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Vitaly Citovsky

Publications and source records attributed to Vitaly Citovsky.

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

How pollen tubes grow.

Sexual reproduction of flowering plants depends on delivery of the sperm to the egg, which occurs through a long, polarized projection of a pollen cell, called the pollen tube. The pollen tube grows exclusively at its tip, and this growth is distinguished by very fast rates and reaches extended lengths. Thus, one of the most fascinating aspects of pollen biology is the question of how enough cell wall material is produced to accommodate such rapid extension of pollen tube, and how the cell wall deposition and structure are regulated to allow for rapid changes in the direction of growth. This review discusses recent advances in our understanding of the mechanism of pollen tube growth, focusing on such basic cellular processes as control of cell shape and growth by a network of cell wall-modifying enzymes, molecular motor-mediated vesicular transport, and intracellular signaling by localized gradients of second messengers.

Arabidopsis↗
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

Suppressor of RNA silencing encoded by Tomato yellow leaf curl virus-Israel.

The Israeli isolate of Tomato yellow leaf curl geminivirus (TYLCV-Is) is a major tomato pathogen, causing extensive crop losses both in the New and Old World. Surprisingly, however, little is known about the molecular mechanisms of TYLCV-Is interactions with tomato cells. Here, we have identified a TYLCV-Is protein, V2, which acts as a suppressor of RNA silencing and which is unrelated to presently known viral suppressors. Specifically, V2, but not other proteins of TYLCV-Is, inhibited RNA silencing of a reporter transgene, GFP. This inhibition elevated the cellular levels of the GFP transcript and the GFP protein, but it had no apparent effect on the accumulation of GFP-specific short interfering RNAs (siRNAs), suggesting that TYLCV-Is V2 targets a step in the RNA silencing pathway which is subsequent to the Dicer-mediated cleavage of dsRNA. Visualization of the sub-cellular localization of TYLCV-Is V2 in plant protoplasts and tissues showed that this protein is associated with cytoplasmic strands and inclusion bodies in the cortical regions of the cell.

Begomovirus↗
PubMed2006

Agrobacterium-mediated genetic transformation of tea leaf explants: effects of counteracting bactericidity of leaf polyphenols without loss of bacterial virulence.

Tea is one of the major crops in Asia and Africa, and its improvement by genetic modification is important for economy of many tea-producing regions. Although somatic embryos derived from cotyledon explants have been transformed with Agrobacterium, the leaves of several commercially important tea cultivars have remained recalcitrant to transformation, largely due to bactericidal effect of polyphenols that are exuded by tea leaves in vitro. Moreover, the commonly used polyphenol adsorbents and antioxidants cannot overcome this problem. Leaf explants, however, are more desirable than cotyledon-derived somatic embryos, especially when it is necessary to further improve a selected elite and also retain its superior traits. Thus, we developed a procedure for Agrobacterium-mediated genetic transformation of tea leaf explants which is based on the presence of L-glutamine in the co-cultivation medium. We then showed that the transformation process is facilitated via a protective action of L-glutamine against bactericidal effects of leaf polyphenols without affecting the bacterial virulence (vir) gene expression.

Anti-Bacterial Agents↗
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

Pollen-specific pectin methylesterase involved in pollen tube growth.

Pollen tube elongation in the pistil is a crucial step in the sexual reproduction of plants. Because the wall of the pollen tube tip is composed of a single layer of pectin and, unlike most other plant cell walls, does not contain cellulose or callose, pectin methylesterases (PMEs) likely play a central role in the pollen tube growth and determination of pollen tube morphology. Thus, the functional studies of pollen-specific PMEs, which are still in their infancy, are important for understanding the pollen development. We identified a new Arabidopsis pollen-specific PME, AtPPME1, characterized its native expression pattern, and used reverse genetics to demonstrate its involvement in determination of the shape of the pollen tube and the rate of its elongation.

Arabidopsis↗
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

Nuclear export of African swine fever virus p37 protein occurs through two distinct pathways and is mediated by three independent signals.

Nucleocytoplasmic shuttling activity of the African swine fever virus p37 protein, a major structural protein of this highly complex virus, has been recently reported. The systematic characterization of the nuclear export ability of this protein constituted the major purpose of the present study. We report that both the N- and C-terminal regions of p37 protein are actively exported from the nucleus to the cytoplasm of yeast and mammalian cells. Moreover, experiments using leptomycin B and small interfering RNAs targeting the CRM1 receptor have demonstrated that the export of p37 protein is mediated by both the CRM1-dependent and CRM1-independent nuclear export pathways. Two signals responsible for the CRM1-mediated nuclear export of p37 protein were identified at the N terminus of the protein, and an additional signal was identified at the C-terminal region, which mediates the CRM1-independent nuclear export. Interestingly, site-directed mutagenesis revealed that hydrophobic amino acids are critical to the function of these three nuclear export signals. Overall, our results demonstrate that two distinct pathways contribute to the strong nuclear export of full-length p37 protein, which is mediated by three independent nuclear export signals. The existence of overlapping nuclear export mechanisms, together with our observation that p37 protein is localized in the nucleus at early stages of infection and exclusively in the cytoplasm at later stages, suggests that the nuclear transport ability of this protein may be critical to the African swine fever virus replication cycle.

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

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

Identification of an interactor of cadmium ion-induced glycine-rich protein involved in regulation of callose levels in plant vasculature.

Cadmium-induced glycine-rich protein (cdiGRP) is a cell wall-associated factor that increases callose levels in plant vasculature. To better understand the cdiGRP/callose regulation system, we identified a tobacco protein, GrIP (cdiGRP-interacting protein, GrIP), that associates with cdiGRP and localizes at the plant cell wall. Constitutive overexpression of GrIP enhanced the accumulation of the cdiGRP protein and callose in vasculature-associated cells with or without treatment with cadmium ions. That GrIP gene expression was not affected by cadmium ions indicated that GrIP does not directly modulate the callose levels induced by the treatment. Instead, GrIP most likely functions by further elevating the accumulated amount of cdiGRP, the expression of which is up-regulated by the cadmium ions. Interestingly, the levels of cdiGRP mRNA were not affected by constitutive expression of GrIP, demonstrating that the enhancement in cdiGRP protein accumulation by GrIP overexpression occurs posttranslationally. Collectively, these observations suggest that GrIP interacts with cdiGRP and increases its level of accumulation; in turn, the elevated amounts of cdiGRP induce callose deposits in the plant cell walls. Therefore, GrIP and cdiGRP represent sequentially acting factors in a biochemical pathway that regulates callose accumulation in the plant vasculature.

Blotting, Western↗
PubMed2005

Effects of calreticulin on viral cell-to-cell movement.

Cell-to-cell tobacco mosaic virus movement protein (TMV MP) mediates viral spread between the host cells through plasmodesmata. Although several host factors have been shown to interact with TMV MP, none of them coresides with TMV MP within plasmodesmata. We used affinity purification to isolate a tobacco protein that binds TMV MP and identified it as calreticulin. The interaction between TMV MP and calreticulin was confirmed in vivo and in vitro, and both proteins were shown to share a similar pattern of subcellular localization to plasmodesmata. Elevation of the intracellular levels of calreticulin severely interfered with plasmodesmal targeting of TMV MP, which, instead, was redirected to the microtubular network. Furthermore, in TMV-infected plant tissues overexpressing calreticulin, the inability of TMV MP to reach plasmodesmata substantially impaired cell-to-cell movement of the virus. Collectively, these observations suggest a functional relationship between calreticulin, TMV MP, and viral cell-to-cell movement.

Amino Acid Sequence↗
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↗