides an assay for detection of protein-protein interactions, and for the subcellular localization of the interacting protein partners. To facilitate the application of BiFC to plant research, we designed a series of vectors for easy construction of N-terminal and C-terminal fusions of the target protein to the yellow spectral variant of the green fluorescent protein fragments. These vectors carry constitutive expression cassettes with an expanded multi-cloning site. In addition, these vectors facilitate the assembly of BiFC expression cassettes into Agrobacterium multi-gene expression binary plasmids for co-expression of interacting partners and additional autofluorescent proteins that may serve as internal transformation controls and markers of subcellular compartments. We demonstrate the utility of these vectors for the analysis of specific protein-protein interactions in various cellular compartments, including the nucleus, plasmodesmata, and chloroplasts of different plant species and cell types.

Biological Assay↗
PubMed2006

CHRD, a plant member of the evolutionarily conserved YjgF family, influences photosynthesis and chromoplastogenesis.

Studies on the carotenoid-overaccumulating structures in chromoplasts have led to the characterization of proteins termed plastid lipid-associated proteins (PAPs), involved in the sequestration of hydrophobic compounds. Here we characterize the PAP CHRD, which, based on sequence homology, belongs to a highly conserved group of proteins, YER057c/YjgF/UK114, involved in the regulation of basic and vital cellular processes in bacteria, yeast and animals. Two nuclear genes were characterized in tomato plants: one (LeChrDc) is constitutively expressed in various tissues and the other (LeChrDi) is induced by stress in leaves and is upregulated by developmental cues in floral tissues. Using RNAi and antisense approaches, we show their involvement in biologically significant processes such as photosynthesis. The quantum yield of photosynthetic electron flow in transgenic tomato leaves with suppressed LeChrDi/c expression was 30-50% of their control, non-transgenic counterparts and was ascribed to lower PSI activity. Transgenic flowers with suppressed LeChrDi/c also accumulated up to 30% less carotenoids per unit protein as compared to control plants, indicating an interrelationship between PAPs and floral-specific carotenoid accumulation in chromoplasts. We suggest that CHRD's role in the angiosperm reproductive unit may be a rather recent evolutionary development; its original function may have been to protect the plant under stress conditions by preserving plastid functionality.

Amino Acid Sequence↗
PubMed2006

Agrobacterium-mediated genetic transformation of plants: biology and biotechnology.

Agrobacterium-mediated genetic transformation is the dominant technology used for the production of genetically modified transgenic plants. Extensive research aimed at understanding and improving the molecular machinery of Agrobacterium responsible for the generation and transport of the bacterial DNA into the host cell has resulted in the establishment of many recombinant Agrobacterium strains, plasmids and technologies currently used for the successful transformation of numerous plant species. Unlike the role of bacterial proteins, the role of host factors in the transformation process has remained obscure for nearly a century of Agrobacterium research, and only recently have we begun to understand how Agrobacterium hijacks host factors and cellular processes during the transformation process. The identification of such factors and studies of these processes hold great promise for the future of plant biotechnology and plant genetic engineering, as they might help in the development of conceptually new techniques and approaches needed today to expand the host range of Agrobacterium and to control the transformation process and its outcome during the production of transgenic plants.

Biotechnology↗
PubMed2006

On tracks and locomotives: the long route of DNA to the nucleus.

Molecular motors have prominent functions in organelle transport, cytoskeletal organization, division and motility. The dyneins are one of the three families of cytoskeleton-based molecular motors and they travel along the cytoplasmic microtubule network towards the minus end of the microtubule. This directed movement is used by DNA viruses to deliver their infectious genome and proteins to the host cell nucleus. In recent studies, it has been hypothesized that Agrobacterium species use a similar pathway to deliver their infectious unit--a large complex between single-stranded DNA and proteins--to the host cell nucleus and that a karyophilic protein carrier that can deliver synthetic DNA to the nucleus is also driven by a dynein motor. These studies shed light on the mechanism of Agrobacterium-mediated genetic transformation and could lead to new methods for the efficient transfection of synthetic DNA.

Active Transport, Cell Nucleus↗
PubMed2006

Mammalian cells.

Agrobacterium most likely can transform virtually all known plant species, and experimental protocols for Agrobacterium-mediated genetic transformation of yet more plant species, ecotypes, and cultivars are published almost on a daily basis. Interestingly, the Agrobacterium host range is not limited to the plant kingdom, and it has been shown to transform many species of fungi and even prokaryotes. The ability of Agrobacterium to genetically transform HeLa cells further widens the range of potential hosts of Agrobacterium to include humans and perhaps other animal species. Furthermore, because mammalian cells significantly differ from plant cells, they provide a useful experimental system for identification and functional characterization of plant-specific factors involved in the transformation process. Here, we present basic procedures for transfection and Agrobacterium-mediated genetic transformation of mammalian cells. We also demonstrate the use of mammalian cells for studies of the cellular components of the genetic transformation pathway.

Agrobacterium tumefaciens↗
PubMed2005

Involvement of KU80 in T-DNA integration in plant cells.

In Agrobacterium-mediated genetic transformation of plant cells, the bacterium exports a well defined transferred DNA (T-DNA) fragment and a series of virulence proteins into the host cell. Following its nuclear import, the single-stranded T-DNA is stripped of its escorting proteins, most likely converts to a double-stranded (ds) form, and integrates into the host genome. Little is known about the precise mechanism of T-DNA integration in plants, and no plant proteins specifically associated to T-DNA have been identified. Here we report the direct involvement of KU80, a protein that binds dsT-DNA intermediates. We show that ku80-mutant Arabidopsis plants are defective in T-DNA integration in somatic cells, whereas KU80-overexpressing plants exhibit increased susceptibility to Agrobacterium infection and increased resistance to DNA-damaging agents. The direct interaction between dsT-DNA molecules and KU80 in planta was confirmed by immunoprecipitation of KU80 dsT-DNA complexes from Agrobacterium-infected plants. Transformation of KU80-overexpressing plants with two separate T-DNA molecules resulted in an increased rate of extrachromosomal T-DNA to T-DNA recombination, indicating that KU80 bridges between dsT-DNAs and double-strand breaks. This last result further supports the notion that integration of T-DNA molecules occurs through ds intermediates and requires active participation of the host's nonhomologous end-joining repair machinery.

Antigens, Nuclear↗
PubMed2005

Agrobacterium is not alone: gene transfer to plants by viruses and other bacteria.

Agrobacterium-mediated genetic transformation is the most widely used technology for obtaining the overexpression of recombinant proteins in plants. However, complex patent issues related to the use of Agrobacterium as a tool for plant genetic engineering and the general requirement of establishing transgenic plants can create obstacles in using this technology for speedy research and development and for agricultural improvements in many plant species. Recent studies addressing these issues have shown that virus-based vectors can be efficiently used for high transient expression of foreign proteins in transfected plants and that non-Agrobacterium bacterial species can be used for the production of transgenic plants, laying the foundation for alternative tools for future plant biotechnology.

Gene Transfer Techniques↗
PubMed2005

A case of promiscuity: Agrobacterium's endless hunt for new partners.

Agrobacterium tumefaciens is a phytopathogenic bacterium that induces the 'crown gall' disease in plants by transfer and integration of a segment of its tumor-inducing (Ti) plasmid DNA into the genome of numerous plant species that represent most of the higher plant families. Recently, it has been shown that, under laboratory conditions, the host range of Agrobacterium can be extended to non-plant eukaryotic organisms. These include yeast, filamentous fungi, cultivated mushrooms and human cultured cells. In this article, we present Agrobacterium-mediated transformation of non-plant organisms as a source of new protocols for genetic transformation, as a unique tool for genomic studies (insertional mutagenesis or targeted DNA integration) and as a useful model system to study bacterium-host cell interactions. Moreover, better knowledge of the DNA-transfer mechanisms from bacteria to eukaryotic organisms can also help in understanding horizontal gene transfer--a driving force throughout biological evolution.

DNA, Bacterial↗
PubMed2005

Towards targeted mutagenesis and gene replacement in plants.

Advances in the development of biotechnological tools for plant gene disruption and repair have lagged behind the rapid progress made in whole-genome sequencing of many model and crop plant species. Plant DNA-repair machinery predominantly uses non-homologous end-joining (NHEJ), making the homologous recombination (HR)-based methods, which have proved fruitful for gene targeting in non-plant systems, unsuitable for use in plant systems. Two recent reports describe successful targeted mutagenesis and gene targeting in Arabidopsis by either harnessing the plant NHEJ machinery using site-specific induction of double-strand breaks (DSBs), or by activation of a HR pathway through overexpression of a yeast DNA recombination gene in transgenic plants. These reports provide a foundation from which new technologies for site-specific genome alterations in plant species can be developed.

Arabidopsis↗
PubMed2005

Uncoupling of the functions of the Arabidopsis VIP1 protein in transient and stable plant genetic transformation by Agrobacterium.

Agrobacterium-mediated genetic transformation of plants, a unique example of transkingdom DNA transfer, requires the presence of several proteins encoded by the host cell. One such cellular factor is VIP1, an Arabidopsis protein proposed to interact with and facilitate import of the bacterial DNA-protein transport (T) complexes into the plant cell nucleus. Thus, VIP1 is required for transient expression of the bacterial DNA, an early step in the transformation process. However, the role of VIP1 in subsequent transformation events leading to the stable expression of bacterial DNA was unexplored. Here, we used reverse genetics to dissect VIP1 functionally and demonstrate its involvement in the stable genetic transformation of Arabidopsis plants by Agrobacterium. Our data indicate that the ability of VIP1 to interact with the VirE2 protein component of the T-complex and localize to the cell nucleus is sufficient for transient genetic transformation, whereas its ability to form homomultimers and interact with the host cell H2A histone in planta is required for tumorigenesis and, by implication, stable genetic transformation.

Active Transport, Cell Nucleus↗
PubMed2005

pSAT vectors: a modular series of plasmids for autofluorescent protein tagging and expression of multiple genes in plants.

Autofluorescent protein tags represent one of the major and, perhaps, most powerful tools in modern cell biology for visualization of various cellular processes in vivo. In addition, advances in confocal microscopy and the development of autofluorescent proteins with different excitation and emission spectra allowed their simultaneous use for detection of multiple events in the same cell. Nevertheless, while autofluorescent tags are widely used in plant research, the need for a versatile and comprehensive set of vectors specifically designed for fluorescent tagging and transient and stable expression of multiple proteins in plant cells from a single plasmid has not been met by either the industrial or the academic communities. Here, we describe a new modular satellite (SAT) vector system that supports N- and C-terminal fusions to five different autofluorescent tags, EGFP, EYFP, Citrine-YFP, ECFP, and DsRed2. These vectors carry an expanded multiple cloning site that allows easy exchange of the target genes between different autofluorescence tags, and expression of the tagged proteins is controlled by constitutive promoters, which can be easily replaced with virtually any other promoter of interest. In addition, a series of SAT vectors has been adapted for high throughput Gateway recombination cloning. Furthermore, individual expression cassettes can be assembled into Agrobacterium binary plasmids, allowing efficient transient and stable expression of multiple autofluorescently tagged proteins from a single vector following its biolistic delivery or Agrobacterium-mediated genetic transformation.

Base Sequence↗
PubMed2005

Exploring cargo transport mechanics in the type IV secretion systems.

Type IV secretion systems (T4SSs) are used by various bacteria to deliver protein and DNA molecules to a wide range of target cells. These include systems that are directly involved in pathogenesis, such as the secretion of pertussis toxin by Bordetella pertussis into human cells and the delivery of single-stranded DNA (ssDNA) into plants by Agrobacterium. These complex systems are composed of proteins that span the bacterial cytoplasm. The Agrobacterium T4SS is composed of 12 virulence proteins and delivers its transferred ssDNA and several virulence protein substrates to a variety of eukaryotic cells. Recent studies on the Agrobacterium T4SS have revealed new information on the localization and structure of its proteins in the bacteria, the biochemical properties of its transport signal, the route of a DNA substrate through the secretion system, and the initial point of contact of the system with its host. These findings have expanded our knowledge and understanding of the still mostly obscure structure and function of the T4SSs.

Bacteria↗
PubMed2005

A versatile vector system for multiple gene expression in plants.

Today, cloning vectors that have been specifically designed to facilitate the fusion, overexpression or down-regulation of a variety of genes in plant cells are available from various sources. In most cases, their basic design allows the cloning of a single target gene, typically under a specific promoter, in parallel with the expression of selection and/or marker genes from the same vector. However, new and versatile systems now exist that expand the user's choice to a large number of promoters and terminators, and various autofluorescent tags confer the ability to express multiple genes from a single transformation vector.

Cloning, Molecular↗
PubMed2004

The VirE3 protein of Agrobacterium mimics a host cell function required for plant genetic transformation.

To genetically transform plants, Agrobacterium exports its transferred DNA (T-DNA) and several virulence (Vir) proteins into the host cell. Among these proteins, VirE3 is the only one whose biological function is completely unknown. Here, we demonstrate that VirE3 is transferred from Agrobacterium to the plant cell and then imported into its nucleus via the karyopherin alpha-dependent pathway. In addition to binding plant karyopherin alpha, VirE3 interacts with VirE2, a major bacterial protein that directly associates with the T-DNA and facilitates its nuclear import. The VirE2 nuclear import in turn is mediated by a plant protein, VIP1. Our data indicate that VirE3 can mimic this VIP1 function, acting as an 'adapter' molecule between VirE2 and karyopherin alpha and 'piggy-backing' VirE2 into the host cell nucleus. As VIP1 is not an abundant protein, representing one of the limiting factors for transformation, Agrobacterium may have evolved to produce and export to the host cells its own virulence protein that at least partially complements the cellular VIP1 function necessary for the T-DNA nuclear import and subsequent expression within the infected cell.

Bacterial Proteins↗